WO2009011778A1 - βMETHOD FOR IDENTIFYING MODULATORS OF LRRTM1, LRRTM2 AND LRRTM4 USEFUL FOR TREATING ALZHEIMER'S DISEASE - Google Patents

Abstract

Methods for identifying modulators of LRRTM1, LRRTM2 and LRRTM4 are described. The methods are particularly useful for identifying analytes that stimulate LRRTM1or LRRTM2 activity or inhibit LRRTM4 activity such that they antagonize processing of amyloid precursor protein (APP) to Aβ peptide (Aβ). Such methods can be used to identify analyties for treating Alzheimer disease.

Description

TITLE OF THE INVENTION

METHOD FOR IDENTIFYING MODULATORS OF LRRTMl, LRRTM2 AND LRRTM4

USEFUL FOR TREATING ALZHEIMER'S DISEASE

BACKGROUND OF THE INVENTION

( 1 ) Field of the Invention

The present invention relates to methods for identifying modulators of LRRTMl , LRRTM2 and LRRTM4. The methods are particularly useful for identifying analytes that antagonize LRRTMl's, LRRTM2's and LRRTM4's effect on processing of amyloid precursor protein (APP) to Aβ peptide (Aβ) and are thus useful for identifying analytes that can be used for treating Alzheimer disease.

(2) Description of Related Art Alzheimer's disease is a common chronic neurodegenerative disease, characterized by a progressive loss of memory and behavioral abnormalities, as well as an impairment of other cognitive functions that often leads to dementia and death. It ranks as the fourth leading cause of death in industrialized societies after heart disease, cancer, and stroke. The incidence of Alzheimer's disease is high, with over 4 million patients affected in the United States and perhaps 17 to 25 million worldwide. Moreover, the number of sufferers is expected to grow as the population ages.

Alzheimer's disease (AD) is characterized by the presence of large numbers of insoluble deposits, known as amyloid plaques, in the brains of those affected. Amyloid plaques are composed predominantly of amyloid beta (referred to herein interchangeably as "Aβ peptide" or "Aβ") (Glenner and Wong, Biochem. Biophvs. Res. Comm. 120: 885- 890) that is thought to cause or contribute to the disease process (Yankner, Science 250: 279-282 (1990); Mattson et al., J. Neurosci. 12: 379-389 (1992); Games et al., Nature 373: 523-527 (1995); LaFerla et al., Nature Genetics 9: 21-29 (1995)). This is based on findings that mutations in APP, leading to elevated Aβ production, are associated with heritable forms of AD (see, for example Goate et al., Nature 349:704-706 (1991); Chartier-Harlan et al., Nature 353:844-846 (1991); Murrel et al., Science 254: 97-99 (1991); Mullan et al., Nature Genetics 1 : 345-347 (1992)) and that presenilin-1 (PSl) and presenilin-2 (PS2) related familial early-onset AD (FAD) shows disproportionately increased production of Aβ 1-42, the 42 amino acid isoform of Aβ, as opposed to Aβl-40, the 40 amino acid isoform (Scheuner et al., Nature Medicine 2: 864-870 (1996)), the longer isoform being more prone to aggregation associated with the formation of Aβ (Jarrett et al.. Biochemistry 32:4693-4697 (1993); Kuo et al., J. Biol. Chem. 271 : 4077-4081 (1996)). See, Selkoe, J. Neuropathol. Exp. Neurol. 53: 438-447 (1994) for a review of the central role of amyloid plaques in AD. APP is actually a family of polypeptides produced by alternative splicing from a single gene. Predominant forms of APP are known as APP695, APP751 , and APP770, with the subscripts referring to the number of amino acids in each splice variant (Ponte et al., Nature 331 : 525-527 (1988); Tanzi et al., Nature 331 : 528-530 (1988); Kitaguchi et al., Nature 331 : 530- 532(1988)). APP is a ubiquitous membrane-spanning (type 1) glycoprotein that undergoes proteolytic cleavage by at least two pathways (Selkoe, Trends Cell Biol. 8: 447-453 (1998)). In one pathway, cleavage by an enzyme known as α-secretase occurs (Kuentzel et al., Biochem. J. 295:367-378 (1993)). This cleavage by α-secretase occurs within the Aβ portion of APP, thus precluding the formation of Aβ. In an alternative proteolytic pathway, cleavage of the Met596- Asp597 bond (numbered according to the 695 amino acid protein) by β-secretase occurs.

Cleavage by β-secretase generates the N- terminus of the Aβ peptide. The C-terminus is formed by cleavage by a second enzyme activity known as γ-secretase and is a heterogeneous collection of cleavage sites in that γ-secretase activity occurs over a short stretch of APP amino acids rather than at a single site results in Aβ peptides ranging from 39 to 43 amino acids. While Aβl-40 and Aβl-42 are the predominate forms of Aβ generated by γ-secretase, Aβl-42 is more prone to aggregation than Aβl-40 and its production is closely associated with the development of Alzheimer's disease (Sinha and Lieberburg, Proc. Natl. Acad. Sci. USA 96: 1 1049-11053 (1999)). The bond cleaved by γ-secretase is situated within the transmembrane domain of APP. For a review of APP and its processing, see Selkoe, Trends Cell. Biol. 8: 447-453 (1998). While abundant evidence suggests that extracellular accumulation and deposition of Aβ peptide is a central event in the etiology of Alzheimer's disease, recent studies have also proposed that increased intracellular accumulation of Aβ peptide or Aβ containing C-terminal fragments of APP may play a role in the pathophysiology of Alzheimer's disease. For example, over-expression of APP harboring mutations which cause familial Alzheimer's disease results in the increased intracellular accumulation of C99, the carboxy-terminal 99 amino acids of APP containing Aβ peptide in neuronal cultures. Moreover, evidence suggests that intra- and extracellular Aβ peptide are formed in distinct cellular pools in hippocampal neurons and that a common feature associated with two types of familial Alzheimer's disease mutations in APP ("Swedish" and "London") is an increased intracellular accumulation of Aβ42 peptide. Thus, based on these studies and earlier reports implicating extracellular Aβ peptide accumulation in Alzheimer's disease pathology, it appears that altered APP catabolism may be involved in disease progression.

Much interest has focused on the possibility of inhibiting the development of amyloid plaques as a means of preventing or ameliorating the symptoms of Alzheimer's disease. To that end, a promising strategy is to inhibit the activity of β- and γ- secretase, the two enzymes that together are responsible for producing Aβ. This strategy is attractive because, if the formation of amyloid plaques is a result of the deposition of Aβ, inhibiting the activity of one or both of the two secretases would intervene in the disease process at an early stage, before late- stage events such as inflammation or neuronal loss occur. Such early stage intervention is expected to be particularly beneficial (see, for example, Citron, Molecular Medicine Today 6:392-397 (2000)).

To that end, various assays have been developed that are directed to the identification of substances that may interfere with the production of Aβ or its deposition into amyloid plaques. U.S. Patent No. 5,441,870 is directed to methods of monitoring the processing of APP by detecting the production of amino terminal fragments of APP. U.S. Patent No. 5,605,811 is directed to methods of identifying inhibitors of the production of amino terminal fragments of APP. U.S. Patent No. 5,593,846 is directed to methods of detecting soluble Aβ by the use of binding substances such as antibodies. U.S. Patent No. 7,196,163 describes using

APP with modified β-secretase cleavage sites to monitor β-secretase activity. Esler et al., Nature Biotechnology 15: 258-263 (1997) describe an assay for monitoring the deposition of Aβ from solution onto a synthetic analogue of an amyloid plaque. The assay is suitable for identifying substances that could inhibit the deposition of Aβ, but is not suitable for identifying substances, such as inhibitors of β- or γ-secretase, that would prevent the formation of Aβ peptide.

Various groups have cloned and sequenced cDNAs encoding a protein believed to be β-secretase (Vassar et al., Science 286: 735-741 (1999); Hussain et al., MoI. Cell. Neurosci. 14: 419- 427 (1999); Yan et al., Nature 402: 533-537 (1999); Sinha et al., Nature 402: 537-540 (1999); Lin et al., Proc. Natl. Acad. Sci. USA 97: 1456-1460 (2000)). U.S. Patent Nos. 6,828,1 17 and 6,737,510 disclose a β-secretase, which the inventors call aspartyl protease 2 (Asp2), variant Asp-2(a) and variant Asp-2(b), respectively, and U. S Pat. No. 6,545,127 discloses a catalytically active enzyme known as memapsin. Hong et al., Science 290: 150-153 (2000) determined the crystal structure of the protease domain of human β-secretase complexed with an eight-residue peptide-like inhibitor at 1.9 angstrom resolution. Compared to other human aspartic proteases, the active site of human β-secretase is more open and less hydrophobic, contributing to the broad substrate specificity of human β-secretase (Lin et al., Proc. Natl. Acad. Sci. USA 97: 1456-1460 (2000)).

