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  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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  • A61K38/46—Hydrolases (3)

Definitions

• This application relates to compositions and methods for inhibiting or reducing the activity, signaling, and/or function of leukocyte-common antigen related (LAR) family of phosphatases and to methods and compositions for inhibiting ⁇ -amyloidosis and treating Alzheimer's disease.
• LAR leukocyte-common antigen related
• AD Alzheimer's disease
• ⁇ -amyloid ( ⁇ ) peptides in the brain, a process also known as ⁇ - amyloidosis, which is often accompanied by neuroinflammation and formation of neurofibrillary tangles containing Tau, a microtubule binding protein.
• ⁇ peptides mainly derive from sequential cleavage of neuronal amyloid precursor protein (APP) by the ⁇ - and ⁇ -secretases.
• APP neuronal amyloid precursor protein
• molecular regulation of the amyloidogenic secretase activities remains poorly understood, hindering the design of therapeutics to specifically target the APP amyloidogenic pathway.
• Embodiments described herein relate to methods of inhibiting and/or reducing ⁇ -amyloid accumulation and/or Tau aggregation in a subject in need thereof.
• the methods include administering to the subject a therapeutic agent that inhibits one or more of catalytic activity, signaling, and function of the LAR family phosphatases.
• the LAR family phosphatase is a receptor protein tyrosine phosphatase sigma ( ⁇ )
• the therapeutic agent includes a therapeutic peptide having an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to about 10 to about 20 consecutive amino acids of the wedge domain of ⁇ .
• therapeutic agent can include a therapeutic peptide selected from the group consisting of SEQ ID NOs: 9-13 andl6.
• the LAR family phosphatase is a receptor protein tyrosine phosphatase sigma ( ⁇ )
• the therapeutic agent can include a therapeutic peptide at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 16.
• the therapeutic peptide can include, for example, a conservative substitution of an amino acid of at least one, two, three, or four of residue 4, 5, 6, 7, 9, 10, 12, or 13 of SEQ ID NO: 16.
• the therapeutic agent is administered systemically or locally to the subject or to a neural cell, glial cell, glial progenitor cell, or a neural progenitor cell.
• the therapeutic agent includes a transport moiety that is linked to the therapeutic peptide and facilitates uptake of the therapeutic peptide by the cell.
• the transport moiety can be an HIV Tat transport moiety.
• the cell is in a subject being treated, and the therapeutic agent is administered locally or systemically to the subject being treated.
• the therapeutic peptide is expressed by a cell of the subject.
• Embodiments herein also relate to methods of treating diseases, disorders, and/or conditions associated with ⁇ -amyloid accumulation and/or Tau aggregation in a subject in need thereof.
• the methods include administering to the subject a therapeutic agent that inhibits one or more of catalytic activity, signaling, and function of the LAR family phosphatases.
• the disease, disorder, and/or condition includes at least one of a disease, disorder, and/or condition of the nervous system.
• the disease, disorder, and/or condition of the nervous system includes at least one of a neurological disorder, neuropsychiatric disorder, neural injury, neural toxicity disorder, a neuropathic pain, and neural degenerative disorders.
• the neurological disorder can include at least one of Alzheimer's disease or dementias related to Alzheimer's disease.
• FIGs. l(A-I) illustrate images and immunoblots showing ⁇ is an APP binding partner in the brain.
• A-F Colocalization of ⁇ (A) and APP (B) in hippocampal CA1 neurons of adult rat is shown by confocal imaging. Nuclei of CA1 neurons are stained with DAPI (C).
• D Merge of three channels. Scale bar, 50 ⁇ .
• E Zoom-in image of the soma layer in D. Arrows, intensive colocalization of ⁇ and APP in the initial segments of apical dendrites; arrow heads, punctates of colocalization in the perinuclear regions. Scale bar, 20 ⁇ .
• F Zoom-in image of the very fine grained punctates in the axonal compartment in D. Arrows points to the colocalization of ⁇ and APP in axons projecting perpendicular to the focal plane. Scale bar, 10 ⁇ .
• G Schematic diagram of ⁇ expressed on cell surface as a two-subunit complex. ⁇ is post-translationally processed into an extracellular domain (ECD) and a transmembrane-intracellular domain (ICD). These two subunits associate with each other through noncovalent bond. Ig-like, immunoglobulin-like domains; FNIII-like, fibronectin Ill-like domains; Dl and D2, two phosphatase domains.
• H Co- immunoprecipitation (co-IP) of ⁇ and APP from mouse forebrain lysates.
• Left panels expression of ⁇ and APP in mouse forebrains.
• Right panels IP using an antibody specific for the C-terminus (C-term) of APP.
• Full length APP (APP FL) is detected by anti- APP C-term antibody.
• H ⁇ co-IP with APP from forebrain lysates of wild type but not ⁇ -deficient mice (Balb/c background), detected by an antibody against ⁇ -ECD.
• I ⁇ co-IP with APP from forebrain lysates of wild type but not APP knockout mice (B6 background), detected by an antibody against ⁇ -ICD. Dotted lines in I indicate lanes on the same western blot exposure that were moved adjacent to each other. Images shown are representatives of at least three independent experiments.
