WO2008051326A2 - Identification of contactins and l1- cams as ligands for the amyloid precursor protein - Google Patents

Identification of contactins and l1- cams as ligands for the amyloid precursor protein Download PDF

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WO2008051326A2
WO2008051326A2 PCT/US2007/018428 US2007018428W WO2008051326A2 WO 2008051326 A2 WO2008051326 A2 WO 2008051326A2 US 2007018428 W US2007018428 W US 2007018428W WO 2008051326 A2 WO2008051326 A2 WO 2008051326A2
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app
contactin
binding
protein
aplp2
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PCT/US2007/018428
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WO2008051326A3 (en
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Miriam E. Osterfield
John G. Flanagan
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President And Fellows Of Harvard College
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention relates to molecules that bind amyloid precursor protein and to the biological function of amyloid precursor protein.
  • the invention particularly relates to methods and products for preventing and treating Alzheimer's disease and abnormal production of amyloid ⁇ .
  • the invention also relates to methods for screening and identifying small molecule drugs and other agents that may affect processing or function of amyloid precursor protein.
  • the beta-amyloid precursor protein has been linked to several processes that might be important for the development and function of the nervous system, including neuronal survival and apoptosis, cell adhesion and migration, neurite outgrowth, synaptogenesis, and synaptic plasticity (Mattson, 1997; also, see below).
  • APP Amyloid Precursor Protein
  • AD Alzheimer's Disease
  • beta-amyloid which is produced from APP by proteolytic processing.
  • APP itself may have beneficial roles in normal neural function which are interrupted in AD.
  • APP APP protein
  • a number of extracellular binding partners for APP have previously been identified (metal ions, heparin, collagen, F-spondin, Notch, Nogo receptor, and others) but the functional significance of these interactions, and the biological role of APP, generally remain unclear.
  • agents capable of binding to APP and modulating (e.g., promoting or inhibiting) APP function particularly by altering the proteolytic processing of APP.
  • Such agents would be beneficial for the treatment and prevention of development and progression of diseases, such as AD.
  • the invention provides novel interactions of APP with extracellular binding partners in two molecular families: Contactins (particularly Contactin-3 and -4) and Ll family protiens (particularly Ll and NgCAM). These interactions can regulate processing of APP. As disclosed herein, the invention also provides that APP, contactin-4 and NgCAM can collaborate in regulating the development of neural connections. The invention also includes methods to identify compounds that modulate the binding of APP to these extracellular binding partners, as well as compounds that modulate APP processing. The methods and compositions of the invention are also useful for preventing and/or treating A ⁇ -accumulation- associated disorders, such as AD. According to one aspect of the invention, methods for modulating APP processing are provided.
  • the methods include contacting a mammalian cell with an agent that modulates the amount or rate of binding of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2) to contactins and/or Ll family proteins.
  • the agent is expressed by the cell or exogenously added so as to contact the cell.
  • the agents include one or more contactins such as contactin-3. contactin-4 and contactin-5, or Ll family proteins, such as Ll and Ng-CAM, or fragments thereof that bind to APP, APLPl or APLP2.
  • the agent is a fusion protein including one or more contactins, Ll family proteins or fragments.
  • the fusion protein is an Fc fusion protein.
  • the agent is an antibody or an antigen-binding fragment thereof.
  • the antibody is selected from an antibody or antigen-binding fragment thereof that binds a contactin or a Ll family protein, preferably contactin-3 contactin-4, contactin-5, Ll or Ng-CAM 5 or, an antibody or antigen- binding fragment thereof binds APP, APLPl or APLP2.
  • the antibody or antigen-binding fragment thereof binds to a complex of a contactin or an Ll family protein and APP, APLPl or APLP2.
  • the antibody or antigen-binding fragment thereof is a bispecific antibody.
  • the methods include agents that block or disrupt the binding of APP, APLPl or APLP2 to contactins and/or Ll family proteins and agents that promote or stabilize the binding of APP, APLPl or APLP2 to contactins and/or Ll family proteins.
  • the methods also include agents that can reduce processing of APP to ⁇ - amyloid.
  • methods for treating or preventing include agents that block or disrupt the binding of APP, APLPl or APLP2 to contactins and/or Ll family proteins and agents that promote or stabilize the binding of APP, APLPl or APLP2 to contactins and/or Ll family proteins.
  • methods for treating or preventing include agents that can reduce processing of APP to ⁇ - amyloid.
  • the methods include administering to a subject an effective amount of an agent that modulates the amount or rate of binding of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2) to contactins and/or Ll family proteins.
  • the agents include one or more contactins or Ll family proteins or fragments thereof that bind to APP 5 APLPl or APLP2.
  • the contactin is contactin-3 contactin-4, contactin-5 or a combination thereof
  • the Ll-CAM is Ll or Ng- CAM
  • the fragment is preferably a fragment of contactin-3, contactin-4, Ll or Ng- CAM that binds to APP, APLPl or APLP2, or a combination of such fragments.
  • the agent is a fusion protein, preferably an Fc fusion protein, including one or more contactins, Ll family proteins or fragments thereof.
  • the agent is an antibody or an antigen-binding fragment thereof.
  • the agents include an antibody or antigen- binding fragment thereof that binds a contactin or a Ll family protein, particularly contactin-3 contactin-4, contactin-5, Ll or Ng-CAM 5 as well as APP 5 APLPl or APLP2.
  • the antibody or antigen-binding fragment thereof binds a complex of a contactin or an Ll family protein and APP 5 APLPl or APLP2.
  • the methods include antibody or antigen-binding fragment thereof that is a bispecific antibody.
  • the agent blocks or disrupts the binding of APP, APLPl or APLP2 to contactins and/or Ll family proteins.
  • the agent promotes or stabilizes the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs.
  • the methods includes an agent that reduces processing of APP to ⁇ -amyloid.
  • the methods include a vectorized agent that crosses the blood brain barrier. According to the invention, the methods are used for various subject, where a preferred subject is a human.
  • compositions include one or more contactins and/or Ll family proteins and/or one fragments thereof that bind to APP, APLPl or APLP2, and a pharmaceutically acceptable carrier.
  • the contactin is contactin-3 contactin- 4, contactin-5 or a combination thereof
  • the Ll-CAM is Ll or Ng-CAM.
  • the pharmaceutical composition includes a fragment of contactin-3, contactin- 4, Ll or Ng-CAM that binds to APP, APLPl or APLP2, or a combination of such fragments.
  • some embodiments include a pharmaceutical composition that is a fusion protein, including one or more contactins, Ll-CAMs or fragments thereof. In some cases, the composition includes an Fc fusion protein.
  • the pharmaceutical compositions include one or more antibodies that bind a contactin, an Ll family protein, APP, APLPl or APLP2 or antigen- binding fragments thereof, where preferred contactin or Ll-CAM is contactin-3 contactin-4, contactin-5, Ll or Ng-CAM.
  • the pharmaceutical composition includes an antibody or antigen-binding fragment thereof that binds a complex of a contactin or a Ll-CAM and APP, APLPl or APLP2.
  • the antibody or antigen-binding fragment thereof is a bispecific antibody.
  • the pharmaceutical composition in some embodiments includes an agent that blocks or disrupts the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs. In other embodiments, the pharmaceutical composition includes an agent that promotes or stabilizes the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs. In some embodiments of the foregoing composition, the agent reduces processing of APP to ⁇ -amyloid. In some cases, the pharmaceutical composition according to the foregoing includes a vectorized agent that crosses the blood brain barrier of a subject, preferably a human.
  • APP amyloid precursor protein
  • APLPl amyloid precursor like protein 1
  • APLP2 amyloid precursor like protein 2
  • These methods include providing a reaction mixture that comprises (1) APP, APLPl, APLP2 and/or a fragment thereof that binds to contactins and/or Ll-CAMS, and (2) contactins, Ll- CAMs and/or a fragment thereof that binds to APP, APLPl, APLP2; contacting the reaction mixture with a test compound; determining a level of binding of APP, APLPl, APLP2 and/or a fragment thereof with contactins, Ll-CAMs and/or a fragment thereof in the absence and in the presence of the test compound; and comparing the level of binding of APP, APLPl, APLP2 or fragment thereof with contactins, Ll-CAMs and/or a fragment thereof in the absence and in the presence of the test compound, wherein a test compound that modulates the binding relative to the level of binding in the absence of the test compound is a compound that modulates the binding of APP, APLPl , APLP2 or fragment thereof with
  • the test compound is a small molecule; an antibody that binds to APP, APLPl, APLP2 a contactin or a Ll-CAM, or an antigen-binding fragment thereof; or a fragment of APP, APLPl , APLP2, a contactin or a Ll- CAM, where preferably the contactins and/or Ll-CAMs is selected from the group consisting of: contactin-3, contactin-4, contactin-5, Ll and Ng-CAM.
  • methods for identifying compounds that modulate the processing of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (AP LP2) include: providing a reaction mixture that comprises (1) contactins, Ll-CAMs and/or a fragment thereof that binds to APP, APLPl, APLP2, and (2) APP, APLPl, APLP2 and/or a fragment thereof that binds to contactins and/or Ll family proteins; contacting the reaction mixture with a test compound; determining a level of processing of APP, APLPl or APLP2 in the absence and in the presence of the test compound; and comparing the processing in the absence and in the presence of the test compound, wherein a test compound that modulates processing of APP, APLPl or APLP2 relative to the level of processing in the absence of the test compound is a compound that modulates the processing of APP, APLPl or APLP2.
  • the test compound is a small molecule; an antibody that binds to APP, APLPl, APLP2, a contactin or a Ll family protein, or an antigen-binding fragment thereof; a fragment of APP, APLPl or APLP2, a contactin or a Ll family protein, where preferably the contactins and/or Ll-CAMs is selected from the group consisting of: contactin-3, contactin-4, contactin-5, Ll and Ng-CAM.
  • the processing of APP to A ⁇ is reduced.
  • FIG. 1 Major proteolytic fragments of APP released in the amyloidogenic or non- amyloidogenic pathway. Not drawn to scale. N-terminus of APP is extracellular or luminal (up), and C-terminus is intracellular (down). Location of cleavage sites, according to APP695 amino acid numbering, is as follows: ⁇ 596-597, ⁇ 612-613, ⁇ (4 o) 636-637, ⁇ (4 2) 638- 639, ⁇ 645-646. The Ab region of APP is indicated in light gray.
  • FIG. 1 APP binds to embryonic chicken brain. AP-tagged probes were used in situ to detect binding sites for APP.
  • A Binding of AP-mAPP compared to AP alone; ventral view of E12-E13 brains. Olfactory bulbs are indicated by arrows, tecta by arrowheads.
  • B Binding of AP-mAPP to E9, ElO, and El 6 brains; dorsal view. Anterior (A) and posterior (P) extremes of tecta are indicated.
  • FIG. 5 Affinity purification of APP binding proteins from olfactory bulbs.
  • Supernatants from PI-PLC treated olfactory bulbs were affinity purified against AP- or AP-mAPP- conjugated beads. Arrows indicate bands specific for AP-mAPP sample; the lower band was analyzed by tandem mass spectrometry.
  • Figure 7 Identification of an APP interacting protein from tecta.
  • Figure 8 Identification of a panel of GPI-anchored olfactory bulbs proteins.
  • A Silver stained 2D gels of supernatants from olfactory bulbs after mock or PI-PLC treatment. Spots circled in the PI-PLC gel were excised and analyzed by tandem mass spectrometry.
  • Figure 9 Identification of a panel of tectal surface proteins.
  • FIG. 10 APP and related protein APLPl bind to specific contactin family members. Media containing Fc-fusion proteins were normalized for concentration, then bound to
  • Protein A beads Media containing AP fusion protein was normalized for AP activity, then exposed to these beads. AP activity retained by the beads after washing is shown.
  • FIG. 13 APP interacts with NgCAM.
  • A Western Blots of immunoprecipitates of AP or AP-mAPP after reversible crosslinking to tecta; the protocol used is the same as in Figure 6. Detection was by Avidin-HRP (for biotinylated proteins), or one of two different anti-NgCAM antibodies.
  • FIG. 14 APLPl binds to olfactory bulbs. AP in situs were used to detect binding of AP- APLPl or AP-APLPl Nterm compared to AP alone. Ventral view of E 12-El 3 brains; olfactory bulbs are indicated by arrows.
  • APLP2 binds to embryonic chick brains and an endothelial cell line.
  • A AP in situs were used to detect binding of AP-APLP2 middle; ventral view of E 12-El 3 brains, with magnified view of indicated region shown below
  • B AP in situs were used to detect binding of AP-APLP2 middle to RF/6A (rhesus monkey retinal choroid endothelial) cells, compared to binding of AP alone or AP-mAPP(l 99-345).
  • Fc Anti-HA western blot of cell lysates from 293T cells co-transfected with APP-HA and the indicated construct;
  • Fc refers to the pSecTaglg plasmid, which encodes secreted Fc.
  • Figure 20 Effect on RGC axon outgrowth of reagents directed against APP or APP cleavage.
  • FIG. 22 RGC outgrowth on NgCAM-Fc substrates that also contain the indicated Fc fusion protein.
  • amyloid precursor protein is a type 1 transmembrane protein with a large extracellular domain and a relatively small intracellular domain.
  • the cytoplasmic domain is ⁇ vell conserved among APP family members in both vertebrates and invertebrates, as are two extracellular domains termed El and E2 (Daigle and Li, 1993).
  • El and E2 two extracellular domains termed El and E2 (Daigle and Li, 1993).
  • the terms, "amyloid-beta precursor protein”, “beta-amyloid precursor protein” and “amyloid precursor protein” are used interchangeably.
  • amyloid-beta precursor protein or "beta-amyloid precursor protein” may be used to distinguish the precursor species that specifically give rise to the beta form of the peptide, as opposed to the term “amyloid precursor protein”, which is used generally to encompass all eventual peptide products (without prejudice for any particular products and/or intermediates, e.g., alpha, beta, gamma, and epsilon).
  • ⁇ and ⁇ as in “ ⁇ - amyloid” and “beta-amyloid" are used herein interchangeably.
  • ⁇ ", “ ⁇ ” and “ ⁇ ” may be expressed as “alpha”, "gamma” and “epsilon", respectively.
  • the extracellular portion of APP can be divided structurally into several parts: El, which contains the growth factor-like domain, or GFLD (amino acids 23-128), and the copper binding domain, or CuBD (amino acids 124-189); the acidic or anionic domain; E2, which is also called the central APP domain or CAPPD (289-479); the linker region (507- 589); and finally the N-terminal portion of the Abeta region (Reinhard et al., 2005).
  • GFLD growth factor-like domain
  • CuBD amino acids 124-189
  • E2 which is also called the central APP domain or CAPPD (289-479
  • the linker region (507- 589)
  • the N-terminal portion of the Abeta region Reinhard et al., 2005.
  • Several splice forms of APP have been reported, but the three major isoforms, called APP695, APP751, and APP770, according to the number of amino acids in the unprocesse
  • APP695 is the splice variant used in this study, so all amino acid numbering here is given accordingly.
  • APP751 only differs from APP695 by the inclusion of exon 7, which encodes an extracellular Kunitz protease inhibitor (KPI) domain, while APP770 includes both the KPI domain and exon 8, the OX-2 domain (Coulson et al., 2000).
  • KPI domain has been studied for its interaction with the blood coagulation cascade (Van Nostrand et al., 1992), and its interaction with LRP can promote the endocytosis and degration of APP (Kounnas et al., 1995).
  • APP751 and APP770 (sometimes referred to collectively as APP-KPI) are not distinguished from each other.
  • APP695 expression predominates, while in non- neuronal cells, the KPI containing forms are more abundant (Neve et al., 1988; Tanzi et al., 1987).
  • Vertebrate genomes have been found to contain two additional genes that are closely related to APP, called amyloid precursor like protein 1 (APLPl) and amyloid precursor like protein 2 (APLP2).
  • APLPl amyloid precursor like protein 1
  • APLP2 amyloid precursor like protein 2
  • APLP2 shares a higher sequence homology with APP695 than does APLPl 5 and APLP2 also has alternative splicing that allows for addition of a KPI domain. Neither APLP contains an OX-2 like domain (Coulson et al., 2000).
  • APP was originally identified as the source of the beta-amyloid peptide (also called Abeta).
  • beta-amyloid As used herein, the terms “beta-amyloid”, “ ⁇ -amyloid”, “Abeta” and “A ⁇ ” are intended to mean the same.
  • Abeta is a major component of the plaques seen in the brains of patients with Alzheimer's disease (Kang et al., 1987).
  • Abeta is derived from APP proteolytically, by cleavage at the sites termed beta and gamma (see Figure 1). Cleavage of APP by beta-secretase, that is, the beta-site directed protease, releases a large extracellular fragment called APPsbeta.
  • CTFbeta transmembrane carboxy-terminal fragment
  • gamma secretase cleavage site CTFbeta
  • Abeta peptides are usually 40 or, less commonly, 42 amino acids long.
  • cleavage at the epsilon sites releases the APP intracellular domain fragment (AICD). Because of the generation of Abeta, this is termed the amyloidogenic pathway.
  • the first cleavage event is at the alpha site, located between the beta and gamma sites, generating CTFalpha and the large extracellular fragment APPsalpha, and precluding the formation of Abeta.
  • Gamma and epsilon cleavage also occur in this pathway, so AICD is still released, but instead of Abeta, the shorter, apparently non-pathogenic, p3 fragment is formed (Vetrivel and Thinakaran, 2006). Since the processing of APP is thought to be central to both the pathology and physiology associated with APP, much work has been done on the secretases responsible for APP cleavage.
  • processing of APP is intended to include proteolytic cleavage of the precursor at one or more sites recognized by specific secretases, which gives rise to various peptide intermediates and products that are discussed in more detail below (see Figure 1).
  • the invention is directed to modulating the process of APP processing which can be used for a variety of applications.
  • ADAM disintegrin and metalloprotease
  • ADAM9/MDC9 (Koike et al., 1999) have been shown confer alpha secretase activity when overexpressed in cells. Studies in mutant mice have shown that none of these enzymes by itself is required for all alpha cleavage events, and it is likely that more than one enzyme may act as an alpha secretase in vivo (Allinson et al., 2003).
  • beta secretase resulted in the independent identification of the membrane-associated aspartic protease BACEl (beta-site APP-cleaving enzyme 1 ; also called Asp2 and memapsin 2) by several groups (Hussain et al., 1999; Lin et al., 2000; Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999). Genetic knockouts have clearly shown BACEl to be the primary beta secretase in the brain (Cai et al., 2001 ; Luo et al., 2001 ; Roberds et al., 2001).
  • BACEl membrane-associated aspartic protease BACEl
  • BACE2 a homologous protein, has also been shown to possess beta secretase activity in vitro (Farzan et al., 2000; Hussain et al., 2000) and both BACEl and 2 must be removed in order to completely inhibit glial production of Abeta (Dominguez et al., 2005).
  • BACE2 has also been shown to cleave APP in a non-amyloidogenic fashion after residues 19 or 20 of the Abeta domain (Farzan et al., 2000; Fluhrer et al., 2002; Yan et al., 2001), a few amino acids downstream of alpha cleavage, and RNAi -mediated reduction of BACE2 has been shown in some instances to increase production of Abeta (Basi et al., 2003).
  • Gamma secretase A complex of four proteins, presenilin (1 or 2), Aph-1, nicastrin, and Pen-2 is thought to constitute gamma secretase (De Strooper, 2003).
  • Gamma secretase has been found to be required for cleavage not only at the gamma site, primarily after amino acids 636 or 638, but also for cleavage at the epsilon site, either before or after Leu-645 (Gu et al., 2001; Sastre et al., 2001; Weidemann et al., 2002; Yu et al., 2001).
  • APP proteolysis is generally thought to occur along a "pathway", in that the first cleavage event, either alpha or beta, is thought to stimulate gamma cleavage by generating a transmembrane protein with a short extracellular protein stub that can be recognized by gamma secretase (Struhl and Adachi, 2000), probably through interaction with nicastrin (Shah et al., 2005). How epsilon cleavage, with its production of AICD, fits into either pathway is not completely clear, but the identification of C-terminally extended Abeta fragments (Qi-Takahara et al., 2005) and the processing of such fragments into either
  • Abeta40 or Abeta42 depending on the location of epsilon cleavage (Funamoto et al., 2004), suggests a model where epsilon cleavage normally precedes gamma cleavage. If gamma secretase cleavage is stimulated by either alpha or beta cleavage, and is preceded by epsilon cleavage, one might expect the amyloidogenic and non-amyloidogenic pathways to generate AICD equally well. However, AICD levels seem to be positively (Kume et al., 2004) or negatively (Hoe et al., 2005) correlated with alpha cleavage under different conditions. Whether the AICD levels in these studies reflect changes in production, or stability or activity, is unclear.
  • AD Alzheimer's disease
  • a potential link between APP and AD was apparent immediately upon the identification of APP, since the APP-derived Abeta peptide had already been identified as a major component of the AD plaques (Kang et al., 1987).
  • the subsequent discovery that several families with familial, dominantly-inherited AD carried mutations in APP suggested a causal role of Abeta in AD.
  • mutations in presenillins 1 and 2 the genes implicated in the majority of familial AD cases, modulated gamma secretase cleavage further strengthened the case for the direct involvement of Abeta.
  • mice deficient for APP have been generated and studied. These mice are viable and fertile, but have been found to have some abnormalities, including increased copper levels in the liver and cortex (White et al., 1999), decreased locomotor activity and grip strength (Zheng et al., 1995), and increased susceptibility to induced seizures (Steinbach et al., 1998). In addition, although the brains of these mice seem to develop fairly normally, a defect in corpus collosum formation has been seen in some strain backgrounds, suggesting a potential role in axon outgrowth or guidance (Magara et al., 1999).
  • APPL Drosophila
  • Drosophila APPL is the single member of the APP family of proteins. Flies deficient in APPL are viable and fertile, but exhibit subtle behavioral defects (Luo et al., 1992), increased neuronal excitability (Li et al., 2004), and defects in axonal transport (Gunawardena and Goldstein, 2001) and neuromuscular synaptic bouton formation (Torroja et al., 1999).
  • Abeta has been widely studied for its role in Alzheimer's disease, but it is found in the blood and cerebrospinal fluid even non-diseased states (Seubert et al., 1993), and there is evidence it may have physiological functions. It has been observed that many of the biological activities exhibited by Abeta, ranging from effects on ion channel currents to influences on learning and memory, oppose the activities exhibited by APPsalpha, suggesting that alternative cleavage of APP at either the alpha or beta site may allow for the promotion of an effect both by promoting one cues while simultaneously repressing an opposing cue (Turner et al., 2003).
  • Abeta may cause cell death by permeabilizing plasma membranes (Arispe et al., 1993; Quist et al., 2005; Singer and Dewji, 2006), however, it may also cause cell death as well as other non-pathological responses simply by signaling through cell surface receptors. In fact, Abeta interactions with a wide range cell surface proteins, including the NMDA receptor, the P75 neurotrophin receptor, integrins, and APP itself, have been described (Verdier et al., 2004).
  • APPsalpha as a ligand: APPsalpha is a constitutively produced, but its production can be increased by physiological stimuli, such as metabotropic glutamate receptor stimulation (Allinson et al., 2003; Lee et al., 1995). Many studies have demonstrated that APPsalpha application can result in changed cellular behaviors, including the stimulation of neurite outgrowth or changes in LTP (Mattson, 1997). These effects suggest that APPsalpha may act as a physiological ligand. Since multiple domains of APP have been shown to have biological effects, it is possible that APPsalpha may signal through more than one receptor. A cell surface receptor capable of mediating APPsalpha induced signaling has not yet been identified.
  • the methods of the invention in some aspects involve the use of compounds that can modulate the amount of APP-derived peptides.
  • Some aspects of the invention is based on the recognition that the alpha cleavage of the precursor precludes subsequent beta processing (see Figure 1); therefore, the relative amount of processed peptides derived from the two pathways of the APP processing that gives rise to either plaque-forming beta peptides or non- plaque-associated peptides is likely to affect pathogenesis.
  • the term “Abeta production” means the generation of Abeta in a cell, tissue or subject.
  • the term “subject” means any mammal that may be in need of treatment with the Abeta production-modulating compound of the invention. Subjects include by are not limited to: humans, non-human primates, cats, dogs, sheep, pigs, horses, cows, rodents such as mice, hamsters and rats.
  • the term "Abeta processing-modulating compound” means a compound that modulates the processing (e.g., proteolysis) of APP by forming a complex with APP and/or its intermediate peptide products.
  • the “complex” means physical association of molecules. Such association may be direct interaction between two or more binding partners or indirect interaction mediated by one or more adapter molecules. Such binding may be stable or transient. The binding (complex formation) or the stability of the complex may in turn be modulated by agents that can either promote or inhibit the binding. In some embodiments of the invention, direct binding of extracellular matrix proteins to APP affects APP processing, thereby modulating the amount and/or rate of Abeta production.
  • APP was thought likely to be a receptor based on its predicted transmembrane structure (Kang et al., 1987).
  • the cytoplasmic domain did not contain obvious signal transduction motifs, such as kinase domains, but potential pathways through which APP might signal were suggested by the identification of cytoplasmic binding partners.
  • One well-studied cytoplasmic partner is Fe65, an adaptor protein which may be involved in APP-regulated cell motility or migration (Guenette et al., 2006; Sabo et al., 2001), as well as transcriptional control (see below).
  • cytoplasmic partners include the scaffolding protein JIP-Ib (JNK-interacting protein-1; (Matsuda et al., 2001), Disabled, an adaptor protein that can regulate the actin cytoskeleton (Suetsugu et al., 2004; Trommsdorff et al., 1998), and adaptor protein Mint/Xl 1 (Borg et al., 1996; McLoughlin and Miller, 1996).
  • APP may also signal through trimeric G protein G(o); however, varying results from different experimental systems leave it unresolved as to whether APP acts to increase or decrease G(o) signaling (Brouillet et al., 1999; Nishimoto et al., 1993; Okamoto et al., 1995).
  • AICD detected in the nucleus without reducing transactivation (Cao and Sudhof, 2004).
  • the role of gamma secretase cleavage in APP signaling is still controversial, as there is evidence both for (Cao and Sudhof, 2004) and against (Hass and Yankner, 2005) it being essential.
  • APP signaling has been shown to act via Tip60 that is phosphorylated at a CDK site, suggesting a model in which APP may activate Tip60 by recruiting it to the plasma membrane, allowing for its subsequent phosphorylation (Hass and Yankner, 2005).
  • gamma secretase cleavage may simply be one mechanism to inhibit the sequestration of Fe65 at the plasma membrane, and it has been proposed that APP phosphorylation may be an alternative mechanism for releasing Fe65 (Nakaya and Suzuki, 2006).
  • Full-length APP has also been proposed to signal through Fe65 by both inhibiting an intramolecular interaction in Fe65 and allowing for the recruitment of a unidentified membrane localized binding partner critical to the activity of Fe65 (Cao and Sudhof, 2004).
  • Extracellular binding partners for APPsalpha or APP, and related functions Extracellular binding partners for APPsalpha or APP, and related functions:
  • APP has been suggested to play a role in cell adhesion and motility (Herms et al., 2004; Sabo et al., 2003). APP binds to several extracellular matrix components, including heparan sulfate (Multhaup, 1994; Small et al., 1994), collagen (Beher et al., 1996), laminin (Narindrasorasak et al., 1992), and fibulin 1 (Ohsawa et al., 2001). Such cell-substratum interactions may be important to APP's role in neurite outgrowth and cell motility.
  • cell-cell adhesion may be mediated by trans- dimers of APP 3 or trans interaction between APP and either APLPl or APLP2 (Soba et al., 2005).
  • Secreted forms of APP containing the kunitz-type protease inhibitor (BCPI) domain initially identified and studied under the name protease nexin II (Oltersdorf et al., 1989; Van Nostrand et al., 1989) (Van Nostrand et al., 1989), bind and inhibit blood coagulation factor XIa (Van Nostrand et al., 1992).
  • BCPI kunitz-type protease inhibitor
  • APP695 does not contain the KPI domain, but does can bind heparin, which inhibits blood coagulation (Petitou et al., 2003) and high molecular weight kininogen, which is also involved in blood coagulation (Das et al., 2002).
  • APP can reduce copper (II) to copper (I) (Multhaup et al., 1996) and is required for normal copper metabolism (White et al., 1999).
  • APP lipoprotein receptors scavenger receptor A
  • LRP lipoprotein receptors scavenger receptor A
  • LRP also binds and promotes the internalization of APPs, but only of KPI-containing forms (Kounnas et al., 1995). It is interesting to note that LRP participates in a range of biological processes beyond its role in lipoprotein metabolism, including cell motility and proliferation (Lillis et al., 2005), raising the possibility of additional functions for an APP- LRP interaction.
  • NgR Nogo-66 receptor
  • amyloid-beta precursor protein is a transmembrane protein with widespread expression in embryos as well as adults. Based on its transmembrane structure, APP was initially predicted to be a cell-surface receptor (Kang et al., 1987), and in fact, evidence has accumulated to suggest that APP and its proteolytic products may play multiple roles in cell-cell communication. As disclosed herein, it is now shown that not only does full-length APP function as a receptor, but the secreted ectodomain APPsalpha functions as a ligand for an unidentified cell surface receptor.
  • APP For several binding partners, full-length APP appears to have more than one interacting domain. The list above only includes domains within APPsalpha.
  • the invention provides candidate APP binding proteins.
  • the invention further includes a characterization of APP interaction with two of these proteins, namely, contactin 4 and NgCAM, and also identifies an interaction between APP and contactin 3. All three of these proteins belong to the NCAM family of neurally expressed IgCAMs.
  • the NCAM family itself is divided into four subfamilies, referred to according their founding members: NCAM (neural cell adhesion molecule), DCC (deleted in colorectal cancer), Ll, and contactin.
  • Ig immunoglobulin
  • FnIII fibronectin type III
  • GPI-anchor or transmembrane domain Vaughn and Bjorkman Neuron 1996.
  • Contactin family members are characterized by six Ig domains, four FnIII domains and a glycosylphosphotidylinositol (GPI) membrane anchor.
  • contactin 3 also called BIG-I or PANG
  • contactin 4 also called BIG-2
  • the terms “contactin 3” and “contactin-3” are used interchangeably; similarly, the terms “contactin 4" and “contactin-4”, as well as the terms “contactin 5" and “contactin-5”, are used interchangeably.
  • contactins 3 and 4 are both located on the short arm of human chromosome 3, that is, on 3p (Luo et al., 2005; Zeng et al., 2002).
  • CNTN4 has been suggested to be involved in 3p deletion syndrome, a disorder in which the patient exhibits growth and mental retardation as well as distinctive facial features.
  • CNTN4 falls within the minimum chromosomal region implicated in this disorder, and one patient with 3p deletion syndrome has been identified in which a translocation breakpoint disrupts the contactin 4 transcript (Fernandez et al., 2004).
  • Contactin 3 and 4 are expressed in many regions throughout the brain, and both proteins, when presented as Fc fusions, have been shown to support outgrowth of neurites from hippocampal neurons (Yoshihara et al., 1995; Yoshihara et al., 1994).
  • contactin 1 F3/F11
  • contactin 2 axonin-1 /Tag- 1 /TAX-I
  • contactin 1 and 2 have been shown to bind a partially overlapping list of cell-surface or extracellular matrix proteins, including Ll family members NgCAM and NrCAM, the chondroitin sulfate proteoglycan phosphacan/RPTPb, tenascin, and bl-integrin (Sonderegger, 1997).
  • Contactins 1 and 2 can both signal through fyn kinase; however, since they are GPI- anchored proteins, and thus without a cytoplasmic domain, activation of fyn through a direct interaction can be excluded, and it is not known how this signal is transduced into the cell (Kunz et al., 1996; Zisch et al., 1995).
  • Contactin 1 and 2 have both been implicated in cell migration and axon guidance, as well as in the organization of specialized domains in myelinated axons, with contactin-2 being required at the juxtaparanode and contactin 1 being required at the septate junctions and in the node of Ranvier (FaIk et al., 2002).
  • NgCAM neuroneuron-glial cell adhesion molecule
  • Ng-CAM neuron-glial cell adhesion molecule
  • Ll Ll
  • NrCAM NgCAM-related cell adhesion molecule
  • CHLl Close Homolog of Ll
  • NgCAM is now generally accepted to be the chicken homolog of mammalian Ll, although the relatively low sequence homology between these two proteins (43% identity between chicken NgCAM and human Ll, versus 78% identity between chicken and human NrCAM) initially led to some controversy over this point.