Ghosh et al., J. Am. Chem. Soc. 122: 3522-3523 (2000) disclosed two inhibitors of β-secretase, OM99-1 and OM99-2, that are modified peptides based on the β-secretase cleavage site of the Swedish mutation of APP (SEVNL/DAEFR, with "/" indicating the site of cleavage). OM99-1 has the structure VNL⁺AAEF (with "L* A" indicating the uncleavable hydroxyethylene transition-state isostere of the LA peptide bond) and exhibits a Ki towards recombinant β-secretase produced in E. coli of 6.84x 10-8 M±2.72* 10-9 M. OM99-2 has the structure EVNL⁺AAEF (with "L*A" indicating the uncleavable hydroxyethylene transition-state isostere of the LA peptide bond) and exhibits a Ki towards recombinant β-secretase produced in E. coli of 9.58x 10-9 M±2.86x l0-10 M. OM99-1 and OM99-2, as well as related substances, are described in International Patent Publication WOO 100665. Currently, most drug discovery programs for Alzheimer's disease have targeted either aceytlcholinesterase or the secretases directly responsible for APP processing. While acetylcholinesterase inhibitors are marketed drugs for Alzheimer's disease they have limited efficacy and do not have disease modifying properties. Secretase inhibitors, on the other hand, have been plagued either by mechanism-based toxicity (γ-secretase inhibitors) or by extreme difficulties in identifying small molecule inhibitors with appropriate pharmacokinetic properties to allow them to become drugs (β-secretase inhibitors). Identifying novel factors involved in APP processing would expand the range of targets for Alzheimer's disease treatments and therapy.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for identifying modulators of LRRTMl , LRRTM2 and LRRTM4. The methods are particularly useful for identifying analytes that either stimulate LRRTMl or LRRTM2 or that inhibit LRRTM4, which, in turn, inhibits the processing of APP to Aβ and, as such, are useful for identifying analytes that can be used for treating Alzheimer disease.

Therefore, in one embodiment, the present invention provides a method for screening for analytes that antagonize the processing of APP to Aβ, either by stimulating LRRTMl or LRRTM2 or by inhibiting LRRTM4, comprising providing recombinant cells which ectopically expresses LRRTMl, LRRTM2 or LRRTM4 and APP; incubating the cells in a culture medium under conditions for expression of LRRTMl, LRRTM2 or LRRTM4 and APP with an analyte; removing the culture medium from the recombinant cells; and determining the amount of at least one processing product of APP in the medium, where said product is selected from the group consisting of sAPPβ and Aβ, wherein a decrease in the amount of said product with the analyte as compared to the amount of said product in medium produced in said recombinant cells without the analyte indicates that the analyte is an antagonist of the processing of the APP to Aβ peptide, i.e. an LRRTMl or LRRTM2 agonist or an LRRTM4 antagonist. In further aspects of the method, the recombinant cells each comprises a first nucleic acid that encodes LRRTMl, LRRTM2 or LRRTM4 operably linked to a first heterologous promoter and a second nucleic acid that encodes an APP operably linked to a second heterologous promoter. In preferred aspects of the present invention, the APP is APP NFEV. In preferred aspects, the method includes a control which comprises providing recombinant cells that ectopically express APP but not LRRTMl, LRRTM2 or LRRTM4.

The present invention further provides a method for screening for analytes that antagonize processing of APP to Aβ, either by stimulating LRRTM lor LRRTM2 or by inhibiting LRRTM4, comprising providing recombinant cells which ectopically express LRRTMl, LRRTM2 or LRRTM4 and a recombinant APP, which comprises APP fused to a transcription factor that when removed from APP during processing produces an active transcription factor, and a reporter gene operably linked to a promoter inducible by the transcription factor; incubating the cells in a culture medium under conditions for expression of

LRRTMl, LRRTM2 or LRRTM4 and recombinant APP with an analyte; and determining expression of the reporter gene, wherein a decrease in expression of the reporter gene with the analyte as compared to expression of the reporter gene in recombinant cells in medium without the analyte indicates that the analyte is an antagonist of the processing of the APP to Aβ peptide, i.e. an LRRTMl or LRRTM2 agonist or an LRRTM4 antagonist.

In further aspects of the method, the recombinant cells each comprise a first nucleic acid that encodes LRRTMl, LRRTM2 or LRRTM4 operably linked to a first heterologous promoter, a second nucleic acid that encodes the recombinant APP fused to a transcription factor that when removed from APP during processing produces an active transcription factor, and a third nucleic acid that encodes a reporter gene operably linked to a promoter responsive to the transcription factor, wherein expression of said recombinant cells comprises proteolytic fragments of the recombinant APP. In light of the analytes that can be identified using the above methods, the present invention further provides a method for treating Alzheimer's disease in an individual which comprises providing to the individual an effective amount of an agonist of LRRTMl or

LRRTM2 or an antagonist of LRRTM4 activity.

Further still, the present invention provides a method for identifying an individual who has Alzheimer's disease or is at risk of developing Alzheimer's disease comprising obtaining a sample from the individual and measuring the amount of LRRTMl, LRRTM2 or

LRRTM4 in the sample.

Further still, the present invention provides for the use of a modulator of

LRRTMl, LRRTM2 or LRRTM4 for the manufacture of a medicament for the treatment of Alzheimer's disease.

Further still, the present invention provides for the use of an activating antibody specific for LRRTMl, LRRTM2 or an inactivating antibody specific for LRRTM4 for the manufacture of a medicament for the treatment of Alzheimer's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a nucleic acid sequence encoding human LRRTMl (SEQ ID NO: 1).

Figure 2 is an amino acid sequence of human LRRTMl (SEQ ID NO: 2).

Figure 3 is a nucleic acid sequence encoding human LRRTM2 (SEQ ID NO: 3).

Figure 4 is an amino acid sequence of human LRRTM2 (SEQ ID NO: 4). Figure 5 is a nucleic acid sequence encoding human LRRTM4 (SEQ ID NO: 5).

Figure 6 is an amino acid sequence of human LRRTM4 (SEQ ID NO: 6). Figure 7 is a graph showing the relative expression of APP secreted metabolites expressed as a percent of the mean control non-silencing siRNA value of 100 following RNAi knockdown of LRRTM 1.

Figure 8 is a graph showing the relative expression of APP secreted metabolites expressed as a percent of the mean control non-silencing siRNA value of 100 following RNAi knockdown of LRRTM2.

Figure 9 is a graph showing the relative expression of APP secreted metabolites expressed as a percent of the mean control non-silencing siRNA value of 100 following RNAi knockdown of LRRTM4. Figure 10 is a western blot (Figure 10A) and a graph (Figure 10B) showing the

C-terminal fragments (CTFs) of APP_NFEV following RNAi knockdown of LRRTMl .

Figure 1 1 is a western blot (Figure 1 IA) and a graph (Figure 1 IB) showing the C-terminal fragments (CTFs) of APP NFEV following RNAi knockdown of LRRTM2.

Figure 12 is a western blot (Figure 12A) and a graph (Figure 12B) showing the C-terminal fragments (CTFs) of APP_NFEV following RNAi knockdown of LRRTM4.

Figures 13A-13C show the tissue distribution of LRRTMl mRNA in various human tissues.

Figures 14A-14C show the tissue distribution of LRRTM2 mRNA in various human tissues. Figures 15A-15C show the tissue distribution of LRRTM4 mRNA in various human tissues.

Figure 16 is a phylogeny tree showing the relationship of LRRTMl, LRRTM2 and LRRTM4 relative to LRRTM3 (NOAHlO) and the NOGO receptors.

Figure 17 is an alignment of the amino acid sequences of human LRRTMl (SEQ ID NO: 2), human LRRTM3 (SEQ ID NO: 7) and mouse LRRTMl (SEQ ID NO: 8).

Figure 18 is an alignment of the amino acid sequences of human LRRTM2 (SEQ ID NO: 4), human LRRTM3 (SEQ ID NO: 7) and mouse LRRTM2 (SEQ ID NO: 9).

Figure 19 is an alignment of the amino acid sequences of human LRRTM4 (SEQ ID NO: 6), human LRRTM3 (SEQ ID NO: 7) and mouse LRRTM4 (SEQ ID NO: 10).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term "analyte" refers to a compound, chemical, agent, composition, antibody, peptide, aptamer, nucleic acid, or the like, which can modulate the activity of LRRTMl , LRRTM2 or LRRTM4.