• FIGs. 2(A-I) illustrate a schematic diagram, immunoblots, and graphs showing genetic depletion of ⁇ reduces ⁇ -amyloidogenic products of APP.
• A Schematic diagram showing amyloidogenic processing of APP by the ⁇ - and ⁇ -secretases.
• Full length APP APP FL
• sAPP soluble N-terminal
• CTF C-terminal fragments.
• APP CTF can be further processed by ⁇ -secretase into a C-terminal intracellular domain (AICD) and an ⁇ peptide.
• Aggregation of ⁇ is a definitive pathology hallmark of AD.
• ⁇ deficiency reduces the level of an APP CTF at about 15 KD in mouse forebrain lysates, without affecting the expression of APP FL.
• Antibody against the C- terminus of APP recognizes APP FL and CTFs of both mouse and human origins.
• C and D The 15 KD APP CTF is identified as CTF by immunoprecipitation (IP) followed with western blot analysis, using a pair of antibodies as marked in the diagram (A).
• IP immunoprecipitation
• A Antibodies against amino acids 1-16 of ⁇ (anti- ⁇ 1-16) detect CTF but not CTFa, as the epitope is absent in CTFa.
• C Mouse endogenous CTF level is reduced in ⁇ - deficient mouse brains.
• G and H ⁇ deposition in the hippocampus of 10-month old TgAPP-SwDI mice. ⁇ (green) is detected by immunofluorescent staining using anti- ⁇ antibodies clone 6E10 (G) and clone 4G8 (H). DAPI staining is shown. ⁇ deficiency significantly decreases ⁇ burden in the brains of TgAPP-SwDI mice.
• H Upper panels, the stratum oriens layer between dorsal subiculum (DS) and CAl (also shown with arrows in G); middle panels, oriens layer between CAl and CA2; lower panels, the hilus of dentate gyrus (DG, also shown with arrow heads in g).
• Total integrated density of ⁇ in DG hilus was normalized to the area size of the hilus to yield the average intensity as show in the bar graph. Mean value of each group was normalized to that of 16 month old TgAPP-SwDI mice expressing wild type ⁇ . All p values, Student's t test, 2-tailed. Error bars, SEM.
• Figs. 3(A-C) illustrate an immunoblot, graph, and plot showing lower affinity between BACEl and APP in ⁇ -deficient brains.
• A Coimmunoprecipitation experiments show nearly equal BACE1-APP association in wild type and ⁇ - deficient mouse brains under mild detergent condition (1% NP40). However, in ⁇ -deficient brains, BACE1- APP association detected by co-immunoprecipitation is more vulnerable to increased detergent stringency as compared to that in wild type brains.
• Panels of blots show full length APP (APP FL) pulled down with an anti-BACEl antibody from mouse forebrain lysates.
• NP40 Nonidet P-40, non-ionic detergent.
• Figs. 4(A-F) illustrate immunoblots showing ⁇ does not generically modulate ⁇ and ⁇ secretases. Neither expression levels of the secretases or their activities on other major substrates are affected by ⁇ depletion.
• Mouse forebrain lysates with or without ⁇ were analyzed by western blot.
• a and B ⁇ deficiency does not change expression level of BACEl (A) or ⁇ -secretase subunits (B).
• Presenilinl and 2 (PS 1/2) are the catalytic subunits of ⁇ -secretase, which are processed into Nterminal and C-terminal fragments (NTF and CTF) in their mature forms.
• Nicastrin, Presenilin Enhancer 2 (PEN2), and APH1 are other essential subunits of ⁇ -secretase.
• C ⁇ deficiency does not change the level of Neuregulinl (NGR1)
• CTF the C-terminal cleavage product by BACE1.
• NRG1 FL full length Neuregulinl.
• D The level of Notch cleavage product by ⁇ - secretase is not affected by ⁇ deficiency.
• TMIC Notch transmembrane/intracellular fragment, which can be cleaved by ⁇ -secretase into a C-terminal intracellular domain NICD (detected by an antibody against Notch C-terminus in the upper panel, and by an antibody specific for ⁇ - secretase cleaved NICD in the lower panel).
• E Actin loading control for A and C.
• F Actin loading control for B and D. All images shown are representatives of at least three independent experiments. All images shown are representatives of at least three independent experiments.
• FIGs. 5(A-I) illustrate images and a graph showing ⁇ deficiency attenuates reactive astrogliosis in APP transgenic mice.
• Expression level of GFAP a marker of reactive astrocytes, is suppressed in the brains of TgAPP-SwDI mice by ⁇ depletion.
• Representative images show GFAP and DAPI staining of nuclei in the brains of 9-month old TgAPP-SwDI mice.
• A-D Dentate gyrus (DG) of the hippocampus; scale bars, 100 ⁇ .
• E-H Primary somatosensory cortex; scale bars, 200 ⁇ .
• I ImageJ quantification of GFAP level in DG hilus from TgAPP-SwDI mice aged between 9 to 11 months.