  • NgCAM/Ll has been shown to bind a variety of molecules, including some contactin
  • homophilic trans interactions of contactin 2 may be capable of mediating cell adhesion, if not neurite outgrowth (Kunz et al., 2002), and trans interactions between NgCAM and contactin 1, or NrCAM and contactin 2, have been reported (Sonderegger, 1997).
  • Mice in which Ll expression is eliminated exhibit severe defects, including enlarged ventricles and defects in the corticospinal tract and corpus collosum (Demyanenko et al., 1999).
  • mice in which the sixth Ig domain of Ll has been deleted appear normal, though embryonic lethality in some genetic backgrounds. Based on the previous mapping of binding domains in Ll 3 this implies that neither Ll homophilic interactions nor Ll interactions with contactin 1 and 2 are absolutely required.
  • CSPGs chodroitin sulfate proteoglycans
  • EphBs An interaction between ephrin-Bs and EphBs is also thought to involve repulsion, but in this case, ventrally expressed EphBs are thought to prevent dorsal axons from bypassing the optic disk (Birgbauer et al., 2000; Birgbauer et al., 2001).
  • Antibody blocking studies have implicated the IgCAMs neurolin and Ll in intraretinal guidance (Brittis et al., 1995; Ott et al., 1998) possibly through a role in axon fasciculation. Once RGC axons reach the optic disk, they exit the retina and enter the optic nerve.
  • Netrin and its receptor DCC are both genetically required for RGC axons to exit the eye and enter the optic nerve (excellentr et al., 1997).
  • laminin-1 can convert the effect of netrin- 1 from attraction to repulsion, combined with the co-expression of netrin and laminin at the retinal surface around the optic disk, led to the proposal that netrin and laminin act together to drive axons out of the retina and into the optic nerve head, where netrin is expressed in the absence of laminin (Hopker et al., 1999).
  • the RGC axons grow towards and enter the tectum in a manner dependent upon FGF signaling (McFarlane et al., 1996; McFarlane et al., 1995).
  • RGC axons must find appropriate synaptic partners.
  • the retinotectal projection is arranged topographically; that is to say, the spatial arrangement of RGCs in the retina is reflected in the spatial arrangement of their synapses in the tectum.
  • the temporal-nasal axis of the retina maps onto the anterior-posterior axis of the tectum using gradients of ephrinAs and EphAs as spatial cues
  • the dorsal-ventral axis of the retina maps onto the ventral-dorsal axis of the tectum using gradients of ephrinBs, EphBs, Wnt3, and Ryk as cues (Flanagan, 2006). Additional details about topographic mapping in the retinotectal projection, including the role of axon-axon competition, is discussed below. Topographic location is not the only criterion according to which axons choose synaptic partners.
  • RGC axons In chicken, RGC axons initially enter the tectum and find their topographically correct locations within the most superficial layer, the stratum opticum (SO). Axons then turn or branch orthogonally to the anterior-posterior and dorsal ventral axes, and each RGC axon must synapse in one particular retinorecipient layer, SGFS (stratum griseum et fibrosum superficiale) layer B, D, or F.
  • SGFS stratum griseum et fibrosum superficiale
  • RGCs There are different types of RGCs that can be distinguished by accumulation of distinct neuropeptides, and choice of retinorecipient layer seems to correlate at least partly with this classification (Kuljis and Receive, 1988).
  • Biochemically distinct RGC populations can form in retinas that are prevented from innervating the tectum, and tectal lamina form normally in the absence of innervation, suggesting that the subtype of an RGC is not determined based on the layer in which it has already formed synapses, but rather that subtype is determined independently of synapse formation, and the choice of synaptic partners is guided complementary cues (Yamagata and Sanes, 1995). Some tectal cues which aid RGC axons choosing appropriate lamina have been identified, including versican and N- cadherin, but laminar choice is still not completely understood (Inoue and Sanes, 1997; Yamagata and Sanes, 2005).
  • contactin 4 and NgCAM as APP interacting proteins.
  • APP binds to contactin 4 and contactin 3 through what is most likely a direct physical interaction. Domains sufficient for this binding are amino acids 18-205 of APP, which essentially corresponds to the El domain, and a domain including the four fibronectin type 3 repeats of contactin 3 or 4. NgCAM interacts physically with APP, but a direct interaction was not demonstrated.
  • NgCAM interacts indirectly with APP, or requires the presence of an additional co-factor.
  • Other possible explanations include a binding specificity of APP for NgCAM with specific post-translational modifications or of a different splice form, or a relatively low affinity direct interaction between APP and NgCAM that is undetectable by the methods we used. None of these alternative explanations alone easily explains all of the data here, but they may still be relevant factors.
  • Contactin 4 and NgCAM each can dramatically affect the levels of APP and APP CTFalpha seen in transfected cells, although whether the effect is to increase or decrease these levels varies between experiments. This may be due to different ratios of cis-to-trans interactions, or a biphasic response to increasing expression levels (either physiological or due to a dominant negative effect).
  • APP, NgCAM, and contactin 4 are expressed in chick embryos, at least at the level of RNA, in the both the olfactory epithelium and the olfactory bulb. These genes are also expressed, along with contactin 3, in the retina and the tectum. The partially overlapping regions of expression seen suggest that these proteins may have the opportunity to interact in vivo, and suggest that interactions among these proteins might play a role in the development of the olfactory and visual systems.
  • NgCAM supports outgrowth of retinal ganglion cell (RGC) axons, and this outgrowth can be modified by APP- or contactin 4-related reagents.
  • RRC retinal ganglion cell
  • APP signaling an anti-APP antibody and gamma secretase inhibitor DAPT
  • DAPT gamma secretase inhibitor
  • contactin 4-Fc inhibits outgrowth on NgCAM but not laminin. Based on the observation that Fc fusions of contactins 1, 2, 3, and 6 also inhibited outgrowth on NgCAM, and in light of published studies implicating contactin 2 as a co-receptor in NgCAM- promoted neurite outgrowth (Buchstaller et al., 1996), a preferred interpretation is that contactin 4-Fc is binding to NgCAM and blocking access of axonal receptors to NgCAM. However, it remains formally possible that NgCAM and contactin 4 act together to generate an inhibitory signal. Finally, we showed that APPsalpha, and in particular the El domain, enhances
  • contactin 4 uses its fibronectins domains to bind APP, this could potentially allow for the formation of a trimolecular complex among APP 5 contactin 4, and NgCAM.
  • APP may interact with NgCAM indirectly, a model in which contactin 4 acts as a bridge between APP and NgCAM is not sufficient to explain all of our results.
  • NgCAM can be crosslinked to AP -m APP(199-345), a protein in which APP 's binding domain for contactin 4 is not present.
  • contactin 4 could still potentially be required for a direct interaction between APP and NgCAM, if, for example, contactin 4 binding relieved inhibitory intramolecular interactions or otherwise changed the conformation of NgCAM or APP.
  • modulating or modulation of APP processing refers to an increase or decrease in the rate and/or amount of APP processing to generate various intermediate and/or final peptide products.
  • the methods include contacting a mammalian cell with an agent that modulates the amount or rate of binding of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2) to contactins and/or Ll-CAMs.
  • an agent means a molecule, molecular compound, a composition or formulation having a desired biological effect.
  • An agent may be naturally occurring or synthetic.
  • An agent may be in a substantially pure form or may be a component of a mixture.
  • An agent may be provided in a soluble form (e.g., solution, medium, buffer, etc.) or in an insoluble form (e.g., as a substrate, cell surface molecule, on a solid support, etc.).
  • specific configuration of the presentation of a binding molecule(s) such as in cis verses in trans, may be considered in an assay or treatment.
  • the agent is expressed by the cell or exogenously added so as to contact the cell.
  • exogenously added means that the agent or an equivalent thereof is applied to a target from an external source, e.g., as a composition containing the agent.
  • the agents include one or more contactins such as contactin-3, contactin-4 and contactin-5, or Ll-CAMs such as Ng-CAM, or fragments thereof that bind to APP, APLPl or APLP2.
  • the agent is a fusion protein including one or more contactins, Ll-CAMs or fragments.
  • a fusion protein is a recombinant protein containing more than one segments of peptides fused together, often with a short linker or spacer sequence separating between them, such that when expressed the they form one continuous peptide.
  • a target peptide can be fused to a variety of tags, labels, purification moieties, and the like.
  • fusion proteins include but are not limited to: GST- fusion proteins, Flag-fusion proteins, MBP-fusion proteins, His 6 fusion proteins, Myc fusion proteins, Fc fusion proteins, chitin fusion proteins, AP-fusion proteins, transferrin fusion proteins, GFP fusion proteins, GIu fusion proteins, HA fusion proteins, and others. Such techniques are well known in the art.
  • the fusion protein is an Fc fusion protein, e.g., a fusion of a Fc molecule and one of the polypeptides mentioned herein.
  • the agent is an antibody or an antigen-binding fragment thereof (see below).
  • the antibody is selected from an antibody or antigen-binding fragment thereof that binds a contactin or a Ll-CAM, preferably contactin-3 contactin-4, contactin-5 or Ng-CAM, or, an antibody or antigen-binding fragment thereof binds APP,
  • the antibody or antigen-binding fragment thereof binds to a complex of a contactin or a Ll-CAM and APP, APLPl or APLP2.
  • the antibody or antigen-binding fragment thereof is a bispecific antibody.
  • the term "bispecific antibody” refers to an engineered antibody with two different binding sites that recognize two different antigens. Various bispecific antibodies and methods of generating such antibodies are known in the art.
  • the methods include agents that block or disrupt the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs.
  • an agent that "blocks" the binding of two or more molecules or molecular complexes can interfere with the normal binding.
  • an agent may interfere with the binding between two proteins by binding to a first protein, thereby either creating a physical hindrance to the binding site on the first protein against a second protein; alternatively, the binding of the agent to a first protein may cause a conformational change to the first protein which results in unfavorable structural changes that render reduced binding to its normal binding partner(s).
  • An agent that "disrupts" the binding of two or more molecules or molecular complexes may promote the dissociation of an already bound complex.
  • an agent that can block or disrupt the binding may cause a biochemical or cellular environment that renders the binding unfavorable.
  • An agent may exert both blocking and disrupting effects.
  • the invention describes agents that promote or stabilize the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs.
  • an agent that "promotes" the binding of one or more molecules or molecular complexes favors association of binding partner(s) such that an association/binding constant of the reaction is increased in the presence of such agent.
  • An agent that "stabilizes" the binding of one or more molecules or molecular complexes acts on an existing (bound) complex as substrate to enhance the association, such that a dissociation constant of the reaction is lowered in the presence of such agent.
  • An agent may exert both promoting and stabilizing effects.
  • the methods also include agents that can reduce processing of APP to ⁇ -amyloid.
  • antibodies and/or antigen-binding fragments thereof that specifically bind to APP, APLPl or APLP2, contactins and/or Ll-CAMs, or to protein complex of these molecules, are useful in additional screening methods.
  • the antibodies of the present invention thus are prepared by any of a variety of methods, including administering protein, fragments of protein, cells expressing the protein or fragments thereof and the like to an animal to induce polyclonal antibodies.
  • antibodies that specifically bind to the above-mentioned proteins are available commercially. The production of monoclonal antibodies is according to techniques well known in the art.
  • an antibody from which the pFc 1 region has been enzymatically cleaved, or which has been produced without the pFc' region designated an F9(ab') 2 fragment
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd Fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • CDRs complementarity determining regions
  • Frs framework regions
  • non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody.
  • This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. See, e.g., U.S. patents 4,816,567, 5,225,539, 5,585,089, 5,693,762 and 5,859,205.
  • PCT International Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin. Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as "chimeric" antibodies.
  • Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (HAMA) responses when administered to humans.
  • HAMA human anti-mouse antibody
  • the present invention also provides for F(ab') 2 , Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or Fr and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab') 2 fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or nonhuman sequences.
  • the present invention also includes so-called single chain antibodies.
  • polypeptides of numerous size and type that bind specifically to APP, APLPl, APLP2, contactins or Ll-CAMs, complexes thereof, or fragments thereof.
  • polypeptides may be derived also from sources other than antibody technology.
  • polypeptide-binding agents can be provided by degenerate peptide libraries, which can be readily prepared in solution, in immobilized form or as phage display libraries.
  • Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptoids and non-peptide synthetic moieties.
  • RRC retinal ganglion cell
  • SC superior colliculus
  • the temporal-nasal axis of the retina maps onto the anterior-posterior axis of the tectum, with neurons from nasal positions forming synapses in the posterior tectum, while the dorsal -ventral axis of the retina maps onto the ventral- dorsal axis of the tectum, with ventral RGCs forming synapses in the dorsal tectum.
  • Sperry first proposed that the formation of topographic maps could be guided by a gradient of a label across the target field in conjunction with a gradient of a complementary label in the projecting field (Sperry, 1963).
  • Ephrin-A2 or -A5 expression was found in a high posterior, low anterior gradient in the tectum, while EphA3 or EphA5 was found in a high temporal, low nasal gradient in the retina (Cheng et al., 1995; Drescher et al. 5 1995; Feldheim et al., 2000; Feldheim et al., 1998; Monschau et al., 1997; Zhang et al., 1996).
  • ephrin-As were found to be capable of repelling RGC axons (Drescher et al., 1995;
  • a topographic map cannot be completely determined by one gradient in the target field and one in the projecting field, because all axons would be expected to map to one end. In the case described above, for example, all axons might be expected to map temporally to avoid repulsion by ephrin-As in the posterior tectum.
  • An additional counterbalancing force is thought to be required (Gierer, 1983; Prestige and Willshaw, 1975), which could be in the form of multiple gradients, attractive and repulsive cues from a single gradient, or axon competition. Mapping of the dorsal-ventral axis in the retinotectal system provides an example of the first type of counterbalance.
  • a gradient of Wnt3 in the tectum acts overall to repel axons toward the lateral tectum.
  • Topographic differences in the degree to which axons are repelled seem to be determined by the relative levels of Frizzleds, uniformly expressed attractive Wnt3 receptors, and Ryk, a repulsive receptor expressed in a ventral high retinal gradient (Schmitt et al., 2006).
  • Evidence for the second kind type of counterbalancing force, attractive and repulsive cues from a single gradient, has been found in the anterior-posterior axis of the retinotectal system.
  • retinotectal mapping The third type of counterbalancing force, axon-axon competition, is also clearly implicated in retinotectal mapping.
  • One line of evidence comes from numerous studies in which retinas or tecta were surgically manipulated to create a mismatch between the number or identity of the RGC axons entering the tectum and the region of the tectum available for innervation. These studies showed that the retinotectal map could expand or contract to fill the entire tectal region available, suggesting the map was not determined simply by matching specified concentrations of topographic markers (recently reviewed in Goodhill and Richards, 1999).
  • Axon-axon competition is a common feature in the development of many types of neuronal connections, and several general models for axon-axon competition have been proposed (van Ooyen, 2001). Electrical activity plays an important role in some of these models, and electrical activity clearly plays a role in the refinement of visual maps (Torborg and Feller, 2005). However, topographic maps expand or contract appropriately in goldfish with partial ablations of the retina or tectum, even under electrical blockade by tetrodotoxin, implying that the competition driving the initial rough formation of the topographic map is activity independent (Meyer and Wolcott, 1987).
  • Some types of models that could be potentially be relevant include a competition for limiting neurotrophic factors, a competition for physical space, and direct negative interactions between axons (van Ooyen, 2001). None of these models has been definitively implicated in the retinotectal projection, although BDNF and Ll have been proposed as limiting factors (Flanagan, 2006). In the first case, this idea is based on the observation that axon arborizations increase or decrease in response to increases or decreases in available BDNF (Cohen-Cory and Fraser, 1995). In the second case, a function for Ll in competition is suggested by the observation that the superior colliculus is incompletely innervated, with large patches of low or nonexistent innervation in the anterior SC, in Ll deficient mice. However, other possible interpretations, including a requirement for Ll in axonal remodeling, have been suggested (Demyanenko and Maness, 2003).
  • Ll protein in the mouse SC like NgCAM protein in the chicken tectum, seems to be largely RGC axon-derived (Lemmon and McLoon, 1986; Lyckman et al., 2000; Yamagata and Sanes, 1995), Ll may be a more likely candidate for contributing to axonal competition as a mediator of direct negative signals between axons than as a limiting factor for axons compete.
  • Ll-Ll homophilic interactions generally appear to be positive rather than negative in nature, raising the possibility that negative signals involving Ll may involve alternative receptors or ligands.
  • Fasciclin II Fasciclin II
  • NCAM ortholog Fasciclin II
  • APPL the downstream function of APPL
  • Drosophila APP ortholog possibly by recruiting APP to the cell surface, although the signaling mechanism is not completely clear. It may be interesting to investigate whether competition among RGC axons may work under a similar principle, for example, with axons measuring relative amounts of NgC AM/LI, provided by neighboring axons, through an APP dependent mechanism.
  • NgCAM expressed on RGC axons is of a different splice form than the our recombinant NgCAM 3 or that it is post-translationally modified differently in RGCs than in 293T cells.
  • NgCAM has been shown to bind to the first four Ig domains of contactin 2 (Rader et al., 1996), but using the Fc/AP binding method described in this chapter, we were unable to detect this interaction (figure 3-3A).
  • the theory of such a low affinity interaction between APP and NgCAM cannot on its own account for the strong binding of AP-APP to embryonic chick tecta.
  • CTFalpha levels were seen to change, often, but not always, in conjunction with APP levels. Why CTFalpha levels are sometimes increased, sometimes decreased, in response to contactin 4 or NgCAM is unclear. Some possible explanations we have considered are variations in cell density, which might change the ratio of cis to trans interactions, or variations in expression level, which might affect oligomerization states of the transfected proteins or result in an excess of transfected APP over the levels of any endogenous proteins (for example, proteases) involved in a response.
  • endogenous proteins for example, proteases
  • contactin 4 and NgCAM fusion proteins may be acting essentially as dominant negatives in the cases where CTFalpha levels are decreased may be suggested by the observation that expression of NrCAM, which might be predicted to be capable of binding endogenous contactins, also resulted in decreased CTFalpha levels.
  • NrCAM which might be predicted to be capable of binding endogenous contactins
  • CTFalpha is usually thought of as an intermediate product which, shortly after its generation by alpha secretase activity, would be expected to be degraded by gamma secretase activity.
  • the invention provides methods and compositions useful for the prevention and/or treatment of Abeta-accumulation-associated disease or disorder, such as Alzheimer's disease.
  • treating or “treatment” of a disease or disorder refers to improving, or remedying of a condition.
  • preventing or “prevention” of a disease or disorder refers to hindering the occurrence of disease in a susceptible population (e.g., a subject at risk of developing a disease or disorder), and/or arresting or retarding the progress of a disease.
  • the invention in some aspects provides methods for treating or preventing Alzheimer's disease in a subject.
  • the term "subject” means any mammal that may be in need of treatment with the compounds of the invention that modulate APP processing.
  • Subjects include but are not limited to: humans, non-human primates, cats, dogs, sheep, pigs, horses, cows, rodents such as mice, hamsters, and rats.
  • Particular subjects to which the present invention can be applied are subjects at risk for or known to have an APP processing-associated disorder.
  • Such disorders may include, but are not limited to:
  • Alzheimer's disease and any other diseases associated with abnormal APP processing including overproduction of A ⁇ or reduced clearance of A ⁇ such as Down's syndrome, cerebrovascular amyloidosis, inclusion body myositis and hereditary inclusion body myopathies, any disease associated with abnormal BACE activity, ischemia, oxidative stress, head trauma, stroke, hypoglycemia, and any neurodegenerative disorder with abnormal APP processing.
  • diseases associated with abnormal APP processing including overproduction of A ⁇ or reduced clearance of A ⁇ such as Down's syndrome, cerebrovascular amyloidosis, inclusion body myositis and hereditary inclusion body myopathies, any disease associated with abnormal BACE activity, ischemia, oxidative stress, head trauma, stroke, hypoglycemia, and any neurodegenerative disorder with abnormal APP processing.
  • the evaluation of the treatment also may be based upon an evaluation of the symptoms or clinical end-points of the associated disease.
  • the subjects to which the methods of the invention are applied are already diagnosed as having a particular condition or disease.
  • the measurement will represent the diagnosis of the condition or disease.
  • the subjects will already be undergoing drug therapy for an APP processing-associated disorder (e.g. Alzheimer's disease), while in other instances the subjects will be without present drug therapy for such a disorder.
  • an APP processing-associated disorder e.g. Alzheimer's disease
  • the methods as disclosed in the invention include administering to a subject a therapeutically effective amount of an agent that modulates the amount or rate of binding of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2) to contactins and/or Ll-CAMs.
  • APP amyloid precursor protein
  • APLPl amyloid precursor like protein 1
  • APLP2 amyloid precursor like protein 2
  • the amount of a treatment may be varied for example by increasing or decreasing the amount of a therapeutic composition, by changing the therapeutic composition administered, by changing the route of administration, by changing the dosage timing and so on.
  • the effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration, and the like factors within the knowledge and expertise of the health practitioner. For example, an effective amount can depend upon the degree to which an individual has abnormal levels and/or activity of APP processing.
  • a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
  • the therapeutically effective amount of a pharmacological compositions of the invention is that amount effective to modulate A ⁇ accumulation, the rate and/or the level of APP processing to reduce, prevent, or eliminate the associated disorder.
  • testing can be performed to determine plasma A ⁇ levels in a subject. Additional tests useful for monitoring the onset, progression, and/or remission, of A ⁇ accumulation-associated disorders such as those described above herein, are well known to those of ordinary skill in the art.
  • an effective amount is the amount of a pharmacological agent of the invention that modulate the rate and/or amount of APP processing that diminishes the disorder, as determined by the aforementioned tests.
  • compositions used in the foregoing methods preferably are sterile and contain an effective amount of a pharmacological agent for producing the desired response in a unit of weight or volume suitable for administration to a patient.
  • the doses of pharmacological agents administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
  • the dosage of a pharmacological agent of the invention may be adjusted by the individual physician or veterinarian, particularly in the event of any complication.
  • a therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg, and most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days.
  • Various modes of administration will be known to one of ordinary skill in the art which effectively deliver the pharmacological agents of the invention to a desired tissue, cell, or bodily fluid.
  • the administration methods include: topical, intravenous, oral, inhalation, intracavity, intrathecal, intrasynovial, buccal, sublingual, intranasal, transdermal, intravitreal, subcutaneous, intramuscular and intradermal administration.
  • the agents include one or more contactins or Ll-CAMs or fragments thereof that bind to APP, APLPl or APLP2.
  • the contactin is contactin-3 contactin-4, contactin-5 or a combination thereof
  • the Ll-CAM is Ll or Ng-CAM, wherein the fragment is preferably a fragment of contactin-3, contactin-4, Ll or Ng-CAM that binds to APP, APLPl or APLP2, or a combination of such fragments.
  • the agent is a fusion protein, preferably an Fc fusion protein, including one or more contactins, Ll-CAMs or fragments.
  • the agent is an antibody or an antigen- binding fragment thereof.
  • the agents include an antibody or antigen-binding fragment thereof that binds a contactin or a Ll-CAM, particularly contactin-3 contactin-4, contactin-5, Ll or Ng-CAM, as well as APP, APLPl or APLP2.
  • the antibody or antigen-binding fragment thereof binds a complex of a contactin or a Ll-CAM and APP, APLPl or APLP2.
  • the methods include antibody or antigen-binding fragment thereof that is a bispecific antibody.
  • the agent blocks or disrupts the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs. In other embodiments of the methods, the agent promotes or stabilizes the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs. In some embodiments of the foregoing, the methods includes an agent that reduces processing of APP to ⁇ -amyloid. According to some embodiments, the methods include a vectorized agent that crosses the blood brain barrier. According to the invention, the methods are used for various subject, where a preferred subject is a human.
  • vectorized refers to engineered moieties or modifications to a subject agent or compound for the purpose of delivering the composition to a target site in a cell or a tissue.
  • vectorized agents are produced by covalently linking a compound to a moiety which promotes delivery from the circulation to a predetermined destination in the body.
  • vectorized agents competent for transcytosis across the blood- brain barrier are useful for targeting the brain of a subject for therapeutic purposes. Examples of vectorized molecules which can traverse the blood-brain barrier are found in the prior art (Bickel et al., Proc. Natl. Acad. Sci.
  • antibodies are linked to another macromolecule, the antibodies being the agent which promotes delivery of the macromolecules.
  • an agent is an antibody which is directed towards a cell surface component, such as a receptor, which is transported away from the cell surface.
  • antibodies which confer the ability to trancytose the blood-brain barrier include, without limitation, anti-insulin receptor antibodies, and also anti- transferrin receptors (Saito et al., Proc. Natl. Acad. Sci. USA 92: 10227-31 (1995); Pardridge et al., Pharm. Res.
  • This first antibody is covalently linked to an antibody which binds f-amyloid.
  • ligands which bind these receptors e.g., insulin, transferrin, or low density lipoprotein
  • a vector moiety can be chemically attached to the agent to facilitate its delivery into the central nervous system.
  • the moiety can be genetically engineered into the agent as an integral component.
  • This vector component can be for example, an anti- transferrin receptor antibody or anti-insulin receptor antibody which binds the receptors present on the brain capillary endothelial cells (Bickel et al., Proc. Natl. Acad. Sci. USA 90: 2618-22 (1993); Pardridge et al., J. Pharmacol. Exp. Ther. 259: 66-70 (1991); Saito et al., Proc. Natl. Acad. Sci. USA 92: 10227-31(1995); Friden et al., J.
  • compositions also referred to herein as therapeutic compounds and/or pharmaceutical compounds. These include one or more contactins and/or Ll-CAMs and/or one fragments thereof that bind to APP, APLPl or APLP2, and a pharmaceutically acceptable carrier.
  • compositions of the present invention are administered in pharmaceutically acceptable preparations.
  • Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • the characteristics of the carrier will depend on the route of administration.
  • the therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be oral, intravenous, intrathecal, intraperitoneal, intramuscular, intranasal, intracavity, subcutaneous, intradermal, or transdermal.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the therapeutic agent, which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butane diol.
  • a non-toxic parenterally-acceptable diluent or solvent for example as a solution in 1, 3-butane diol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of i ⁇ jectables.
  • Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • compositions suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the therapeutic agent.
  • Other compositions include suspensions in aqueous liquors or non-aqueous liquids such as a syrup, an elixir, or an emulsion.
  • the invention provides a composition of the above-described agents for use as a medicament, methods for preparing the medicament and methods for the sustained release of the medicament in vivo.
  • Delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the therapeutic agent of the invention, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer-based systems such as polylactic and polyglycolic acid, poly(lactide-glycolide), copolyoxalates, polyanhydrides, polyesteramides, polyorthoesters, polyhydroxy butyric acid, and polycaprolactone. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S.
  • Nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di- and tri-glycerides; phospholipids; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like.
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di- and tri-glycerides; phospholipids; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like.
  • Specific examples include, but are not limited to: (a) erosional systems in which the polysaccharide is contained in a form within a matrix, found in U.S. Patent Nos.
  • the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient.
  • exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. PCT/US95/03307 (Publication No. WO 95/24929, entitled “Polymeric Gene Delivery System.”
  • PCT7TJS95/03307 describes a biocompatible, preferably biodegradable polymeric matrix for containing an exogenous gene under the control of an appropriate promoter. The polymeric matrix is used to achieve sustained release of the exogenous gene in the patient.
  • the compound(s) of the invention is encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US95/03307.
  • the polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the compound is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the compound is stored in the core of a polymeric shell).
  • Other forms of the polymeric matrix for containing the compounds of the invention include films, coatings, gels, implants, and stents.
  • the size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted.
  • the size of the polymeric matrix device further is selected according to the method of delivery that is to be used.
  • the polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material that is bioadhesive, to further increase the effectiveness of transfer when the devise is administered to a vascular surface.
  • the matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.
  • Both non-biodegradable and biodegradable polymeric matrices can be used to deliver agents of the invention of the invention to the subject.
  • Biodegradable matrices are preferred.
  • Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred.
  • the polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable.
  • the polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.
  • the agents of the invention are delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix.
  • exemplary synthetic polymers that can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose prop
  • non-biodegradable polymers examples include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.
  • biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
  • Bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein by reference, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly (hexylmethacry late), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
  • long-term sustained release implant may be particularly suitable for treatment of established neurological disorder conditions as well as subjects at risk of developing a neurological disorder.
  • "Long-term" release means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days.
  • the implant may be positioned at or near the site of the neurological damage or the area of the brain or nervous system affected by or involved in the neurological disorder.
  • Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above.
  • the contactin is contactin-3 contactin-4, contactin-5 or a combination thereof
  • the Ll-CAM is Ll or Ng-CAM.
  • the pharmaceutical composition includes a fragment of contactin-3, contactin-4, Ll or Ng-CAM that binds to APP, APLPl or APLP2, or a combination of such fragments.
  • some embodiments include a pharmaceutical composition that is a fusion protein, including one or more contactins, Ll- CAMs or fragments. In some cases, the composition includes an Fc fusion protein.
  • the pharmaceutical compositions include one or more antibodies that bind a contactin, a Ll-CAM, APP, APLPl or APLP2 or antigen-binding fragments thereof, where preferred contactin or Ll-CAM is contactin-3 contactin-4, contactin-5. Ll or Ng-CAM.
  • the pharmaceutical composition includes an antibody or antigen-binding fragment thereof that binds a complex of a contactin or a Ll-CAM and APP, APLPl or APLP2.
  • the antibody or antigen-binding fragment thereof is a bispecific antibody.
  • the pharmaceutical composition in some embodiments includes an agent that blocks or disrupts the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs. In other embodiments, the pharmaceutical composition includes an agent that promotes or stabilizes the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs. In some embodiments of the foregoing composition, the agent reduces processing of APP to ⁇ -amyloid. In some cases, the pharmaceutical composition according to the foregoing includes a vectorized agent that crosses the blood brain barrier of a subject, preferably a human.
  • the invention is also useful for providing screening methods for identifying and/or isolating molecules or compounds that elicit modulatory effects on APP processing.
  • methods for identifying compounds that modulate the binding of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2) with contactins and/or Ll-CAMs are provided.
  • These methods include providing a reaction mixture that comprises (1) APP, APLPl, APLP2 and/or a fragment thereof that binds to contactins and/or Ll -CAMS, and (2) contactins, Ll- CAMs and/or a fragment thereof that binds to APP, APLPl 3 APLP2; contacting the reaction mixture with a test compound; determining a level of binding of APP, APLPl, APLP2 and/or a fragment thereof with contactins, Ll-CAMs and/or a fragment thereof in the absence and in the presence of the test compound; and comparing the level of binding of APP, APLPl, APLP2 or fragment thereof with contactins, Ll-CAMs and/or a fragment thereof in the absence and in the presence of the test compound, wherein a test compound that modulates the binding relative to the level of binding in the absence of the test compound is a compound that modulates the binding of APP, APLPl, APLP2 or fragment thereof with
  • the test compound is a small molecule.
  • small molecules encompass numerous chemical classes, although typically they are organic compounds.
  • the candidate test compounds are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500.
  • Candidate compounds comprise functional chemical groups necessary for structural interactions with proteins and/or nucleic acid molecules, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups.
  • the candidate compounds can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups.
  • Candidate compounds also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like.
  • the agent typically is a DNA or RNA molecule, although modified nucleic acid molecules as defined herein are also contemplated.
  • Candidate test compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means. Further, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the agents.
  • reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce non-specific or background interactions of the reaction components.
  • reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.
  • the test compound is an antibody that binds to APP, APLPl, APLP2 a contactin or a Ll-CAM, or an antigen-binding fragment thereof; or a fragment of APP, APLPl , APLP2, a contactin or a Ll-CAM, where preferably the contactins and/or Ll-CAMs is selected from the group consisting of: contactin-3, contactin-4, contactin-5, Ll and Ng-CAM.