The term "LRRTM" refers to a leucine rich repeat transmembrane neuronal gene. Specifically, it can refer to either LRRTMl, or LRRTM2, or LRRTM4. The term "LRRTMl" refers to "leucine rich repeat transmembrane neuronal 1" (GenBank Accession No. NM_ 1788839 (SEQ ID NO: 1) and NP_849161 (SEQ ID NO: 2)). Human LRRTMl is 39.3% identical with human LRRTM3 (GenBank Accession No. NP_821079 (SEQ ID NO: 7)) and 96.9% identical with mouse LRRTMl (GenBank Accession No. NP 083153 (SEQ ID NO: 8)). The term further includes mutants, variants, alleles, and polymorphs of LRRTMl . Where appropriate, the term further includes fusion proteins comprising all or a portion of the amino acid sequence of LRRTMl fused to the amino acid sequence of a heterologous peptide or polypeptide, for example, hybrid immuoglobulins comprising the amino acid sequence of LRRTMl or LRRTMl without the transmembrane region fused at its C-terminus to the N-terminus of an immunoglobulin constant region amino acid sequence (See, for example, U.S. Patent No. 5,428,130 and related patents).

The term "LRRTM2" refers to "leucine rich repeat transmembrane neuronal 2" (GenBank Accession No. NM_015564 (SEQ ID NO: 3) and NP_056370 (SEQ ID NO: 4)). Human LRRTM2 is 39.4% identical with human LRRTM3 (GenBank Accession No. NP 821079 (SEQ ID NO: 7)) and 97.79% identical with mouse LRRTM2 (GenBank Accession No. NP_821072 (SEQ ID NO: 9)). The term further includes mutants, variants, alleles, and polymorphs of LRRTM2. Where appropriate, the term further includes fusion proteins comprising all or a portion of the amino acid sequence of LRRTM2 fused to the amino acid sequence of a heterologous peptide or polypeptide, for example, hybrid immuoglobulins comprising the amino acid sequence of LRRTM2 or LRRTM2 without the transmembrane region fused at its C-terminus to the N-terminus of an immunoglobulin constant region amino acid sequence (See, for example, U.S. Patent No. 5,428,130 and related patents).

The term "LRRTM4" refers to "leucine rich repeat transmembrane neuronal 4" (GenBank Accession Nos. NM_024993 (SEQ ID NO: 5) and NP_079269 (SEQ ID NO: 6)). Human LRRTM4 is 55.0% identical with human LRRTM3 (GenBank Accession No.

NP_821079 (SEQ ID NO: 7)) and 99.8% identical with mouse LRRTM4 (GenBank Accession No. AA067552 (SEQ ID NO: 10)). The term further includes mutants, variants, alleles, and polymorphs of LRRTMl . Where appropriate, the term further includes fusion proteins comprising all or a portion of the amino acid sequence of LRRTMl fused to the amino acid sequence of a heterologous peptide or polypeptide, for example, hybrid immuoglobulins comprising the amino acid sequence of LRRTMl or LRRTMl without the transmembrane region fused at its C-terminus to the N-terminus of an immunoglobulin constant region amino acid sequence (See, for example, U.S. Patent No. 5,428,130 and related patents).

The terms "LRRTMl, LRRTM2 and LRRTM4" and "LRRTMl, LRRTM2 or LRRTM4" are used interchangeably depending on context and are intended to refer to each gene individually and not collectively. For example, reference to a modulator of LRRTMl , LRRTM2 and LRRTM4 means a modulator of a single LRRTM, i.e. LRRTMl or LRRTM2 or LRRTM4, not a modulator of all three genes. The term "APP_NFEV" refers to an amyloid precursor protein (APP) molecule of the 695 isoform having an optimized β-secretase cleavage site comprising the amino acid sequence NFEV (SEQ ID NO: 1 1) in lieu of the wild type sequence KMDA (SEQ ID NO: 12) at amino acid residues 596-598. NFEV has been described in U.S. Patent Nos. 7,132,401 and 7,196,163 and in US03/05458, which published as WO 03/072041. In a preferred embodiment used herein, the molecule further includes HA, Myc and FLAG sequences at amino acid position 289 and a K612V mutation.

The term "APP_NFEV metabolites" refers to the metabolites produced upon cleavage of APP_NFEV by β-secretase, for example, sAPPβ NF, EV40 and EV42. sAPPβ NF is a unique sAPPβ-like fragment having the amino acids asparagine and phenylalanine, respectively, at its C-terminus as compared to the wild-type C-terminus of lysine and methionine. Similarly, EV40 and EF42 are unique Aβ40-like and Aβ42-like peptides having glutamic acid and valine at its N-terminus as compared to the wild-type N-terminus of aspartic acid and alanine.

The leucine-rich repeat transmembrane neuronal 1 , 2 and 4 proteins (herein after referred to as LRRTMl, LRRTM2 and LRRTM4) are neuronal associated proteins that Applicants have discovered to have a role in processing of APP to Aβ peptide (Aβ). LRRTMl, LRRTM2 and LRRTM4 are part of a gene family known as leucine-rich repeat transmembrane neuronal (LRRTMs) proteins that have been previously cloned and sequenced (Lauren et al.,

Genomics 81 (4): 41 1-421 (2003); Genome Res., 13 (10): 2265-2270 (2003)). LRRTM proteins may function in adhesion, in protein-protein interactions, and as a receptor-binding ligand. LRRTMl, LRRTM2 and LRRTM4 are related to LRRTM3, a neuron-specific activator of β- secretase and therapeutic target for AD (see, co-pending application PCT/US2006/016407, which published as WO 2006/1 19095).

A defining characteristic of Alzheimer's disease (AD) is the deposition of aggregated plaques containing Aβ in the brains of affected individuals. While ongoing drug discovery efforts have focused on identifying antagonists of β-secretase and γ-secretase mediated cleavage of APP, the complicated nature of AD suggests that efficacious treatments and therapies for Alzheimer's disease might comprise other targets for modulating APP processing. Applicants' discovery that LRRTMl, LRRTM2 and LRRTM4 have a role in processing APP to Aβ suggests that LRRTMl, LRRTM2 and LRRTM4 are other targets for which modulators of and, in particular, agonists are expected to provide efficacious treatments or therapies for AD, either alone or in combination with one or more other modulators of APP processing, for example, antagonists selected from the group consisting of β-secretase and γ-secretase.

Therefore, in light of Applicants' discovery, identifying molecules which target activity or expression of LRRTMl , LRRTM2 or LRRTM4 would be expected to lead to treatments or therapies for AD. Expression or activity of LRRTMl, LRRTM2 and LRRTM4 may also be useful as a diagnostic marker for identifying individuals who have AD or who are at risk of developing AD.

LRRTMl, LRRTM2 and LRRTM4 were identified by screening an siRNA library for siRNA that inhibited APP processing. As described in Example 1, a library of about 15,200 siRNA pools, each targeting a single gene, was transfected individually into recombinant cells ectopically expressing a recombinant APP molecule having an optimized β-secretase cleavage site (APP_NFEV), having HA, Myc, and FLAG sequences at amino acid position 289, and having a K612V mutation. Metabolites of APP_NFEV produced during APP β/γ-secretase or α-secretase processing are sAPPβ with NF at the C-terminus ("sAPPβ_NF"), EV40, and EV42 or sAPPα. The fragments, sAPPβ_NF, sAPPα, EV40, and EV42 were detected by an immunodetection method that used antibodies specific for the various APP_NFEV metabolites. Expression levels were determined relative to a non-silencing siRNA control. Following the second round of screening, which consisted of about 1600 siRNAs performed in triplicate repeats, siRNAs were identified that targeted an mRNA encoding LRRTMl, LRRTM2 or LRRTM4, polypeptides with structural similarities to the NOGO receptor family of axon guidance genes, and that consistently altered processing of APP to sAPPβNF, EV40, and EV42. The nucleic acids encoding these polypeptides, herein designated as LRRTMl (SEQ ID NO: 1), LRRTM2 (SEQ ID NO: 3) and LRRTM4 (SEQ ID NO: 5), were found to have 31.2%, 34.9% and 65%, respectively, sequence identity to human LRRTM3 (GenBank Accession Nos. NMJ7801 or AYl 82027 (SEQ ID NO: 7)) (Lauren et aL, Genomics 81 : 41 1-421 (2003)) and 80.1%, 56.2% and 95% sequence identify to mouse LRRTMl (SEQ ID NO: 8), LRRTM2 (SEQ ID NO: 9) and LRRTM4 (SEQ ID NO: 10).

The nucleic acid sequences encoding human LRRTM 1 , LRRTM2 and LRRTM4 (SEQ ID NOS: 1 , 3 and 5) are shown in Figures 1 , 3 and 5, respectively, and the amino acid sequence for human LRRTMl , LRRTM2 and LRRTM4 (SEQ ID NOS: 2, 4, and 6) are shown in Figures 2, 4, and 6, respectively. The mRNAs encoding LRRTMl, LRRTM2 and LRRTM4 were found to be expressed in regions of the brain subject to AD pathology, Example 3, Figures 14, 15, and 16, respectively.