• APP-SwDI(-)PTPo(+/+) non- trans genie wild type littermates (expressing ⁇ but not the human APP transgene).
• Total integrated density of GFAP in DG hilus was normalized to the area size of the hilus to yield average intensity as shown in the bar graph.
• Figs. 6(A-G) illustrates images and a graph showing ⁇ deficiency protects APP transgenic mice from synaptic loss. Representative images show immunofluorescent staining of presynaptic marker Synaptophysin in the mossy fiber terminal zone of CA3 region. A-F, Synaptophysin, red; DAPI, blue. Scale bars, 100 ⁇ . G, ImageJ quantification of Synaptophysin expression level in CA3 mossy fiber terminal zone from mice aged between 9 to 11 months. Total integrated density of Synaptophysin in CA3 mossy fiber terminal zone was normalized to the area size to yield average intensity as shown in the bar graph.
• Figs. 7(A-H) illustrates a schematic diagram, images, and graph showing ⁇ deficiency mitigates Tau pathology in TgAPP-SwDI mice.
• A Schematic diagram depicting distribution pattern of Tau aggregation detected by immunofluorescent staining using an anti- Tau antibody, in brains of 9 to 11 month-old TgAPP-SwDI transgenic mice.
• Aggregated Tau is found most prominently in the molecular layer of piriform and entorhinal cortex, and occasionally in hippocampal regions in APPSwDI(+)PTPo(+/+) mice.
• B ⁇ deficiency diminishes Tau aggregation.
• Bar graph shows quantification of Tau aggregation in coronal brain sections from 4 pairs of age- and sexmatched APP-SwDI(+)PTPo(+/+) and APP- SwDI(+)PTPo(-/-) mice of 9 to 11 month-old. For each pair, the value from APP- SwDI(+)PTPo(-/-) sample is normalized to the value from APP-SwDI(+)PTPo(+/+) sample, p value, Student's t test, 2-tailed. Error bar, SEM.
• C D, Representative images of many areas with Tau aggregation in APP-SwDI(+)PTPo(+/+) brains.
• C and F Hippocampal regions.
• D-H Piriform cortex.
• E Staining of a section adjacent to d, but without primary antibody (no 1° Ab).
• H no Tau aggregates are detected in aged-matched non- trans genie wild type littermates (expressing ⁇ but not the human APP transgene). Arrows points to Tau aggregates. Scale bars, 50 ⁇ .
• Figs. 8(A-C) illustrate graphs showing ⁇ deficiency rescues behavioral deficits in TgAPP-SwDI mice.
• A In the Y-maze assay, performance of spatial navigation is scored by the percentage of spontaneous alternations among total arm entries. Values are normalized to that of non-transgenic wild type APP-SwDI(-)PTPo(+/+) mice within the colony. Compared to non-transgenic wild type mice, APP-SwDI(+)PTPo(+/+) mice show deficit of shortterm spatial memory, which is rescued by genetic depletion of ⁇ in APP- SwDI(+)PTPo(-/-) mice.
• Ages of all genotype groups are similarly distributed between 4 and 11 months.
• B, C Novel object test.
• NO novel object.
• FO familiar object. Attention to NO is measured by the ratio of NO exploration to total object exploration (NO+FO) in terms of exploration time (B) and visiting frequency (C). Values are normalized to that of non- transgenic wild type mice.
• APP-SwDI(+)PTPo(+/+) mice showed decreased interest in NO compared to wild type APPSwDI(-)PTPo(+/+) mice.
• the deficit is reversed by ⁇ depletion in APP-SwDI(+)PTPo(-/-) mice.
• APPSwDI(-)PTPo(+/+), n 28 (19 females and 9 males);
• Ages of all groups are similarly distributed between 4 and 11 months. All p values, Student's t test, 2-tailed. Error bars, SEM.
• Fig. 9 illustrates a graph showing ⁇ deficiency restores short-term spatial memory in TgAPP-SwDI mice.
• performance of spatial navigation is scored by the percentage of spontaneous alternations among total arm entries. The raw values shown here are before normalization in Fig. 6A.
• APP-SwDI(+)PTPo(+/+) mice show deficit of short-term spatial memory, which is rescued by genetic depletion of ⁇ .
• Figs. lO(A-D) illustrate graphs showing ⁇ deficiency enhances novelty exploration by TgAPP-SwDI mice.
• NO novel object.
• FO familiar object.
• a and B in novel object test, NO preference is measured by the ratio between NO and FO exploration, where NO/FO >1 indicates preference for NO.
• C and D attention to NO is additionally measured by the discrimination index, NO/(NO+FO), the ratio of NO exploration to total object exploration (NO+FO).
• the raw values shown here in c and d are before normalization in Fig. 6B and C. Mice of this colony show a low baseline of the NO/(NO+FO)
• discrimination index likely inherited from their parental Balb/c line.
• the discrimination index is slightly above 0.5 (chance value), similar to what was previously reported for the Balb/c wild type mice 27.
• a sole measurement of the discrimination index may not reveal the preference for NO as does the NO/FO ratio.