  • APP amyloid precursor protein
  • APLPl amyloid precursor like protein 1
  • APLP2 amyloid precursor like protein 2
  • These methods include: providing a reaction mixture that comprises (1) contactins, Ll-CAMs and/or a fragment thereof that binds to APP, APLPl, APLP2, and (2) APP, APLPl, APLP2 and/or a fragment thereof that binds to contactins and/or Ll-CAMS; contacting the reaction mixture with a test compound; determining a level of processing of APP, APLPl or APLP2 in the absence and in the presence of the test compound; and comparing the processing in the absence and in the presence of the test compound, wherein a test compound that modulates processing of APP, APLPl or APLP2 relative to the level of processing in the absence of the test compound is a compound that modulates the processing of APP, APLPl or APLP2.
  • the test compound is a small molecule (see above); an antibody that binds to APP, APLPl, APLP2, a contactin or a Ll-CAM, or an antigen-binding fragment thereof; a fragment of APP, APLPl or APLP2, a contactin or a Ll-CAM, where preferably the contactins and/or Ll-CAMs is selected from the group consisting of: contactin-3, contactin-4, contactin-5, Ll and Ng-CAM.
  • the processing of APP to A ⁇ is reduced. Changes in relative or absolute rate and/or amount of APP processing of greater than 0.1% may indicate an abnormality.
  • the change in the rate and/or amount of APP processing, which indicates an abnormality is greater than 0.2%, greater than 0.5%, greater than 1.0%, 2.0%, 3.0% , 4.0%, 5.0%, 7.0%, 10%,. 15%, 20%, 25%, 30%, 40%, 50%, or more, where greater changes correlate with greater efficacy of the modulatory compound.
  • Example 1 Purification of candidate APP binding proteins
  • the approach which was taken to study APP in cell-cell signaling was to first identify novel extracellular binding partners, then to examine these for the ability to act as a functional receptor or ligand for APP.
  • alkaline-phosphatase tagged APPsalpha was used as an affinity probe to visualize the expression pattern of binding partners in embryonic chick brains, and the domains of APP required for this interaction were determined.
  • the invention describes the identification of a family of proteins that bind to APP.
  • the invention also includes the recognition that binding of APP to extracellular binding partners leads to the modulation of APP processing. This can be used for a variety of applications including therapeutic interventions in diseases and screening systems for effector molecules and agents that may affect APP processing.
  • APtag4 has been described previously (Flanagan et al., 2000).
  • AP-mAPP was constructed by inserting the sequence encoding amino acids 18 through 612 of mouse APP695 into APtag4. This portion of APP was obtained by PCR amplification with Taq polymerase from mouse midbrain-derived cDNA using the primer pairs tccactcgcacacggagcactcgg [SEQ ID NO:1] plus cggacgtacttcttcagcatgttg [SEQ ID NO:2], followed by cctccggactggaggtacccactgatgg [SEQ ID NO:3] plus gtaattctcgaggtccaggcg [SEQ ID NO:4] to amplify base pairs 190 through 1329, and the primer pair ctggacctcgagaattac [SEQ ID NO:5] plus ccctctagattatttttgatggcggacttc [SEQ ID NO:6] to amplify base pairs 1329 to 1974 with a stop codon added
  • the first PCR product was cut with BspEI and Xhol, the second with Xhol and Xbal, and both were inserted by three-piece ligation into APtag4 cut with BspEI and Xbal.
  • Deletion constructs of AP-mAPP were made by PCR amplification of selected portions of APP, using Pfu as the polymerase and AP-mAPP as the template, adding a BspEI site on the 5' end, and a stop codon followed by an Xbal site on the 3' end to allow insertion into APtag4.
  • Amino acids included in these constructs are as follows: 18-65, 18-124, 18-156, 18-205, 18-293, 18-345, 18-480, 66-612, 125-612, 157-612, 199-612, 294-612, 346-612, and 481-612.
  • AP fusion proteins were produced by transfecting 293T cells with the appropriate plasmids, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Conditioned medium was collected four to six days after transfection, filtered, and supplemented with 20 mM HEPES (pH 7.0) and 0.05% sodium azide.
  • the cell culture medium used was Dulbecco's Modified Eagle Medium (Invitrogen) with 10% calf serum, supplemented with glutamine and Penicillin/Streptomycin solution (both from Biowhittaker). The cultured supernatants were used without purification for all experiments.
  • HBSS Hank's balanced salt solution
  • HBAH was HBSS with 0.5 mg/ml bovine serum albumin, 0.05% sodium azide, and 20 mM HEPES (pH 7.0) added.
  • HH was HBSS with 20 mM HEPES (pH 7.0).
  • Lysis buffer was 0.5% sodium deoxycholate, 0.5% Triton Xl 14, and 0.05% SDS in phosphate buffered saline. Lysis buffer plus protease inhibitors additionally included 5 mM EDTA plus a cOmplete protease inhibitor cocktail tablet (Roche), dissolved to the manufacturer's recommended concentration.
  • Wash buffer was 0.1% Triton X-100 in phosphate buffered saline.
  • High salt wash buffer was IM NaCl, 20 mM HEPES (pH 7.0), and 0.1% Triton XlOO.
  • Elution solution was 100 mM glycine, 150 mM NaCl, pH 3.0.
  • Anti-AP beads were made essentially as previously described (Flanagan et al., 2000). Briefly, CNBr-Sepharose beads (Amersham Biosciences) were washed with 500 ml ImM HCl, then conjugated to monoclonal anti-human placental alkaline phosphatase antibody MIA1801 (Seradyn) in the presence of 0.25 M sodium phosphate pH 8.3, allowing lmg antibody per ImI packed beads. After an overnight incubation at 4 degrees C, the reaction was stopped by adding ethanolamine (pH 8.0) to 333 mM and incubating an additional 4-5 hours. Beads were then washed with 0.5 M sodium phosphate (pH 8.3), followed by several washes of Lysis buffer.
  • AP and AP-mAPP beads were made by a similar procedure to that for anti-AP beads, except that concentrated AP or AP-mAPP cultured supernatants were used in place of antibody. These supernatants were produced by transfected 293T cells grown in Opti-MEM with ITS-A, an insulin, transferrin, and selenium supplement (Invitrogen), and were concentrated approximately 12-fold using a Centricon device with a 10 kDa molecular weight cut-off (Millipore).
  • AP fusion proteins were used as in situ probes for staining tissue according to published protocols (Flanagan et al., 2000). Embryonic chick brains were dissected, washed with HBAH 5 then incubated in AP fusion proteins for at least 90 minutes. After 6 washes in HBAH, brains were fixed for 2 minutes (in 40% acetone, 8% formalin, 20 mM HEPES, pH 7.0), washed three times in HBS (150 mM NaCl, 20 mM HEPES, pH 7.0), then incubated in HBS overnight at 65 degrees Celsius to inactivate endogenous phosphatase activity.
  • AP developing buffer 100 mM NaCl, 5mM MgCl 2 , 100 mM Tris-HCl, pH 9.5
  • Phosphatidylinositol-specific phospholipase C was obtained from Molecular
  • olfactory bulbs from El 3 chick embryos were washed with HH, then transferred to 5 U/ml PI- PLC in HH (or simply HH only for mock-treatment) and incubated at 37 degrees for 30 minutes. The supernatant was collected and in some cases stored at 4 degrees up to several days. For 2D gels, this supernatant was desalted into 2D sample buffer using spin columns (Pierce), and electrophoresis was carried out as described below.
  • the supernatant of PI-PLC treated olfactory bulbs was cleared by spinning at 10O 5 OOOg at 4 degrees, then divided and incubated with either AP or AP-mAPP beads overnight at 4 degrees.
  • the beads were then washed extensively with cold HH, and bound proteins were eluted with Elution solution, buffered with 100 mM Tris pH 8.0 and precipitated with 10% trichloroacetic acid.
  • the precipitated proteins were centrifuged to pellet, washed with acetone, air-dried, and boiled in SDS-PAGE sample buffer supplemented with 100 mM Tris pH 8.0.
  • tecta were dissected from El 3 chick embryos (approximately 100 embryos for small-scale experiments, approximately 1,200 for Figure 7), rinsed in HBSS, and biotinylated with 0.2 mg/ml EZ-link NHS-LC-Biotin (Pierce) in HH for 45 minutes. The biotinylation reaction was quenched by adding 50 mM Tris pH 7.5 then incubating 20 minutes.
  • tecta were then washed in HBAH, incubated in AP fusion-containing conditioned media (AP, AP-mAPP, or AP-mAPP 199-345) for at least 90 minutes, then washed first six times in ice-cold HBAH, then six times in ice-cold HBSS.
  • Reversible chemical crosslinking was performed by exposing tecta to 0.5 mg/ml DTSSP (Pierce) in HH for 45 minutes (at room temperature), and the crosslinking reaction was quenched by adding 50 mM Tris pH 7.5 then incubating 20 minutes.
  • Tecta were partially lysed for 45 minutes on ice in Lysis buffer plus protease inhibitors.
  • Remaining tectal debris was pelleted and the supernatant was, in some cases, stored at 4 degrees for up to several days before further processing.
  • the lysate was cleared by spinning at 100,000g at 4 degrees, then incubated overnight at 4 degrees with anti-AP beads.
  • the beads were washed several times, first with ice-cold Lysis buffer, then with ice-cold Wash buffer. Bound proteins were eluted with Elution solution and buffered with 100 mM Tris pH 8.0.
  • the eluted proteins were precipitated with 10% trichloroacetic acid, pelleted, washed with acetone, air-dried, and boiled in SDS-PAGE sample buffer supplemented with 100 mM Tris pH 8.0 before undergoing gel electrophoresis.
  • SDS-PAGE sample buffer supplemented with 100 mM Tris pH 8.0 before undergoing gel electrophoresis.
  • the eluted proteins were boiled for 15 minutes in 100 mM DTT to cleave the DTSSP crosslinker, incubated with 9.25 mg/ml iodoacetamide (15 minutes, room temperature) to prevent disulfide bonds from re-forming, then precipitated with 10% trichloroacetic acid.
  • the precipitated proteins were centrifuged to pellet, washed with acetone, air-dried, and resuspended in 2D sample buffer as described below.
  • tecta dissected from approximately 800 El 3 chick embryos were rinsed in HBSS, then reversibly biotinylated with 1 mg/ml Sulfo-NHS- SS-Biotin (Pierce) in HH for 1 hour.
  • the biotinylation reaction was quenched by the addition of 50 mM Tris pH 7.5 for a 20 minute incubation, followed by extensive washing in HBSS.
  • Tectal lysis and clearing of the lysate were as in the preceding protocol, then the lysate was incubated overnight at 4 degrees with NeutrAvidin beads (Pierce).
  • the beads were washed several times, first with Lysis buffer, then Wash buffer, then High salt wash buffer, then finally with Wash buffer again.
  • the beads were then incubated in 100 mM DTT at 65 degrees for 40 minutes to reduce the disulfide bond in the biotinylation reagent.
  • the supernatant was collected, and proteins were precipitated with 10% trichloroacetic acid.
  • the precipitated proteins were centrifuged to pellet, washed with acetone, air-dried, and boiled in SDS-PAGE sample buffer supplemented with 100 mM Tris pH 8.0.
  • the ZOOM IPGRunner system (Invitrogen) was used, with isoelectric focusing done on pH 3-10 strips followed by SDS-PAGE on 4-20% Tris-glycine gels.
  • the sample buffer for isoelectric focusing was 7M urea, 2M thiourea, and 4% CHAPS (from 2D insoluble protein sample prep kit, Pierce), with the addition of carrier ampholytes (Invitrogen), 20 mM dithiothreitol, and 0.02 mg/ml bromophenol blue.
  • the TMpred program was used to search for hydrophobic regions that might correspond to transmembrane domains, signal peptides, or GPI anchorage signals (at www.ch.embnet.org/software/TMPRED form.html).
  • the big-PI Predictor program of the IMP Bioinformatics Group in Vienna, Austria was also used to predict GPI anchorage sites.
  • AP-mAPP a fusion protein with Alkaline Phosphatase fused to the N-terminus of APPsa, to use as an affinity probe.
  • This approach has been previously used to identify several novel receptors or ligands for orphan cell-surface proteins (Flanagan and Cheng, 2000).
  • AP-mAPP binds to embryonic chick brains, with. strong midbrain and olfactory bulb signals ( Figure 2A).
  • the binding pattern of AP-mAPP to chick tectum coincides with the expected location of retinal ganglion cell (RGC) axons.
  • RRC retinal ganglion cell
  • SR-A does not seem to be expressed by neurons in neonatal or adult mice, or in humans, though there is expression elsewhere in the brain, including in microglia, macrophages, and perivascular sites, (Bell et al., 1994; Christie et al., 1996), so this binding pattern still seemed likely to be due to an unidentified binding partner for APP.
  • PI-PLC phosphatidylinositol-specific phospholipase C
  • Figure 4B PI-PLC cleaves GPI anchorages, so the olfactory bulb binding partner for APP appeared most likely to be a GPI-anchored protein, or possibly a soluble molecule tightly associated with a GPI-anchored protein.
  • the interaction with a different APP domain and the insensitivity of APP binding to PI-PLC treatment in the tectum suggested that this structure expresses a distinct, non-GPI linked APP binding partner.
  • the olfactory bulb-expressed binding partner for APP seemed likely to be a GPI anchored molecule, and the affinity purification above had already given some idea of its molecular weight. Therefore, we decided to identify the major GPI-anchored proteins of greater than 50 kD expressed on the olfactory bulb. To do this, we dissected chick olfactory bulbs and subjected them to either PI-PLC or mock treatment. The supernatants were run on 2D gels, and several spots specific to the PI-PLC-treated samples were analyzed by tandem mass spectroscopy (Figure 8A).
  • the purified proteins were separated by SDS-PAGE, and bands corresponding to the molecular weights of bands detected in previous co-IPs (Figure 9B, compare to Figure 6B) were cut out and analyzed by tandem mass spectroscopy. The sequences of the identified proteins were analyzed for potential transmembrane domains or signal peptides. The candidate tectal surface proteins identified are listed in Figure 9C.
  • a fusion protein of APPsalpha tagged with Alkaline Phosphatase was produced. This probe was found to bind to embryonic chick brains. Deletion analysis revealed that amino acids 18-205 of APP were sufficient for strong binding to olfactory bulbs, as well as weaker binding throughout the brain. In contrast, amino acids 199-293 were sufficient for binding to RGC axons, although the larger region of 199-345 exhibited stronger binding. This suggested the presence of at least two distinct binding partners.
  • IgTag2Eco was constructed by Andrew Bergemann, and includes a polylinker followed by the Fc fragment of human IgG (Aruffo et al., 1990) in a pcDNAl (Invitrogen) backbone.
  • pSecTaglg was constructed by Mitsu Hattori by inserting the Hindlll-Xhol fragment of IgTag2Eco into pSecTag2C (Invitrogen), creating a vector encoding the Fc fragment downstream of a signal peptide.
  • NgCAM-Fc plasmid To construct the NgCAM-Fc plasmid, pSCT-NgCAM (kindly provided by P. Sonderegger, (Buchstaller et al., 1996) was cut with Xbal and RsrII, and this fragment was inserted into pSecTaglg that had been cut with Nhel and RsrII. The resulting plasmid was then cut with BgI II, blunt ended, then cut with RsrII, and the RsrII-FspI fragment of pSCT- NgCAM was then inserted. This resulted in a construct predicted to encode the first 1 134 amino acids of NgCAM fused upstream of Fc. The NrCAM-Fc construct was kindly provided by M. Grumet (Lustig et al., 1999).
  • Fc fusions of all six human contactin family members were made by inserting the portion of the gene from the start codon through the predicted GPI anchorage site into IgTag2Eco.
  • the NCBI accession number, restriction sites added by PCR for cloning, and predicted GPI site used are as follows: contactin 1 (NM_001843, HindIII + RsrII, 993); contactin 2 (NM_005076, HindIII + BgI II, 1012); contactin 3 ([see below], RsrII + BgI II, 1002); contactin 4 (NMJ 75607, Rsr II + BamHI, 1000); contactin 5 (NM_014361, EcoRV + EcoRI, 1072); contactin 6 (NMJ) 14461, HindIII + Rsr TI, 999).
  • the sequence available for human contactin 3 did not appear to include the 5' end of the gene, so the 5' primer for PCR (ccccggaccgaaaatgatgtttccatggaaacagttg) [SEQ ID NO:7] was designed based on a stretch of human chromosome 3 genomic sequence highly homologous to the 5 1 end of rat contactin 3 (NMJ 19329).
  • the assembled full-length contactin 3 sequence encoded a protein of 3068 amino acids.
  • the six contactin-Fc clones that were chosen agreed with the NCBI sequence listed above at the predicted amino acid level.
  • contactin 3-Fc construct N- terminal to XM_039627 was confirmed by checking that two independent clones agreed in sequence for this region.
  • Deletion constructs for contactin 2, 3, or 4-Fc were constructed by amplifying the desired fragments by PCR, adding an Sfi I site to the 5' end and either an EcoRV site (for contactin 3) or a BamHI site (for contactin 4) to the 3' end.
  • pSecTaglg was partially digested with Sfi I and completely digested with either BamHI or EcoRV, and the appropriately digested contactin fragments were inserted. All of these plasmids were verified by sequencing.
  • the fragments used encode the following amino acid numbers: c2Igl-4 (31-
  • All AP-APP or AP-APLP constructs were constructed by amplifying the chosen portion by PCR, adding a BspEI site to the 5' end and an Xba I site preceded by a stop codon to the 3' end, and inserting into the corresponding sites in APtag4.
  • NCBI accession numbers used as reference for human sequences were Y00264 for APP, NM_005166 for APLPl, and L27631 for APLP2.
  • "Full length" constructs were designed to begin after the predicted signal peptide and end at the alpha cleavage site (for APP) or the homologous region. The fragments used encode the following regions by amino acid number: AP-APP (18-612), AP- APLPl (34-567), AP-APLPl Nterm (34-221), AP-APLP2 middle (215-419).
  • APP-HA full length human APP was amplified by PCR and inserted into pcDNA3.1 Zeo+. The 5' end was preceded by an Kozak sequence and a HindIII site for cloning, and the 3' end was followed by an HA tag, an Xbal cloning site (encoding amino acids S and R) 5 and a stop codon, followed by Notl and Apal sites. Deletion constructs of APP-HA were made by PCR, with the 3' primer designed to preserve the Apa I cloning site as well as the C-terminal tag (HA + Ser +Arg) and stop codon.
  • the 5' primer included a single start codon immediately upstream of the indicated cleavage site, and the PCR product was inserted into pcDNA3.1 Zeo+, using an added HindII site and Apal.
  • the 5' primer was designed to insert the first APP-derived codon immediately after the Sfil site in pSecTag2A; after signal peptide cleavage, the resulting proteins are predicted to begin with AAQPA followed by the indicated portion of APP.
  • the first APP-derived amino acid for each deletion constructs follows: beta 597, alpha 613, gamma(40) 637, epsilon 646.
  • the PCR construct listed above were verified by sequencing. Antibodies:
  • Antibodies for testing candidate tectal proteins included anti-PSA NCAM (Amersham), anti-chick NCAM-I (Amersham), anti-tenascin (Chemicon), anti-neogenin (R&D systems), anti-contactin 1 (BD Biosciences).
  • Anti-HA was from Roche, and IRdye800 conjugated anti-human Fc and anti-rat Fc were from Rockland or VWR.
  • Binding assays were carried out as previously described (Flanagan and Cheng, 2000). For preliminary cell binding assays, 293T cells were transfected with plasmids expected to encode full-length expression constructs of candidates. One day after transfection, cells were dislodged from the plates by pipetting, briefly incubated in HBAH to block non-specific interactions, then incubated in AP fusion supernatant. Cells were washed seven times with ice-cold HBAH than lysed, and AP activity was measured colorometrically. For binding assays using Fc- and AP- fusion proteins, Opti-MEM based supernatants containing contactin-Fc fusion protein were incubated with protein A beads. AP fusion proteins supernatants, normalized for AP activity, were incubated with the beads, and the AP activity retained by the beads after washing was measured.
  • Fc fusions were subjected to an anti-Fc Western blot, and quantitated using Li-COR Odyssey scanner and software.
  • 293T cells were grown in 6 well dishes and transfected using TransIT-LTl (Minis Bio Corp) according to manufacturer's protocols, using 6 microliters of transfection reagent per well. Cells were collected for analysis one day after transfection. Plates were pre-chilled on ice 5 minutes, then lysed in 200 microliters of 1% Triton XlOO, 10 mM Tris pH 8.0, plus 140 mM NaCl for 20 minutes. Lysates were centrifuged 5 minutes to clear, combined with sample buffer, and boiled 5 minutes.
  • the co-IP shown in Figure 13B differed from the protocol used above in that BS3, a non-cleavable crosslinker, was used instead of DTSSP, a crosslinker that is cleaved under reducing conditions.
  • This change was made to examine the molecular weight of complexes containing NgCAM and APP. The observed spread of signal over a wide range of molecular weights is difficult to interpret clearly, but may indicate the presence of additional molecules of varying weights in the crosslinked NgCAM-APP complexes.
  • contactin 4 and NgCAM fusion constructs used lack any membrane attachment or cytoplasmic domains, which suggested to us that these proteins may be able to affect APP or CTFalpha levels when presented in trans to APP.
  • contactin 4 or NgCAM protein presented in trans rather than by co-transfection, consistently results in changed levels of full-length APP or CTFalpha.
  • Figure 17C 5 we show an example in which APP- HA CTFalpha levels were increased in cells exposed to supernatants containing clustered NgCAM-Fc in conjunction with contactin 4 presented either as a clustered supernatant or through transfection.
  • Example 3 APP. NgCAM. and contactin 4 in the visual system
  • the invention further characterizes biological relevance of the protein-protein interactions. Some regions in the nervous system of the developing chicken embryo for expression of these genes were examined. Based on both expression patterns and previously reported functional roles of Ll , the mammalian NgCAM ortholog, possible functional interactions of APP 5 contactin 4, and NgCAM in the.retinotectal system were then investigated.
  • Plasmids All plasmids for in situ probes were constructed with a pBluescript II SK(-) vector backbone (Stratagene). The following fragments were inserted: APP nucleotides 471 to 1218, using endogenous Pstl sites; NgCAM nucleotides 2827 to 3754, using endogenous Sacl and BamHI sites; contactin 3 nucleotides 2465 to 3277, using endogenous Pstl sites; contactin 4 nucleotides 21 to 818 of chESTl 18118 (from the BBSRC ChickEST database at www .chick.umist.ac.uk), using Pstl sites added by PCR.
  • the NgCAM fragment was cut from pSCT-NgCAM, a kind gift from Sonderegger (Buchstaller et al., 1996), while other fragments were generated by RT-PCR from chick tectal cDNA. Except for contactin 4, the above nucleotide numbering is given according to the coding region; see accession number Z75013 for NgCAM sequence information, accession number XM_414433 for contactin 3, and a combination of ChEST590c2 (from the BBSRC ChickEST database) and accession number AF042098 to construct the chick APP695 sequence.
  • the PCR primers used were tctagaatggtgctgctgtgtggacc [SEQ ID NO: 8] and ctgcagtatgacactcagttctgcac [SEQ ID NO: 9], based on the sequence in chESTl 18118.
  • the resulting PCR product was checked, and the predicted translation is 84% identical to amino acids 140 to 402 of human contactin 4 (accession number NM_175607). This translated sequence is only 69% identical to the corresponding region of human contactin 3, by comparison.
  • Plasmids used to produce soluble Fc proteins are described in above. Plasmids used to produce AP fusion proteins (including APtag4 for AP alone) are described above.
  • the plasmids were linearized with a restriction enzyme, and transcription, was carried out with T3 or T7 polymerase (Promega) according to manufacturer's protocol, except using ImM each ATP, CTP, and GTP, with 0.65mM UTP and 0.35 mM digoxigenin-UTP (Roche). Restriction enzymes and polymerase used were as follows: APP antisense EcoRI/T3, sense BamHI/T7; NgCAM antisense Sad (followed by blunt ending)/T7, sense BamHI/T3; contactin 3 antisense Spel/T7, EcoRI/T3; contactin 4 antisense Xbal/T7, sense EcoRI/T3.
  • Embryonic day 11 chick embryo heads were prepared for in situs by fixation in 4% paraformaldehyde in Phosphate Buffered Saline, then were embedded and frozen in OCT before being cut into 10 micron thick sections. Section in situs were carried out according to standard protocols. Detection of probe was with alkaline phosphatase conjugated anti-digoxigenin antibody (Roche), visualized using 5-bromo-4- chloro-3-indoxyl phosphate and nitrotetrazolium blue (Biosynth International) as substrates.
  • Retinal outgrowth assays were performed by dissecting embryonic day 6 chick retinas, mounting them on a polycarbonate filter (Sartorius) with the RGC layer up, cutting into strips 300 microns wide, then laying these strips RGC side down on coated coverslips, and culturing for two days.
  • the media used for culturing retinal explants was based on a combination of media types used in slightly different assays (Yamagata and Sanes, 1995), (Walter et al., 1987) and consisted of 47.6% Neurobasal media, 37.5% DMEM/F12, 4.8% fetal bovine serum, 2.4% chick serum, 1% B27, 0.15% methyl cellulose, 0.14% glucose, 20 mM HEPES pH7.0, 10 mM glutamine, 71 U/ml Penicillin, 71 microg/ml Streptomycin, and 1.76 microg/ml glutamate.
  • Brain extract made by homogenizing E6 chick brains in HBSS and centrifuging to remove debris, was added to the media fresh before use, allowing extract from one brain for every 5 mis of media.
  • Anti-APP antibody was goat anti-APP amino acids 44-63 (Calbiochem) and was used at a 1 : 100 dilution.
  • DAPT Calbiochem was used at 0.5 micromolar.
  • Axons were stained by incubating in 33 micromolar carboxy fluorescein diacetate, succinimidyl ester (Invitrogen) for 10 minutes, and visualized under FITC illumination. Outgrowth varied somewhat between experiments, so figures here represent only one or two individual experiments that exemplify the trends seen in other experiments. Outgrowth was measured by counting individual axons. The p-values given are according to the Student's t- test.
  • Purified Fc fusion proteins were produced by calcium phosphate or Lipofectamine 2000 (Invitrogen) mediated transfection of 293T cells with the appropriate plasmid.
  • the cells were maintained in Opti-MEM plus ITS-A, an insulin, transferring, and selenium supplement (Invitrogen), and the supernatants were collected several days later. After filtration, the supernatants were incubated with Protein A Sepharose beads (4 Fast Flow, Amersham Biosciences), at approximately a one-thousandth volume. The beads were washed several times with cold 10OmM Tris pH8.0 then 10 mM Tris pH 8.0.
  • Bound proteins were eluted in 150 mM NaCl plus 10OmM, pH 3.0, then dialyzed against HBSS (VWR or Invitrogen) using a 10,000 molecular weight cut-off dialysis cassette (Pierce).
  • AP fusion proteins were produced by calcium phosphate mediated transfection of 293T cells with APtag4, AP-mAPP, or AP-mAPP( 18-205). The cells were then maintained in Opti-MEM plus ITS-A for several days. Supernatants were not subjected to purification, but were filtered, then concentrated 10-fold using Amicon Centricon devices with a 10,000 molecular weight cut-off (Millipore).
  • APP interacts physically with contactin 3, contactin 4, and NgCAM.
  • Rikke Egelund carried out RNA in situs to determine the expression pattern of these genes in El 1 chick embryos.
  • NgCAM protein expression in the tectum has previously been examined, and it appears quite specific for the SO (Yamagata and Sanes, 1995), a layer which is composed mostly of RGC axons, so we decided to examine the effect of our proteins of interest on RGC axons cultured in vitro.
  • anti-APP Since gamma secretase activity is required for Notch signaling and affects the processing of many additional proteins, we also tried using an anti-APP antibody as a potentially more specific inhibitor. As can be seen in Figure 2OB, anti-APP also promoted RGC outgrowth on NgCAM-Fc substrates (p ⁇ 0.05). IfNgCAM stimulated outgrowth by signaling through APP, DAPT or antibody treatment might have been expected to decrease outgrowth on NgCAM-Fc. The increase in outgrowth seen under these conditions suggests that APP signaling may in fact act to reduce or inhibit outgrowth.
  • NgCAM has an inhibitory effect on RGC outgrowth through its interaction with APP, while promoting outgrowth through some other receptor. If the DAPT and antibody used above blocked APP signaling in response to NgCAM, one might expect that DAPT and anti-APP would fail to promote outgrowth on another substrate. However, RGC axon outgrowth from explants grown on laminin was also promoted by DAPT (pO.OOOl; Figure 20C). Preliminary experiments testing the specificity of anti-APP promoted outgrowth for NgCAM substrates are not conclusive, though it appears that anti-APP may also promote outgrowth on laminin. In addition, it was tested whether NgCAM may signal through contactin 4, this time using a dominant negative approach.
  • NgCAM and contactin 4 were interested in the expression of NgCAM and contactin 4 to determine whether their expression patterns correlated well with binding of APP to embryonic chick brains discussed above.
  • AP-mAPP( 18-205) was found to bind particularly strongly to olfactory bulbs, although binding above background was detected throughout the brain. This is consistent with the ability of AP-m APP(18-205) to bind contactin 4 (see Example 3), and the expression of contactin 4 in the olfactory bulb and the OE (since ORN axons project from the OE to the olfactory bulb).
  • NgCAM RNA in RGCs shown here and published observations of NgCAM protein in the tectal SO layer (Yamagata et al., 1995) both correlate well with the AP-APP(199-345) tectal binding described in Example 1. Another reason we were interested in the expression patterns of APP, NgCAM, and contactin 4 was to identify biological contexts in which interactions among these proteins may exist. These genes are expressed, at least at the level of RNA, in the olfactory and retinotectal systems.
  • contactin 4 may bind directly to NgCAM, acting as a blocking reagent to prevent outgrowth mediated by axonal proteins that recognize the same, or an overlapping, region of NgCAM.
  • the combined actions of NgCAM and contactin 4 may generate an inhibitory signal; as contactin 4 does not interfere with outgrowth on laminin, an inhibitory signal cannot be attributed to contactin 4 acting alone. Since we saw a similar inhibitory effect on NgCAM induced outgrowth with all of the other contactin family members we tested, since contactins 1 and 2 have already been shown to bind NgCAM
  • APPsalpha in the form of AP-mAPP
  • N-terminal El domain has already been shown to promote neurite outgrowth in other systems (Ohsawa et al., 1997; Small et al., 1994), so this was not entirely surprising.
  • AP-mAPP (18-205) binds to contactin 4, one possibility is that APP El signaling that promotes axon outgrowth may be mediated by axonal or cell-surface contactin 4.
  • Contactin 4-Fc seems to inhibit APPsalpha stimulated outgrowth, but since contactin 4 also blocks outgrowth on NgCAM, we cannot draw any firm conclusions.
  • One future experiment could be to use deletion constructs of contactin 4-Fc as blocking reagents, with the first four Ig domains as a potential NgCAM- specific blocking reagent (by analogy to NgCAM's binding to the first four domains of contactin 2 (Rader et al., 1996), and the fibronectin domains as a potential APPsalpha-blocking reagent.
  • Another option in principle, could be to study the role of contactin 4 in APPsalpha-stimulated growth in the absence of NgCAM, but we have so far failed to see APPsalpha enhance growth on laminin. More definitive experiments could involve RNAi-mediated knock-down of contactin 3 and/or contactin 4 or, if similar results were seen with mouse RGCs, mice deficient in these genes.
  • ADAMs family members as amyloid precursor protein alpha-secretases J Neurosci Res, 74, 342-352.
  • Alzheimer disease amyloid beta protein forms calcium channels in bilayer membranes: blockade by tromethamine and aluminum. Proc Natl Acad Sci U S A, 90, 567-571.
  • amyloid precursor protein interacts with Go heterotrimeric protein within a cell compartment specialized in signal transduction. J Neurosci, 19, 1717-1727.
  • Ng-CAM origin of the polypeptides and relation to the Ig superfamily. J Cell Biol, 112(5), 1017-29.
  • BACE2 a beta - secretase homolog, cleaves at the beta site and within the amyloid-beta region of the amyloid-beta precursor protein.
  • Alzheimer's disease Trends Pharmacol Sci, 12, 383-388. Hass, M.R. and Yankner, B. A. (2005) A ⁇ gamma ⁇ -secretase-independent mechanism of signal transduction by the amyloid precursor protein. J Biol Chem, 280, 36895-36904. Hatten, M.E. (1999) Central nervous system neuronal migration. Annu Rev Neurosci, 22, 511-539.