In light of Applicants' discovery, LRRTMl, LRRTM2 or LRRTM4, or modified mutants or variants thereof, are useful for identifying analytes that either stimulate LRRTMl or LRRTM2 or inhibit LRRTM4 which, in turn, antagonize processing of APP to produce Aβ. These analytes can be used to treat patients afflicted with AD. LRRTMl, LRRTM2 or LRRTM4 can also be used to help diagnose AD by assessing genetic variability within the locus. LRRTMl, LRRTM2 or LRRTM4 can be used alone or in combination with acetylcholinesterase inhibitors, NMDA receptor partial agonists, secretase inhibitors, amyloid-reactive antibodies, and other treatments for AD.

The present invention provides methods for identifying LRRTMl , LRRTM2 and LRRTM4 modulators by contacting LRRTMl, LRRTM2 or LRRTM4 with a substance that inhibits or stimulates LRRTMl, LRRTM2 or LRRTM4 expression and determining whether expression of LRRTMl, LRRTM2 or LRRTM4 polypeptides or nucleic acid molecules encoding an LRRTMl, LRRTM2 or LRRTM4 are modified. The present invention also provides methods for identifying modulators that either agonize LRRTM l's or LRRTM2's or antagonize LRRTM4's inhibitory effect on processing APP to Aβ or formation of amyloid plaques in tissues where LRRTMl, LRRTM2 or LRRTM4 are localized or co-expressed. For example, LRRTMl, LRRTM2 or LRRTM4 protein can be expressed in cell lines that also express APP and the effect of the modulator on Aβ production can be monitored using standard biochemical assays with Aβ-specific antibodies or by mass spectrophotometric techniques. Modulators for LRRTMl , LRRTM2 and LRRTM4 are identified by screening for a reduction in the release of Aβ. Both small molecules and larger biomolecules that modulate LRRTMl, LRRTM2 and LRRTM4-mediated processing of APP to Aβ can be identified using such an assay. The methods described herein can also be used for identifying either agonists of LRRTMl 's or LRRTM2's or an antagonist LRRTM4's effect on the processing APP to Aβ and are amenable to high throughput screening. In addition, the methods disclosed in U.S. Patent Nos. 7,132,401 and 7,196,163 can be adapted for use as assays to identify antagonists of LRRTMl, LRRTM2 and LRRTM4 activity.

A mammalian LRRTMl, LRRTM2 or LRRTM4 cDNA, encompassing the first through the last predicted codon contiguously, was amplified from brain total RNA with sequence-specific primers by reverse-transcription polymerase chain reaction (RT-PCR). The amplified sequence was cloned into a pCMV-SPORT6 (Invitrogen, Carlsbad, CA) vector. The fidelity of the sequence and the ability of the plasmid to encode full-length LRRTMl, LRRTM2 and LRRTM4 was validated by DNA sequencing of the LRRTMl, LRRTM2 and LRRTM4 plasmids (pcDNA LRRTMl, pcDNA_LRRTM2 and pcDNA_LRRTM4, respectively). Commercially available mammalian expression vectors which are suitable for recombinant LRRTMl, LRRTM2 and LRRTM4 expression include, but are not limited to, pcDNA3.neo (Invitrogen, Carlsbad, CA), pcDNA3.1 (Invitrogen, Carlsbad, CA), pcDNA3.1/Myc-His (Invitrogen), pCI-neo (Promega, Madison, WI), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Biolabs, Beverly, MA), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene, La Jolla, CA), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-l(8-2) (ATCC 371 10), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), 1ZD35 (ATCC 37565), pMClneo (Stratagene), pcDNA3.1, pCR3.1 (Invitrogen), EBO-pSV2-neo (ATCC 37593), pCI.neo (Promega), pTRE (Clontech, Palo Alto, CA), pVl Jneo, pIRESneo (Clontech, Palo Alto, CA), pCEP4 (Invitrogen,), pSCl 1, and pSV2-dhfr (ATCC 37146). The choice of vector will depend upon the cell type in which it is desired to express LRRTMl, LRRTM2 or LRRTM4, as well as on the level of expression desired, co-transfection with expression vectors encoding APP NFEV, and the like.

Cells transfected with a plasmid vector comprising APP NFEV, for example the HEK293T/APP_NFEV cells used to detect LRRTMl, LRRTM2 and LRRTM4 activity in the siRNA screening experiment described in Example 1 , are used as described in Example 1 with the following modifications. Cells were co-transfected with a plasmid expression vector comprising APP NFEV operably linked to a heterologous promoter and a plasmid expression vector comprising LRRTMl, LRRTM2 or LRRTM4 operably linked to a heterologous promoter. Alternatively, the HEK293T/APP_NFEV cells described in Example 1 were transfected with a plasmid expression vector comprising LRRTMl , LRRTM2 or LRRTM4 operably linked to a heterologous promoter. The promoter comprising the plasmid expression vector can be a constitutive promoter or an inducible promoter. Preferably, the assay includes a negative control comprising the expression vector without LRRTMl, LRRTM2 or LRRTM4.

After the cells have been transfected, the transfected or co-transfected cells are incubated with an analyte being tested for its ability to either agonize LRRTMl's or LRRTM2's or antagonize LRRTM4's effect on processing of APP to Aβ. The analyte is assessed for an effect on LRRTMl, LRRTM2 or LRRTM4 transfected or cotransfected cells that is minimal or absent in the negative control cells. In general, the analyte is added to the cell medium the day after the transfection and the cells are incubated for one to 24 hours with the analyte. In particular embodiments, the analyte is serially diluted and each dilution provided to a culture of the transfected or co-transfected cells. After the cells have been incubated with the analyte, the medium is removed from the cells and assayed for secreted sAPPα, sAPPβ, Aβ40, and Aβ42, generally, or in the present instance sAPPα, sAPPβ NF, EV40, and EV42 as described in Example 1. Briefly, the antibodies specific for each of the metabolites are used to detect the metabolites in the medium. Preferably, the cells are assessed for viability.

Analytes that alter the secretion of EV40, EV42, sAPPα, and/or sAPPβ NF in the presence of LRRTMl, LRRTM2 or LRRTM4 are considered to be modulators of LRRTMl, LRRTM2 or LRRTM4 and potential therapeutic agents for LRRTMl, LRRTM2 or LRRTM4- related diseases. For example, using APP NFEV, agonists of LRRTMl or LRRTM2 are expected to result in a decrease in the amount of secreted EV40 and EV42 in the medium, whereas an antagonist might be expected to cause an increase in the amount of secreted EV40 and EV42 in the medium. Similarly, agonists of LRRTM4 are expected to result in a decrease in the amount of secreted EV40, EV42 and sAPPβ_NF, whereas an antagonist might be expected to cause an increase in EV40, EV42, and sAPPβ NF in the medium. An antagonist of LRRTM4 might further result in a decrease in the amount of secreted sAPPα in the medium.

Analytes that alter the secretion of one or more of EV40, EV42, sAPPα, or sAPPβ NF in the presence of LRRTMl, LRRTM2 or LRRTM4 protein are considered to be modulators of LRRTMl, LRRTM2 or LRRTM4 and are potentially useful as therapeutic agents for LRRTMl, LRRTM2 or LRRTM4-related diseases: Direct modulation of LRRTMl, LRRTM2 or LRRTM4 can be confirmed using binding assays using full-length LRRTMl, LRRTM2 or LRRTM4, an extracellular or intracellular domain thereof, or a LRRTMl , LRRTM2 or LRRTM4 fusion protein comprising the intracellular or extracellular domain coupled to a C-terminal FLAG, or other epitopes. A cell-free binding assay using full-length LRRTMl, LRRTM2 or LRRTM4, an extracellular or intracellular domain thereof, a LRRTMl, LRRTM2 or LRRTM4 fusion protein, or membranes containing LRRTMl, LRRTM2 or LRRTM4 integrated therein and a labeled-analyte can be performed and the amount of labeled analyte bound to the LRRTMl, LRRTM2 or LRRTM4 determined. The present invention further provides a method for measuring the ability of an analyte to modulate the level of LRRTMl, LRRTM2 or LRRTM4 mRNA or protein in a cell. In this method, a cell that expresses LRRTMl, LRRTM2 or LRRTM4 is contacted with a candidate compound and the amount of LRRTMl, LRRTM2 or LRRTM4 mRNA or protein in the cell is determined. This determination of LRRTMl, LRRTM2 or LRRTM4 level may be made using any of the above-described immunoassays or techniques disclosed herein. The cell can be any LRRTMl, LRRTM2 or LRRTM4 expressing cell, such as a cell transfected with an expression vector comprising LRRTMl, LRRTM2 or LRRTM4 operably linked to its native promoter or a cell taken from a brain tissue biopsy from a patient.