• the NO/(NO+FO) index is most commonly used as it provides a normalization of the NO exploration to total object exploration activity.
• Figs. 1 l(A-C) illustrate ⁇ deficiency improves behavioral performance of TgAPP-SwInd mice.
• A Performance of spatial navigation is scored by the percentage of spontaneous alternations among total arm entries in the Y-maze assay.
• APPSwInd(+)PTPo(+/+) mice showed improved short-term spatial memory.
• Ages of both genotype groups are similarly distributed between 4 and 11 months.
• Figs. 12 illustrates an immunoblot showing the effects ISP in combination with a ⁇ -secretase inhibitor on APP processing compared to a ⁇ -secretase inhibitor administered alone or a BACE1 inhibitor administered in combination with a ⁇ -secretase inhibitor.
• a, b, and c means a, b, c, ab, ac, be, or abc.
• the use of "or” herein is the inclusive or.
• administering includes dispensing, delivering or applying an active compound in a pharmaceutical formulation to a subject by any suitable route for delivery of the active compound to the desired location in the subject (e.g., to thereby contact a desired cell such as a desired neuron), including administration into the cerebrospinal fluid or across the blood- brain barrier, delivery by either the parenteral or oral route, intramuscular injection, subcutaneous or intradermal injection, intravenous injection, buccal administration, transdermal delivery and administration by the rectal, colonic, vaginal, intranasal or respiratory tract route.
• the agents may, for example, be administered to a comatose, anesthetized or paralyzed subject via an intravenous injection or may be administered intravenously to a pregnant subject to stimulate axonal growth in a fetus.
• Specific routes of administration may include topical application (such as by eyedrops, creams or erodible formulations to be placed under the eyelid), intraocular injection into the aqueous or the vitreous humor, injection into the external layers of the eye, such as via subconjunctival injection or subtenon injection, parenteral administration or via oral routes.
• antibody includes human and animal mAbs, and preparations of polyclonal antibodies, synthetic antibodies, including recombinant antibodies (antisera), chimeric antibodies, including humanized antibodies, anti-idiotopic antibodies and derivatives thereof.
• a portion or fragment of an antibody refers to a region of an antibody that retains at least part of its ability (binding specificity and affinity) to bind to a specified epitope.
• epitope or “antigenic determinant” refers to a site on an antigen to which antibody paratope binds.
• Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
• An epitope typically includes at least 3, at least 5, or 8 to 10, or about 13 to 15 amino acids in a unique spatial conformation.
• Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., 66 EPITOPE MAPPING
• central nervous system (CNS) neurons include the neurons of the brain, the cranial nerves and the spinal cord.
• a "chimeric protein” or “fusion protein” is a fusion of a first amino acid sequence encoding a polypeptide with a second amino acid sequence defining a domain (e.g., polypeptide portion) foreign to and not substantially homologous with the domain of the first polypeptide.
• a chimeric protein may present a foreign domain, which is found (albeit in a different protein) in an organism, which also expresses the first protein, or it may be an "interspecies", “intergenic”, etc. fusion of protein structures expressed by different kinds of organisms.
• contacting neurons or “treating neurons” refers to any mode of agent delivery or “administration,” either to cells or to whole organisms, in which the agent is capable of exhibiting its pharmacological effect in neurons.
• Contacting neurons includes both in vivo and in vitro methods of bringing an agent of the invention into proximity with a neuron. Suitable modes of administration can be determined by those skilled in the art and such modes of administration may vary between agents.
• an "effective amount" of an agent or therapeutic peptide is an amount sufficient to achieve a desired therapeutic or pharmacological effect, such as an amount that is capable of inhibiting ⁇ -amyloid accumulation of Tau aggregation.
• An effective amount of an agent as defined herein may vary according to factors such as the disease state, age, and weight of the subject, and the ability of the agent to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects of the active compound are outweighed by the therapeutically beneficial effects.
• a "therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
• a therapeutic result may be, e.g., lessening of symptoms, prolonged survival, improved mobility, and the like.
• a therapeutic result need not be a "cure.”
• the term "expression” refers to the process by which nucleic acid is translated into peptides or is transcribed into RNA, which, for example, can be translated into peptides, polypeptides or proteins. If the nucleic acid is derived from genomic DNA, expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA. For heterologous nucleic acid to be expressed in a host cell, it must initially be delivered into the cell and then, once in the cell, ultimately reside in the nucleus.
• the term "genetic therapy” involves the transfer of heterologous DNA to cells of a mammal, particularly a human, with a disorder or conditions for which therapy or diagnosis is sought.
• the DNA is introduced into the selected target cells in a manner such that the heterologous DNA is expressed and a therapeutic product encoded thereby is produced.
• the heterologous DNA may in some manner mediate expression of DNA that encodes the therapeutic product; it may encode a product, such as a peptide or RNA that in some manner mediates, directly or indirectly, expression of a therapeutic product.
• Genetic therapy may also be used to deliver nucleic acid encoding a gene product to replace a defective gene or supplement a gene product produced by the mammal or the cell in which it is introduced.