  • mice with combined gene knock-outs reveal essential and partially redundant functions of amyloid precursor protein family members. JNenrosci, 20, 7951-7963. Hebert, S.S., Serneels, L., Tolia, A. 5 Craessaerts, K., Derks, C, Filippov, M.A., Muller, U. and De Strooper, B. (2006) Regulated intramembrane proteolysis of amyloid precursor protein and regulation of expression of putative target genes. EMBO Rep, 7, 739-745.
  • Alzheimer's disease, beta-amyloid protein and zinc J Nutr, 130, 1488S-1492S. Hussain, L, Powell, D., Howlett, D.R., Tew, D.G., Meek, T.D., Chapman, C, Gloger. I.S., Murphy, K.E., Southan, CD., Ryan, D.M., Smith, T.S., Simmons, D.L., Walsh, F.S., Dingwall, C. and Christie, G. (1999) Identification of a novel aspartic protease (Asp 2) as beta-secretase. MoI Cell Neurosci, 14, 419-427.
  • the precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature, 325, 733-736. Kimberly, W.T., Zheng, J.B., Guenette, S. Y. and Selkoe, DJ. (2001)
  • the intracellular domain of the beta-amyloid precursor protein is stabilized by Fe65 and translocates to the nucleus in a notch-like manner.
  • Membrane-anchored metalloprotease MDC9 has an alpha-secretase activity responsible for processing the amyloid precursor protein. Biochem J, 343 Pt 2, 371-375.
  • LDL receptor-related protein a multifunctional ApoE receptor, binds secreted beta-amyloid precursor protein and mediates its degradation.
  • Alzheimer's disease amyloid precursor protein interacts with phosphotyrosine-binding domain proteins in the yeast two-hybrid system. FEBS Lett, 397, 197-200.
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  • Fibulin-1 binds the amino-terminal head of beta-amyloid precursor protein and modulates its physiological function.
  • J Neurochem, 16, 1411-1420. Okamoto, T., Takeda, S., Murayama, Y., Ogata, E. and Nishimoto, I. (1995) Ligand- dependent G protein coupling function of amyloid transmembrane precursor. J Biol Chem, 270, 4205-4208. Oltersdorf, T., Fritz, L.C, Schenk, D.B., Lieberburg, I., Johnson-Wood, K.L., Beattie, E.C.,
  • the secreted form of the Alzheimer's amyloid precursor protein with the Kunitz domain is protease nexin-II.
  • Amyloid beta protein implications for the mechanism of intramembrane cleavage by gamma-secretase. J Neurosci, 25, 436-445. Quist, A., Doudevski, I., Lin, H., Azimova, R., Ng, D., Frangione, B., Kagan, B., Ghiso, J. and LaI, R. (2005) Amyloid ion channels: a common structural link for protein- misfolding disease. Proc Natl Acad Sci USA, 102, 10427-10432.
  • McConlogue, L. (2001) BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics.
  • a heparin-binding domain in the amyloid protein precursor of Alzheimer's disease is involved in the regulation of neurite outgrowth. JNeurosci, 14, 21 17-2127. Soba, P., Eggert, S., Wagner, K., Zentgraf, H., Siehl, K., Kreger, S., Lower, A., Langer, A., Merdes, G., Paro, R., Masters, C.L., Muller, U., Kins, S. and Beyreuther, K.
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Abstract

The invention relates to APP regulation by extracellular binding partners. More specifically, the invention porvides methods and compositions for treating or preventing diseases, such as Alzheimer's disease, mediated by Contactins and/or Ll family of proteins. The invention further provides methods for identifying molecules that modulate the binding and/or processing of APP and other related molecules.

Description

IDENTIFICATION OF CONT ACTINS AND Ll-CAMS AS LIGANDS FOR THE AMYLOID PRECURSOR PROTEIN
Government Support
This work was funded in part by grant number HD29417, from the National Institutes of Health, and grant number EYl 1559 from the National Institutes of Health. Accordingly, the United States Government may have certain rights to this invention.
Related Application
This application claims the benefit under 35 U. S. C. § 119(e) of United States provisional patent application serial number 60/839,114, filed August 21, 2006, the contents of which are incorporated herein by reference in their entirety.
Field of the Invention
The invention relates to molecules that bind amyloid precursor protein and to the biological function of amyloid precursor protein. The invention particularly relates to methods and products for preventing and treating Alzheimer's disease and abnormal production of amyloid β. The invention also relates to methods for screening and identifying small molecule drugs and other agents that may affect processing or function of amyloid precursor protein.
Background of the Invention
The development of a functional vertebrate brain is a complex process, involving the production of many different types of neurons and glial cells and the migration of these cells to their proper locations. The function of the brain depends on the network of connections among neurons, so the outgrowth and guidance of axons and dendrites to sometimes quite distant targets, and the subsequent formation of synapses between specific neurons, must also be highly regulated. Although much has been discovered about the molecular mechanisms driving various aspects of neural development (Chilton, 2006; Dickson, 2002; Guillemot et al., 2006; Hatten, 1999; Marin and Rubenstein, 2003), much remains to be understood. The beta-amyloid precursor protein (APP) has been linked to several processes that might be important for the development and function of the nervous system, including neuronal survival and apoptosis, cell adhesion and migration, neurite outgrowth, synaptogenesis, and synaptic plasticity (Mattson, 1997; also, see below). In particular, the Amyloid Precursor Protein (APP) is implicated as a causative agent of Alzheimer's Disease (AD). In AD, neural function is thought to be impaired by beta-amyloid, which is produced from APP by proteolytic processing. Also, APP itself may have beneficial roles in normal neural function which are interrupted in AD. Although the normal functions of APP are not well understood, it has long been considered that it may be a cell surface receptor, in which case its processing and function should be regulated by extracellular ligand(s). A number of extracellular binding partners for APP have previously been identified (metal ions, heparin, collagen, F-spondin, Notch, Nogo receptor, and others) but the functional significance of these interactions, and the biological role of APP, generally remain unclear.
Therefore, it would be of interest to identify agents capable of binding to APP and modulating (e.g., promoting or inhibiting) APP function, particularly by altering the proteolytic processing of APP. Such agents would be beneficial for the treatment and prevention of development and progression of diseases, such as AD.
Summary of the Invention
The invention provides novel interactions of APP with extracellular binding partners in two molecular families: Contactins (particularly Contactin-3 and -4) and Ll family protiens (particularly Ll and NgCAM). These interactions can regulate processing of APP. As disclosed herein, the invention also provides that APP, contactin-4 and NgCAM can collaborate in regulating the development of neural connections. The invention also includes methods to identify compounds that modulate the binding of APP to these extracellular binding partners, as well as compounds that modulate APP processing. The methods and compositions of the invention are also useful for preventing and/or treating Aβ-accumulation- associated disorders, such as AD. According to one aspect of the invention, methods for modulating APP processing are provided. The methods include contacting a mammalian cell with an agent that modulates the amount or rate of binding of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2) to contactins and/or Ll family proteins. In some embodiments of the foregoing methods, the agent is expressed by the cell or exogenously added so as to contact the cell. In some embodiments of the invention, the agents include one or more contactins such as contactin-3. contactin-4 and contactin-5, or Ll family proteins, such as Ll and Ng-CAM, or fragments thereof that bind to APP, APLPl or APLP2. In certain embodiments of the method, the agent is a fusion protein including one or more contactins, Ll family proteins or fragments. In other embodiments of the foregoing methods, the fusion protein is an Fc fusion protein.
In still other embodiments of the methods, wherein the agent is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is selected from an antibody or antigen-binding fragment thereof that binds a contactin or a Ll family protein, preferably contactin-3 contactin-4, contactin-5, Ll or Ng-CAM5 or, an antibody or antigen- binding fragment thereof binds APP, APLPl or APLP2. In some embodiments, the antibody or antigen-binding fragment thereof binds to a complex of a contactin or an Ll family protein and APP, APLPl or APLP2. In some circumstances, the antibody or antigen-binding fragment thereof is a bispecific antibody. In some embodiments of the invention, the methods include agents that block or disrupt the binding of APP, APLPl or APLP2 to contactins and/or Ll family proteins and agents that promote or stabilize the binding of APP, APLPl or APLP2 to contactins and/or Ll family proteins. The methods also include agents that can reduce processing of APP to β- amyloid. According to another aspect of the invention, methods for treating or preventing
Alzheimer's disease are provided. The methods include administering to a subject an effective amount of an agent that modulates the amount or rate of binding of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2) to contactins and/or Ll family proteins. In some embodiments of the methods, the agents include one or more contactins or Ll family proteins or fragments thereof that bind to APP5 APLPl or APLP2. In preferred embodiments, the contactin is contactin-3 contactin-4, contactin-5 or a combination thereof, and the Ll-CAM is Ll or Ng- CAM, wherein the fragment is preferably a fragment of contactin-3, contactin-4, Ll or Ng- CAM that binds to APP, APLPl or APLP2, or a combination of such fragments. In some embodiments of the methods, the agent is a fusion protein, preferably an Fc fusion protein, including one or more contactins, Ll family proteins or fragments thereof. In other embodiments of the methods, the agent is an antibody or an antigen-binding fragment thereof. For example, the agents include an antibody or antigen- binding fragment thereof that binds a contactin or a Ll family protein, particularly contactin-3 contactin-4, contactin-5, Ll or Ng-CAM5 as well as APP5 APLPl or APLP2. In some embodiments of the methods, the antibody or antigen-binding fragment thereof binds a complex of a contactin or an Ll family protein and APP5 APLPl or APLP2. In some embodiments, the methods include antibody or antigen-binding fragment thereof that is a bispecific antibody. According to some embodiments of the methods, the agent blocks or disrupts the binding of APP, APLPl or APLP2 to contactins and/or Ll family proteins. In other embodiments of the methods, the agent promotes or stabilizes the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs. In some embodiments of the foregoing, the methods includes an agent that reduces processing of APP to β-amyloid. According to some embodiments, the methods include a vectorized agent that crosses the blood brain barrier. According to the invention, the methods are used for various subject, where a preferred subject is a human.
According to yet another aspect of the invention, pharmaceutical compositions are provided. These include one or more contactins and/or Ll family proteins and/or one fragments thereof that bind to APP, APLPl or APLP2, and a pharmaceutically acceptable carrier. In preferred embodiments of the compositions, the contactin is contactin-3 contactin- 4, contactin-5 or a combination thereof, and the Ll-CAM is Ll or Ng-CAM. In some embodiments, the pharmaceutical composition includes a fragment of contactin-3, contactin- 4, Ll or Ng-CAM that binds to APP, APLPl or APLP2, or a combination of such fragments. According to the invention, some embodiments include a pharmaceutical composition that is a fusion protein, including one or more contactins, Ll-CAMs or fragments thereof. In some cases, the composition includes an Fc fusion protein.
In certain embodiments, the pharmaceutical compositions include one or more antibodies that bind a contactin, an Ll family protein, APP, APLPl or APLP2 or antigen- binding fragments thereof, where preferred contactin or Ll-CAM is contactin-3 contactin-4, contactin-5, Ll or Ng-CAM. In some embodiments, the pharmaceutical composition includes an antibody or antigen-binding fragment thereof that binds a complex of a contactin or a Ll-CAM and APP, APLPl or APLP2. In certain embodiments of the foregoing compositions, the antibody or antigen-binding fragment thereof is a bispecific antibody. The pharmaceutical composition in some embodiments includes an agent that blocks or disrupts the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs. In other embodiments, the pharmaceutical composition includes an agent that promotes or stabilizes the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs. In some embodiments of the foregoing composition, the agent reduces processing of APP to β-amyloid. In some cases, the pharmaceutical composition according to the foregoing includes a vectorized agent that crosses the blood brain barrier of a subject, preferably a human.
In another aspect of the invention, methods for identifying compounds that modulate the binding of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2) with contactins and/or Ll-CAMs are provided. These methods include providing a reaction mixture that comprises (1) APP, APLPl, APLP2 and/or a fragment thereof that binds to contactins and/or Ll-CAMS, and (2) contactins, Ll- CAMs and/or a fragment thereof that binds to APP, APLPl, APLP2; contacting the reaction mixture with a test compound; determining a level of binding of APP, APLPl, APLP2 and/or a fragment thereof with contactins, Ll-CAMs and/or a fragment thereof in the absence and in the presence of the test compound; and comparing the level of binding of APP, APLPl, APLP2 or fragment thereof with contactins, Ll-CAMs and/or a fragment thereof in the absence and in the presence of the test compound, wherein a test compound that modulates the binding relative to the level of binding in the absence of the test compound is a compound that modulates the binding of APP, APLPl , APLP2 or fragment thereof with contactins, Ll- CAMs and/or a fragment thereof.
In some embodiments of the foregoing methods, the test compound is a small molecule; an antibody that binds to APP, APLPl, APLP2 a contactin or a Ll-CAM, or an antigen-binding fragment thereof; or a fragment of APP, APLPl , APLP2, a contactin or a Ll- CAM, where preferably the contactins and/or Ll-CAMs is selected from the group consisting of: contactin-3, contactin-4, contactin-5, Ll and Ng-CAM.
In yet another aspect of the invention, methods for identifying compounds that modulate the processing of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (AP LP2) are provided. These methods include: providing a reaction mixture that comprises (1) contactins, Ll-CAMs and/or a fragment thereof that binds to APP, APLPl, APLP2, and (2) APP, APLPl, APLP2 and/or a fragment thereof that binds to contactins and/or Ll family proteins; contacting the reaction mixture with a test compound; determining a level of processing of APP, APLPl or APLP2 in the absence and in the presence of the test compound; and comparing the processing in the absence and in the presence of the test compound, wherein a test compound that modulates processing of APP, APLPl or APLP2 relative to the level of processing in the absence of the test compound is a compound that modulates the processing of APP, APLPl or APLP2. According to some embodiments of the foregoing methods, the test compound is a small molecule; an antibody that binds to APP, APLPl, APLP2, a contactin or a Ll family protein, or an antigen-binding fragment thereof; a fragment of APP, APLPl or APLP2, a contactin or a Ll family protein, where preferably the contactins and/or Ll-CAMs is selected from the group consisting of: contactin-3, contactin-4, contactin-5, Ll and Ng-CAM. In certain embodiments of the methods, the processing of APP to Aβ is reduced. These and other aspects of the invention are described in further detail below in connection with the detailed description of the invention.
Brief Description of the Drawings
Figure 1. Major proteolytic fragments of APP released in the amyloidogenic or non- amyloidogenic pathway. Not drawn to scale. N-terminus of APP is extracellular or luminal (up), and C-terminus is intracellular (down). Location of cleavage sites, according to APP695 amino acid numbering, is as follows: β 596-597, α 612-613, γ(4o) 636-637, γ(42) 638- 639, ε 645-646. The Ab region of APP is indicated in light gray.
Figure 2. APP binds to embryonic chicken brain. AP-tagged probes were used in situ to detect binding sites for APP.
(A) Binding of AP-mAPP compared to AP alone; ventral view of E12-E13 brains. Olfactory bulbs are indicated by arrows, tecta by arrowheads. (B) Binding of AP-mAPP to E9, ElO, and El 6 brains; dorsal view. Anterior (A) and posterior (P) extremes of tecta are indicated.
(C) Binding of AP-mAPP to brain from embryo with single enucleated eye. Tectum contralateral to enucleated eye is indicated by arrow.
(D) Striated AP-mAPP staining of a tectum under greater magnification. Anterior (A) and posterior (P) extremes of tectum are indicated.
Figure 3. Identification of a tectal binding domain in the APP ectodomain.
(A) Fusion protein constructs, with AP fused various portions of APPsalpha, are indicated schematically and by amino acids (numbered according to APP695) included. Full length AP-mAPP is 18-612. Binding was scored positive (+) if staining in ElO chick brains included striated staining in the anterior of the tectum similar to that shown in Figure 2D.
(B) Binding of AP-mAPP( 199-293) and AP-APP(199-345) to tecta; ventral view of E12-E13 brains. Figure 4. Characterization of APP binding to olfactory bulbs.
(A) Binding of AP-mAPP(l 8-205) compared to AP only control; ventral view of E12-E13 brains. Olfactory bulbs are indicated by arrows.
(B) Binding of AP-mAPP to brains pretreated with PI-PLC, or mock treated; ventral view of E 12-El 3 brains. Olfactory bulbs are indicated by arrows.
Figure 5. Affinity purification of APP binding proteins from olfactory bulbs. Supernatants from PI-PLC treated olfactory bulbs were affinity purified against AP- or AP-mAPP- conjugated beads. Arrows indicate bands specific for AP-mAPP sample; the lower band was analyzed by tandem mass spectrometry.
Figure 6. Crosslinker-based affinity purification of APP interacting proteins from tecta.
(A) Outline of purification scheme. Details are provided in the methods section.
(B) Western blot of sample from small-scale purification using NeutrAvidin-HRP to detect biotin-conjugated proteins.
Figure 7. Identification of an APP interacting protein from tecta.
(A) Silver stained 2D gels from large-scale tectal affinity purification. Arrows indicate areas of gels excised for analysis by tandem mass spectrometry. The samples were combined for analysis, and the proteins identified were AP, APP, and contactin 4.
(B) Western blot of 2D gels from small-scale tectal affinity purification. Arrow indicates spot used to guide location of excision from silver stained gels.
Figure 8. Identification of a panel of GPI-anchored olfactory bulbs proteins. (A) Silver stained 2D gels of supernatants from olfactory bulbs after mock or PI-PLC treatment. Spots circled in the PI-PLC gel were excised and analyzed by tandem mass spectrometry.
(B) List of candidate GPI-anchored proteins identified from Figure 8A. A larger list of proteins was identified by tandem mass spectrometry, but only those that appeared likely to be GPI-anchored (by analysis of the protein sequence; see text) are included here.
Figure 9. Identification of a panel of tectal surface proteins.
(A) Outline of purification scheme. Details are provided in the methods section. (B) Silver stain of purified biotinylated tectal proteins. Arrows indicate regions of lane excised for tandem mass spectrometry analysis. The choice of regions to analyze was guided by the location of biotinylated bands seen in tectal APP affinity purifications (see Figure 6B).
(C) List of candidate tectal surface proteins identified from Figure 8B. A larger list of proteins was identified by tandem mass spectrometry, but only those that appeared likely to be secreted or expressed on the cell surface (by analysis of the protein sequence; see text) are included here.
Figure 10. APP and related protein APLPl bind to specific contactin family members. Media containing Fc-fusion proteins were normalized for concentration, then bound to
Protein A beads. Media containing AP fusion protein was normalized for AP activity, then exposed to these beads. AP activity retained by the beads after washing is shown.
Figure 11. Saturation binding and Scatchard analysis of APP and APLPl binding to contactin family members.
(A) Saturation curves were generated by carrying out binding assays as in Figure 10, except varying the concentration of AP fusion protein exposed to the beads.
(B) Points on the Scatchard plots shown here were generated from the raw data graphed in (A). The line was generated by least squares fitting. (C) The KD of each interaction was calculated from -I/slope, using the sjope of the lines in
(B).
(D) Dotted lines added by hand indicate potential for interpreting APP binding as biphasic. The solid line is the fitted line from (B), which corresponds to interpretation as a single binding site.
Figure 12. Identification of domains sufficient for APP-contactin 4 interaction.
(A) Results of binding assay, done according to method of Figure 10, except that Fc fusion protein containing media was used without normalizing for concentration: expression levels of Fc fusion proteins used can be seen in (B). Fc constructs included the specified portions of contactin 2, 3, or 4. AP fusion constructs are as indicated in the figure.
(B) Western blot against human Fc, on Fc fusion protein-containing media used in (A).
Figure 13. APP interacts with NgCAM. (A) Western Blots of immunoprecipitates of AP or AP-mAPP after reversible crosslinking to tecta; the protocol used is the same as in Figure 6. Detection was by Avidin-HRP (for biotinylated proteins), or one of two different anti-NgCAM antibodies.
(B) Western Blots of immunoprecipitates of AP, AP-mAPP(199-345), or AP-mAPP after irreversible crosslinking to tecta; except for the use of BS3 instead of DTSSP as crosslinker, the protocol is the same as in (A). Detection was by mouse anti-NgCAM.
Figure 14. APLPl binds to olfactory bulbs. AP in situs were used to detect binding of AP- APLPl or AP-APLPl Nterm compared to AP alone. Ventral view of E 12-El 3 brains; olfactory bulbs are indicated by arrows.
Figure 15. APLP2 binds to embryonic chick brains and an endothelial cell line. (A) AP in situs were used to detect binding of AP-APLP2 middle; ventral view of E 12-El 3 brains, with magnified view of indicated region shown below (B) AP in situs were used to detect binding of AP-APLP2 middle to RF/6A (rhesus monkey retinal choroid endothelial) cells, compared to binding of AP alone or AP-mAPP(l 99-345).
Figure 16. Co-expression of tagged contactin 4 constructs affects levels of APP CTF alpha.
(A) Anti-HA western blot of cell lysates from 293T cells co-transfected with APP-HA and the indicated construct; Fc refers to the pSecTaglg plasmid, which encodes secreted Fc.
(B) Anti-HA western blot of cell lysates, either co-expressing APP-HA and contactin 4-AP, or expressing an N-terminally deleted version of APP-HA. These constructs begin in APP- HA after the cleavage site indicated, but are preceded by either a starting methionine or a signal peptide. Diagram shows relative location of cleavage sites on APP, but is not drawn to scale.
Figure 17. Effects of contactin 4 and NgCAM on APP CTFalpha levels.
(A) Anti-HA western blot of cell lysates from 293T cells co-transfected with APP-HA and the indicated construct. One microgram of each plasmid was used; for transfections with only one plasmid (other than APP-HA) indicated, one microgram of pcDNA3.1 Zeo+ was also added.
(B) Anti-HA western blot of cell lysates from 293T cells co-transfected with APP-Ha and the indicated construct. One microgram of each plasmid was used, except that 0.5 micrograms each of contactin 4-Fc and NgCAM-Fc were used in the final lane. (C) Anti-HA western blot of cell lysates from 293T cells. Cells were co-transfected with APP-HA and either contactin 4- AP or pcDNA3.1 Zeo+. Transfected cells were incubated overnight with the clustered Fc fusion proteins indicated; microgram total of Fc fusion added was the same for each well.
Figure 18. APP, NgCAM, and contactin 4 are expressed in the olfactory system.
(A) RNA in situ hybridization to detect expression (antisense) above background (sense). Olfactory bulb indicated by arrows.
(B) RNA in situ hybridization. Olfactory epithelium indicated by arrows.
Figure 19. APP, NgCAM, contactin 3, and contactin 4 are expressed in the visual system
(A) RNA in situ hybridization in the tectum to detect expression (antisense) above background (sense).
(B) RNA in situ hybridization. RGC layer of the retina indicated by arrows. The retina is bounded on the opposite side of the RGC layer by the pigmented epithelium (indicated by arrowheads).
Figure 20. Effect on RGC axon outgrowth of reagents directed against APP or APP cleavage. (A) Effect of gamma secretase inhibitor DAPT, compared to DMSO control, on RGC outgrowth on NgCAM-Fc substrates. Quantitation is number of axons per retinal explant. For DMSO explants n=8, DAPT, n= 10; p<0.05 . Error bars indicate SEM.
(B) Effect of anti-APP antibody versus control. For anti-APP n=12, control n=12. Control includes both nothing added (n=6) and goat serum (n=6) conditions, as these control conditions resulted in similar outgrowth (mean nothing added=28.5, mean goat serum=33.5). For comparing anti-APP to control, p<0.05.
(C) Effect of gamma secretase inhibitor DAPT, compared to DMSO control, on RGC outgrowth on laminin substrates. For DMSO explants n=8, DAPT n=8; pO.0001.
Figure 21. Contactin-Fc fusion proteins inhibit outgrowth on NgCAM-Fc.
(A) RGC outgrowth on NgCAM-Fc or laminin substrates with or without the addition of contactin 4-Fc to the substrate.
(B) RGC outgrowth on NgCAM-Fc substrates that also contain the indicated Fc fusion protein. Figure 22. Interactions among NgCAM, contactin 4, and APPsalpha in promoting RGC axon outgrowth. Quantitation of number of axons per retinal explant in each of six . conditions, with substrates containing the indicated proteins. For both "AP only" conditions n=12, for all other conditions n=6. Error bars indicate SEM. In the context of NgCAM, the differences between AP only versus AP-mAPP(l 8-205) (p<0.02) and AP only versus AP- mAPP (p<0.002) are statistically significant. In the context of NgCAM + contactin 4-Fc, neither difference rises to statistical significance, although a trend is apparent in comparing AP only to AP-mAPP (p=0.56). A difference also appears to exist between NgCAM versus NgCAM + contactin 4-Fc outgrowth in the context of both AP-mAPP(l 8-205) (p=0.166) and AP-mAPP (p=0.063), although neither is statistically significant.
Detailed Description of the Invention
APP structure:
The amyloid precursor protein (APP) is a type 1 transmembrane protein with a large extracellular domain and a relatively small intracellular domain. The cytoplasmic domain is Λvell conserved among APP family members in both vertebrates and invertebrates, as are two extracellular domains termed El and E2 (Daigle and Li, 1993). As used herein, the terms, "amyloid-beta precursor protein", "beta-amyloid precursor protein" and "amyloid precursor protein", are used interchangeably. In some cases, "amyloid-beta precursor protein" or "beta-amyloid precursor protein" may be used to distinguish the precursor species that specifically give rise to the beta form of the peptide, as opposed to the term "amyloid precursor protein", which is used generally to encompass all eventual peptide products (without prejudice for any particular products and/or intermediates, e.g., alpha, beta, gamma, and epsilon). In addition, the notation "β" and "beta" (as in "β- amyloid" and "beta-amyloid") are used herein interchangeably. Similarly, "α", "γ" and "ε" may be expressed as "alpha", "gamma" and "epsilon", respectively.
The extracellular portion of APP can be divided structurally into several parts: El, which contains the growth factor-like domain, or GFLD (amino acids 23-128), and the copper binding domain, or CuBD (amino acids 124-189); the acidic or anionic domain; E2, which is also called the central APP domain or CAPPD (289-479); the linker region (507- 589); and finally the N-terminal portion of the Abeta region (Reinhard et al., 2005). Several splice forms of APP have been reported, but the three major isoforms, called APP695, APP751, and APP770, according to the number of amino acids in the unprocessed protein, differ only in the extracellular domain. APP695 is the splice variant used in this study, so all amino acid numbering here is given accordingly. APP751 only differs from APP695 by the inclusion of exon 7, which encodes an extracellular Kunitz protease inhibitor (KPI) domain, while APP770 includes both the KPI domain and exon 8, the OX-2 domain (Coulson et al., 2000). The KPI domain has been studied for its interaction with the blood coagulation cascade (Van Nostrand et al., 1992), and its interaction with LRP can promote the endocytosis and degration of APP (Kounnas et al., 1995). In contrast, the physiological significance of the OX-2 domain has not been thoroughly examined and, in many studies, the functions of APP751 and APP770 (sometimes referred to collectively as APP-KPI) are not distinguished from each other. In neurons, APP695 expression predominates, while in non- neuronal cells, the KPI containing forms are more abundant (Neve et al., 1988; Tanzi et al., 1987). Vertebrate genomes have been found to contain two additional genes that are closely related to APP, called amyloid precursor like protein 1 (APLPl) and amyloid precursor like protein 2 (APLP2). APLP2 shares a higher sequence homology with APP695 than does APLPl5 and APLP2 also has alternative splicing that allows for addition of a KPI domain. Neither APLP contains an OX-2 like domain (Coulson et al., 2000).
Proteolysis:
APP was originally identified as the source of the beta-amyloid peptide (also called Abeta). As used herein, the terms "beta-amyloid", "β-amyloid", "Abeta" and "Aβ" are intended to mean the same. Abeta is a major component of the plaques seen in the brains of patients with Alzheimer's disease (Kang et al., 1987). Abeta is derived from APP proteolytically, by cleavage at the sites termed beta and gamma (see Figure 1). Cleavage of APP by beta-secretase, that is, the beta-site directed protease, releases a large extracellular fragment called APPsbeta. Cleavage of the remaining transmembrane carboxy-terminal fragment (CTFbeta) at the gamma cleavage site generates Abeta. The exact location of gamma secretase cleavage varies, but the resulting Abeta peptides are usually 40 or, less commonly, 42 amino acids long. In addition, cleavage at the epsilon sites releases the APP intracellular domain fragment (AICD). Because of the generation of Abeta, this is termed the amyloidogenic pathway. In the non-amyloidogenic pathway, in contrast, the first cleavage event is at the alpha site, located between the beta and gamma sites, generating CTFalpha and the large extracellular fragment APPsalpha, and precluding the formation of Abeta. Gamma and epsilon cleavage also occur in this pathway, so AICD is still released, but instead of Abeta, the shorter, apparently non-pathogenic, p3 fragment is formed (Vetrivel and Thinakaran, 2006). Since the processing of APP is thought to be central to both the pathology and physiology associated with APP, much work has been done on the secretases responsible for APP cleavage.
As used herein "processing" of APP is intended to include proteolytic cleavage of the precursor at one or more sites recognized by specific secretases, which gives rise to various peptide intermediates and products that are discussed in more detail below (see Figure 1).
The invention is directed to modulating the process of APP processing which can be used for a variety of applications.
Inhibitor studies implicated zinc metalloproteases in alpha secretase activity (Roberts et al., 1994), and in fact three members of the ADAM (a disintegrin and metalloprotease) family, ADAM17/TACE (Slack et al., 2001), ADAMlO (Lammich et al., 1999) and
ADAM9/MDC9 (Koike et al., 1999) have been shown confer alpha secretase activity when overexpressed in cells. Studies in mutant mice have shown that none of these enzymes by itself is required for all alpha cleavage events, and it is likely that more than one enzyme may act as an alpha secretase in vivo (Allinson et al., 2003). The search for a beta secretase resulted in the independent identification of the membrane-associated aspartic protease BACEl (beta-site APP-cleaving enzyme 1 ; also called Asp2 and memapsin 2) by several groups (Hussain et al., 1999; Lin et al., 2000; Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999). Genetic knockouts have clearly shown BACEl to be the primary beta secretase in the brain (Cai et al., 2001 ; Luo et al., 2001 ; Roberds et al., 2001). BACE2, a homologous protein, has also been shown to possess beta secretase activity in vitro (Farzan et al., 2000; Hussain et al., 2000) and both BACEl and 2 must be removed in order to completely inhibit glial production of Abeta (Dominguez et al., 2005). However, the exact function of BACE2 in APP processing remains unclear, since BACE2 has also been shown to cleave APP in a non-amyloidogenic fashion after residues 19 or 20 of the Abeta domain (Farzan et al., 2000; Fluhrer et al., 2002; Yan et al., 2001), a few amino acids downstream of alpha cleavage, and RNAi -mediated reduction of BACE2 has been shown in some instances to increase production of Abeta (Basi et al., 2003).
A complex of four proteins, presenilin (1 or 2), Aph-1, nicastrin, and Pen-2 is thought to constitute gamma secretase (De Strooper, 2003). Gamma secretase has been found to be required for cleavage not only at the gamma site, primarily after amino acids 636 or 638, but also for cleavage at the epsilon site, either before or after Leu-645 (Gu et al., 2001; Sastre et al., 2001; Weidemann et al., 2002; Yu et al., 2001).
APP proteolysis is generally thought to occur along a "pathway", in that the first cleavage event, either alpha or beta, is thought to stimulate gamma cleavage by generating a transmembrane protein with a short extracellular protein stub that can be recognized by gamma secretase (Struhl and Adachi, 2000), probably through interaction with nicastrin (Shah et al., 2005). How epsilon cleavage, with its production of AICD, fits into either pathway is not completely clear, but the identification of C-terminally extended Abeta fragments (Qi-Takahara et al., 2005) and the processing of such fragments into either
Abeta40 or Abeta42, depending on the location of epsilon cleavage (Funamoto et al., 2004), suggests a model where epsilon cleavage normally precedes gamma cleavage. If gamma secretase cleavage is stimulated by either alpha or beta cleavage, and is preceded by epsilon cleavage, one might expect the amyloidogenic and non-amyloidogenic pathways to generate AICD equally well. However, AICD levels seem to be positively (Kume et al., 2004) or negatively (Hoe et al., 2005) correlated with alpha cleavage under different conditions. Whether the AICD levels in these studies reflect changes in production, or stability or activity, is unclear.