The present invention further provides a method of determining whether an individual has a LRRTMl, LRRTM2 or LRRTM4-associated disorder or a predisposition for a LRRTMl, LRRTM2 or LRRTM4-associated disorder. The method includes providing a tissue or serum sample from an individual and measuring the amount of LRRTMl, LRRTM2 or LRRTM4 in the tissue sample. The amount of LRRTMl, LRRTM2 or LRRTM4 in the sample is then compared to the amount of LRRTMl, LRRTM2 or LRRTM4 in a control sample. An alteration in the amount of LRRTM 1 , LRRTM2 or LRRTM4 in the sample relative to the amount of LRRTMl, LRRTM2 or LRRTM4 in the control sample indicates the subject has a LRRTMl , LRRTM2 or LRRTM4-associated disorder. A control sample is preferably taken from a matched individual, that is, an individual of similar age, sex, or other general condition but who is not suspected of having an LRRTMl, LRRTM2 or LRRTM4 related disorder. In another aspect, the control sample may be taken from the subject at a time when the subject is not suspected of having a condition or disorder associated with abnormal expression of LRRTMl , LRRTM2 or LRRTM4.

Other methods for identifying inhibitors of LRRTMl , LRRTM2 and LRRTM4 can include blocking the interaction between LRRTMl, LRRTM2 or LRRTM4 and the enzymes involved in APP processing or trafficking using standard methodologies for analyzing protein- protein interaction such as fluorescence energy transfer or scintillation proximity assay. Surface Plasmon Resonance can be used to identify molecules that physically interact with purified or recombinant LRRTMl, LRRTM2 or LRRTM4. As LRRTMl, LRRTM2 and LRRTM4 are likely involved in cell adhesion, inhibitors of LRRTMl, LRRTM2 and LRRTM4 can be discovered by blocking LRRTMl, LRRTM2 and LRRTM4-dependent cell adhesion (created by co-culturing cells expressing LRRTMl, LRRTM2 or LRRTM4 with a suitable adherent partner cell line or by monitoring adhesion to specific chemical or biological substrates). In accordance with yet another embodiment of the present invention, there are provided antibodies having specific affinity for LRRTM 1 , LRRTM2 or LRRTM4 or an epitope thereof. The term "antibodies" is intended to be a generic term which includes polyclonal antibodies, monoclonal antibodies, Fab fragments, single VH chain antibodies such as those derived from a library of camel or llama antibodies or camelized antibodies (Nuttall et al., Curr. Pharm. Biotechnol. 1 : 253-263 (2000); Muyldermans, J. Biotechnol. 74: 277-302 (2001)), and recombinant antibodies. The term "recombinant antibodies" is intended to be a generic term which includes single polypeptide chains comprising the polypeptide sequence of a whole heavy chain antibody or only the amino terminal variable domain of the single heavy chain antibody (VH chain polypeptides) and single polypeptide chains comprising the variable light chain domain (VL) linked to the variable heavy chain domain (VH) to provide a single recombinant polypeptide comprising the Fv region of the antibody molecule (scFv polypeptides) (See, Schmiedl et al., J. Immunol. Meth. 242: 101-114 (2000); Schultz et al., Cancer Res. 60: 6663- 6669 (2000); Dϋbel et al., J. Immunol. Meth. 178: 201-209 (1995); and U.S. Patent No. 6,207,804 to Huston et al.). Construction of recombinant single VH chain or scFv polypeptides which are specific against an analyte can be obtained using currently available molecular techniques such as phage display (de Haard et al., J. Biol. Chem. 274: 18218-18230 (1999); Saviranta et al., Bioconjugate 9: 725-735 (1999); de Greeff et al., Infect. Immun. 68: 3949-3955 (2000)) or polypeptide synthesis. In further embodiments, the recombinant antibodies include modifications such as polypeptides having particular amino acid residues, ligands or labels, including, but not limited to, horseradish peroxidase, alkaline phosphatase, fluors and the like. Still further embodiments include fusion polypeptides which comprise the above polypeptides fused to a second polypeptide, such as a polypeptide comprising protein A or G.

The antibodies specific for LRRTMl, LRRTM2 or LRRTM4 can be produced by methods known in the art. For example, see the methods for producing polyclonal and monoclonal antibodies described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988). LRRTMl, LRRTM2 and LRRTM4 or fragments thereof can be used as immunogens for generating such antibodies. Alternatively, synthetic peptides based on LRRTMl, LRRTM2 and LRRTM4 can be prepared (using commercially available synthesizers) and used as immunogens. Amino acid sequences can be analyzed by methods well known in the art to determine whether they encode hydrophobic or hydrophilic domains of the corresponding polypeptide. Altered antibodies such as chimeric, humanized, camelized, CDR-grafted, or bifunctional antibodies can also be produced by methods well known in the art. Such antibodies can also be produced by hybridoma, chemical synthesis or recombinant methods. See, for example, Sambrook et al., supra, and Harlow and Lane, supra. Both anti-peptide and anti-fusion protein antibodies can be used. See, for example, Bahouth et al., Trends Pharmacol. Sci. 12: 338 (1991); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, N. Y. (1989). Antibodies so produced can be used for immunoaffinity or affinity chromatography purification of LRRTMl, LRRTM2 or LRRTM4 and LRRTMl, LRRTM2 or LRRTM4/ligand or analyte complexes. Accordingly, contemplated herein are compositions comprising a carrier and an amount of an antibody having specificity for LRRTMl, LRRTM2 or LRRTM4 effective to block naturally occurring LRRTMl, LRRTM2 or LRRTM4 from binding its ligand or for effecting the processing of APP to Aβ.

Therefore, in another aspect, the present invention further provides pharmaceutical compositions that antagonize LRRTMl, LRRTM2 or LRRTM4 and affect processing of APP to Aβ. Such compositions include a LRRTMl, LRRTM2 or LRRTM4 nucleic acid, a LRRTMl, LRRTM2 or LRRTM4 peptide, a fusion protein comprising LRRTMl, LRRTM2 or LRRTM4, or a fragment thereof coupled to a heterologous peptide or protein or fragment thereof, an antibody specific for LRRTMl, LRRTM2 or LRRTM4, or combinations thereof, and a pharmaceutically acceptable carrier or diluent.

In a still further aspect, the present invention provides a kit for in vitro diagnosis of disease by detection of LRRTMl, LRRTM2 or LRRTM4 in a biological sample from a patient. A kit for detecting LRRTMl , LRRTM2 or LRRTM4 preferably includes a primary antibody capable of binding to LRRTMl, LRRTM2 or LRRTM4 and a secondary antibody conjugated to a signal-producing label, the secondary antibody being capable of binding an epitope different from, i.e., spaced from, that to which the primary antibody binds. Such antibodies can be prepared by methods well-known in the art. This kit is most suitable for carrying out a two-antibody sandwich immunoassay, e.g., two-antibody sandwich ELISA.

LRRTMl, LRRTM2 and LRRTM4 can be used to identify endogenous brain proteins that bind thereto using biochemical purification, genetic interaction, or other techniques common to those skilled in the art. These proteins or their derivatives can subsequently be used to either stimulate LRRTMl or LRRTM2 or to inhibit LRRTM4 activity and thus be used to treat Alzheimer's disease. Additionally, polymorphisms in LRRTMl, LRRTM2 or LRRTM4 RNA or in the genomic DNA in and around LRRTMl, LRRTM2 or LRRTM4 could be used to diagnose patients at risk for Alzheimer's disease or to identify likely responders in clinical trials.

The following examples are intended to promote a further understanding of the present invention. EXAMPLE 1

LRRTMl, LRRTM2 and LRRTM4 were identified in a screen of an siRNA library for modulators of APP processing. A cell plate was prepared by plating HEK293T/APP_NFEV cells to the wells of a 384-well Corning PDL-coated assay plate at a density of about 2,000 cells per well in 40 μL DMEM containing 10% fetal bovine serum (FBS) and antibiotics. The cell plate was incubated overnight at 37°C in 5% CO2- HEK293T/APP_NFEV cells are a sublcone of HEK293T cells stably transformed with the APP_NFEV plasmid described in U.S. Patent No. 7,196,163. In brief, the APP molecule used therein encoded a human APP695, modified at amino acid positions 595- 598 by substitution of the amino acid sequence NFEV (SEQ ID NO: 1 1) for the endogenous KMDA (SEQ ID NO: 12) amino acid sequence, which comprises the β-secretase cleavage site. The plasmid therein was also modified at amino acid position 289 by an in-frame insertion of HA, Myc, and FLAG epitope amino acid sequences. Thus, β-secretase cleaves the modified cleavage site between amino acids F and E of the modified site NFEV (SEQ ID NO: 11). Maintenance of the plasmid within the sublcone is achieved by culturing the cells in the presence of the antibiotic puromycin.

The next day, the cells in each of the wells of the cell plate were transfected with an siRNA library as follows. Oligofectamine™ (Invitrogen, Inc., Carlsbad, CA) was mixed with Opti-MEM® (Invitrogen, Inc., Carlsbad, CA) at a ratio of 1 to 40 and 20 μL of the mixture was added to each well of a 384-well plate. To each well of the plate, 980 nL of a particular 10 μM siRNA species was added and the plate incubated for ten minutes at room temperature. Afterwards, five μL of each the siRNA/Oligofectamine™/Opti-MEM® mixtures was added to a corresponding well in the cell plate containing the HEK293/APP_NFEV cells. The cell plate was incubated for 24 hours at 37°C in 5% CO2- Controls were provided which contained non- silencing siRNA or an siRNA that inhibited β-secretase.