• the introduced nucleic acid may encode a therapeutic compound that is not normally produced in the mammalian host or that is not produced in therapeutically effective amounts or at a therapeutically useful time.
• the heterologous DNA encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof.
• gene refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and
• heterologous nucleic acid sequence is typically DNA that encodes RNA and proteins that are not normally produced in vivo by the cell in which it is expressed or that mediates or encodes mediators that alter expression of endogenous DNA by affecting transcription, translation, or other regulatable biochemical processes.
• a heterologous nucleic acid sequence may also be referred to as foreign DNA. Any DNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which it is expressed is herein encompassed by heterologous DNA.
• heterologous DNA include, but are not limited to, DNA that encodes traceable marker proteins, such as a protein that confers drug resistance, DNA that encodes therapeutically effective substances and DNA that encodes other types of proteins, such as antibodies.
• Antibodies that are encoded by heterologous DNA may be secreted or expressed on the surface of the cell in which the heterologous DNA has been introduced.
• homology and “identity” are used synonymously throughout and refer to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous or identical at that position. A degree of homology or identity between sequences is a function of the number of matching or homologous positions shared by the sequences.
• neurological disorder includes a disease, disorder, or condition which directly or indirectly affects the normal functioning or anatomy of a subject's nervous system.
• parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
• systemic administration means the administration of a compound, drug or other material other than directly into a target tissue (e.g. , the central nervous system), such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
• target tissue e.g. , the central nervous system
• the term “patient” or “subject” or “animal” or “host” refers to any mammal.
• the subject may be a human, but can also be a mammal in need of veterinary treatment, e.g., domestic animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, fowl, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).
• domestic animals e.g., dogs, cats, and the like
• farm animals e.g., cows, sheep, fowl, pigs, horses, and the like
• laboratory animals e.g., rats, mice, guinea pigs, and the like.
• peripheral nervous system includes the neurons which reside or extend outside of the CNS.
• PNS is intended to include the neurons commonly understood as categorized in the peripheral nervous system, including sensory neurons and motor neurons.
• polynucleotide sequence and “nucleotide sequence” are also used interchangeably herein.
• peptide or “polypeptide” are used interchangeably herein and refer to compounds consisting of from about 2 to about 90 amino acid residues, inclusive, wherein the amino group of one amino acid is linked to the carboxyl group of another amino acid by a peptide bond.
• a peptide can be, for example, derived or removed from a native protein by enzymatic or chemical cleavage, or can be prepared using conventional peptide synthesis techniques (e.g., solid phase synthesis) or molecular biology techniques (see Sambrook et al., MOLECULAR CLONING: LAB. MANUAL (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989)).
• a "peptide” can comprise any suitable L- and/or D-amino acid, for example, common a-amino acids (e.g., alanine, glycine, valine), non-a-amino acids (e.g., P-alanine, 4-aminobutyric acid, 6aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g., citrulline, homocitruline, homoserine, norleucine, norvaline, ornithine).
• the amino, carboxyl and/or other functional groups on a peptide can be free (e.g., unmodified) or protected with a suitable protecting group.
• Suitable protecting groups for amino and carboxyl groups and means for adding or removing protecting groups are known in the art. See, e.g., Green & Wuts, PROTECTING GROUPS IN ORGANIC SYNTHESIS (John Wiley & Sons, 1991).
• the functional groups of a peptide can also be derivatized (e.g., alkylated) using art-known methods.
• Peptides can be synthesized and assembled into libraries comprising a few too many discrete molecular species. Such libraries can be prepared using well-known methods of combinatorial chemistry, and can be screened as described herein or using other suitable methods to determine if the library comprises peptides which can antagonize CSPG- ⁇ interaction. Such peptide antagonists can then be isolated by suitable means.
• peptidomimetic refers to a protein-like molecule designed to mimic a peptide.
• Peptidomimetics typically arise either from modification of an existing peptide, or by designing similar systems that mimic peptides, such as peptoids and ⁇ -peptides. Irrespective of the approach, the altered chemical structure is designed to advantageously adjust the molecular properties such as, stability or biological activity. These modifications involve changes to the peptide that do not occur naturally (such as altered backbones and the incorporation of nonnatural amino acids).
• progenitor cells are cells produced during differentiation of a stem cell that have some, but not all, of the characteristics of their terminally-differentiated progeny. Defined progenitor cells, such as “neural progenitor cells,” are committed to a lineage, but not to a specific or terminally differentiated cell type.
• stem cell means a cell that can undergo self-renewal (i.e., progeny with the same differentiation potential) and also produce progeny cells that are more restricted in differentiation potential.
• a stem cell would also encompass a more differentiated cell that has dedifferentiated, for example, by nuclear transfer, by fusions with a more primitive stem cell, by introduction of specific transcription factors, or by culture under specific conditions.
• a polynucleotide sequence (DNA, RNA) is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that polynucleotide sequence.
• the term "operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the polynucleotide sequence to be expressed, and maintaining the correct reading frame to permit expression of the polynucleotide sequence under the control of the expression control sequence, and production of the desired polypeptide encoded by the polynucleotide sequence.