Alzheimer's disease:
Alzheimer's disease (AD) is a neurodegenerative disease that is morphologically characterized by the presence in the brain of intracellular tangles and extracellular plaques. A potential link between APP and AD was apparent immediately upon the identification of APP, since the APP-derived Abeta peptide had already been identified as a major component of the AD plaques (Kang et al., 1987). The subsequent discovery that several families with familial, dominantly-inherited AD carried mutations in APP suggested a causal role of Abeta in AD. Furthermore, the realization that mutations in presenillins 1 and 2, the genes implicated in the majority of familial AD cases, modulated gamma secretase cleavage further strengthened the case for the direct involvement of Abeta. In particular, several of the disease-causing presenillin mutation were found to have the effect of increasing the ratio of the more fϊbrillogenic Abeta42 to Abeta 40 (Scheuner et al., 1996). Combined, this suggested that Abeta aggregates were central to AD pathology.
Currently, the most widely accepted models for the etiology of AD are variations on what is known as the amyloid cascade hypothesis (Hardy and Allsop, 1991; Selkoe, 1999). In the original version of this model, the accumulation of excess Abeta42 results in the formation of plaques, which leads to inflammatory responses, oxidative stress, neurofibrillary tangle formation, and widespread neuronal dysfunction and death. More recently, attention has shifted away from the effects of plaques, partially based on the observation that plaque load does not correlate well with the degree of dementia in AD. Instead, focus has shifted to low molecular weight oligomers of Abeta and their potential to cause both cell death and reversible impairments of neuronal function (Klein et al., 2001 ; Lesne et al., 2006).
Roles in normal physiology:
Genetic requirements for APP and APLPs:
Although perhaps most of the work on APP has focused on its role in disease, a considerable amount has also been done trying to understand the normal physiological role of APP. One valuable approach to understanding the function of a gene is to remove it, and accordingly, mice deficient for APP have been generated and studied. These mice are viable and fertile, but have been found to have some abnormalities, including increased copper levels in the liver and cortex (White et al., 1999), decreased locomotor activity and grip strength (Zheng et al., 1995), and increased susceptibility to induced seizures (Steinbach et al., 1998). In addition, although the brains of these mice seem to develop fairly normally, a defect in corpus collosum formation has been seen in some strain backgrounds, suggesting a potential role in axon outgrowth or guidance (Magara et al., 1999).
One common difficulty in studying a gene's function through genetic deletion arises when multiple genes have redundant functions. The presence of two APP homologues, amyloid precursor like protein 1 (APLPl) and amyloid precursor like protein 2 (APLP2) in mammalian genomes suggested that redundancy might be relevant to this case. Generation of double knock-outs confirmed this, as mice deficient in both APP and APLP2 were found to die shortly after birth. In addition, APLP1/APLP2 deficient mice were similarly found to be not viable, though APP/ APLPl mice were viable. Although these results show that APP family members are necessary for survival, the specific defects causing lethality were not clear (Heber et al., 2000).
Triple knockout mice missing all three APP family members exhibit a partially pentrant defect in cortical morphology reminiscent of human type 2 (or cobblestone) lissencephaly, with clusters of ectopic neuroblasts apparent in a majority of the animals (Herms et al., 2004). This strongly suggests that APP family members may function in ■ cortical neurons during normal migration, although the migration defects may be due to abnormalities in extracellular cues rather than in the cells themselves. The apparently normal expression patterns of known cues reelin and chondroitin sulfate proteoglycan in these mice suggest a cell autonomous effect, although the reduced number of Cajal-Retzius cells, which are required for normal neuronal migration (Soriano and Del Rio, 2005) do indicate that non- autonomous effects can't be excluded. It is interesting to note that the cortical defects in these mice are only partially penetrant, but all of the triple-knockout animals die perinatally, and the exact reasons APP, APLPl5 and APLP2 are jointly required for viability remain mysterious. Invertebrate model organisms have also been used to probe the function of APP. In
Drosophila, APPL is the single member of the APP family of proteins. Flies deficient in APPL are viable and fertile, but exhibit subtle behavioral defects (Luo et al., 1992), increased neuronal excitability (Li et al., 2004), and defects in axonal transport (Gunawardena and Goldstein, 2001) and neuromuscular synaptic bouton formation (Torroja et al., 1999). Although APPL and APP appear to have diverged somewhat both structurally (Coulson et al., 2000) and functionally (Simons et al., 2002), some functions clearly remain intact, as seen by the ability of human APP to rescue behavioral defects in APPL deficient flies (Luo et al., 1992).
Biochemical and other in vitro experiments have generated a large list of potential functions for APP and its proteolytic fragments. Many of these functions await genetic validation, as redundancy among APP family members and possible pleiotropic roles complicate genetic analysis. Below we will review some of the evidence for multiple function APP, but also the proteolytic derivatives Abeta, APPsalpha, and AICD.
Abeta:
Abeta has been widely studied for its role in Alzheimer's disease, but it is found in the blood and cerebrospinal fluid even non-diseased states (Seubert et al., 1993), and there is evidence it may have physiological functions. It has been observed that many of the biological activities exhibited by Abeta, ranging from effects on ion channel currents to influences on learning and memory, oppose the activities exhibited by APPsalpha, suggesting that alternative cleavage of APP at either the alpha or beta site may allow for the promotion of an effect both by promoting one cues while simultaneously repressing an opposing cue (Turner et al., 2003). Abeta may cause cell death by permeabilizing plasma membranes (Arispe et al., 1993; Quist et al., 2005; Singer and Dewji, 2006), however, it may also cause cell death as well as other non-pathological responses simply by signaling through cell surface receptors. In fact, Abeta interactions with a wide range cell surface proteins, including the NMDA receptor, the P75 neurotrophin receptor, integrins, and APP itself, have been described (Verdier et al., 2004). Direct genetic evidence for the role of Abeta may be difficult to obtain, since APP knockout mice would be missing APP and proteolytic products other than Abeta, and reductions in gamma or beta secretase would be expected to affect the function of other target proteins.
APPsalpha as a ligand: APPsalpha is a constitutively produced, but its production can be increased by physiological stimuli, such as metabotropic glutamate receptor stimulation (Allinson et al., 2003; Lee et al., 1995). Many studies have demonstrated that APPsalpha application can result in changed cellular behaviors, including the stimulation of neurite outgrowth or changes in LTP (Mattson, 1997). These effects suggest that APPsalpha may act as a physiological ligand. Since multiple domains of APP have been shown to have biological effects, it is possible that APPsalpha may signal through more than one receptor. A cell surface receptor capable of mediating APPsalpha induced signaling has not yet been identified. The methods of the invention in some aspects involve the use of compounds that can modulate the amount of APP-derived peptides. Some aspects of the invention is based on the recognition that the alpha cleavage of the precursor precludes subsequent beta processing (see Figure 1); therefore, the relative amount of processed peptides derived from the two pathways of the APP processing that gives rise to either plaque-forming beta peptides or non- plaque-associated peptides is likely to affect pathogenesis. As used herein, the term "Abeta production" means the generation of Abeta in a cell, tissue or subject. As used herein, the term "subject" means any mammal that may be in need of treatment with the Abeta production-modulating compound of the invention. Subjects include by are not limited to: humans, non-human primates, cats, dogs, sheep, pigs, horses, cows, rodents such as mice, hamsters and rats.
As used herein, the term "Abeta processing-modulating compound" means a compound that modulates the processing (e.g., proteolysis) of APP by forming a complex with APP and/or its intermediate peptide products. As used herein, the "complex" means physical association of molecules. Such association may be direct interaction between two or more binding partners or indirect interaction mediated by one or more adapter molecules. Such binding may be stable or transient. The binding (complex formation) or the stability of the complex may in turn be modulated by agents that can either promote or inhibit the binding. In some embodiments of the invention, direct binding of extracellular matrix proteins to APP affects APP processing, thereby modulating the amount and/or rate of Abeta production.
APP as a receptor:
Beginning with its initial identification, APP was thought likely to be a receptor based on its predicted transmembrane structure (Kang et al., 1987). The cytoplasmic domain did not contain obvious signal transduction motifs, such as kinase domains, but potential pathways through which APP might signal were suggested by the identification of cytoplasmic binding partners. One well-studied cytoplasmic partner is Fe65, an adaptor protein which may be involved in APP-regulated cell motility or migration (Guenette et al., 2006; Sabo et al., 2001), as well as transcriptional control (see below). Other cytoplasmic partners include the scaffolding protein JIP-Ib (JNK-interacting protein-1; (Matsuda et al., 2001), Disabled, an adaptor protein that can regulate the actin cytoskeleton (Suetsugu et al., 2004; Trommsdorff et al., 1998), and adaptor protein Mint/Xl 1 (Borg et al., 1996; McLoughlin and Miller, 1996). APP may also signal through trimeric G protein G(o); however, varying results from different experimental systems leave it unresolved as to whether APP acts to increase or decrease G(o) signaling (Brouillet et al., 1999; Nishimoto et al., 1993; Okamoto et al., 1995).
One appealing potential mechanism for APP signaling was suggested on the basis of an analogy drawn between APP and Notch (Chan and Jan, 1998). In the Isreal model for Notch signaling, binding to the ligand Delta would activate metalloprotease-dependent cleavage of Notch at extracellular site 2, and intracellular or transmembrane cleavage at site 3, releasing the Notch intracellular domain, which would be free to function in transcription (Logeat et al., 1998). If APP is to function in a similar manner, binding of a ligand might promote alpha and gamma cleavage, freeing the intracellular domain to participate in transcription.
The first evidence for this model was that overexpression of APP, Fe65, and Tip60, a histone acetyltransferase found to interact with Fe65, could jointly activate transcription of luciferase reporter construct, although this required the addition of a DNA binding domain, such as Gal4, fused to either APP or Tip60 (Cao and Sudhof, 2001). A reasonable assumption was that an unidentified DNA binding factor not present in this experimental paradigm might be recruited in promoting transcription of physiological targets. Subsequently, the detection of Fe65 stabilized AICD in the cell nucleus provided further evidence for a direct role of AICD in transcription (Kimberly et al., 2001 ; Kinoshita et al., 2002). However, experiments using the above experimental paradigm have suggested differences from the Notch model. The importance of AICD translocating to the nucleus has been questioned, since addition of a nuclear export signal greatly reduces the amount of
AICD detected in the nucleus without reducing transactivation (Cao and Sudhof, 2004). The role of gamma secretase cleavage in APP signaling is still controversial, as there is evidence both for (Cao and Sudhof, 2004) and against (Hass and Yankner, 2005) it being essential. APP signaling has been shown to act via Tip60 that is phosphorylated at a CDK site, suggesting a model in which APP may activate Tip60 by recruiting it to the plasma membrane, allowing for its subsequent phosphorylation (Hass and Yankner, 2005). Another possibility is that gamma secretase cleavage may simply be one mechanism to inhibit the sequestration of Fe65 at the plasma membrane, and it has been proposed that APP phosphorylation may be an alternative mechanism for releasing Fe65 (Nakaya and Suzuki, 2006). Full-length APP has also been proposed to signal through Fe65 by both inhibiting an intramolecular interaction in Fe65 and allowing for the recruitment of a unidentified membrane localized binding partner critical to the activity of Fe65 (Cao and Sudhof, 2004).
The experimental paradigm developed by Cao and Sudhof has been extremely valuable, but it will be necessary to study the normal signaling of APP to verify any of the above models. Identifying physiological transcription targets of APP signaling would be an important first step, and in fact several targets have been proposed, including Kail, GSK3beta, TipβO, BACE, and APP (Baek et al., 2002; von Rotz et al., 2004), although these targets are still controversial (Hass and Yankner, 2005; Hebert et al., 2006). Additionally, studying physiological APP signaling would be aided by the identification of a ligands that regulate its signaling. Several molecules that bind the extracellular domain of APP have been identified, but so far, only F-spondin has been proposed to function as a ligand capable of inducing signaling, though surprisingly, it appears to downregulate rather than upregulate APP dependent transcription (Ho and Sudhof, 2004; Hoe et al., 2005).
Extracellular binding partners for APPsalpha or APP, and related functions:
Based on both overexpression and genetic deletion studies, APP has been suggested to play a role in cell adhesion and motility (Herms et al., 2004; Sabo et al., 2003). APP binds to several extracellular matrix components, including heparan sulfate (Multhaup, 1994; Small et al., 1994), collagen (Beher et al., 1996), laminin (Narindrasorasak et al., 1992), and fibulin 1 (Ohsawa et al., 2001). Such cell-substratum interactions may be important to APP's role in neurite outgrowth and cell motility. In addition, cell-cell adhesion may be mediated by trans- dimers of APP3 or trans interaction between APP and either APLPl or APLP2 (Soba et al., 2005). Secreted forms of APP containing the kunitz-type protease inhibitor (BCPI) domain, initially identified and studied under the name protease nexin II (Oltersdorf et al., 1989; Van Nostrand et al., 1989) (Van Nostrand et al., 1989), bind and inhibit blood coagulation factor XIa (Van Nostrand et al., 1992). APP695 does not contain the KPI domain, but does can bind heparin, which inhibits blood coagulation (Petitou et al., 2003) and high molecular weight kininogen, which is also involved in blood coagulation (Das et al., 2002).
The metal ions zinc and copper both have binding sites near the N-terminus of APP, as well as additional sites in the Abeta peptide (Turner et al., 2003). APP can reduce copper (II) to copper (I) (Multhaup et al., 1996) and is required for normal copper metabolism (White et al., 1999). Whether APP is important in zinc metabolism is less clear, but zinc appears to modulate some APP activities, increasing binding to heparin (Bush et al., 1993) and, for KPI-containing APP isoforms, increasing the inhibition of factor XIa (Komiyama et al., 1992). The interaction of APP with metal ions may be relevant to the oxidative damage found in Alzheimer's disease, since Abeta interactions with copper (II) and iron (III) promote formation of hydrogen peroxide, while zinc (II) quenches its formation (Huang et al., 2000). APP has additionally been shown to interact with the lipoprotein receptors scavenger receptor A (SR-A) and LRP. SR-A binds to secreted APP (APPs) derived from both APP695 and the KPI-containing splice forms and promotes its internalization (Santiago-Garcia et al., 2001). LRP also binds and promotes the internalization of APPs, but only of KPI-containing forms (Kounnas et al., 1995). It is interesting to note that LRP participates in a range of biological processes beyond its role in lipoprotein metabolism, including cell motility and proliferation (Lillis et al., 2005), raising the possibility of additional functions for an APP- LRP interaction.
More recently, several extracellular binding partners for APP have been identified that do not fit in the above functional classifications, but look like traditional ligands or cell- surface receptors. Nogo-66 receptor (NgR) binds the APP695 extracellular domain, and mice deficient for NgR show decreased levels of sAPPalpha and sAPP beta, implying that NgR normally acts to reduce both alpha- and beta-secretase cleavage of APP, possibly by sterically inhibiting secretase access (Park et al., 2006). In addition, several groups have recently reported physical interaction or signaling cross-talk between APP and Notch family members (Chen et al., 2006; Fassa et al., 2005; Fischer et al., 2005; Oh et al., 2005). Finally, as mentioned above, F-spondin has been proposed to be an APP ligand, and it appears to reduce beta-cleavage and downregulate APP dependent transcription (Ho and Sudhof, 2004; Hoe et al., 2005).
The amyloid-beta precursor protein (APP) is a transmembrane protein with widespread expression in embryos as well as adults. Based on its transmembrane structure, APP was initially predicted to be a cell-surface receptor (Kang et al., 1987), and in fact, evidence has accumulated to suggest that APP and its proteolytic products may play multiple roles in cell-cell communication. As disclosed herein, it is now shown that not only does full-length APP function as a receptor, but the secreted ectodomain APPsalpha functions as a ligand for an unidentified cell surface receptor.
As discussed above, several binding partners for the APP extracellular domain are known. In many cases, the domain of APP involved in binding was characterized, as summarized below:
Figure imgf000023_0001
For several binding partners, full-length APP appears to have more than one interacting domain. The list above only includes domains within APPsalpha.
A variety of biological functions have been attributed to APPsalpha , and in some cases the relevant domain has been determined (Mattson, 1997). There appear to be three major domains implicated in APPsalpha function: the N-terminal El region, from approximately amino acids 23 to 189 (Morimoto et al., 1998a; Morimoto et al., 1998b; Ohsawa et al., 1997; Ohsawa et al., 1999; Small et al., 1994), the RERMS peptide from 328- 332 (Jin et al., 1994; Ninomiya et al., 1994; Roch et al., 1994) and the more C-terminal heparin binding domains (for example, (Furukawa et al., 1996), see also (Mattson, 1997)).
As described above, the invention provides candidate APP binding proteins. The invention further includes a characterization of APP interaction with two of these proteins, namely, contactin 4 and NgCAM, and also identifies an interaction between APP and contactin 3. All three of these proteins belong to the NCAM family of neurally expressed IgCAMs. The NCAM family itself is divided into four subfamilies, referred to according their founding members: NCAM (neural cell adhesion molecule), DCC (deleted in colorectal cancer), Ll, and contactin. Members of these subfamilies have similar structures, with a specific number of immunoglobulin (Ig) domains followed by another specified number of fibronectin type III (FnIII) and either a GPI-anchor or transmembrane domain (Vaughn and Bjorkman Neuron 1996). Some of the functions attributed to contactins and to NgCAM are briefly discussed below.
Contactin 3 and contactin 4:
Contactin family members are characterized by six Ig domains, four FnIII domains and a glycosylphosphotidylinositol (GPI) membrane anchor. There are six known members of the contactin family, of which contactin 3 (also called BIG-I or PANG) and contactin 4 (also called BIG-2) are the most closely related pair (Ogawa et al., 1996). The terms "contactin 3" and "contactin-3" are used interchangeably; similarly, the terms "contactin 4" and "contactin-4", as well as the terms "contactin 5" and "contactin-5", are used interchangeably.
The genes encoding contactins 3 and 4 (CNTN3 and CNTN4, respectively) are both located on the short arm of human chromosome 3, that is, on 3p (Luo et al., 2005; Zeng et al., 2002). CNTN4 has been suggested to be involved in 3p deletion syndrome, a disorder in which the patient exhibits growth and mental retardation as well as distinctive facial features. CNTN4 falls within the minimum chromosomal region implicated in this disorder, and one patient with 3p deletion syndrome has been identified in which a translocation breakpoint disrupts the contactin 4 transcript (Fernandez et al., 2004). Contactin 3 and 4 are expressed in many regions throughout the brain, and both proteins, when presented as Fc fusions, have been shown to support outgrowth of neurites from hippocampal neurons (Yoshihara et al., 1995; Yoshihara et al., 1994).
Although the functions of contactin 3 and 4 are not well characterized, contactin 1 (F3/F11) and contactin 2 (axonin-1 /Tag- 1 /TAX-I) have been more extensively studied. Contactin 1 and 2 have been shown to bind a partially overlapping list of cell-surface or extracellular matrix proteins, including Ll family members NgCAM and NrCAM, the chondroitin sulfate proteoglycan phosphacan/RPTPb, tenascin, and bl-integrin (Sonderegger, 1997). Contactins 1 and 2 can both signal through fyn kinase; however, since they are GPI- anchored proteins, and thus without a cytoplasmic domain, activation of fyn through a direct interaction can be excluded, and it is not known how this signal is transduced into the cell (Kunz et al., 1996; Zisch et al., 1995). Contactin 1 and 2 have both been implicated in cell migration and axon guidance, as well as in the organization of specialized domains in myelinated axons, with contactin-2 being required at the juxtaparanode and contactin 1 being required at the septate junctions and in the node of Ranvier (FaIk et al., 2002).
NgCAM:
NgCAM (neuron-glial cell adhesion molecule) (or "Ng-CAM") is a member of the Ll family, which also includes Ll, NrCAM (NgCAM-related cell adhesion molecule), neurofascin, and CHLl (Close Homolog of Ll), and whose members are characterized by six Ig domains, five FnIII domains, a transmembrane domain, and a conserved cytoplasmic tail (Holm et al., 1996; Hortsch, 1996). NgCAM is now generally accepted to be the chicken homolog of mammalian Ll, although the relatively low sequence homology between these two proteins (43% identity between chicken NgCAM and human Ll, versus 78% identity between chicken and human NrCAM) initially led to some controversy over this point.
NgCAM/Ll has been shown to bind a variety of molecules, including some contactin
1 or 2 binding proteins, as well as contactins 1 and 2 themselves (Sonderegger, 1997). Although there appears to be rather promiscuous binding between Ll and contactin family members in vitro, these interactions do not appear to be interchangeable physiologically. For example, antibody blocking experiments in the spinal cord show that the function of contactin
2 in axon guidance specifically involves NgCAM, while contactin 1 function specifically involves NrCAM, in spite of the ubiquitous expression of NrCAM and NgCAM throughout the affected regions (Perrin et al., 2001). Different combinations and topological presentations of these proteins also may induce different biological effects. For example, a combination of biochemical and in vitro culture experiments has led to the model that contactin 2 and NgCAM interact heterophilically in cis, and together mediate neurite outgrowth through a homophilic trans interaction with NgCAM (Buchstaller et al., 1996; Lemmon et al., 1989; Stoeckli et al., 1996). However, homophilic trans interactions of contactin 2 may be capable of mediating cell adhesion, if not neurite outgrowth (Kunz et al., 2002), and trans interactions between NgCAM and contactin 1, or NrCAM and contactin 2, have been reported (Sonderegger, 1997). Mice in which Ll expression is eliminated exhibit severe defects, including enlarged ventricles and defects in the corticospinal tract and corpus collosum (Demyanenko et al., 1999). In contrast, mice in which the sixth Ig domain of Ll has been deleted appear normal, though embryonic lethality in some genetic backgrounds. Based on the previous mapping of binding domains in Ll3 this implies that neither Ll homophilic interactions nor Ll interactions with contactin 1 and 2 are absolutely required. However, the importance of strain background suggests some of these interactions may be important, but can be compensated for by redundant mechanisms (Itoh et al., 2004). The projection of retinal ganglion cell (RGC) axons from the retina to their midbrain target, the tectum (the superior colliculus (SC) in mammals), has been an important model system for studying axon guidance. RGC axons migrate a relatively long distance to reach their synaptic targets, and this journey can be divided into several sequential steps, each with distinct guidance cues. In the first step of their journey, RGC axons navigate centrally towards the optic fissure or optic disk, where they exit the retina. It appears that there are multiple, partially redundant cues that guide RGC axons to the optic disk, although the logic behind these cues differ (Oster et al., 2004). For example, chodroitin sulfate proteoglycans (CSPGs) are thought to drive axons towards the central retina by inhibit growth towards the periphery, where CSPGs are expressed (Brittis et al., 1992). An interaction between ephrin-Bs and EphBs is also thought to involve repulsion, but in this case, ventrally expressed EphBs are thought to prevent dorsal axons from bypassing the optic disk (Birgbauer et al., 2000; Birgbauer et al., 2001). Antibody blocking studies have implicated the IgCAMs neurolin and Ll in intraretinal guidance (Brittis et al., 1995; Ott et al., 1998) possibly through a role in axon fasciculation. Once RGC axons reach the optic disk, they exit the retina and enter the optic nerve. Netrin and its receptor DCC are both genetically required for RGC axons to exit the eye and enter the optic nerve (Deiner et al., 1997). The demonstration that laminin-1 can convert the effect of netrin- 1 from attraction to repulsion, combined with the co-expression of netrin and laminin at the retinal surface around the optic disk, led to the proposal that netrin and laminin act together to drive axons out of the retina and into the optic nerve head, where netrin is expressed in the absence of laminin (Hopker et al., 1999).
The guidance of RGC axons from the optic nerve head to the tectum is not completely understood, though several molecular players have been identified. Netrin and L 1 are expressed in the optic nerve, where they are generally presumed to contribute positively to axon outgrowth and fasciculation, although their function specifically in the optic nerve has not been demonstrated (Deiner et al., 1997; Rager et al., 1996). In addition, Sema5A surrounds the optic nerve, and antibody perturbation experiments suggest that Sema5 A may function to prevent the straying of RGC axons from the optic nerve (Oster et al., 2003). In an analogous way, expression of slit proteins appears to channel RGC axons into the optic chiasm by repelling them from surrounding areas (Plump et al., 2002). Before reaching the tectum, RGC axons encounter the optic chiasm, a midline structure. Ephrin B2 expressed here appears to be both necessary and sufficient to deflect ipsilaterally projecting RGC axons, which express EphBl, from the chiasm (Nakagawa et al., 2000; Williams et al., 2003). The question of how contralateral^ projecting axons (which in vertebrates without binocular vision is essentially all of the axons) are guided across the chiasm is not well understood. Expression of NrCAM, an Ll-family protein, on RGC axons is required for some axons to project contralateral^, but since a substantial contralateral projection still exists in the absence of NrCAM, it is likely that other molecules, either related or not, act redundantly (Williams et al., 2006). After crossing (or being deflected from) the chiasm, the RGC axons grow towards and enter the tectum in a manner dependent upon FGF signaling (McFarlane et al., 1996; McFarlane et al., 1995).
Once in the tectum, RGC axons must find appropriate synaptic partners. The retinotectal projection is arranged topographically; that is to say, the spatial arrangement of RGCs in the retina is reflected in the spatial arrangement of their synapses in the tectum. Briefly, the temporal-nasal axis of the retina maps onto the anterior-posterior axis of the tectum using gradients of ephrinAs and EphAs as spatial cues, and the dorsal-ventral axis of the retina maps onto the ventral-dorsal axis of the tectum using gradients of ephrinBs, EphBs, Wnt3, and Ryk as cues (Flanagan, 2006). Additional details about topographic mapping in the retinotectal projection, including the role of axon-axon competition, is discussed below. Topographic location is not the only criterion according to which axons choose synaptic partners. In chicken, RGC axons initially enter the tectum and find their topographically correct locations within the most superficial layer, the stratum opticum (SO). Axons then turn or branch orthogonally to the anterior-posterior and dorsal ventral axes, and each RGC axon must synapse in one particular retinorecipient layer, SGFS (stratum griseum et fibrosum superficiale) layer B, D, or F. There are different types of RGCs that can be distinguished by accumulation of distinct neuropeptides, and choice of retinorecipient layer seems to correlate at least partly with this classification (Kuljis and Karten, 1988). Biochemically distinct RGC populations can form in retinas that are prevented from innervating the tectum, and tectal lamina form normally in the absence of innervation, suggesting that the subtype of an RGC is not determined based on the layer in which it has already formed synapses, but rather that subtype is determined independently of synapse formation, and the choice of synaptic partners is guided complementary cues (Yamagata and Sanes, 1995). Some tectal cues which aid RGC axons choosing appropriate lamina have been identified, including versican and N- cadherin, but laminar choice is still not completely understood (Inoue and Sanes, 1997; Yamagata and Sanes, 2005).
Antibody perturbation experiments have suggested that NgCAM and Ll are required for intraretinal guidance (Brittis et al.s 1995; Schlosshauer and Dutting, 1991). Furthermore, in mice deficient for Ll , the topographic aspect of the retinocollicular map is severely perturbed (Demyanenko and Maness, 2003). With these observations in mind, we were particularly interested to begin examining potential roles for NgCAM or contactin 4 interactions with APP in the visual system.
The work presented here identified contactin 4 and NgCAM as APP interacting proteins.
APP binds to contactin 4 and contactin 3 through what is most likely a direct physical interaction. Domains sufficient for this binding are amino acids 18-205 of APP, which essentially corresponds to the El domain, and a domain including the four fibronectin type 3 repeats of contactin 3 or 4. NgCAM interacts physically with APP, but a direct interaction was not demonstrated.
One possibility is that NgCAM interacts indirectly with APP, or requires the presence of an additional co-factor. Other possible explanations include a binding specificity of APP for NgCAM with specific post-translational modifications or of a different splice form, or a relatively low affinity direct interaction between APP and NgCAM that is undetectable by the methods we used. None of these alternative explanations alone easily explains all of the data here, but they may still be relevant factors.
Contactin 4 and NgCAM each can dramatically affect the levels of APP and APP CTFalpha seen in transfected cells, although whether the effect is to increase or decrease these levels varies between experiments. This may be due to different ratios of cis-to-trans interactions, or a biphasic response to increasing expression levels (either physiological or due to a dominant negative effect).
APP, NgCAM, and contactin 4 are expressed in chick embryos, at least at the level of RNA, in the both the olfactory epithelium and the olfactory bulb. These genes are also expressed, along with contactin 3, in the retina and the tectum. The partially overlapping regions of expression seen suggest that these proteins may have the opportunity to interact in vivo, and suggest that interactions among these proteins might play a role in the development of the olfactory and visual systems.
NgCAM supports outgrowth of retinal ganglion cell (RGC) axons, and this outgrowth can be modified by APP- or contactin 4-related reagents.
Putative blocking reagents of APP signaling (an anti-APP antibody and gamma secretase inhibitor DAPT) increase outgrowth on NgCAM, suggesting that signaling mediated by axonal or cell-surface APP may inhibit RGC axon outgrowth. The observation that DAPT also increases outgrowth on laminin, argues against the hypothesis that this APP- mediated signaling is in response to NgCAM; however, although exogenously provided NgCAM is not required for DAPT to stimulate outgrowth, it remains possible that endogenously expressed NgCAM. or contactin 3 or 4, might inhibit outgrowth through APP.
Contactin 4-Fc inhibits outgrowth on NgCAM but not laminin. Based on the observation that Fc fusions of contactins 1, 2, 3, and 6 also inhibited outgrowth on NgCAM, and in light of published studies implicating contactin 2 as a co-receptor in NgCAM- promoted neurite outgrowth (Buchstaller et al., 1996), a preferred interpretation is that contactin 4-Fc is binding to NgCAM and blocking access of axonal receptors to NgCAM. However, it remains formally possible that NgCAM and contactin 4 act together to generate an inhibitory signal. Finally, we showed that APPsalpha, and in particular the El domain, enhances
NgCAM-supported RGC axon outgrowth. Since the El domain binds to contactin 4, APPsalpha could potentially be stimulating outgrowth through an interaction with RGC- expressed contactin 4. A trend was seen in which contactin 4-Fc appeared to inhibit APPsalpha-enhanced outgrowth, but the previously described inhibition of NgCAM- supported outgrowth by contactin 4-Fc prevented a definitive interpretation of this experiment.
Potential interactions among APP, contactin 4, and NgCAM:
As previously discussed, the data presented herein strongly support a model in which APP binds directly with contactin 4, but APP interactions with NgCAM may be either direct or indirect. In light of studies demonstrating binding between NgCAM and contactin 1 or 2, our observation that contactin 4-Fc, as well as Fc fusions of contactins 1 , 2, 3, and 6, inhibits outgrowth on NgCAM suggests that contactin 4 may also bind to NgCAM. The domains required for NgCAM-contactin 2 binding have been determined, and since the first four Ig domains of contactin 2 are sufficient for this interaction (Rader et al., 1996), one might expect the same region of contactin 4 to interact with NgCAM. Since contactin 4 uses its fibronectins domains to bind APP, this could potentially allow for the formation of a trimolecular complex among APP5 contactin 4, and NgCAM. Although APP may interact with NgCAM indirectly, a model in which contactin 4 acts as a bridge between APP and NgCAM is not sufficient to explain all of our results. In particular, NgCAM can be crosslinked to AP -m APP(199-345), a protein in which APP 's binding domain for contactin 4 is not present. However, contactin 4 could still potentially be required for a direct interaction between APP and NgCAM, if, for example, contactin 4 binding relieved inhibitory intramolecular interactions or otherwise changed the conformation of NgCAM or APP.
The data presented suggests a complex model of interaction involving APP, contactin 4, and NgCAM. For example, in our studies on the effects of NgCAM and contactin 4 on APP processing or stability (see Example 2), we never saw an effect on APP when contactin 4 was presented alone in trans. This suggested the possibility that APP and contactin 4 may interact in cis, while NgCAM might potentially signal in trans to an APP-contactin 4 complex. In contrast, a model in which contactin 4 acts in trans as a receptor for APPsalpha is consistent with, though not proven by, the outgrowth assays provided herein. However, there are many different topological arrangements in which these three proteins could potentially interact, and it may be that more than one of these arrangements is biologically relevant.
The invention, therefore, provides methods for modulating APP processing. As used herein, "modulating" or "modulation" of APP processing refers to an increase or decrease in the rate and/or amount of APP processing to generate various intermediate and/or final peptide products.