On the next day, for each of the wells of the cell plate, the siRNA and Oligofectamine™/Opti-MEM® mixture was removed and replaced with 70 μL DMEM containing 10% FBS and MERCK compound A (see, WO 2003093252, for the preparation of spirocyclic [1 ,2,5] thiadiazole derivatives as γ-secretase inhibitors for treatment of Alzheimer's disease, Collins et al.), a γ-secretase inhibitor, given at a final concentration equal to its IC50 in cell-based enzyme assays. The cell plate was incubated for 24 hours at 37°C in 5% CO2- On the next day, for each of the wells of the cell plate, 64 μL of the medium (conditioned medium) was removed and transferred to four 384-well REMP plates in 22, 22, 10, and 10 μL aliquots for subsequent use in detecting sAPPα, EV42, EV40, sAPPβ NF using the AlphaScreen™ (PerkinElmer, Wellesley, MA) detection technology. Viability of the cells was determined by adding 40 μL 10% AlamarBlue (Serotec, Inc., Raleigh, NC) in DMEM containing 10% FBS to each of the wells of the cell plate with the conditioned medium removed. The cell plate was then incubated at 37°C for two hours. The Acquest™ (Molecular Devices

Corporation, Sunnyvale, CA) plate reader was used to assay fluorescence intensity (ex. 545 nm, em. 590 nm) as a means to confirm viability of the cells. Assays for detecting and measuring sAPPβJSfF, EV42, EV40, and sAPPα were detected using antibodies as follows. In general, detection-specific volumes (8 or 0.5 μL) were transferred to a 384-well, white, small-volume detection plate (Greiner Bio-One, Monroe, NC). In the case of the smaller volume, 7.5 μL of assay medium was added for a final volume of eight μL per well. One μL of an antibody/donor bead mixture (see below) was dispensed into the solution, and one μL antibody/acceptor bead mixture was added. Plates were incubated in the dark for 24 hours at 4°C. The plates were then read using AlphaQuest™ (PerkinElmer, Wellesley, MA) instrumentation. In all protocols, the plating medium was DMEM (Invitrogen, La Jolla, CA; Cat. No. 21063-029); 10% FBS, the AlphaScreen™ buffer was 50 mM HEPES, 150 mM NaCl, 0.1% BSA, 0.1% Tween-20, pH 7.5, and the AlphaScreen™ Protein A kit was used.

Anti-NF antibodies and anti-EV antibodies were prepared as taught in U.S. Patent No.7,196,163. β-secretase cleaves between amino acids F and E of the NFEV cleavage site of APP NFEV to produce an sAPPβ peptide with NF at the C-terminus (sAPPβ NF) and an EV40 or EV42 peptide with amino acids E and V at the N-terminus. Anti-NF antibodies bind the C- terminal neo-epitope NF at the C-terminus of the sAPPβ peptide produced by β-secretase cleavage of the NFEV sequence of APP_NFEV. Anti-EV antibodies bind the N-terminal neo- epitope EV at the N-terminus of EV40 and EV42 produced by β-secretase cleavage of the NFEV sequence of APP_NFEV. Anti-Bio-G2-10 and anti-Bio-G2-l 1 antibodies are available from the Genetics Company, Zurich, Switzerland. Anti-Bio-G2-l l antibodies bind the neo-epitope generated by the γ-secretase cleavage of Aβ or EV peptides at the 42 amino acid position. Anti- Bio-G2-10 antibodies bind the neo-epitope generated by the γ-secretase cleavage of Aβ or EV peptides at the 40 amino acid position. Anti-6E10 antibodies are commercially available from Signet Laboratories, Inc., Dedham, MA. Anti-6E10 antibodies bind an epitope within amino acids 1 to 17 of the N-terminal region of the Aβ, EV40 and EV42 peptides and also bind sAPPα because the same epitope resides in amino acids 597 to 614 of sAPPα. Bio-M2 anti-FLAG antibodies are available from Sigma-Aldrich, St. Louis, MO.

Detecting sAPPβ: An AlphaScreen™ assay for detecting sAPPβ NF produced from cleavage of APP NFEV at the β-secreatase cleavage site was performed as follows. Conditioned medium for each well was diluted 32-fold into a final volume of eight μL. As shown in Table 1 , biotinylated-M2 anti-FLAG antibody, which binds the FLAG epitope of the APP NFEV, was captured on streptavidin-coated donor beads by incubating a mixture of the antibody and the streptavidin coated beads for one hour at room temperature in AlphaScreen™ buffer. The amount of antibody was adjusted such that the final concentration of antibody in the detection reaction was 3 nM. Anti-NF antibody was similarly captured separately on protein-A acceptor beads in AlphaScreen™ buffer and used at a final concentration of 1 nM (Table 1). The donor and acceptor beads were each used at final concentrations of 20 μg/mL. Table 1

Detecting EV42: Conditioned medium for each well was used neat (volume eight μL). As shown in Table 2, anti-Bio-G2-l 1 antibody was captured on streptavidin-coated donor beads by incubating a mixture of the antibody and the streptavidin coated beads for one hour at room temperature in ALPHASCREEN buffer. The amount of antibody was adjusted such that the final concentration of antibody in the detection reaction was 20 nM. Anti-EV antibody was similarly captured separately on protein-A acceptor beads in AlphaScreen™ buffer and used at a final concentration of 5 nM (Table 2). The donor and acceptor beads were used at a final concentration of 20 μg/mL.

Table 2

Detecting EV40: Conditioned medium for each well was diluted four-fold into a final volume eight μL. As shown in Table 3, anti-Bio-G2-10 antibody was captured on streptavidin-coated donor beads by incubating a mixture of the antibody and the streptavidin coated beads for one hour at room temperature in AlphaScreen™ buffer. The amount of antibody was adjusted such that the final concentration of antibody in the detection reaction was 20 nM. Anti-EV antibody was similarly captured separately on prόtein-A acceptor beads in AlphaScreen™ buffer and used at a final concentration of 5 nM. The donor and acceptor beads were used at a final concentration of 20 μg/mL. Table 3

Detecting sAPPα: Conditioned medium for each well was diluted four-fold into a final volume eight μL. As shown in Table 4, Bio-M2 anti-FLAG antibody was captured on streptavidin-coated donor beads by incubating a mixture of the antibody and the streptavidin coated beads for one hour at room temperature in AlphaScreen™ buffer. Anti-6E10 antibody acceptor beads were obtained from the manufacturer (PerkinElmer, Inc., which makes the beads and conjugates antibody 6E10 to them). Antibody 6E10 (made by Signet Laboratories, Inc., a Covance Company, Dedham, MA) was used at a final concentration of 30 μg/ml. The donor beads were used at a final concentration of 20 μg/mL.

Table 4

About 15,200 single replicate pools of siRNAs were tested for modulation of sAPPβ, sAPPα, EV40 and EV42 by the AlphaScreen™ immunodetection method as described above. Based on the profile from this primary screen, 1 ,622 siRNA were chosen for an additional round of screening in triplicate. An siRNA was defined as "secretase-like" if a significant decrease in sAPPβ, EV40 and EV42 was detected, as well as either no change or an increase in sAPPα.

As least one siRNA was identified which inhibited an mRNA having a nucleotide sequence encoding LRRTMl, LRRTM2 or LRRTM4, the nucleotide sequences of which are set forth in GenBank Accession Nos. NM_178839 (SEQ ID NO: 1), NM_015564 (SEQ ID NO: 3), and NM_024993 (SEQ ID NO: 5), respectively, which were also described by Lauren et al., Genomics 81 : 411-421 (2003). The amino acid sequences for LRRTM 1, LRRTM2, and LRRTM4 are set forth in GenBank Accession Nos. NP_849161 (SEQ ID NO: 2), NP_056379 (SEQ ID NO: 4), and NP_079269 (SEQ ID NO: 6), respectively.

Compared to control non-silencing siRNAs (set to 100%), the LRRTMl siRNA pool significantly increased EV42 220% and EV40 153% (Figure 7). Similarly, the LRRTM2 siRNA pool 158% and EV40 141% and there was no change in APPα (Figure 8). In contrast, the LRRTM4 siRNA pool decreased EV 42 22.0% and EV 40 20.0% (Figure 9). Because of their roles in APP processing, LRRTMl, LRRTM2 and LRRTM4 appear to have a role in the establishment or progression of Alzheimer's disease.