• tissue-specific promoter means a nucleic acid sequence that serves as a promoter, i.e., regulates expression of a selected nucleic acid sequence operably linked to the promoter, and which affects expression of the selected nucleic acid sequence in specific cells of a tissue, such as cells of epithelial cells.
• tissue-specific promoter means a nucleic acid sequence that serves as a promoter, i.e., regulates expression of a selected nucleic acid sequence operably linked to the promoter, and which affects expression of the selected nucleic acid sequence in specific cells of a tissue, such as cells of epithelial cells.
• the term also covers so-called “leaky” promoters, which regulate expression of a selected nucleic acid primarily in one tissue, but cause expression in other tissues as well.
• transfection is used to refer to the uptake of foreign DNA by a cell.
• a cell has been "transfected” when exogenous DNA has been introduced inside the cell membrane.
• transfection techniques are generally known in the art. See, e.g., Graham et al., Virology 52:456 (1973); Sambrook et al., Molecular Cloning: A Laboratory Manual (1989); Davis et al., Basic Methods in Molecular Biology (1986); Chu et al., Gene 13: 197 (1981).
• exogenous DNA moieties such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.
• the term captures chemical, electrical, and viral-mediated transfection procedures.
• transcriptional regulatory sequence is a generic term used throughout the specification to refer to nucleic acid sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.
• transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequence), which controls the expression of the recombinant gene in a cell-type in which expression is intended.
• the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences, which control transcription of the naturally occurring form of a protein.
• vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• Preferred vectors are those capable of one or more of, autonomous replication and expression of nucleic acids to which they are linked.
• Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
• wild type refers to the naturally-occurring polynucleotide sequence encoding a protein, or a portion thereof, or protein sequence, or portion thereof, respectively, as it normally exists in vivo.
• nucleic acid refers to polynucleotides, such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
• DNA deoxyribonucleic acid
• RNA ribonucleic acid
• the term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double- stranded polynucleotides.
• the agents, compounds, compositions, antibodies, etc. used in the methods described herein are considered to be purified and/or isolated prior to their use.
• Purified materials are typically "substantially pure", meaning that a nucleic acid, polypeptide or fragment thereof, or other molecule has been separated from the components that naturally accompany it.
• the polypeptide is substantially pure when it is at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and other organic molecules with which it is associated naturally.
• a substantially pure polypeptide may be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in a cell that does not normally express that protein, or by chemical synthesis.
• isolated materials have been removed from their natural location and environment.
• the domain or fragment is substantially free from amino acid sequences that flank the protein in the naturally-occurring sequence.
• isolated DNA means DNA has been substantially freed of the genes that flank the given DNA in the naturally occurring genome.
• isolated DNA encompasses, for example, cDNA, cloned genomic DNA, and synthetic DNA.
• portion when referring to a polypeptide of the present invention include any polypeptide that retains at least some biological activity referred to herein (e.g., inhibition of an interaction such as binding).
• Polypeptides as described herein may include portion, fragment, variant, or derivative molecules without limitation, as long as the polypeptide still serves its function.
• Polypeptides or portions thereof of the present invention may include proteolytic fragments, deletion fragments and in particular, or fragments that more easily reach the site of action when delivered to an animal.
• Embodiments described herein relate to methods of inhibiting and/or reducing ⁇ -amyloid accumulation and/or Tau aggregation in a subject in need thereof, ⁇ -amyloid accumulation and Tau aggregation are hallmarks of Alzheimer's disease, yet their underlying molecular mechanisms remain obscure.
• neuronal receptor ⁇ mediates both ⁇ -amyloid and Tau pathogenesis in two mouse models.
• ⁇ binds to ⁇ - amyloid precursor protein (APP).
• APP ⁇ - amyloid precursor protein
• a small peptide mimetic of the wedge shaped domain (i.e., wedge domain) of the intracellular catalytic domain of ⁇ can suppress APP amyloidogenic processing by BACE1 , to a similar degree as ⁇ depletion.
• a peptide mimetic of the wedge domain of ⁇ when delivered to a subject in need thereof can inhibit both ⁇ -amyloid and Tau pathogenesis, actively suppress ⁇ -amyloid compared to passive amyloid immunotherapies, and regulate APP processing without affecting other substrates of the secretases, thus providing a safer therapy than secretase inhibitors.
• a therapeutic agent that inhibits one or more of catalytic activity, signaling, and function of a LAR family phosphatases, such as ⁇ can be administered to a subject in need thereof to inhibit and/or reduce ⁇ -amyloid accumulation and/or Tau aggregation in the subject in need thereof and/or treat Alzheimer' s disease and/or dementias related to Alzheimer's disease.