Accordingly, the methods include contacting a mammalian cell with an agent that modulates the amount or rate of binding of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2) to contactins and/or Ll-CAMs. As used herein, "an agent" means a molecule, molecular compound, a composition or formulation having a desired biological effect. An agent may be naturally occurring or synthetic. An agent may be in a substantially pure form or may be a component of a mixture. An agent may be provided in a soluble form (e.g., solution, medium, buffer, etc.) or in an insoluble form (e.g., as a substrate, cell surface molecule, on a solid support, etc.). In some circumstances, specific configuration of the presentation of a binding molecule(s), such as in cis verses in trans, may be considered in an assay or treatment.
In some embodiments of the foregoing methods, the agent is expressed by the cell or exogenously added so as to contact the cell. As used herein, "exogenously added" means that the agent or an equivalent thereof is applied to a target from an external source, e.g., as a composition containing the agent.
In some embodiments of the invention, the agents include one or more contactins such as contactin-3, contactin-4 and contactin-5, or Ll-CAMs such as Ng-CAM, or fragments thereof that bind to APP, APLPl or APLP2. In certain embodiments of the method, the agent is a fusion protein including one or more contactins, Ll-CAMs or fragments. As used herein, "a fusion protein" is a recombinant protein containing more than one segments of peptides fused together, often with a short linker or spacer sequence separating between them, such that when expressed the they form one continuous peptide. A target peptide can be fused to a variety of tags, labels, purification moieties, and the like. Some examples of fusion proteins include but are not limited to: GST- fusion proteins, Flag-fusion proteins, MBP-fusion proteins, His6 fusion proteins, Myc fusion proteins, Fc fusion proteins, chitin fusion proteins, AP-fusion proteins, transferrin fusion proteins, GFP fusion proteins, GIu fusion proteins, HA fusion proteins, and others. Such techniques are well known in the art.
In some embodiments of the foregoing methods, the fusion protein is an Fc fusion protein, e.g., a fusion of a Fc molecule and one of the polypeptides mentioned herein.
In other embodiments of the methods, the agent is an antibody or an antigen-binding fragment thereof (see below).
In some embodiments, the antibody is selected from an antibody or antigen-binding fragment thereof that binds a contactin or a Ll-CAM, preferably contactin-3 contactin-4, contactin-5 or Ng-CAM, or, an antibody or antigen-binding fragment thereof binds APP,
APLPl or APLP2. In some embodiments, the antibody or antigen-binding fragment thereof binds to a complex of a contactin or a Ll-CAM and APP, APLPl or APLP2. In some circumstances, the antibody or antigen-binding fragment thereof is a bispecific antibody. The term "bispecific antibody" refers to an engineered antibody with two different binding sites that recognize two different antigens. Various bispecific antibodies and methods of generating such antibodies are known in the art.
In some embodiments of the invention, the methods include agents that block or disrupt the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs. As used herein, an agent that "blocks" the binding of two or more molecules or molecular complexes can interfere with the normal binding. For example, an agent may interfere with the binding between two proteins by binding to a first protein, thereby either creating a physical hindrance to the binding site on the first protein against a second protein; alternatively, the binding of the agent to a first protein may cause a conformational change to the first protein which results in unfavorable structural changes that render reduced binding to its normal binding partner(s). An agent that "disrupts" the binding of two or more molecules or molecular complexes may promote the dissociation of an already bound complex. Alternatively, an agent that can block or disrupt the binding may cause a biochemical or cellular environment that renders the binding unfavorable. An agent may exert both blocking and disrupting effects.
In other embodiments, the invention describes agents that promote or stabilize the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs. As used herein, an agent that "promotes" the binding of one or more molecules or molecular complexes favors association of binding partner(s) such that an association/binding constant of the reaction is increased in the presence of such agent. An agent that "stabilizes" the binding of one or more molecules or molecular complexes acts on an existing (bound) complex as substrate to enhance the association, such that a dissociation constant of the reaction is lowered in the presence of such agent. An agent may exert both promoting and stabilizing effects.
The methods also include agents that can reduce processing of APP to β-amyloid. As used herein, antibodies and/or antigen-binding fragments thereof, that specifically bind to APP, APLPl or APLP2, contactins and/or Ll-CAMs, or to protein complex of these molecules, are useful in additional screening methods. As described herein, the antibodies of the present invention thus are prepared by any of a variety of methods, including administering protein, fragments of protein, cells expressing the protein or fragments thereof and the like to an animal to induce polyclonal antibodies. In addition, antibodies that specifically bind to the above-mentioned proteins are available commercially. The production of monoclonal antibodies is according to techniques well known in the art.
Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc1 region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F9(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd Fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (Frs), which maintain the tertiary structure of the paratope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FRl through FR4) separated respectively by three complementarity determining regions (CDRl through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity.
It is now well-established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. See, e.g., U.S. patents 4,816,567, 5,225,539, 5,585,089, 5,693,762 and 5,859,205. Thus, for example, PCT International Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin. Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as "chimeric" antibodies. Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (HAMA) responses when administered to humans.
Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab')2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or Fr and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or nonhuman sequences. The present invention also includes so-called single chain antibodies.
Thus, the invention involves polypeptides of numerous size and type that bind specifically to APP, APLPl, APLP2, contactins or Ll-CAMs, complexes thereof, or fragments thereof. These polypeptides may be derived also from sources other than antibody technology. For example, such polypeptide-binding agents can be provided by degenerate peptide libraries, which can be readily prepared in solution, in immobilized form or as phage display libraries. Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptoids and non-peptide synthetic moieties.
A wide variety of assays to identify pharmacological agents that modulate the stability of APP/L1-CAM complex and equivalents, and/or modulate the formation of such complex, can be used in accordance with the aspects of the invention.
Topographic mapping and Ll:
The projection of retinal ganglion cell (RGC) axons from the retina to their midbrain target, the tectum (the superior colliculus (SC) in mammals), has long been a major model system for studying the development of topographic maps. In a topographic map, the spatial arrangement of neurons in the projecting field determines the spatial arrangement of their synapses in the target field, with nearest-neighbor relationships maintained. In the retinotectal (or retinocollicular) projection, the temporal-nasal axis of the retina maps onto the anterior-posterior axis of the tectum, with neurons from nasal positions forming synapses in the posterior tectum, while the dorsal -ventral axis of the retina maps onto the ventral- dorsal axis of the tectum, with ventral RGCs forming synapses in the dorsal tectum. Sperry first proposed that the formation of topographic maps could be guided by a gradient of a label across the target field in conjunction with a gradient of a complementary label in the projecting field (Sperry, 1963).
Sperry's chemoaffinity theory was first validated at the molecular level in studies of proteins belonging to the ephrin-A family of GPI-anchored proteins and their receptors in the EphA family of receptor tyrosine kinases. Ephrin-A2 or -A5 expression was found in a high posterior, low anterior gradient in the tectum, while EphA3 or EphA5 was found in a high temporal, low nasal gradient in the retina (Cheng et al., 1995; Drescher et al.5 1995; Feldheim et al., 2000; Feldheim et al., 1998; Monschau et al., 1997; Zhang et al., 1996). Furthermore, ephrin-As were found to be capable of repelling RGC axons (Drescher et al., 1995;
Monschau et al., 1997; Nakamoto et al., 1996), suggesting that topographic mapping in the retinotectal projection was guided by increasing ephrin-A concentrations towards the posterior of the tectum having increasingly repulsive effects on axons with greater expression of EphAs, that is, those in more temporal portions of the retina. The importance of these molecules has been genetically validated by observations of severe defects in retinocollicular topographic mapping in mice in which ephrin-A2 and — A5 are both deficient (Feldheim et al., 2000) or in which EphA5 is disrupted (Feldheim et al., 2004).
A topographic map cannot be completely determined by one gradient in the target field and one in the projecting field, because all axons would be expected to map to one end. In the case described above, for example, all axons might be expected to map temporally to avoid repulsion by ephrin-As in the posterior tectum. An additional counterbalancing force is thought to be required (Gierer, 1983; Prestige and Willshaw, 1975), which could be in the form of multiple gradients, attractive and repulsive cues from a single gradient, or axon competition. Mapping of the dorsal-ventral axis in the retinotectal system provides an example of the first type of counterbalance. Mapping in this axis, as in the anterior-posterior axis, appears to involve gradients of ephrins and Ephs. However, in this case it appears that dorsal RGC axons, which express high levels of ephrin-Bs, are attracted to the ventral tectum by its high levels of EphBs (Mann et al., 2002), while ventral RGC axons, which express high levels of EphBs, are attracted to the dorsal tectum by its high levels of ephrin-Bs ((Hindges et al., 2002). Additionally, a gradient of Wnt3 in the tectum, with high dorsal expression, acts overall to repel axons toward the lateral tectum. Topographic differences in the degree to which axons are repelled seem to be determined by the relative levels of Frizzleds, uniformly expressed attractive Wnt3 receptors, and Ryk, a repulsive receptor expressed in a ventral high retinal gradient (Schmitt et al., 2006). Evidence for the second kind type of counterbalancing force, attractive and repulsive cues from a single gradient, has been found in the anterior-posterior axis of the retinotectal system. Low concentrations of ephrin-A2 have been shown to promote outgrowth, while higher concentration inhibit. The concentration at which ephrin-A2 changes from promoting to inhibiting growth varies across the nasal-temporal axis of the retina, allowing for the possibility of a single ephrin gradient in the tectum specifying different optimal tectal locations for different RGC axons (Hansen et al., 2004).
The third type of counterbalancing force, axon-axon competition, is also clearly implicated in retinotectal mapping. One line of evidence comes from numerous studies in which retinas or tecta were surgically manipulated to create a mismatch between the number or identity of the RGC axons entering the tectum and the region of the tectum available for innervation. These studies showed that the retinotectal map could expand or contract to fill the entire tectal region available, suggesting the map was not determined simply by matching specified concentrations of topographic markers (recently reviewed in Goodhill and Richards, 1999). More recent molecular studies also support a role for axon-axon competition; for example, in mice deficient in both ephrin- A2 and — A5, the topographic order of the retinocollicular projection is severely disturbed, but the SC is still completely innervated (Feldheim et al., 2000), while in mice in which a subset of RGCs overexpress EphA35 synaptic locations of unaffected RGCs are shifted, allowing two neighboring maps, one of overexpressing RGCs and one of unaffected RGCs, to cover the SC (Brown et al., 2000). Axon-axon competition is a common feature in the development of many types of neuronal connections, and several general models for axon-axon competition have been proposed (van Ooyen, 2001). Electrical activity plays an important role in some of these models, and electrical activity clearly plays a role in the refinement of visual maps (Torborg and Feller, 2005). However, topographic maps expand or contract appropriately in goldfish with partial ablations of the retina or tectum, even under electrical blockade by tetrodotoxin, implying that the competition driving the initial rough formation of the topographic map is activity independent (Meyer and Wolcott, 1987). Some types of models that could be potentially be relevant include a competition for limiting neurotrophic factors, a competition for physical space, and direct negative interactions between axons (van Ooyen, 2001). None of these models has been definitively implicated in the retinotectal projection, although BDNF and Ll have been proposed as limiting factors (Flanagan, 2006). In the first case, this idea is based on the observation that axon arborizations increase or decrease in response to increases or decreases in available BDNF (Cohen-Cory and Fraser, 1995). In the second case, a function for Ll in competition is suggested by the observation that the superior colliculus is incompletely innervated, with large patches of low or nonexistent innervation in the anterior SC, in Ll deficient mice. However, other possible interpretations, including a requirement for Ll in axonal remodeling, have been suggested (Demyanenko and Maness, 2003).
Since Ll protein in the mouse SC, like NgCAM protein in the chicken tectum, seems to be largely RGC axon-derived (Lemmon and McLoon, 1986; Lyckman et al., 2000; Yamagata and Sanes, 1995), Ll may be a more likely candidate for contributing to axonal competition as a mediator of direct negative signals between axons than as a limiting factor for axons compete. Ll-Ll homophilic interactions generally appear to be positive rather than negative in nature, raising the possibility that negative signals involving Ll may involve alternative receptors or ligands. In Drosophila, relative levels of Fasciclin II (FasII; the NCAM ortholog) presynaptically versus postsynaptically at neuromuscular junctions regulate the number of synaptic boutons formed, with imbalances leading to decreased bouton numbers, but with parallel changes in pre- and post-synaptic FasII levels in some cases even increasing bouton number. Interestingly, these effects of FasII on bouton number are dependent on the downstream function of APPL, the Drosophila APP ortholog, possibly by recruiting APP to the cell surface, although the signaling mechanism is not completely clear (Ashley et al., 2005). It may be interesting to investigate whether competition among RGC axons may work under a similar principle, for example, with axons measuring relative amounts of NgC AM/LI, provided by neighboring axons, through an APP dependent mechanism.
The work presented herein provides evidence for a physical interaction between several pairs of proteins; for APP with contactin 3, contactin 4, and NgCAM, and for APLPl with contactins 3, 4, and 5 (see Examples). In particular, the binding assays conducted with Fc and AP fusion proteins argue strongly in favor of direct binding between APP or APLPl and their respective contactin family interactors, although a requirement for a cofactor or cofactors present at high levels in 293T cultured supernatants cannot be excluded. In contrast, the crosslinker/co-IP protocol used to demonstrate an interaction between APP and NgCAM does not distinguish between direct and indirect interactions. We have tried using recombinant NgCAM, either full-length or a tagged ectodomain, in binding assays with APP, and have so far failed to detect an interaction. This may suggest that the APP-NgCAM interaction is indirect, although there are several alternative explanations. One possibility is that NgCAM expressed on RGC axons is of a different splice form than the our recombinant NgCAM3 or that it is post-translationally modified differently in RGCs than in 293T cells. Both of these possibilities seem somewhat credible in light of observations that the mammalian NgCAM homolog, Ll , has splice forms in which both homophilic binding and heterophilic binding to contactins 1 and 2 are reduced (De Angelis et al., 2001) and that NCAM, a somewhat more distantly related protein, has binding properties that can be modified by the relatively unique modification of polysialation (reviewed by Bruses and Rutishauser, 2001). One difficulty with both of these explanations is that while they might explain an absence of binding using recombinant NgCAM proteins, it is not obvious why recombinant NgCAM would still be able to affect APP processing or stability.
An additional possible explanation for the lack of apparent binding between AP-APP and NgCAM-Fc, might be that even if a direct interaction exists, it may be of low enough affinity to be undetectable by the method used. It is interesting to note that most studies examining the interaction of NgCAM family members with contactin family members involve aggregation assays with protein coated beads or the binding of protein coated beads to cells expressing a putative binding partner. These experimental approaches may be better suited to examining low affinity interactions, since the potential for multiple low affinity interactions could easily result in relatively high avidity interactions between such beads and cells. In fact, using such a bead-cell binding assay, NgCAM has been shown to bind to the first four Ig domains of contactin 2 (Rader et al., 1996), but using the Fc/AP binding method described in this chapter, we were unable to detect this interaction (figure 3-3A). However, the theory of such a low affinity interaction between APP and NgCAM cannot on its own account for the strong binding of AP-APP to embryonic chick tecta.
The experiments examining the effect of contactin 4 and NgCAM on APP indicate that interaction among these proteins can influence APP processing in some way, as
CTFalpha levels were seen to change, often, but not always, in conjunction with APP levels. Why CTFalpha levels are sometimes increased, sometimes decreased, in response to contactin 4 or NgCAM is unclear. Some possible explanations we have considered are variations in cell density, which might change the ratio of cis to trans interactions, or variations in expression level, which might affect oligomerization states of the transfected proteins or result in an excess of transfected APP over the levels of any endogenous proteins (for example, proteases) involved in a response. The possibility that contactin 4 and NgCAM fusion proteins may be acting essentially as dominant negatives in the cases where CTFalpha levels are decreased may be suggested by the observation that expression of NrCAM, which might be predicted to be capable of binding endogenous contactins, also resulted in decreased CTFalpha levels. In order to better understand the possible effects of contactin 4 or NgCAM on APP signaling, it should be valuable to examine parameters other than APP and CTFalpha levels. After all, CTFalpha is usually thought of as an intermediate product which, shortly after its generation by alpha secretase activity, would be expected to be degraded by gamma secretase activity. Even in the cases where we see CTFalpha levels increase without a concomitant effect on full-length APP5 we can not definitely conclude that alpha cleavage activity is directly upregulated, since changes in the relative degradation rates of APP and CTFalpha could theoretically yield similar results. Potentially informative future studies might include quantifying APPsalpha levels as another measure of alpha secretase cleavage or AICD levels as a more relevant indication of APP signaling, or looking for possible changes in the phosphorylation state or cellular localization of APP, as either may be upstream of changes in APP processing or degradation.
However, there are also other known examples of regulatory modes of signal transduction involving extracellular matrix proteins, particularly those involved in axonal guidance, such that under different contexts they exert opposing cellular effects. One such example is the ephrins, which can inhibit axon outgrowth at high concentrations, but promote outgrowth at low concentrations (Hansen et al., 2004). Another example is that changes in the intracellular cyclic nucleotide levels can convert many extracellular cues between being attractants verses repellents (Song et al., 1998). Therefore, an analogous regulation may exist for APP signaling.
Implications for Alzheimer's disease:
The identification of novel binding partners presents new insights on physiological functions of APP. Data as disclosed herein provide novel tools for intervening the onset and/or progression of Alzheimer's disease, a pathological process in which APP plays a central role. There is some hint at an involvement of NgCAM/Ll, since elevated levels of Ll fragments have been detected in the cerebrospinal fluid of patients with Alzheimer or non- Alzheimer dementia (Strekalova et al., 2006). In addition, contactin 4 maps to chromosome 3p26 near the D3S2387 marker, near a region with suggestive genetic linkage to Alzheimer's disease (Blacker et al., 2003). Accordingly, the invention provides methods and compositions useful for the prevention and/or treatment of Abeta-accumulation-associated disease or disorder, such as Alzheimer's disease. As used herein, "treating" or "treatment" of a disease or disorder refers to improving, or remedying of a condition. As used herein, "preventing" or "prevention" of a disease or disorder refers to hindering the occurrence of disease in a susceptible population (e.g., a subject at risk of developing a disease or disorder), and/or arresting or retarding the progress of a disease.
The invention in some aspects provides methods for treating or preventing Alzheimer's disease in a subject. As used herein, the term "subject" means any mammal that may be in need of treatment with the compounds of the invention that modulate APP processing. Subjects include but are not limited to: humans, non-human primates, cats, dogs, sheep, pigs, horses, cows, rodents such as mice, hamsters, and rats. Particular subjects to which the present invention can be applied are subjects at risk for or known to have an APP processing-associated disorder. Such disorders may include, but are not limited to:
Alzheimer's disease and any other diseases associated with abnormal APP processing, including overproduction of Aβ or reduced clearance of Aβ such as Down's syndrome, cerebrovascular amyloidosis, inclusion body myositis and hereditary inclusion body myopathies, any disease associated with abnormal BACE activity, ischemia, oxidative stress, head trauma, stroke, hypoglycemia, and any neurodegenerative disorder with abnormal APP processing.
As will be appreciated by those of ordinary skill in the art, the evaluation of the treatment also may be based upon an evaluation of the symptoms or clinical end-points of the associated disease. In some instances, the subjects to which the methods of the invention are applied are already diagnosed as having a particular condition or disease. In other instances, the measurement will represent the diagnosis of the condition or disease. In some instances, the subjects will already be undergoing drug therapy for an APP processing-associated disorder (e.g. Alzheimer's disease), while in other instances the subjects will be without present drug therapy for such a disorder. The methods as disclosed in the invention include administering to a subject a therapeutically effective amount of an agent that modulates the amount or rate of binding of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2) to contactins and/or Ll-CAMs.
The amount of a treatment may be varied for example by increasing or decreasing the amount of a therapeutic composition, by changing the therapeutic composition administered, by changing the route of administration, by changing the dosage timing and so on. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration, and the like factors within the knowledge and expertise of the health practitioner. For example, an effective amount can depend upon the degree to which an individual has abnormal levels and/or activity of APP processing.
The factors involved in determining an effective amount are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
The therapeutically effective amount of a pharmacological compositions of the invention is that amount effective to modulate Aβ accumulation, the rate and/or the level of APP processing to reduce, prevent, or eliminate the associated disorder. For example, testing can be performed to determine plasma Aβ levels in a subject. Additional tests useful for monitoring the onset, progression, and/or remission, of Aβ accumulation-associated disorders such as those described above herein, are well known to those of ordinary skill in the art. As would be understood by one of ordinary skill, for some disorders (e.g., Alzheimer's disease) an effective amount is the amount of a pharmacological agent of the invention that modulate the rate and/or amount of APP processing that diminishes the disorder, as determined by the aforementioned tests.
The pharmaceutical compositions used in the foregoing methods preferably are sterile and contain an effective amount of a pharmacological agent for producing the desired response in a unit of weight or volume suitable for administration to a patient.
The doses of pharmacological agents administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. The dosage of a pharmacological agent of the invention may be adjusted by the individual physician or veterinarian, particularly in the event of any complication. A therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg, and most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days. Various modes of administration will be known to one of ordinary skill in the art which effectively deliver the pharmacological agents of the invention to a desired tissue, cell, or bodily fluid. The administration methods include: topical, intravenous, oral, inhalation, intracavity, intrathecal, intrasynovial, buccal, sublingual, intranasal, transdermal, intravitreal, subcutaneous, intramuscular and intradermal administration. The invention is not limited by the particular modes of administration disclosed herein. Standard references in the art (e.g., Remington 's Pharmaceutical Sciences, 18th edition, 1990) provide modes of administration and formulations for delivery of various pharmaceutical preparations and formulations in pharmaceutical carriers. Other protocols which are useful for the administration of pharmacological agents of the invention will be known to one of ordinary skill in the art, in which the dose amount, schedule of administration, sites of administration, mode of administration (e.g., intra-organ) and the like vary from those presented herein.
Administration of pharmacological agents of the invention to mammals other than humans, e.g. for testing purposes or veterinary therapeutic purposes, is carried out under substantially the same conditions as described above. It will be understood by one of ordinary skill in the art that this invention is applicable to both human and animal diseases including AD. Thus, this invention is intended to be used in husbandry and veterinary medicine as well as in human therapeutics.
In some embodiments of the methods, the agents include one or more contactins or Ll-CAMs or fragments thereof that bind to APP, APLPl or APLP2. In preferred embodiments, the contactin is contactin-3 contactin-4, contactin-5 or a combination thereof, and the Ll-CAM is Ll or Ng-CAM, wherein the fragment is preferably a fragment of contactin-3, contactin-4, Ll or Ng-CAM that binds to APP, APLPl or APLP2, or a combination of such fragments. In some embodiments of the methods, the agent is a fusion protein, preferably an Fc fusion protein, including one or more contactins, Ll-CAMs or fragments. In other embodiments of the methods, the agent is an antibody or an antigen- binding fragment thereof. For example, the agents include an antibody or antigen-binding fragment thereof that binds a contactin or a Ll-CAM, particularly contactin-3 contactin-4, contactin-5, Ll or Ng-CAM, as well as APP, APLPl or APLP2. In some embodiments of the methods, the antibody or antigen-binding fragment thereof binds a complex of a contactin or a Ll-CAM and APP, APLPl or APLP2. In some embodiments, the methods include antibody or antigen-binding fragment thereof that is a bispecific antibody.
According to some embodiments of the methods, the agent blocks or disrupts the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs. In other embodiments of the methods, the agent promotes or stabilizes the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs. In some embodiments of the foregoing, the methods includes an agent that reduces processing of APP to β-amyloid. According to some embodiments, the methods include a vectorized agent that crosses the blood brain barrier. According to the invention, the methods are used for various subject, where a preferred subject is a human.
As used herein, the term "vectorized" refers to engineered moieties or modifications to a subject agent or compound for the purpose of delivering the composition to a target site in a cell or a tissue. For example, vectorized agents are produced by covalently linking a compound to a moiety which promotes delivery from the circulation to a predetermined destination in the body. In particular, for use in the instant invention, vectorized agents competent for transcytosis across the blood- brain barrier are useful for targeting the brain of a subject for therapeutic purposes. Examples of vectorized molecules which can traverse the blood-brain barrier are found in the prior art (Bickel et al., Proc. Natl. Acad. Sci. USA 90: 2618-2622 (1993); Broadwell et al., Exp. Neurol. 142: 47-65 (1996)). In some examples, antibodies are linked to another macromolecule, the antibodies being the agent which promotes delivery of the macromolecules. One example of such an agent is an antibody which is directed towards a cell surface component, such as a receptor, which is transported away from the cell surface. Examples of antibodies which confer the ability to trancytose the blood-brain barrier include, without limitation, anti-insulin receptor antibodies, and also anti- transferrin receptors (Saito et al., Proc. Natl. Acad. Sci. USA 92: 10227-31 (1995); Pardridge et al., Pharm. Res. 12: 807-816 (1995); Broadwell et al., Exp. Neurol. 142: 47-65 (1996)). This first antibody is covalently linked to an antibody which binds f-amyloid. Alternatively, coupling the β-amyloid antibodies to ligands which bind these receptors (e.g., insulin, transferrin, or low density lipoprotein) will also produce a vectorized antibody competent for delivery to the brain from the circulation (Descamps et al., Am. J. Physiol. 270: Hl 149- Hl 158 (1996); Duffy et al., Brain Res. 420: 32-38 (1987); Dehouck et al., J. Cell Biol. 138: 877-889 (1997)).
A vector moiety can be chemically attached to the agent to facilitate its delivery into the central nervous system. Alternatively, the moiety can be genetically engineered into the agent as an integral component. This vector component can be for example, an anti- transferrin receptor antibody or anti-insulin receptor antibody which binds the receptors present on the brain capillary endothelial cells (Bickel et al., Proc. Natl. Acad. Sci. USA 90: 2618-22 (1993); Pardridge et al., J. Pharmacol. Exp. Ther. 259: 66-70 (1991); Saito et al., Proc. Natl. Acad. Sci. USA 92: 10227-31(1995); Friden et al., J. Pharm. Exper. Ther. 278: 1491-1498 (1996)) which make up the blood-brain barrier. The resulting vectorized agent will attach to the appropriate receptors on the luminal side of the vessel (Raso et al., J. Biol. Chem. 272: 27623-27628 (1997); Raso et al., J. Biol. Chem. 272: 27618-27622 (1997); Raso, V. Anal. Biochem. 222: 297-304 (1994); Raso et al., Cancer Res. 41 : 2073-2078 (1981); Raso et al., Monoclonal antibodies as cell targeted carriers of covalently and non-covalently attached toxins. In Receptor mediated targeting of drugs, vol. 82. G. Gregoriadis, G. Post, J. Senior and A. Trouet, editors. NATO Advanced Studies Inst., New York. 1 19-138 (1984)). Once bound to the receptor, the vectorized agent passes across the blood-brain barrier by the process of transcytosis. Effectiveness of such delivery system, when administered intravenously, has been demonstrated in the art. For example, vectorized bispecific antibodies that can cross the blood brain barrier are described in published U.S. Patent Application 20050147613, the contents of which are incorporated herein by reference.
According to yet another aspect of the invention, pharmaceutical compositions (also referred to herein as therapeutic compounds and/or pharmaceutical compounds) are provided. These include one or more contactins and/or Ll-CAMs and/or one fragments thereof that bind to APP, APLPl or APLP2, and a pharmaceutically acceptable carrier.
When administered, the pharmaceutical compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The characteristics of the carrier will depend on the route of administration. The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intrathecal, intraperitoneal, intramuscular, intranasal, intracavity, subcutaneous, intradermal, or transdermal.
The therapeutic compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the therapeutic agent, which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of iηjectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
Compositions suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the therapeutic agent. Other compositions include suspensions in aqueous liquors or non-aqueous liquids such as a syrup, an elixir, or an emulsion.
The invention provides a composition of the above-described agents for use as a medicament, methods for preparing the medicament and methods for the sustained release of the medicament in vivo. Delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the therapeutic agent of the invention, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer-based systems such as polylactic and polyglycolic acid, poly(lactide-glycolide), copolyoxalates, polyanhydrides, polyesteramides, polyorthoesters, polyhydroxy butyric acid, and polycaprolactone. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di- and tri-glycerides; phospholipids; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like. Specific examples include, but are not limited to: (a) erosional systems in which the polysaccharide is contained in a form within a matrix, found in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
In one particular embodiment, the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. PCT/US95/03307 (Publication No. WO 95/24929, entitled "Polymeric Gene Delivery System." PCT7TJS95/03307 describes a biocompatible, preferably biodegradable polymeric matrix for containing an exogenous gene under the control of an appropriate promoter. The polymeric matrix is used to achieve sustained release of the exogenous gene in the patient. In accordance with the instant invention, the compound(s) of the invention is encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US95/03307. The polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the compound is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the compound is stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the compounds of the invention include films, coatings, gels, implants, and stents. The size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted. The size of the polymeric matrix device further is selected according to the method of delivery that is to be used. The polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material that is bioadhesive, to further increase the effectiveness of transfer when the devise is administered to a vascular surface. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.
Both non-biodegradable and biodegradable polymeric matrices can be used to deliver agents of the invention of the invention to the subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers. In general, the agents of the invention are delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix. Exemplary synthetic polymers that can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone.
Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof. Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
Bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein by reference, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly (hexylmethacry late), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
Use of a long-term sustained release implant may be particularly suitable for treatment of established neurological disorder conditions as well as subjects at risk of developing a neurological disorder. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days. The implant may be positioned at or near the site of the neurological damage or the area of the brain or nervous system affected by or involved in the neurological disorder. Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above.
In preferred embodiments of the pharmaceutical compositions of the invention, the contactin is contactin-3 contactin-4, contactin-5 or a combination thereof, and the Ll-CAM is Ll or Ng-CAM. In some embodiments, the pharmaceutical composition includes a fragment of contactin-3, contactin-4, Ll or Ng-CAM that binds to APP, APLPl or APLP2, or a combination of such fragments. According to the invention, some embodiments include a pharmaceutical composition that is a fusion protein, including one or more contactins, Ll- CAMs or fragments. In some cases, the composition includes an Fc fusion protein.
In certain embodiments, the pharmaceutical compositions include one or more antibodies that bind a contactin, a Ll-CAM, APP, APLPl or APLP2 or antigen-binding fragments thereof, where preferred contactin or Ll-CAM is contactin-3 contactin-4, contactin-5. Ll or Ng-CAM. In some embodiments, the pharmaceutical composition includes an antibody or antigen-binding fragment thereof that binds a complex of a contactin or a Ll-CAM and APP, APLPl or APLP2. In certain embodiments of the foregoing compositions, the antibody or antigen-binding fragment thereof is a bispecific antibody. The pharmaceutical composition in some embodiments includes an agent that blocks or disrupts the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs. In other embodiments, the pharmaceutical composition includes an agent that promotes or stabilizes the binding of APP, APLPl or APLP2 to contactins and/or Ll-CAMs. In some embodiments of the foregoing composition, the agent reduces processing of APP to β-amyloid. In some cases, the pharmaceutical composition according to the foregoing includes a vectorized agent that crosses the blood brain barrier of a subject, preferably a human.
The invention is also useful for providing screening methods for identifying and/or isolating molecules or compounds that elicit modulatory effects on APP processing. In one aspect of the invention, methods for identifying compounds that modulate the binding of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2) with contactins and/or Ll-CAMs are provided. These methods include providing a reaction mixture that comprises (1) APP, APLPl, APLP2 and/or a fragment thereof that binds to contactins and/or Ll -CAMS, and (2) contactins, Ll- CAMs and/or a fragment thereof that binds to APP, APLPl3 APLP2; contacting the reaction mixture with a test compound; determining a level of binding of APP, APLPl, APLP2 and/or a fragment thereof with contactins, Ll-CAMs and/or a fragment thereof in the absence and in the presence of the test compound; and comparing the level of binding of APP, APLPl, APLP2 or fragment thereof with contactins, Ll-CAMs and/or a fragment thereof in the absence and in the presence of the test compound, wherein a test compound that modulates the binding relative to the level of binding in the absence of the test compound is a compound that modulates the binding of APP, APLPl, APLP2 or fragment thereof with contactins, Ll- CAMs and/or a fragment thereof. In some embodiments of the foregoing methods, the test compound is a small molecule. As used herein, small molecules encompass numerous chemical classes, although typically they are organic compounds. In some embodiments, the candidate test compounds are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500. Candidate compounds comprise functional chemical groups necessary for structural interactions with proteins and/or nucleic acid molecules, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups. The candidate compounds can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Candidate compounds also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Where the compound is a nucleic acid molecule, the agent typically is a DNA or RNA molecule, although modified nucleic acid molecules as defined herein are also contemplated.