EXAMPLE 2

In order to confirm the biological function of LRRTMl, LRRTM2 and LRRTM4 in inhibiting the proteolysis of APP, the C-terminal products of β-secretase and α-secretase (βCTF NF and αCTF, respectively) were examined by western blot with antibodies directed to the C-terminal region of APP in HEK293 cells expressing APP NFEV and either control non- targeting siRNAs, BACEl siRNAs, or LRRTMl, LRRTM2 or LRRTM4 siRNAs. As shown in Figures 1OA, 1 IA, and 12 A, BACEl siRNAs significantly reduce the amount of βCTF NF detected in the cell lysate as expected, serving as a control for the experiment. LRRTMl and LRRTM2 siRNAs significantly increased the proportion of βCTF_NF to αCTF without decreasing the amount of αCTF (for LRRTM 1 ) or total amount of CTFs (for LRRTM2) (Figures 1OB and 1 IB). For LRRTM4, the siRNAs significantly reduced βCTF_NF and αCTF (Figure 12B). Thus, LRRTMl, LRRTM2 and LRRTM4 inhibition by specific siRNAs demonstrate a role in the processing of APP and concurrently that LRRTMl, LRRTM2 and LRRTM4 appear to have a role in the establishment of progression of Alzheimer's disease.

EXAMPLE 3

Because LRRTMl, LRRTM2 and LRRTM4 appear to have a role in APP processing to Aβ and, as such, a role in the progression of AD, expression of LRRTMl, LRRTM2 and LRRTM4 was assayed in a variety of tissues to determine whether LRRTMl, LRRTM2 and LRRTM4 were expressed in the brain.

A proprietary database, the TGI Body Atlas, was used to show that the results of a microarray analysis of the expression of a majority of characterized genes, including LRRTMl, LRRTM2 and, LRRTM4, in the human genome in a panel of different tissues. LRRTMl , LRRTM2 and LRRTM4 mRNA were found to be expressed in the brain and within cortical structures such as the prefrontal and cerebral cortex, which are subject to Aβ deposition and AD pathology. The results for LRRTMl, LRRTM2 and LRRTM4 are summarized in Figures 13, 14, and 15, respectively. The results strengthen the conclusion of the Example 1 that LRRTMl, LRRTM2 and LRRTM4 have a role in APP processing and, as such, a role in the establishment or progression of AD.

EXAMPLE 4

The LRRTM gene family, consisting of LRRTMl, LRRTM2, LTRRTM3 (also known as NOAHlO, see WO 2006/1 19095) and LRRTM4, are members of a larger family of leucine rich region (LRR) containing membrane bound receptors with homology to the Drosophila axon guidance gene slit. The LRR often functions in adhesion, in protein-protein interactions, and as a receptor-binding ligand. The therapeutically relevant LRR containing NOGO receptor blocks axonal regeneration and its ligand NOGO binds BACEl (β-secretase) and modulate Aβ peptide generation (He et ah, Nature Med. 10: 959-965 (2004)). LRRTMl, LRRTM2 and LRRTM4 shares sequence and domain homology to the NOGO receptor and were cloned in an approach to identify additional NOGO-like receptors involved in axonal guidance. Taken together, this data demonstrating sequence homology with other neuronal receptors with known function suggests the possibility that LRRTMl, LRRTM2 and LRRTM4 may be altering Aβ peptide production in a similar manner to the NOGO receptor. See Figure 16 for phylogeny tree and relationship of LRRTMs to the NOGO receptor.

EXAMPLE 5

The results of Examples 1-4 have shown that LRRTMl, LRRTM2 and LRRTM4 have a role in the establishment or progression of Alzheimer's disease. The results suggest that analytes that activate LRRTMl, LRRTM2 or LRRTM4 activity will be useful for the treatment or therapy of AD. Therefore, there is a need for assays to identify analytes that modulate LRRTM 1 , LRRTM2 and LRRTM4 activity, for example, inhibit binding of LRRTM 1 , LRRTM2 or LRRTM4 to its natural ligand and/or to β-secretase. The following is such an assay that can be used to identify analytes that activate LRRTMl, LRRTM2 and LRRTM4 activity.

HEK293T/APP_NFEV cells are transfected with a plasmid encoding the human LRRTMl, LRRTM2 or LRRTM4 or a homolog of the human LRRTMl, LRRTM2 and LRRTM4, for example, the mouse LRRTMl (SEQ ID NO: 8), LRRTM2 (SEQ ID NO: 9) and LRRTM4 (SEQ ID NO: 10), using a standard transfection protocol to produce HEK293T/APP_NFEV/ LRRTMl, LRRTM2 or LRRTM4 cells. For example, HEK293T/ APP NFEV are plated into a 96-well plate at about 8000 cells per well in 80 μL DMEM containing 10%FBS and antibiotics and the cell plate incubated at 37°C at 5% CO2 overnight. On the next day, a mixture of 600 μL Oligofectamine™ and 3000μL Opti-

MEM® is made and incubated at room temperature for five minutes. Next, 23 μL Opti-MEM® is added to each well of a 96-well mixing plate. 50 ng pcDNA_ LRRTMl, LRRTM2 or LRRTM4 and empty control vector (in 1 μL volume) are added into adjacent wells of the mixing plate in an alternating fashion. The mixing plate is incubated at room temperature for five minutes. Next, 6 μL of the Oligofectamine™ mixture is added to each of the wells of the mixing plate and the mixing plate incubated at room temperature for five minutes. After five minutes, 20 μL of the plasmid/Oligofectamine™ mixture is added to the corresponding well in the plate of HEK293/APP NFEV cells plated in the cell plate and the plates incubated overnight at 37°C in 5% Cθ2-

The next day, the medium is removed from each well and replaced with 100 μL DMEM containing 10% FBS. Analytes being assayed for the ability to activate LRRTMl, LRRTM2 or LRRTM4-mediated activation of Aβ secretion are added to each well individually. The analytes are assessed for an effect on the APP processing to Aβ peptide in LRRTMl ,

LRRTM2 or LRRTM4 transfected cells that is either minimal or absent in cells transfected with the vector-alone as follows. The cells are incubated at 37°C at 5% CO2 overnight.

The next day, conditioned media is collected and the amount of sAPPβ NF, sAPPβ, EV42, EV40, and sAPPα in said media is determined as described in Example 1. Analytes that effect a decrease in the amounts of sAPPβ NF, EV42, and EV40 and either an increase or no change in the amount of sAPPα are activators of LRRTMl, LRRTM2 or LRRTM4. Viability of the cells is determined as in Example 1.

EXAMPLE 6 Analytes that alter secretion of EV40, EV42, sAPPα, or sAPPβ_NF, sAPPβ only, or more, in the presence of LRRTMl, LRRTM2 or LRRTM4 are considered to be modulators of LRRTMl, LRRTM2 or LRRTM4 and potential therapeutic agents for treating LRRTMl , LRRTM2 or LRRTM4-related diseases. The following is an assay that can be used to confirm direct inhibition or modulation of LRRTMl, LRRTM2 or LRRTM4. To confirm direct modulation of LRRTM 1 , LRRTM2 or LRRTM4, LRRTM 1 ,

LRRTM2 or LRRTM4 intracellular or extracellular domains are subcloned into expression plasmid vectors such that a fusion protein with C-terminal FLAG epitopes is encoded. These fusion proteins are purified by affinity chromatography, according to manufacturer's instructions, using an Anti-FLAG M2 agarose resin. A LRRTMl, LRRTM2 or LRRTM4 fusion protein is eluted from the Anti-FLAG column by the addition of a FLAG peptide (Asp-Tyr-Lys- Asp-Asp-Asp-Asp-Lys) (Sigma Aldrich, St. Louis, MO) re-suspended in TBS (50 mM Tris HCl pH 7.4, 150 mM NaCl) to a final concentration of 100 μg/ml. Fractions from the column are collected and concentrations of the fusion protein is determined by A280.

A PD-10 column (Amersham, Boston, MA) is used to buffer exchange all eluted fractions containing the LRRTMl, LRRTM2 or LRRTM4-fusion proteins and simultaneously remove excess FLAG peptide. The FLAG- LRRTMl, LRRTM2 or LRRTM4 fusion protein is then conjugated to the S series CM5 chip surface (Biacore™ International AB, Uppsala, Sweden) using amine coupling as directed by the manufacturer. A pH scouting protocol is followed to determine the optimal pH conditions for immobilization. Immobilization is conducted at an empirically determined temperature in PBS, pH 7.4, or another similar buffer following a standard Biacore™ immobilization protocol. The reference spot on the CM5 chip (a non-immobilized surface) serves as background. A third spot on the CM5 chip is conjugated with bovine serum albumin in a similar fashion to serve as a specificity control. Interaction of the putative LRRTMl, LRRTM2 or LRRTM4 modulating analyte, identified in the assay of Example 5, at various concentrations and LRRTMl, LRRTM2 or LRRTM4 are analyzed using the compound characterization wizard on the Biacore™ S51. Binding experiments are completed at 3O⁰C using 50 mM Tris pH 7, 200 uM MnC12 or MgC12 (+ 5% DMSO) or a similar buffer as the running buffer. Prior to each characterization, the instrument is equilibrated three times with assay buffer. Default instructions for characterization are a contact time of 60 seconds, sample injection of 180 seconds and a baseline stabilization of 30 seconds. All solutions are added at a rate of 30 μL/min. Using the BioEvaluation software (Biacore™ International AB, Uppsala, Sweden), each set of sensorgrams derived from the ligand flowing through the LRRTMl , LRRTM2 or LRRTM4-conjugated sensor chip is evaluated and, if binding is observed, an affinity constant determined.