• the activity, signaling, and/or function of the LAR family phosphatases can be suppressed, inhibited, and/or blocked in several ways including: direct inhibition of the activity of the intracellular domain of the LAR family phosphatases (e.g., by using small molecules, peptidomimetics, or dominant negative polypeptides); activation of genes and/or proteins that inhibit one or more of, the activity, signaling, and/or function of the intracellular domain of the LAR family phosphatases (e.g., by increasing the expression or activity of the genes and/or proteins); inhibition of genes and/or proteins that are downstream mediators of the LAR family phosphatases (e.g., by blocking the expression and/or activity of the mediator genes and/or proteins); introduction of genes and/or proteins that negatively regulate one or more of, activity, signaling, and/or function of LAR family phosphatases (e.g., by using recombinant gene expression vectors, recombinant viral vectors or
• the therapeutic agent that inhibits or reduces one or more of the activity, signaling, and/or function of the LAR family phosphatase, such as ⁇ can include an agent that decreases and/or suppresses the activity, signaling, and/or function of the LAR family phosphatase without inhibiting binding to or activation the LAR family phosphatases by proteoglycans, such as CSPG.
• agents can be delivered intracellularly or extracellularly and once delivered to produce a neurosalutory effect.
• the neurosalutary effect can include a response or result favorable to the health or function of a neuron, of a part of the nervous system, or of the nervous system generally. Examples of such effects include improvements in the ability of a neuron or portion of the nervous system to resist insult, to regenerate, to maintain desirable function, to grow or to survive.
• the neurosalutary effect can include producing or effecting such a response or improvement in function or resilience within a component of the nervous system.
• Examples of producing a neurosalutary effect would include stimulating axonal outgrowth after injury to a neuron; rendering a neuron resistant to apoptosis; rendering a neuron resistant to a toxic compound such as ⁇ -amyloid, ammonia, or other neurotoxins; reversing age-related neuronal atrophy or loss of function; reversing age-related loss of cholinergic innervation, reversing and/or reducing dieback, and/or promoting neural sprouting.
• a toxic compound such as ⁇ -amyloid, ammonia, or other neurotoxins
• LAR family of phosphatases One potential mechanism for regulation, modulation, and/or inhibition of LAR family of phosphatases involves dimerization of the intracellular portion of the LAR family of phosphatases.
• receptor tyrosine kinases which are active as dimers and inactive as monomers
• protein tyrosine phosphatases PTPs
• PTPalpha PTP1B
• CD45 protein tyrosine phosphatases
• ligands to LAR family of phosphatases can direct the activation state of LAR family of phosphatase, such as LAR and ⁇ . Therefore, mimicking dimerization with intracellular- targeted therapies can directly inactivate LAR family of phosphatases without alteration of the extracellular matrix or other ligands.
• the therapeutic agent that inhibits or reduces one or more of the activity, signaling, and/or function of the LAR family phosphatase, such as PTPo can include a therapeutic peptide or small molecule that binds to and/or complexes with the intracellular domain of at least one LAR family phosphatase to inhibit the activity, signaling, and/or function of the LAR family phosphatase. Accordingly, therapeutic peptides or small molecules that binds to and/or complexes with the intracellular domain of at least one LAR family phosphatase of neural cells can be used to inhibit ⁇ -amyloid accumulation or Tau aggregation.
• the therapeutic agent can be a peptide mimetic of the wedged shaped domain (i.e., wedge domain) of the intracellular catalytic domain of the LAR family phosphatases.
• wedge domain the intracellular catalytic domain of the LAR family phosphatases.
• Structural and sequence analysis has revealed that all members of the LAR family contain a conserved 24 amino acid wedge-shaped helix- loop-helix motif in the first intracellular catalytic domain that can potentially mediate homo/heterophilic receptor interaction.
• Table 1 lists the amino acid sequences of intracellular portions of the LAR family phosphatase members that contain the wedge domain.
• the 24 amino acid wedge domain of these intracellular portions of LAR family phosphatases is identified by underlining. While the specific structure of the wedge domain is conserved through most LAR family wedge domains, the exact amino acids that make up the wedge domains vary between individual proteins and sub-families.
• Wedge domains of specific LAR family members were found to engage in homophilic interaction or binding with their specific LAR family member.
• the wedge domain of LAR was able to specifically interact with full length LAR, and not other family members such as ⁇ , in pull-down assays.
• in vitro binding assays showed that wedge domain peptides (wedge domain + HIV-TAT) of PTPmu and LAR specifically homophillically aggregated instead of binding promiscuously with each other.
• the wedge domain of LAR was unable to bind to sigma, showing specificity even between similar family members.
• the therapeutic agent can be a peptide mimetic of the wedge shaped domain (i.e., wedge domain) of the intracellular catalytic domain of ⁇ , such as described, for example, in WO 2013/155103A1, which is herein incorporated by reference in its entirety.
• Peptide mimetics of the wedge domain of the ⁇ when expressed in cells (e.g., neural cells) or conjugated to an intracellular transport moiety can be used to abolish ⁇ signaling in a neural cell and inhibit ⁇ -amyloid accumulation and Tau aggregation.
• Binding of these therapeutic peptides to ⁇ intact wedge domain can potentially: (i) interfere with the ability for ⁇ to interact with target proteins, such as phosphatase targets; (ii) interfere with activity promoting intermolecular interactions between ⁇ and another domain contained in ⁇ , such as the catalytically inactive second phosphatase domain D2; prevent access of proteins to the active phosphatase site; (iii) out- compete normal interactors of the wedge domain; and/or (iv) sterically inhibit phosphatase activity.