Candidate test compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means. Further, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the agents.
A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.
In some embodiments of the foregoing methods, the test compound is an antibody that binds to APP, APLPl, APLP2 a contactin or a Ll-CAM, or an antigen-binding fragment thereof; or a fragment of APP, APLPl , APLP2, a contactin or a Ll-CAM, where preferably the contactins and/or Ll-CAMs is selected from the group consisting of: contactin-3, contactin-4, contactin-5, Ll and Ng-CAM.
In yet another aspect of the invention, methods for identifying compounds that modulate the processing of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2) are provided. In some cases, the methods are used to screen for compounds that increase the rate and/or amount of APP processing. In other cases, the methods are used to screen for compounds that reduce the rate and/or amount of APP processing. These methods include: providing a reaction mixture that comprises (1) contactins, Ll-CAMs and/or a fragment thereof that binds to APP, APLPl, APLP2, and (2) APP, APLPl, APLP2 and/or a fragment thereof that binds to contactins and/or Ll-CAMS; contacting the reaction mixture with a test compound; determining a level of processing of APP, APLPl or APLP2 in the absence and in the presence of the test compound; and comparing the processing in the absence and in the presence of the test compound, wherein a test compound that modulates processing of APP, APLPl or APLP2 relative to the level of processing in the absence of the test compound is a compound that modulates the processing of APP, APLPl or APLP2. According to some embodiments of the foregoing methods, the test compound is a small molecule (see above); an antibody that binds to APP, APLPl, APLP2, a contactin or a Ll-CAM, or an antigen-binding fragment thereof; a fragment of APP, APLPl or APLP2, a contactin or a Ll-CAM, where preferably the contactins and/or Ll-CAMs is selected from the group consisting of: contactin-3, contactin-4, contactin-5, Ll and Ng-CAM. In certain embodiments of the methods, the processing of APP to Aβ is reduced. Changes in relative or absolute rate and/or amount of APP processing of greater than 0.1% may indicate an abnormality. Preferably, the change in the rate and/or amount of APP processing, which indicates an abnormality, is greater than 0.2%, greater than 0.5%, greater than 1.0%, 2.0%, 3.0% , 4.0%, 5.0%, 7.0%, 10%,. 15%, 20%, 25%, 30%, 40%, 50%, or more, where greater changes correlate with greater efficacy of the modulatory compound.
Examples
Example 1 : Purification of candidate APP binding proteins
The approach which was taken to study APP in cell-cell signaling was to first identify novel extracellular binding partners, then to examine these for the ability to act as a functional receptor or ligand for APP. To this end, alkaline-phosphatase tagged APPsalpha was used as an affinity probe to visualize the expression pattern of binding partners in embryonic chick brains, and the domains of APP required for this interaction were determined. Thus, the invention describes the identification of a family of proteins that bind to APP. The invention also includes the recognition that binding of APP to extracellular binding partners leads to the modulation of APP processing. This can be used for a variety of applications including therapeutic interventions in diseases and screening systems for effector molecules and agents that may affect APP processing.
Methods
Plasmids:
APtag4 has been described previously (Flanagan et al., 2000).
AP-mAPP was constructed by inserting the sequence encoding amino acids 18 through 612 of mouse APP695 into APtag4. This portion of APP was obtained by PCR amplification with Taq polymerase from mouse midbrain-derived cDNA using the primer pairs tccactcgcacacggagcactcgg [SEQ ID NO:1] plus cggacgtacttcttcagcatgttg [SEQ ID NO:2], followed by ccctccggactggaggtacccactgatgg [SEQ ID NO:3] plus gtaattctcgaggtccaggcg [SEQ ID NO:4] to amplify base pairs 190 through 1329, and the primer pair ctggacctcgagaattac [SEQ ID NO:5] plus ccctctagattatttttgatggcggacttc [SEQ ID NO:6] to amplify base pairs 1329 to 1974 with a stop codon added. The first PCR product was cut with BspEI and Xhol, the second with Xhol and Xbal, and both were inserted by three-piece ligation into APtag4 cut with BspEI and Xbal. Deletion constructs of AP-mAPP were made by PCR amplification of selected portions of APP, using Pfu as the polymerase and AP-mAPP as the template, adding a BspEI site on the 5' end, and a stop codon followed by an Xbal site on the 3' end to allow insertion into APtag4. Amino acids included in these constructs are as follows: 18-65, 18-124, 18-156, 18-205, 18-293, 18-345, 18-480, 66-612, 125-612, 157-612, 199-612, 294-612, 346-612, and 481-612.
AP fusion proteins:
AP fusion proteins were produced by transfecting 293T cells with the appropriate plasmids, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Conditioned medium was collected four to six days after transfection, filtered, and supplemented with 20 mM HEPES (pH 7.0) and 0.05% sodium azide. For applications other than making protein-conjugated beads, the cell culture medium used was Dulbecco's Modified Eagle Medium (Invitrogen) with 10% calf serum, supplemented with glutamine and Penicillin/Streptomycin solution (both from Biowhittaker). The cultured supernatants were used without purification for all experiments.
Buffers, solutions, and protein-conjugated beads:
Most of the chemicals used were from Sigma. Hank's balanced salt solution (HBSS) was purchased from VWR or Invitrogen. HBAH was HBSS with 0.5 mg/ml bovine serum albumin, 0.05% sodium azide, and 20 mM HEPES (pH 7.0) added. HH was HBSS with 20 mM HEPES (pH 7.0). Lysis buffer was 0.5% sodium deoxycholate, 0.5% Triton Xl 14, and 0.05% SDS in phosphate buffered saline. Lysis buffer plus protease inhibitors additionally included 5 mM EDTA plus a cOmplete protease inhibitor cocktail tablet (Roche), dissolved to the manufacturer's recommended concentration. Wash buffer was 0.1% Triton X-100 in phosphate buffered saline. High salt wash buffer was IM NaCl, 20 mM HEPES (pH 7.0), and 0.1% Triton XlOO. Elution solution was 100 mM glycine, 150 mM NaCl, pH 3.0.
Anti-AP beads were made essentially as previously described (Flanagan et al., 2000). Briefly, CNBr-Sepharose beads (Amersham Biosciences) were washed with 500 ml ImM HCl, then conjugated to monoclonal anti-human placental alkaline phosphatase antibody MIA1801 (Seradyn) in the presence of 0.25 M sodium phosphate pH 8.3, allowing lmg antibody per ImI packed beads. After an overnight incubation at 4 degrees C, the reaction was stopped by adding ethanolamine (pH 8.0) to 333 mM and incubating an additional 4-5 hours. Beads were then washed with 0.5 M sodium phosphate (pH 8.3), followed by several washes of Lysis buffer.
AP and AP-mAPP beads were made by a similar procedure to that for anti-AP beads, except that concentrated AP or AP-mAPP cultured supernatants were used in place of antibody. These supernatants were produced by transfected 293T cells grown in Opti-MEM with ITS-A, an insulin, transferrin, and selenium supplement (Invitrogen), and were concentrated approximately 12-fold using a Centricon device with a 10 kDa molecular weight cut-off (Millipore).
AP in situs:
AP fusion proteins were used as in situ probes for staining tissue according to published protocols (Flanagan et al., 2000). Embryonic chick brains were dissected, washed with HBAH5 then incubated in AP fusion proteins for at least 90 minutes. After 6 washes in HBAH, brains were fixed for 2 minutes (in 40% acetone, 8% formalin, 20 mM HEPES, pH 7.0), washed three times in HBS (150 mM NaCl, 20 mM HEPES, pH 7.0), then incubated in HBS overnight at 65 degrees Celsius to inactivate endogenous phosphatase activity. Brains were then rinsed in AP developing buffer (100 mM NaCl, 5mM MgCl2, 100 mM Tris-HCl, pH 9.5), and AP activity was visualized in AP developing buffer plus 0.1 mg/ml 5-bromo-4- chloro-3-indoxyl phosphate and 0.5 mg/ml nitrotetrazolium blue (Biosynth International).
Protein purification from olfactory bulbs: Phosphatidylinositol-specific phospholipase C (PI-PLC) was obtained from Molecular
Probes. For purifications, olfactory bulbs from El 3 chick embryos (approximately 600 embryos per experimental condition) were washed with HH, then transferred to 5 U/ml PI- PLC in HH (or simply HH only for mock-treatment) and incubated at 37 degrees for 30 minutes. The supernatant was collected and in some cases stored at 4 degrees up to several days. For 2D gels, this supernatant was desalted into 2D sample buffer using spin columns (Pierce), and electrophoresis was carried out as described below. For affinity purification, the supernatant of PI-PLC treated olfactory bulbs was cleared by spinning at 10O5OOOg at 4 degrees, then divided and incubated with either AP or AP-mAPP beads overnight at 4 degrees. The beads were then washed extensively with cold HH, and bound proteins were eluted with Elution solution, buffered with 100 mM Tris pH 8.0 and precipitated with 10% trichloroacetic acid. The precipitated proteins were centrifuged to pellet, washed with acetone, air-dried, and boiled in SDS-PAGE sample buffer supplemented with 100 mM Tris pH 8.0.
Protein purification from tecta:
For purification of APP-binding proteins, tecta were dissected from El 3 chick embryos (approximately 100 embryos for small-scale experiments, approximately 1,200 for Figure 7), rinsed in HBSS, and biotinylated with 0.2 mg/ml EZ-link NHS-LC-Biotin (Pierce) in HH for 45 minutes. The biotinylation reaction was quenched by adding 50 mM Tris pH 7.5 then incubating 20 minutes. The tecta were then washed in HBAH, incubated in AP fusion-containing conditioned media (AP, AP-mAPP, or AP-mAPP 199-345) for at least 90 minutes, then washed first six times in ice-cold HBAH, then six times in ice-cold HBSS. Reversible chemical crosslinking was performed by exposing tecta to 0.5 mg/ml DTSSP (Pierce) in HH for 45 minutes (at room temperature), and the crosslinking reaction was quenched by adding 50 mM Tris pH 7.5 then incubating 20 minutes. Tecta were partially lysed for 45 minutes on ice in Lysis buffer plus protease inhibitors. Remaining tectal debris was pelleted and the supernatant was, in some cases, stored at 4 degrees for up to several days before further processing. The lysate was cleared by spinning at 100,000g at 4 degrees, then incubated overnight at 4 degrees with anti-AP beads. The beads were washed several times, first with ice-cold Lysis buffer, then with ice-cold Wash buffer. Bound proteins were eluted with Elution solution and buffered with 100 mM Tris pH 8.0. For standard SDS- PAGE, the eluted proteins were precipitated with 10% trichloroacetic acid, pelleted, washed with acetone, air-dried, and boiled in SDS-PAGE sample buffer supplemented with 100 mM Tris pH 8.0 before undergoing gel electrophoresis. For 2D gel analysis, the eluted proteins were boiled for 15 minutes in 100 mM DTT to cleave the DTSSP crosslinker, incubated with 9.25 mg/ml iodoacetamide (15 minutes, room temperature) to prevent disulfide bonds from re-forming, then precipitated with 10% trichloroacetic acid. The precipitated proteins were centrifuged to pellet, washed with acetone, air-dried, and resuspended in 2D sample buffer as described below.
For purification of tectal surface proteins, tecta dissected from approximately 800 El 3 chick embryos were rinsed in HBSS, then reversibly biotinylated with 1 mg/ml Sulfo-NHS- SS-Biotin (Pierce) in HH for 1 hour. The biotinylation reaction was quenched by the addition of 50 mM Tris pH 7.5 for a 20 minute incubation, followed by extensive washing in HBSS. Tectal lysis and clearing of the lysate were as in the preceding protocol, then the lysate was incubated overnight at 4 degrees with NeutrAvidin beads (Pierce). The beads were washed several times, first with Lysis buffer, then Wash buffer, then High salt wash buffer, then finally with Wash buffer again. The beads were then incubated in 100 mM DTT at 65 degrees for 40 minutes to reduce the disulfide bond in the biotinylation reagent. The supernatant was collected, and proteins were precipitated with 10% trichloroacetic acid. The precipitated proteins were centrifuged to pellet, washed with acetone, air-dried, and boiled in SDS-PAGE sample buffer supplemented with 100 mM Tris pH 8.0.
Gel electrophoresis and related methods:
For 2D electrophoresis, the ZOOM IPGRunner system (Invitrogen) was used, with isoelectric focusing done on pH 3-10 strips followed by SDS-PAGE on 4-20% Tris-glycine gels. The sample buffer for isoelectric focusing was 7M urea, 2M thiourea, and 4% CHAPS (from 2D insoluble protein sample prep kit, Pierce), with the addition of carrier ampholytes (Invitrogen), 20 mM dithiothreitol, and 0.02 mg/ml bromophenol blue.
Silver staining of gels was done according to published protocols (Shevchenko et al., 1996). Coomassie staining was performed using the Colloidal Blue kit from Invitrogen. For Western Blots, biotin was recognized with horseradish peroxidase conjugated NeutrAvidin (Pierce) at 2 micrograms/ml, followed by chemiluminescent detection.
Protein sequence analysis:
The TMpred program was used to search for hydrophobic regions that might correspond to transmembrane domains, signal peptides, or GPI anchorage signals (at www.ch.embnet.org/software/TMPRED form.html). The big-PI Predictor program of the IMP Bioinformatics Group in Vienna, Austria (mendel.imp.ac.at/gpi/gpi_server.html) was also used to predict GPI anchorage sites.
Results
Characterization of APP binding sites on embryonic chick brains: As a first step to search for novel extracellular binding partners for APP, we generated
AP-mAPP, a fusion protein with Alkaline Phosphatase fused to the N-terminus of APPsa, to use as an affinity probe. This approach has been previously used to identify several novel receptors or ligands for orphan cell-surface proteins (Flanagan and Cheng, 2000). AP-mAPP binds to embryonic chick brains, with. strong midbrain and olfactory bulb signals (Figure 2A). The binding pattern of AP-mAPP to chick tectum coincides with the expected location of retinal ganglion cell (RGC) axons. That is, binding is first observed on the extreme anterior surface of the tectum at embryonic day 9 (E9) and stretches to cover the entire tectum by El 6 (Figure 2B). Furthermore, in three one-eyed chick embryos (one naturally occurring, the other two surgically enucleated) that appeared otherwise normal, AP- mAPP binding was absent in the tectum contralateral to (and therefore normally innervated by) the missing eye (Figure 2C). This suggests that the putative APP binding partner is localized to, or is upregulated in the tectum by, RGC axons. Based on the striated appearance of the staining (Figure 2D), we favor the idea of AP-mAPP binding directly to RGC axons. The domains of APP required for interacting with many of its extracellular binding partners have been mapped, so for comparison, we mapped the domain of APP involved in binding to RGC axons in the tectum. As schematized in Figure 3 A, amino acids 199-293 are sufficient for this binding, although amino acids 199-345 bind more strongly (Figure 3B). This region largely overlaps the domain required for class A scavenger receptor (SR-A) binding, amino acids 191-264 (Santiago-Garcia et al., 2001). However, SR-A does not seem to be expressed by neurons in neonatal or adult mice, or in humans, though there is expression elsewhere in the brain, including in microglia, macrophages, and perivascular sites, (Bell et al., 1994; Christie et al., 1996), so this binding pattern still seemed likely to be due to an unidentified binding partner for APP.
The initial domain analysis of AP-mAPP binding was performed with ElO brains, but olfactory bulbs do not bind APP at this stage. When we tested binding against older brains (El 2 to El 3), which do show olfactory binding, it became clear that the deletion constructs AP-mAPP( 199-293) and AP-mAPP(l 99-345) do not bind olfactory bulbs (compare Figure 3B to 2A), but that AP-mAPP(l 8-205) does (Figure 4A). Furthermore, the binding of AP- mAPP to tecta appears largely unaffected by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), but the binding of AP-APP to chick olfactory bulb is greatly reduced by PI-PLC treatment (Figure 4B). PI-PLC cleaves GPI anchorages, so the olfactory bulb binding partner for APP appeared most likely to be a GPI-anchored protein, or possibly a soluble molecule tightly associated with a GPI-anchored protein. The interaction with a different APP domain and the insensitivity of APP binding to PI-PLC treatment in the tectum suggested that this structure expresses a distinct, non-GPI linked APP binding partner. Biochemical approaches to identifying APP binding partners:
Although there are many potential ways of searching for extracellular binding partners of proteins, two general approaches that have met with particular success have been expression cloning, for example, screening for AP-mAPP binding to cells transfected with an expression library, and biochemical purification, for example, using AP-mAPP as an affinity reagent. Several limitations of expression screening, including the difficulty of identifying a gene if its overexpression results in cell death, as well as a theoretical difficulty if the binding partner being sought is a complex of two or more distinct proteins rather than a single molecule, led me to try to identify binding partners for APP by biochemical purification.
Affinity purifications:
One potential difficulty in affinity purifying a membrane-bound molecule lies in choosing lysis conditions that are strong enough to solubilize the molecule but that do not interfere with its binding to the affinity reagent. Since AP-mAPP( 180-205) binding to olfactory bulbs is PI-PLC sensitive, the olfactory bulb binding partner was likely to be solubilized simply by PI-PLC treatment, probably through cleavage of its own GPI anchorage, though possibly by cleavage of the GPI anchor of an associated protein. Therefore, olfactory bulbs were treated with PI-PLC, and the supernatant was divided for affinity purification against beads conjugated with AP or AP-mAPP. SDS-PAGE analysis followed by silver-staining revealed two faint bands that appeared specific for AP-APP
(Figure 5). The stronger band was analyzed by tandem mass spectrometry, but there was too little data to identify the protein.
The purification of the AP-mAPP (199-345) binding partner in chick tecta was complicated by the observation that tecta had to be exposed to fairly harsh lysis conditions in order to abolish APP binding, and simple affinity purification using tectal lysates from these conditions did not reveal any proteins that bound specifically to AP-mAPP. To circumvent this difficulty, we developed a crosslinking protocol (schematized in Figure 6A) that involved biotinylating tectal surface proteins and exposing these tecta to one of three probes (AP, AP- mAPP( 199-345), AP-APP). After washing away non-bound proteins, tecta were incubated in DTSSP (a reversible, membrane-impermeable crosslinking reagent) then lysed, and the lysates were subjected to an immuno precipitation against AP. Subsequent anti-biotin Western blots revealed bands that were immunoprecipitated with both AP-mAPP and AP- mAPP(l 99-345), though to a lesser degree, but not with AP alone. Two bands of approximate size 150 kD and 120 kD were seen in most experiments; in addition, higher molecular weight bands (between 200 kD and 300 kD) were sometimes seen.
Initial attempts to scale up the above purification for tandem mass spectrometry simply resulted in a smear of proteins on a silver stained gel, but running a 2D gel with similar samples gave cleaner results. As shown in Figure 7 A, each co-IP was run on a 2D gel to be silver stained, and purified AP-tagged probes were also run separately for comparison. A small-scale co-IP was also run on a separate 2D gel for an anti-biotin Western blot (Figure 7B), and the location of a prominent biotinylated spot was used as a guide to determine which region of the silver-stained gels to cut out for analysis. Because of the faint signal on the silver stained gel, I combined the samples from the AP-mAPP and AP-APP (199-345) co-IPs. Analysis by tandem mass spectroscopy identified three proteins with a strong match to the sample submitted: apparent probe contaminants AP and APP, and contactin-4.
Partial purifications: As discussed above, deletion analysis of AP-mAPP binding combined with the specific effect of PI-PLC on olfactory bulb binding led to the hypothesis that APP had two distinct binding partners in the embryonic chick brain. Initial attempts to purify both partners resulted in the identification of only one candidate protein, contactin 4. Since contactin 4 is GPI anchored, but was identified from a tectal, not olfactory bulb, purification, we continued other ongoing efforts at identifying binding partners from both tissue sources. A major problem seemed to be obtaining enough protein for identification by mass spectrometry, but scaling up the affinity purifications much further seemed prohibitively difficult. Several of the purification steps involved, including both eluting from affinity columns and crosslinking, appeared to be fairly inefficient, so we decided to increase protein yield in exchange for a loss of specificity by performing partial purifications, with the hopes of generating a short list of candidate proteins that could subsequently be tested.
The olfactory bulb-expressed binding partner for APP seemed likely to be a GPI anchored molecule, and the affinity purification above had already given some idea of its molecular weight. Therefore, we decided to identify the major GPI-anchored proteins of greater than 50 kD expressed on the olfactory bulb. To do this, we dissected chick olfactory bulbs and subjected them to either PI-PLC or mock treatment. The supernatants were run on 2D gels, and several spots specific to the PI-PLC-treated samples were analyzed by tandem mass spectroscopy (Figure 8A). The sequences of identified proteins were analyzed with software to predict GPI anchors, but were also considered candidates simply if the N- and C- terminal tails appeared strongly hydrophobic (Udenfriend and Kodukula, 1995). The candidate GPI-anchored proteins identified are listed in Figure 8B.
Since the tectal crosslinking protocol described in Figure 6 revealed that AP-mAPP interacted with proteins of specific molecular weights that could be labeled with surface biotinylation, the partial purification applied to tectal proteins relied on just those two criteria. Chick tecta were surface labeled with a cleavable biotin tag, then lysed. Biotinylated proteins were retrieved from this lysate using NeutrAvidin beads (Pierce), and after washing, were released from the beads by cleaving the biotin tag (Figure 9A). The purified proteins were separated by SDS-PAGE, and bands corresponding to the molecular weights of bands detected in previous co-IPs (Figure 9B, compare to Figure 6B) were cut out and analyzed by tandem mass spectroscopy. The sequences of the identified proteins were analyzed for potential transmembrane domains or signal peptides. The candidate tectal surface proteins identified are listed in Figure 9C.
In an attempt to identify novel extracellular binding partners for APP, a fusion protein of APPsalpha tagged with Alkaline Phosphatase was produced. This probe was found to bind to embryonic chick brains. Deletion analysis revealed that amino acids 18-205 of APP were sufficient for strong binding to olfactory bulbs, as well as weaker binding throughout the brain. In contrast, amino acids 199-293 were sufficient for binding to RGC axons, although the larger region of 199-345 exhibited stronger binding. This suggested the presence of at least two distinct binding partners.
The further observation that PI-PLC treatment abolished olfactory bulb, but not RGC, binding suggested that amino acids 18-205 of APP interacted with a GPI anchored protein, while amino acids 199-293 did not. However, this interpretation of the PI-PLC results is definitely not definitive. For example, AP-mAPP (18-205) could be binding a soluble molecule that is anchored to the cell surface through interaction with a separate GPI anchored protein. On the other hand, some GPI anchors are modified in ways that render them resistant to PI-PLC cleavage, so it still remains possible that AP-mAPP (199-293) might interact with such a GPI anchored molecule. It is important to note that these caveats do not detract in any major way from the logic behind the purifications discussed here.
Example 2: Characterization of APP interactions with contactin 4 and NgCAM
Among the list of candidate APP binding proteins identified as described above, a further characterization of APP interaction with two of these proteins, namely, contactin 4 and NgCAM, as well as an interaction between APP and contactin 3, are shown in the example disclosed below.
Materials and Methods Plasmids:
To guide the design of DNA constructs, the following resources were used: bioinformatics.leeds.ac.uk/prot_analysis/Signal.html or www.cbs.dtu.dk/services/SignalP/ for signal peptide prediction, www.ch.embnet.org/software/TMPRED_form.html to predict transmembrane regions, and mendel.imp.ac.at/gpi/gpi_server.html to predict GPI anchorage sites.
IgTag2Eco was constructed by Andrew Bergemann, and includes a polylinker followed by the Fc fragment of human IgG (Aruffo et al., 1990) in a pcDNAl (Invitrogen) backbone. pSecTaglg was constructed by Mitsu Hattori by inserting the Hindlll-Xhol fragment of IgTag2Eco into pSecTag2C (Invitrogen), creating a vector encoding the Fc fragment downstream of a signal peptide.
To construct the NgCAM-Fc plasmid, pSCT-NgCAM (kindly provided by P. Sonderegger, (Buchstaller et al., 1996) was cut with Xbal and RsrII, and this fragment was inserted into pSecTaglg that had been cut with Nhel and RsrII. The resulting plasmid was then cut with BgI II, blunt ended, then cut with RsrII, and the RsrII-FspI fragment of pSCT- NgCAM was then inserted. This resulted in a construct predicted to encode the first 1 134 amino acids of NgCAM fused upstream of Fc. The NrCAM-Fc construct was kindly provided by M. Grumet (Lustig et al., 1999).
Fc fusions of all six human contactin family members were made by inserting the portion of the gene from the start codon through the predicted GPI anchorage site into IgTag2Eco. For each gene, the NCBI accession number, restriction sites added by PCR for cloning, and predicted GPI site used (by residue number) are as follows: contactin 1 (NM_001843, HindIII + RsrII, 993); contactin 2 (NM_005076, HindIII + BgI II, 1012); contactin 3 ([see below], RsrII + BgI II, 1002); contactin 4 (NMJ 75607, Rsr II + BamHI, 1000); contactin 5 (NM_014361, EcoRV + EcoRI, 1072); contactin 6 (NMJ) 14461, HindIII + Rsr TI, 999). The sequence available for human contactin 3 (XM_039627, gi:37549673) did not appear to include the 5' end of the gene, so the 5' primer for PCR (ccccggaccgaaaatgatgtttccatggaaacagttg) [SEQ ID NO:7] was designed based on a stretch of human chromosome 3 genomic sequence highly homologous to the 51 end of rat contactin 3 (NMJ 19329). The assembled full-length contactin 3 sequence encoded a protein of 3068 amino acids. The six contactin-Fc clones that were chosen agreed with the NCBI sequence listed above at the predicted amino acid level. The validity of the contactin 3-Fc construct N- terminal to XM_039627 was confirmed by checking that two independent clones agreed in sequence for this region. Deletion constructs for contactin 2, 3, or 4-Fc were constructed by amplifying the desired fragments by PCR, adding an Sfi I site to the 5' end and either an EcoRV site (for contactin 3) or a BamHI site (for contactin 4) to the 3' end. pSecTaglg was partially digested with Sfi I and completely digested with either BamHI or EcoRV, and the appropriately digested contactin fragments were inserted. All of these plasmids were verified by sequencing. The fragments used encode the following amino acid numbers: c2Igl-4 (31-
413), c2Ig5-6 (414-608), c2FN (609-1012), c3Igl-4 (20-404), c3Ig5-6 (405-597), c3FN (598- 1005), c4Igl-4 (19-404), c4Ig5-6 (405-595), c4FN (596-1000).
All AP-APP or AP-APLP constructs were constructed by amplifying the chosen portion by PCR, adding a BspEI site to the 5' end and an Xba I site preceded by a stop codon to the 3' end, and inserting into the corresponding sites in APtag4. NCBI accession numbers used as reference for human sequences were Y00264 for APP, NM_005166 for APLPl, and L27631 for APLP2. "Full length" constructs were designed to begin after the predicted signal peptide and end at the alpha cleavage site (for APP) or the homologous region. The fragments used encode the following regions by amino acid number: AP-APP (18-612), AP- APLPl (34-567), AP-APLPl Nterm (34-221), AP-APLP2 middle (215-419).
For APP-HA, full length human APP was amplified by PCR and inserted into pcDNA3.1 Zeo+. The 5' end was preceded by an Kozak sequence and a HindIII site for cloning, and the 3' end was followed by an HA tag, an Xbal cloning site (encoding amino acids S and R)5 and a stop codon, followed by Notl and Apal sites. Deletion constructs of APP-HA were made by PCR, with the 3' primer designed to preserve the Apa I cloning site as well as the C-terminal tag (HA + Ser +Arg) and stop codon. To allow cytoplasmic transcription, the 5' primer included a single start codon immediately upstream of the indicated cleavage site, and the PCR product was inserted into pcDNA3.1 Zeo+, using an added HindII site and Apal. In other constructs, the 5' primer was designed to insert the first APP-derived codon immediately after the Sfil site in pSecTag2A; after signal peptide cleavage, the resulting proteins are predicted to begin with AAQPA followed by the indicated portion of APP. The first APP-derived amino acid for each deletion constructs follows: beta 597, alpha 613, gamma(40) 637, epsilon 646. The PCR construct listed above were verified by sequencing. Antibodies:
Antibodies for testing candidate tectal proteins included anti-PSA NCAM (Amersham), anti-chick NCAM-I (Amersham), anti-tenascin (Chemicon), anti-neogenin (R&D systems), anti-contactin 1 (BD Biosciences). Anti-HA was from Roche, and IRdye800 conjugated anti-human Fc and anti-rat Fc were from Rockland or VWR.
Binding assays:
Binding assays were carried out as previously described (Flanagan and Cheng, 2000). For preliminary cell binding assays, 293T cells were transfected with plasmids expected to encode full-length expression constructs of candidates. One day after transfection, cells were dislodged from the plates by pipetting, briefly incubated in HBAH to block non-specific interactions, then incubated in AP fusion supernatant. Cells were washed seven times with ice-cold HBAH than lysed, and AP activity was measured colorometrically. For binding assays using Fc- and AP- fusion proteins, Opti-MEM based supernatants containing contactin-Fc fusion protein were incubated with protein A beads. AP fusion proteins supernatants, normalized for AP activity, were incubated with the beads, and the AP activity retained by the beads after washing was measured.
For normalization, Fc fusions were subjected to an anti-Fc Western blot, and quantitated using Li-COR Odyssey scanner and software.
Signaling assays:
293T cells were grown in 6 well dishes and transfected using TransIT-LTl (Minis Bio Corp) according to manufacturer's protocols, using 6 microliters of transfection reagent per well. Cells were collected for analysis one day after transfection. Plates were pre-chilled on ice 5 minutes, then lysed in 200 microliters of 1% Triton XlOO, 10 mM Tris pH 8.0, plus 140 mM NaCl for 20 minutes. Lysates were centrifuged 5 minutes to clear, combined with sample buffer, and boiled 5 minutes. For Western Blot analysis, samples were subjected to SDS-PAGE using 10% gels in MES buffer (Invitrogen), transferred to a PVDF membrane (Millipore), and detected with anti-HA antibody (rat monoclonal 3F10, Roche) followed by IRdyeSOO conjugated anti-rat antibody (VWR). The blots were scanned using an Odyssey Infrared Imaging System (Li-COR). Results
APP binding to contactin 4:
The biochemical purifications described herein resulted in a list of candidate APP binding proteins. In order to test the olfactory bulb derived candidate genes (see Figure 8B), we amplified several by PCR and inserted them into the pcDNA3.1 Zeo+ expression plasmid, then transfected into 293T cells to test for AP-mAPP binding. The contactin 4 transfected cells were the only ones to exhibit obvious binding, but only approximately three-fold above AP-mAPP binding to control cells. Since one possible explanation was that contactin 4 might be degraded rapidly in cells, we decided to try producing a secreted form in which the GPI anchor was deleted and substituted for by an Fc tag. Using this approach, we found that contactin 4-Fc immobilized on Protein A beads was capable of capturing AP-mAPP protein.
Having identified an interaction between APP and contactin 4, we wondered about the specificity of this interaction. To address this question, we tested all six known human contactin family members for binding to APP and its homolog APLPl, using Fc fusions of the contactin proteins and AP fusions of APP and APLPl (Figure 10). This method showed APP to interact with contactins 3 and 4, while APLPl interacted with contactins 3, 4, and 5. There also appeared to be some binding of APP to contactin 1, but saturation binding experiments, similar to those described below, failed to confirm this interaction.