EXAMPLE 7 This example describes a method for making polyclonal antibodies specific for LRRTMl, LRRTM2 and LRRTM4, or a particular peptide fragment or epitope thereof.

The LRRTMl, LRRTM2 or LRRTM4 is produced as described in Example 1 or a peptide fragment comprising a particular amino acid sequence of LRRTMl, LRRTM2 or LRRTM4 is synthesized and coupled to a carrier such as BSA or KLH. Antibodies are generated in New Zealand white rabbits over a 10-week period. The LRRTMl , LRRTM2 or LRRTM4, or ^" peptide fragment, or epitope is emulsified by mixing with an equal volume of Freund's complete adjuvant and injected into three subcutaneous dorsal sites for a total of about 0.1 mg LRRTMl , LRRTM2 or LRRTM4 per immunization. A booster containing about 0.1 mg LRRTMl , LRRTM2 or LRRTM4 or peptide fragment emulsified in an equal volume of Freund's incomplete adjuvant is administered subcutaneously two weeks later. Animals are bled from the articular artery. The blood is allowed to clot and the serum collected by centrifugation. The serum is stored at 20⁰C.

For purification, the LRRTMl, LRRTM2 or LRRTM4 is immobilized on an activated support. Antisera is passed through the sera column and then washed. Specific antibodies are eluted via a pH gradient, collected, and stored in a borate buffer (0.125M total borate) at -0.25 mg/mL. The anti- LRRTMl, LRRTM2 or LRRTM4 antibody titers are determined using ELISA methodology with free CS 1P5 receptor bound in solid phase (1 pg/well). Detection is obtained using biotinylated anti-rabbit IgG, HRP-SA conjugate, and ABTS. EXAMPLE 8

This example describes a method for making monoclonal antibodies specific for LRRTMl, LRRTM2 and LRRTM4. BALB/c mice are immunized with an initial injection of about 1 μg of purified

LRRTMl, LRRTM2 or LRRTM4 per mouse mixed 1 :1 with Freund's complete adjuvant. After two weeks, a booster injection of about 1 μg of the antigen is injected into each mouse intravenously without adjuvant. Three days after the booster injection serum from each of the mice is checked for antibodies specific for the LRRTMl, LRRTM2 or LRRTM4. The spleens are removed from mice positive for antibodies specific for the

LRRTMl, LRRTM2 or LRRTM4 and washed three times with serum-free DMEM and placed in a sterile Petri dish containing about 20 mL of DMEM containing 20% fetal bovine serum, 1 mM pyruvate, 100 units penicillin, and 100 units streptomycin. The cells are released by perfusion with a 23 gauge needle. Afterwards, the cells are pelleted by low-speed centrifugation and the cell pellet is re-suspended in 5 mL 0.17 M ammonium chloride and placed on ice for several minutes. Then 5 mL of 20% bovine fetal serum is added and the cells pelleted by low-speed centrifugation. The cells are then re-suspended in 10 mL DMEM and mixed with mid-log phase myeloma cells in serum-free DMEM to give a ratio of 3:1. The cell mixture is pelleted by low- speed centrifugation, the supernatant fraction removed, and the pellet allowed to stand for 5 minutes. Next, over a period of 1 minute, 1 mL of 50% polyethylene glycol (PEG) in 0.01 M HEPES, pH 8.1, at 37°C is added. After 1 minute incubation at 37°C, 1 mL of DMEM is added for a period of another 1 minute, then a third addition of DMEM is added for a further period of 1 minute. Finally, 10 mL of DMEM is added over a period of 2 minutes. Afterwards, the cells are pelleted by low-speed centrifugation and the pellet re-suspended in DMEM containing 20% fetal bovine serum, 0.016 mM thymidine, 0.1 hypoxanthine, 0.5 μM aminopterin, and 10% hybridoma cloning factor (HAT medium). The cells are then plated into 96-well plates.

After 3, 5, and 7 days, half the medium in the plates is removed and replaced with fresh HAT medium. After 1 1 days, the hybridoma cell supernatant is screened by an ELISA assay. In this assay, 96-well plates are coated with the LRRTMl, LRRTM2 or LRRTM4. One hundred μL of supernatant from each well is added to a corresponding well on a screening plate and incubated for 1 hour at room temperature. After incubation, each well is washed three times with water and 100 μL of a horseradish peroxide conjugate of goat anti-mouse IgG (H+L), A, M (1 : 1 ,500 dilution) is added to each well and incubated for 1 hour at room temperature. Afterwards, the wells are washed three times with water and the substrate OPD/hydrogen peroxide is added and the reaction is allowed to proceed for about 15 minutes at room temperature. Then 100 μL of 1 M HCl is added to stop the reaction and the absorbance of the wells is measured at 490 nm. Cultures that have an absorbance greater than the control wells are removed to two cm2 culture dishes, with the addition of normal mouse spleen cells in HAT medium. After a further three days, the cultures are re-screened as above and those that are positive are cloned by limiting dilution. The cells in each two cm2 culture dish are counted and the cell concentration adjusted to 1 x 105 cells per mL. The cells are diluted in complete medium and normal mouse spleen cells are added. The cells are plated in 96-well plates for each dilution. After 10 days, the cells are screened for growth. The growth positive wells are screened for antibody production; those testing positive are expanded to 2 cm2 cultures and provided with normal mouse spleen cells. This cloning procedure is repeated until stable antibody producing hybridomas are obtained. The stable hybridomas are progressively expanded to larger culture dishes to provide stocks of the cells. Production of ascites fluid is performed by injecting intraperitoneally 0.5 mL of pristane into female mice to prime the mice for ascites production. After 10 to 60 days, 4.5 x Iθ6 cells are injected intraperitoneally into each mouse and ascites fluid is harvested between 7 and 14 days later.

While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.

Claims

WHAT IS CLAIMED:

1. A method for screening for analytes that antagonize processing of amyloid precursor protein (APP) to Aβ peptide (Aβ) comprising: (a) providing recombinant cells, which express at least one LRRTM, selected from the group consisting of LRRTMl, LRRTM2 and LRRTM4, and APP;

(b)^' incubating the cells of step (a) in a culture medium under conditions suitable for expression of the LRRTM and APP with an analyte;

(c) removing the culture medium of step (b) from cells; and (d) determining the amount of at least one processing product of APP, selected from the group consisting of sAPPβ and Aβ, produced in the medium; wherein a decrease in the amount of the processing product produced in the medium with the analyte, as compared to the amount of the processing product produced in the medium without the analyte, indicates that the analyte is an antagonist of the processing of the APP to Aβ.

2. The method of claim 1 wherein the recombinant cells each comprise a first nucleic acid that encodes an LRRTM, selected from the group consisting of LRRTMl , LRRTM2 and LRRTM4, operably linked to a first heterologous promoter and a second nucleic acid that encodes an APP operably linked to a second heterologous promoter.

3. The method of claim 2 wherein the APP is APP NFEV.

4. The method of claim 1 wherein a control is provided which comprises providing recombinant cells which express APP but not LRRTM.

5. A method for screening for analytes that antagonize processing of amyloid precursor protein (APP) to Aβ peptide (Aβ), comprising:

(a) providing recombinant cells, which express at least one LRRTM, selected from the group consisting of LRRTMl, LRRTM2 and LRRTM4, and a recombinant APP, comprising APP fused to a transcription factor that when removed from APP during processing produces an active transcription factor, and a reporter gene operably linked to a promoter inducible by the transcription factor;

(b) incubating the cells of step (a) in a culture medium under conditions for expression of the LRRTM and the recombinant APP with an analyte; and (c) determining expression of the reporter gene; wherein a decrease in expression of the reporter gene with the analyte, as compared to expression of the reporter gene in recombinant cells in a culture medium without the analyte, indicates that the analyte is an antagonist of the processing of the APP to Aβ peptide.

6. A method for treating Alzheimer's disease in an individual comprising providing to the individual an effective amount of an agonist of LRRTMl or LRRTM2 or an antagonist of LRRTM4 activity.

7. A method for identifying an individual who has Alzheimer's disease or is at risk of developing Alzheimer's disease comprising obtaining a sample from the individual and measuring the amount of LRRTMl, LRRTM2 or LRRTM4 in the sample.

8. The use of an agonist of LRRTMl or LRRTM2 or an antagonist of

LRRTM4 for the manufacture of a medicament for the treatment of Alzheimer's disease.