To study the strength of the above interactions, we generated saturation binding curves by performing binding assays in which the concentration of Fc fusion was fixed, but the initial AP fusion concentration was varied (Figure 1 IA). This set of data was re-graphed as a Scatchard plot (Figure 1 IB), and the dissociation constants (KD) were calculated according to -I/slope for the fitted line. The calculated KDS range from 19nM to 4InM, as listed in Figure 11C. The data points for the APP Scatchard plots do not appear to be as well fitted by a single line as the data points for the APLPl plots. Although this may simply due to measurement error, an alternative possibility is that APP may be recognizing binding sites of different affinity. For binding to two distinct sites, the points in a Scatchard plot would appear to lie along the curve composed by combining the values from two distinct lines, such as those sketched in Figure 1 ID. Since contactin 4 was identified in purifications from both olfactory bulbs and tecta, which exhibit preferential binding for APP amino acids 18-205 and 199-345 respectively, we interested to determine which domain or domains of APP interact with contactin 4. At the same time, we decided to examine the domains of contactin 4 involved in this interaction. The binding assay was done with Fc and AP fusion proteins as above, though less rigorously; Fc fusion proteins were not normalized (expression levels of supernatants used can be seen in Figure 12B), and although AP fusion proteins were normalized for activity, a saturation binding curve was not done. However, the data clearly show that amino acids 18-205 of APP and the fϊbronectin domains of contactin 3 or 4 are sufficient for binding (Figure 12A).
An interaction of APP with NgCAM:
As presented above, we developed a second list of candidate APP binding proteins by selecting for tectal surface expression and specific molecular weights (see Figure 9). Antibodies were available for many of these proteins, so we decided to repeat the crosslinker- based affinity purification described in Figure 6, using candidate antibodies (see methods section for list) instead of avidin-HRP for Western Blot detection. As shown in Figure 13A5 two independent antibodies against NgCAM recognized bands in AP-mAPP, but not in AP alone, immunoprecipitates. One of the bands recognized appears to be approximately the size of the upper band in the 120-150 kD doublet recognized by avidin (see also Figure 6B). Some additional bands (higher in the mouse, lower in the rabbit antibody blot) may also be due to NgCAM; species of 80, 136, 190, and 210 kD have all been reported, with the predominant 80 and 136 kD forms derived from the larger forms by proteolytic cleavage (Burgoon et al., 1991). Similar crosslinker-based affinity purifications done using AP-mAPP (199-345) as an additional probe revealed that NgCAM does in fact co-precipitate with APP amino acids 199-345, the domain shown above to bind RGC axons in the tectum (Figure 13B). The co-IP shown in Figure 13B differed from the protocol used above in that BS3, a non-cleavable crosslinker, was used instead of DTSSP, a crosslinker that is cleaved under reducing conditions. This change was made to examine the molecular weight of complexes containing NgCAM and APP. The observed spread of signal over a wide range of molecular weights is difficult to interpret clearly, but may indicate the presence of additional molecules of varying weights in the crosslinked NgCAM-APP complexes.
The specificity of APP (amino acids 199-345) for binding NgCAM could be partially addressed by the observation that an anti-chicken NCAM antibody did not specifically detect bands in AP-mAPP versus AP immunoprecipitates; antibodies against more closely related chicken proteins were not readily available. The reverse side of the specificity question was addressed by examining the binding of APLPl or APLP2 to embryonic chick brain. An AP fusion to nearly the entire APLPl ectodomain exhibited binding to olfactory bulbs of E 12- 13 chick embryos, but did not appear to bind tectal RGC axons; the binding pattern was essentially the same in an AP fusion containing only the N-terminal portion of APLPl homologous to APP amino acids 18-205 (Figure 14). Furthermore, an AP fusion of the middle domain of APLP2 (roughly homologous to APP (199-345)) did not bind tectal RGC axons, but did reveal a speckled pattern of binding throughout the brain that was reminiscent of blood vessels (Figure 15A). Although we have not determined precisely what these structures are, we did find that the middle domain of APLP2 binds cells of the RF/6A endothelial cell line, while AP-mAPP did not (Figure 15B). These observations do not directly address whether APLPl or APLP2 can interact physically with NgCAM, but they do suggest that the middle domains of APP5 APLPl and APLP2 are likely to have distinct binding specificities.
Effects of contactin 4 and NgCAM on APP stability or processing: Proteolytic cleavage as an early step in APP signal transduction has been widely proposed and studied, although its necessity remains controversial (Hass and Yankner, 2005). We were therefore interested in examining the effect of contactin 4 and NgCAM on APP cleavage. As a first step, we co-transfected 293T cells with APP-HA (full-length APP695 with a C-terminal HA tag) and either contactin 4-Fc or contactin 4- AP. Co-expression of these contactin 4 fusion proteins resulted in the increase of a small HA-tagged fragment of approximately 12 kD (Figure 16A). In several experiments, the amount of full-length APP- HA detected also increased in parallel, although this effect was not seen in all experiments. To identify the 12 kD band, we generated constructs designed to express C-terminal fragments of APP-HA beginning immediately after each of the major cleavage sites, preceded only by an added start codon or a signal peptide with a five amino acid linker. The constructs beginning after gamma or epsilon cleavage were either poorly expressed or degraded, but comparison with the fragments beginning after alpha or beta cleavage clearly show the 12 kD fragment to match the alpha fragment, CTFalpha, in molecular weight.
A majority of similar experiments in which APP-HA was co-transfected with soluble, tagged forms of contactin 4 or NgCAM revealed an increase in CTFalpha levels. An example is shown in Figure 17A, where increased CTF alpha levels can be detected when contactin 4-AP5 NgCAM-Fc5 or both are co-expressed with APP-HA, while control construct NrCAM-Fc appears to even decrease CTFalpha levels. In a smaller, but significant, number of experiments, co-expression of APP-HA with contactin 4 or NgCAM fusion proteins results in a distinct decrease in CTFalpha levels; interestingly, co-expression with both contactin 4 and NgCAM in these experiments generally rescues this decrease, at least partially (Figure 17B). Some possible explanations as to what causes these opposing experimental outcomes will be discussed in the following section.
In the above experiments, the contactin 4 and NgCAM fusion constructs used lack any membrane attachment or cytoplasmic domains, which suggested to us that these proteins may be able to affect APP or CTFalpha levels when presented in trans to APP. We have so far been unable to develop a reliable protocol in which contactin 4 or NgCAM protein presented in trans, rather than by co-transfection, consistently results in changed levels of full-length APP or CTFalpha. However, in Figure 17C5 we show an example in which APP- HA CTFalpha levels were increased in cells exposed to supernatants containing clustered NgCAM-Fc in conjunction with contactin 4 presented either as a clustered supernatant or through transfection.
Example 3: APP. NgCAM. and contactin 4 in the visual system
Based on the evidence that APP interacts physically with both contactin 4 and
NgCAM as disclosed above, the invention further characterizes biological relevance of the protein-protein interactions. Some regions in the nervous system of the developing chicken embryo for expression of these genes were examined. Based on both expression patterns and previously reported functional roles of Ll , the mammalian NgCAM ortholog, possible functional interactions of APP5 contactin 4, and NgCAM in the.retinotectal system were then investigated.
Materials and methods Plasmids: All plasmids for in situ probes were constructed with a pBluescript II SK(-) vector backbone (Stratagene). The following fragments were inserted: APP nucleotides 471 to 1218, using endogenous Pstl sites; NgCAM nucleotides 2827 to 3754, using endogenous Sacl and BamHI sites; contactin 3 nucleotides 2465 to 3277, using endogenous Pstl sites; contactin 4 nucleotides 21 to 818 of chESTl 18118 (from the BBSRC ChickEST database at www .chick.umist.ac.uk), using Pstl sites added by PCR. The NgCAM fragment was cut from pSCT-NgCAM, a kind gift from Sonderegger (Buchstaller et al., 1996), while other fragments were generated by RT-PCR from chick tectal cDNA. Except for contactin 4, the above nucleotide numbering is given according to the coding region; see accession number Z75013 for NgCAM sequence information, accession number XM_414433 for contactin 3, and a combination of ChEST590c2 (from the BBSRC ChickEST database) and accession number AF042098 to construct the chick APP695 sequence. For chick contactin 4, the PCR primers used were tctagaatggtgctgctgtgtggacc [SEQ ID NO: 8] and ctgcagtatgacactcagttctgcac [SEQ ID NO: 9], based on the sequence in chESTl 18118. The resulting PCR product was checked, and the predicted translation is 84% identical to amino acids 140 to 402 of human contactin 4 (accession number NM_175607). This translated sequence is only 69% identical to the corresponding region of human contactin 3, by comparison.
Plasmids used to produce soluble Fc proteins (including pSecTaglg for Fc alone) are described in above. Plasmids used to produce AP fusion proteins (including APtag4 for AP alone) are described above.
In situ hybridization:
To generate the probes, the plasmids were linearized with a restriction enzyme, and transcription, was carried out with T3 or T7 polymerase (Promega) according to manufacturer's protocol, except using ImM each ATP, CTP, and GTP, with 0.65mM UTP and 0.35 mM digoxigenin-UTP (Roche). Restriction enzymes and polymerase used were as follows: APP antisense EcoRI/T3, sense BamHI/T7; NgCAM antisense Sad (followed by blunt ending)/T7, sense BamHI/T3; contactin 3 antisense Spel/T7, EcoRI/T3; contactin 4 antisense Xbal/T7, sense EcoRI/T3. Embryonic day 11 chick embryo heads were prepared for in situs by fixation in 4% paraformaldehyde in Phosphate Buffered Saline, then were embedded and frozen in OCT before being cut into 10 micron thick sections. Section in situs were carried out according to standard protocols. Detection of probe was with alkaline phosphatase conjugated anti-digoxigenin antibody (Roche), visualized using 5-bromo-4- chloro-3-indoxyl phosphate and nitrotetrazolium blue (Biosynth International) as substrates.
Outgrowth assay:
Retinal outgrowth assays were performed by dissecting embryonic day 6 chick retinas, mounting them on a polycarbonate filter (Sartorius) with the RGC layer up, cutting into strips 300 microns wide, then laying these strips RGC side down on coated coverslips, and culturing for two days. The media used for culturing retinal explants was based on a combination of media types used in slightly different assays (Yamagata and Sanes, 1995), (Walter et al., 1987) and consisted of 47.6% Neurobasal media, 37.5% DMEM/F12, 4.8% fetal bovine serum, 2.4% chick serum, 1% B27, 0.15% methyl cellulose, 0.14% glucose, 20 mM HEPES pH7.0, 10 mM glutamine, 71 U/ml Penicillin, 71 microg/ml Streptomycin, and 1.76 microg/ml glutamate. Brain extract, made by homogenizing E6 chick brains in HBSS and centrifuging to remove debris, was added to the media fresh before use, allowing extract from one brain for every 5 mis of media. Anti-APP antibody was goat anti-APP amino acids 44-63 (Calbiochem) and was used at a 1 : 100 dilution. DAPT (Calbiochem) was used at 0.5 micromolar.
Axons were stained by incubating in 33 micromolar carboxy fluorescein diacetate, succinimidyl ester (Invitrogen) for 10 minutes, and visualized under FITC illumination. Outgrowth varied somewhat between experiments, so figures here represent only one or two individual experiments that exemplify the trends seen in other experiments. Outgrowth was measured by counting individual axons. The p-values given are according to the Student's t- test.
Purified Fc fusion proteins were produced by calcium phosphate or Lipofectamine 2000 (Invitrogen) mediated transfection of 293T cells with the appropriate plasmid. The cells were maintained in Opti-MEM plus ITS-A, an insulin, transferring, and selenium supplement (Invitrogen), and the supernatants were collected several days later. After filtration, the supernatants were incubated with Protein A Sepharose beads (4 Fast Flow, Amersham Biosciences), at approximately a one-thousandth volume. The beads were washed several times with cold 10OmM Tris pH8.0 then 10 mM Tris pH 8.0. Bound proteins were eluted in 150 mM NaCl plus 10OmM, pH 3.0, then dialyzed against HBSS (VWR or Invitrogen) using a 10,000 molecular weight cut-off dialysis cassette (Pierce).
AP fusion proteins were produced by calcium phosphate mediated transfection of 293T cells with APtag4, AP-mAPP, or AP-mAPP( 18-205). The cells were then maintained in Opti-MEM plus ITS-A for several days. Supernatants were not subjected to purification, but were filtered, then concentrated 10-fold using Amicon Centricon devices with a 10,000 molecular weight cut-off (Millipore).
For the glass coverslips, all coating steps were carried out either overnight at 4 degrees C or for at least 2 hours at room temperature. Outgrowth was enhanced by including a preliminary step of coating with 20 micrograms/ml poly-L- Lysine. In cases where multiple purified proteins (Fc fusions or laminin) were used, these proteins were mixed to coat the coverslips simultaneously. For coverslips coated with purified proteins plus a supernatant (AP fusion or mock), the coverslips were coated first with the purified proteins, then with the supernatant. All coating steps were followed by two washes in Hank's Balanced Salt Solution (HBSS). Results
Expression of APP, NgCAM, and contactin 3 and 4:
As discussed above, we found that APP interacts physically with contactin 3, contactin 4, and NgCAM. As a first step to investigating possible biological consequences of this interaction, Rikke Egelund carried out RNA in situs to determine the expression pattern of these genes in El 1 chick embryos.
Since we had previously observed AP-mAPP to bind to olfactory bulbs, we were interested to see that APP, NgCAM and contactin 4 are all expressed in the olfactory bulb. Both APP and contactin 4 appear to be expressed superficially in the olfactory bulb, while NgCAM appears to be expressed in a slightly deeper layer (Figure 18A). In addition, all three genes are expressed in the olfactory epithelium (OE). Olfactory receptor neurons (ORNs) located in the OE project axons directly to the olfactory bulb, so we were also interested to see expression of all three genes in the OE (Figure 18B). In contrast, contactin 3 expression was not noted in either the OE or the olfactory bulb. These results suggest that interactions between APP and either NgCAM or contactin 4 might be biologically relevant in the olfactory system.
Expression patterns also suggest that interactions among APP, NgCAM, contactin 3 and contactin 4 may be biologically relevant in the visual system. All four genes are expressed in multiple layers in the tectum (Figure 19A). Although we have not determined precisely which layers show expression, it does appear that the outermost staining for all four genes is found in either the SO or the SFGS. All four genes are also expressed in the RGC layer of the retina, while contactin 4 and APP are also significantly expressed elsewhere in the retina (Figure 19B).
Effects of APP, NgCAM, and contactin 4 on RGC axon outgrowth: It was of interested to examine the role of APP, NgCAM, and contactin 4 in the development of the retinotectal projection, as all three genes appear to be expressed in relevant tissues at the RNA levels, and both NgCAM and contactin 4 protein could be purified from chick tecta, as already discussed above. In addition, the phenotype of the Ll knock-out mouse demonstrates that Ll, the mammalian homolog of NgCAM, is required for the normal development of the equivalent mammalian projection, the retinocollicular projection (Demyanenko and Maness, 2003). The protein expression of NgCAM in the tectum has previously been examined, and it appears quite specific for the SO (Yamagata and Sanes, 1995), a layer which is composed mostly of RGC axons, so we decided to examine the effect of our proteins of interest on RGC axons cultured in vitro.
Retinal explants cultured on NgCAM-Fc coated coverslips were found to exhibit . substantial RGC axon outgrowth, in line with earlier reports of neurite growth on Ll-Fc (Doherty et al., 1995), but coverslips coated with either AP-mAPP or contactin 4-Fc did not promote RGC axon outgrowth at all. Therefore, we initially investigated whether APP might be acting as a receptor for NgCAM by attempting to block APP signaling. Explants grown on NgCAM-Fc in the presence of the gamma secretase inhibitor DAPT exhibited more RGC outgrowth than was seen in the DMSO control (p<0.05; Figure 20A). Since gamma secretase activity is required for Notch signaling and affects the processing of many additional proteins, we also tried using an anti-APP antibody as a potentially more specific inhibitor. As can be seen in Figure 2OB, anti-APP also promoted RGC outgrowth on NgCAM-Fc substrates (p<0.05). IfNgCAM stimulated outgrowth by signaling through APP, DAPT or antibody treatment might have been expected to decrease outgrowth on NgCAM-Fc. The increase in outgrowth seen under these conditions suggests that APP signaling may in fact act to reduce or inhibit outgrowth.
One potential model might be that NgCAM has an inhibitory effect on RGC outgrowth through its interaction with APP, while promoting outgrowth through some other receptor. If the DAPT and antibody used above blocked APP signaling in response to NgCAM, one might expect that DAPT and anti-APP would fail to promote outgrowth on another substrate. However, RGC axon outgrowth from explants grown on laminin was also promoted by DAPT (pO.OOOl; Figure 20C). Preliminary experiments testing the specificity of anti-APP promoted outgrowth for NgCAM substrates are not conclusive, though it appears that anti-APP may also promote outgrowth on laminin. In addition, it was tested whether NgCAM may signal through contactin 4, this time using a dominant negative approach. Contactin 4-Fc added to NgCAM-Fc strongly inhibited outgrowth, as judged by axon length, although the number of RGC axons was not always clearly affected. Outgrowth inhibition by contactin 4-Fc appears to be specific for NgCAM, as contactin 4 did not inhibit outgrowth on laminin (Figure 21 A). However, inhibition of outgrowth on NgCAM is not specific to contactin 4, as Fc fusions of contactins 1, 2, 3, and 6 also inhibited outgrowth on NgCAM3 although Fc alone did not (Figure 21B).
Although no RGC axon outgrowth was observed on AP-mAPP alone, it was found that AP-mAPP enhanced outgrowth on NgCAM-Fc (p<0.002). AP-m APP(18-205) also enhanced outgrowth on NgCAM-Fc (p<0.02), though to an apparently lesser degree. Since AP-mAPP( 18-205) binds contactin 4, this suggests a possible involvement of contactin 4 in this effect. Addition of contactin 4-Fc appeared to reduce outgrowth on NgCAM-Fc supplemented with AP-mAPP or AP-mAPP( 18-205), although these differences were not significantly significant (Figure 22). In this particular experiment, the effect of contactin 4- Fc on NgCAM- induced outgrowth seems to be negligible. However, as discussed previously (Figure 21), experimental conditions with greater outgrowth show that contactin 4-Fc has an inhibitory effect on NgCAM-induced outgrowth, so we cannot conclude from this experiment alone whether contactin 4 may be playing a role specifically in APP-enhanced outgrowth. Potential experiments to address this question will be discussed below.
Discussion
One reason we were interested in the expression of NgCAM and contactin 4 was to determine whether their expression patterns correlated well with binding of APP to embryonic chick brains discussed above. AP-mAPP( 18-205), was found to bind particularly strongly to olfactory bulbs, although binding above background was detected throughout the brain. This is consistent with the ability of AP-m APP(18-205) to bind contactin 4 (see Example 3), and the expression of contactin 4 in the olfactory bulb and the OE (since ORN axons project from the OE to the olfactory bulb). One potential puzzle is why substantial olfactory bulb binding is not seen either with AP-mAPP(l 99-345) or with AP-mAPP after PI- PLC treatment, given the apparently high levels of expression of NgCAM expression in the olfactory bulb. Since we have not detected direct binding between APP and NgCAM in vitro, it is possible that, for example, a specific post-translational modification of NgCAM or an unidentified co-factor required for an APP-NgCAM interaction is not found in the olfactory bulb. However, the AP staining protocol is only very effective at detecting surface binding, and NgCAM expression in the olfactory bulb appears not to be superficial, and therefore may not be detectable by this method. The expression of NgCAM RNA in RGCs shown here and published observations of NgCAM protein in the tectal SO layer (Yamagata et al., 1995) both correlate well with the AP-APP(199-345) tectal binding described in Example 1. Another reason we were interested in the expression patterns of APP, NgCAM, and contactin 4 was to identify biological contexts in which interactions among these proteins may exist. These genes are expressed, at least at the level of RNA, in the olfactory and retinotectal systems. Expression of these genes occurs in both the projecting (OE or retina) and target (olfactory bulb or tectum) fields, and the expression patterns are partially overlapping, but partially distinct, making it difficult to determine from expression alone in which cells APP interactions with NgCAM or contactin 4 are likely to be biologically relevant, or even whether any interactions are likely to occur in cis, in trans (either between distinct populations of cells or between cells of the same type), or both. We chose to begin looking for biologically relevant interactions among these proteins by examining their effects on RGC axons in vitro. Since RGC axons can be grown on NgCAM, we first examined whether NgCAM may signal to RGCs through either APP or contactin 4. Increased outgrowth on NgCAM under conditions expected to decrease APP signaling (addition of an anti-APP antibody or gamma-secretase inhibitor) argues against NgCAM stimulating outgrowth through APP, and actually argues that signaling of APP has an inhibitory affect on RGC axon growth. An important caveat is that it is uncertain whether the above treatments do in fact antagonize APP signaling. The observation that the gamma- secretase inhibitor also stimulates outgrowth on laminin in the absence of exogenously provided NgCAM could suggest that NgCAM is not responsible for an APP-mediated inhibition of outgrowth. However, since NgCAM is expressed in the retina, as is contactin 4, it remains possible that endogenous NgCAM or contactin 4 might inhibit outgrowth through an interaction with APP.
The inhibition of NgCAM-dependent outgrowth by contactin 4-Fc has more than one potential explanation. Contactin 4 may bind directly to NgCAM, acting as a blocking reagent to prevent outgrowth mediated by axonal proteins that recognize the same, or an overlapping, region of NgCAM. Alternatively, the combined actions of NgCAM and contactin 4 may generate an inhibitory signal; as contactin 4 does not interfere with outgrowth on laminin, an inhibitory signal cannot be attributed to contactin 4 acting alone. Since we saw a similar inhibitory effect on NgCAM induced outgrowth with all of the other contactin family members we tested, since contactins 1 and 2 have already been shown to bind NgCAM
(Sonderegger, 1997), and since contactin 2 is thought to be required in at least some cases for NgCAM induced neurite outgrowth (Buchstaller et al., 1996), we favor the first interpretation, although the latter cannot be ruled out.
Finally, we also found that APPsalpha (in the form of AP-mAPP), or just its N- terminal domain (in the form of AP-mAPP (18-205)), stimulates NgCAM dependent outgrowth. The N-terminal El domain has already been shown to promote neurite outgrowth in other systems (Ohsawa et al., 1997; Small et al., 1994), so this was not entirely surprising. However, since AP-mAPP (18-205) binds to contactin 4, one possibility is that APP El signaling that promotes axon outgrowth may be mediated by axonal or cell-surface contactin 4. Contactin 4-Fc seems to inhibit APPsalpha stimulated outgrowth, but since contactin 4 also blocks outgrowth on NgCAM, we cannot draw any firm conclusions. One future experiment could be to use deletion constructs of contactin 4-Fc as blocking reagents, with the first four Ig domains as a potential NgCAM- specific blocking reagent (by analogy to NgCAM's binding to the first four domains of contactin 2 (Rader et al., 1996), and the fibronectin domains as a potential APPsalpha-blocking reagent. Another option, in principle, could be to study the role of contactin 4 in APPsalpha-stimulated growth in the absence of NgCAM, but we have so far failed to see APPsalpha enhance growth on laminin. More definitive experiments could involve RNAi-mediated knock-down of contactin 3 and/or contactin 4 or, if similar results were seen with mouse RGCs, mice deficient in these genes.
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Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. All references disclosed herein are incorporated by reference in their entirety.
What is claimed is:

Claims

Claims
1. A method for modulating APP processing comprising: contacting a mammalian cell with an agent that modulates the amount or rate of binding of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2) to contactins and/or Ll family proteins.
2. The method according to claim 1, wherein said agent is expressed by the cell.
3. The method according to claim 1, wherein said agent is exogenously added so as to contact the cell.
4. The method according to claim 1, wherein said agent comprises one or more contactins or Ll family proteins or fragments thereof that bind to APP5 APLPl or APLP2.
5. The method according to claim 4, wherein said contactin is contactin-3 contactin-4, contactin-5 or a combination thereof.
6. The method according to claim 4, wherein said Ll family protein is Ll or Ng-CAM.
7. The method according to claim 4, wherein said fragment is a fragment of contactin-3, contactin-4, Ll or Ng-CAM that binds to APP, APLPl or APLP2, or a combination of such fragments.
8. The method according to any of claims 4-7, wherein said agent comprises a fusion protein comprising the one or more contactins, Ll family proteins or fragments.
9. The method according to claim 8, wherein said fusion protein is an Fc fusion protein.
10. The method according to claim 1, wherein said agent is an antibody or an antigen- binding fragment thereof.
1 1. The method according to claim 10, wherein the antibody or antigen-binding fragment thereof binds a contactin or a Ll family protein.
12. The method according to claim 11, wherein the contactin or Ll family protein is contactin-3 contactin-4, contactin-5, Ll or Ng-CAM.
13. The method according to claim 10, wherein the antibody or antigen-binding fragment thereof binds APP, APLPl or APLP2.
14. The method according to claim 10, wherein the antibody or antigen-binding fragment thereof binds a complex of a contactin or a Ll family protein and APP, APLPl or APLP2.
15. The method according to any of claims 10-14, wherein the antibody or antigen- binding fragment thereof is a bispecific antibody.
16. The method according to any one of claims 1-15, wherein said agent is an agent that blocks or disrupts the binding of APP, APLPl or APLP2 to contactins and/or Ll family proteins.
17. The method according to any one of claims 1-15, wherein said agent is an agent that promotes or stabilizes the binding of APP, APLPl or APLP2 to contactins and/or Ll family proteins.
1 S. The method according to any one of claims 1-15, wherein said agent is an agent that reduces processing of APP to β amyloid.
19. A method for treating or preventing Alzheimer's disease, comprising: administering to a subject an effective amount of an agent that modulates the amount or rate of binding of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2) to contactins and/or Ll family proteins.
20. The method according to claim 19, wherein said agent comprises one or more contactins or Ll family proteins or fragments thereof that bind to APP, APLPl or APLP2.
21. The method according to claim 20, wherein said contactin is contactin-3 contactin-4., contactin-5 or a combination thereof.
22. The method according to claim 20, wherein said Ll family protein is Ll or Ng-CAM.
23. The method according to claim 20, wherein said fragment is a fragment of contactin- 3, contactin-4, Ll or Ng-CAM that binds to APP, APLPl or APLP2, or a combination of such fragments.
24. The method according to any of claims 20-23, wherein said agent comprises a fusion protein comprising the one or more contactins, Ll family proteins or fragments.
25. The method according to claim 24, wherein said fusion protein is an Fc fusion protein.
26. The method according to claim 19, wherein said agent is an antibody or an antigen- binding fragment thereof.
27. The method according to claim 26, wherein the antibody or antigen-binding fragment thereof binds a contactin or a Ll family protein.
28. The method according to claim 27, wherein the contactin or Ll family protein is contactin-3 contactin-4, contactin-5, Ll or Ng-CAM.
29. The method according to claim 26, wherein the antibody or antigen-binding fragment thereof binds APP, APLPl or APLP2.
30. The method according to claim 26, wherein the antibody or antigen-binding fragment thereof binds a complex of a contactin or a Ll family protein and APP, APLPl or APLP2.
31. The method according to any of claims 26-30, wherein the antibody or antigen- binding fragment thereof is a bispecific antibody.
32. The method according to any one of claims 19-31, wherein said agent is an agent that blocks or disrupts the binding of APP, APLPl or APLP2 to contactins and/or Ll family proteins.
33. The method according to any one of claims 19-315 wherein said agent is an agent that promotes or stabilizes the binding of APP, APLPl or APLP2 to contactins and/or Ll family proteins.
34. The method according to any one of claims 19-31, wherein said agent is an agent that reduces processing of APP to β amyloid.
35. The method according to any one of claims 19-31 , wherein said agent is a vectorized agent that crosses the blood brain barrier.
36. The method according to claim 19, wherein the subject is a human.
37. A pharmaceutical composition comprising: one or more contactins and/or Ll family proteins and/or one fragments thereof that bind to APP, APLPl or APLP2, and a pharmaceutically acceptable carrier.
38. The pharmaceutical composition according to claim 37, wherein said contactin is contactin-3 contactin-4, contactin-5 or a combination thereof.
39. The pharmaceutical composition according to claim 37, wherein said Ll family protein is Ll or Ng-CAM.
40. The pharmaceutical composition according to claim 37, wherein said fragment is a fragment of contactin-3, contactin-4, Ll or Ng-CAM that binds to APP, APLPl or APLP2, or a combination of such fragments.
41. The pharmaceutical composition according to any of claims 37-40, wherein said agent comprises a fusion protein comprising the one or more contactins, Ll family proteins or fragments.
42. The pharmaceutical composition according to claim 41, wherein said fusion protein is an Fc fusion protein.
43. A pharmaceutical composition comprising: one or more antibodies that bind a contactin, a Ll family protein, APP, APLPl or APLP2 or antigen-binding fragments thereof.
44. The pharmaceutical composition according to claim 43, wherein the contactin or Ll family protein is contactin-3 contactin-4, contactin-5, Ll or Ng-CAM.
45. The pharmaceutical composition according to claim 43, wherein the antibody or antigen-binding fragment thereof binds a complex of a contactin or a Ll family protein and APP, APLPl or APLP2.
46. The pharmaceutical composition according to any of claims 37-45, wherein the antibody or antigen-binding fragment thereof is a bispecific antibody.
47. The pharmaceutical composition according to any one of claims 37-46, wherein said agent is an agent that blocks or disrupts the binding of APP, APLPl or APLP2 to contactins and/or Ll family proteins.
48. The pharmaceutical composition according to any one of claims 37-46, wherein said agent is an agent that promotes or stabilizes the binding of APP, APLPl or APLP2 to contactins and/or Ll family proteins.
49. The pharmaceutical composition according to any one of claims 37-46, wherein said agent is an agent that reduces processing of APP to β amyloid.
50. The pharmaceutical composition according to any one of claims 37-46, wherein said agent is a vectorized agent that crosses the blood brain barrier of a subject.
51. The pharmaceutical composition according to claim 50, wherein said subject is a human.
52. A method for identifying compounds that modulate the binding of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2) with contactins and/or Ll-CAMs, the method comprising: providing a reaction mixture that comprises (1) APP, APLPl, APLP2 and/or a fragment thereof that binds to contactins and/or Ll family proteins, and (2) contactins, Ll family proteins and/or a fragment thereof that binds to APP, APLPl, APLP2, contacting the reaction mixture with a test compound, determining a level of binding of APP, APLPl, APLP2 and/or a fragment thereof with contactins, L 1 family proteins and/or a fragment thereof in the absence and in the presence of the test compound, and comparing the level of binding of APP, APLPl5 APLP2 or fragment thereof with contactins, Ll family proteins and/or a fragment thereof in the absence and in the presence of the test compound, wherein a test compound that modulates the binding relative to the level of binding in the absence of the test compound is a compound that modulates the binding of APP5 APLPl, APLP2 or fragment thereof with contactins, Ll family proteins and/or a fragment thereof.
53. The method of claim 52, wherein the test compound is a small molecule.
54. The method of claim 52, wherein the test compound is an antibody that binds to APP, APLPl, APLP2 a contactin or a Ll family protein, or an antigen-binding fragment thereof.
55. The method of claim 52, wherein the test compound is a fragment of APP, APLPl, APLP2, a contactin or a Ll family protein.
56. The method according to any one of claims 52-55, wherein the contactins and/or Ll family proteins is selected from the group consisting of: contactin-3, contactin-4, contactin-5, Ll and Ng-CAM.
57. A method for identifying compounds that modulate the processing of amyloid precursor protein (APP), amyloid precursor like protein 1 (APLPl) or amyloid precursor like protein 2 (APLP2), the method comprising: providing a reaction mixture that comprises (1) contactins, Ll family proteins and/or a fragment thereof that binds to APP, APLPl, APLP2, and (2) APP5 APLPl, APLP2 and/or a fragment thereof that binds to contactins and/or Ll family proteins, contacting the reaction mixture with a test compound, determining a level of processing of APP, APLPl or APLP2 in the absence and in the presence of the test compound, and comparing the processing in the absence and in the presence of the test compound, wherein a test compound that modulates processing of APP, APLPl or APLP2 relative to the level of processing in the absence of the test compound is a compound that modulates the processing of APP, APLPl or APLP2.
58. The method of claim 57, wherein the test compound is a small molecule.
59. The method of claim 57, wherein the test compound is an antibody that binds to APP, APLP 1 , APLP2, a contactin or a Ll family protein, or an antigen-binding fragment thereof.
60. The method of claim 57, wherein the test compound is a fragment of APP, APLPl or APLP2, a contactin or a Ll family protein.
61. The method according to any one of claims 57-60, wherein the contactins and/or Ll family proteins is selected from the group consisting of: contactin-3, contactin-4, contactin-5, Ll and Ng-CAM.
62. The method according to any one of claims 57-61, wherein the processing of APP to Aβ is reduced.
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