• target proteins such as phosphatase targets
• interfere with activity promoting intermolecular interactions between ⁇ and another domain contained in ⁇ , such as the catalytically inactive second phosphatase domain D2 prevent access of proteins to the active phosphatase site
• out- compete normal interactors of the wedge domain and/or (iv) sterically inhibit phosphatase activity.
• the peptide mimetic i.e., therapeutic peptide
• the peptide mimetic can include, consist essentially, and/or consist of about 10 to about 20 amino acids and have an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% identical to an about 10 to about 20 consecutive amino acid portion of the amino acid sequence of the wedge domains of LAR family phosphatases, such as ⁇ .
• the therapeutic peptide can include, consist essentially, and/or consist of about 10 to about 20 amino acids and have an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% identical to about 10 to about 20 consecutive amino acids of the wedge domain of ⁇ .
• the first alpha helix of the wedge domain of ⁇ includes amino acids 1-10, the turn region includes amino acids 11-14, and the second alpha helix includes amino acids 15-24.
• the first alpha helix of the wedge domain of human ⁇ has the amino acid sequence of DMAEHTERLK (SEQ ID NO: 13), the turn has the amino acid sequence of
• the wedge domain also shares sequence homology with the other members of the LAR family, LAR and PTPdelta. It is likely that these amino acids are necessary for the overall structure of the wedge domain. conserveed amino acids include an alanine at position 13, which marks the end of the first alpha helix and the start of the turn, making it likely to be necessary for general wedge size and structure.
• conservative substitutions include the substitution of one non-polar (hydrophobic) residue, such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, and/or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
• one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another
• one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine
• substitution of one basic residue such as lysine, arginine or histidine for another
• one acidic residue such as aspartic acid or glutamic acid
• the unique amino acids to ⁇ were found to be necessary for specificity of wedge domain binding. These include an EH domain in the first alpha helix position 4 and 5 followed by a threonine or a metathione (rat and mouse substitution) at position 6. In the turn, there is a unique serine at position 14 in all higher mammals. Finally, there is a unique leucine at position 17 in the second alpha helix. The potential roles of these unique amino acids will be discussed below.
• the serine residue in the turn at position 14 is of particular interest due to its location in the wedge domain.
• This amino acid, located in the turn between alpha helixes, is slightly extended from the general secondary and tertiary structure of ⁇ , making it available for binding interactions.
• serine due to its hydroxyl group and the polarity it contains, is known to facilitate several homophillic and heterophillic binding events, such as hydrogen binding between adjacent serines.
• Serines are also known to undergo various modifications, such as phosphorylation, making the likelihood of its necessity for specificity high. It is possible that smaller peptides that focus on the turn in the wedge domain and include the conserved serine may offer greater stability with similar function.
• Such peptides can be synthesized as loops, with cysteine's on either end to created di- sulfide bonds.
• the unique amino acids in the first alpha helix include glutamic acid at position 4, histidine at position 5 and threonine or metathione at position 6. Although the histidine is implicated in the consensus wedge domain, it is not found in LAR, PTPdelta, PTPmu or CD45. As all three of these amino acids are either charged or polar, it is likely that either this sequence or one of its components is necessary for ⁇ wedge specificity.
• the second alpha helix contains a unique leucine at position 17.
• Leucines have been implicated as the critical adhesive molecules for the three dimensional structure of leucine zippers. In these molecules, which are structurally similar to wedge domains, leucines of opposing alpha helixes, located at approximately 7 intervals, interact with hydrophobic regions of the opposing alpha helix. As there is also a Leucine in the first alpha helix, located at position 9, it is believed that this unique leucine is necessary for the overall three-dimensional structural integrity of the ⁇ wedge.
• the therapeutic peptide can include, consist essentially of, or consist of about 14 to about 20 amino acids and include the amino acid sequence EHX i ERLKANDS LKL (SEQ ID NO: 16), wherein Xi is T or M.
• a therapeutic peptide including SEQ ID NO: 16 can include at least one, at least two, at least three, at least four, or at least five conservative substitutions so that the therapeutic peptide has an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous to SEQ ID NO: 16.
• the conservative substitutions can be of amino acid residues 4E, 5R, 6L, 7K, 9N, 10D, 12L, or 13K of SEQ ID NO: 16.
• amino acid residue 4E can be substituted with D or Q
• amino acid residue 5R can be substituted with H, L, or K
• amino acid residue 6L can be substituted with I, V, or M
• amino acid residue 7K can be substituted with R or H
• amino acid residue 9N can be substituted with E or D
• amino acid residue 10 D can be substituted with E or N
• amino acid residue 12L can be substituted with I, V, or M
• amino acid residue 13K can be substituted with R or H.
• therapeutic peptides described herein can be subject to other various changes, substitutions, insertions, and deletions where such changes provide for certain advantages in its use.
• therapeutic peptides that bind to and/or complex with a wedge domain of the LAR family phosphatase can correspond to or be substantially homologous with, rather than be identical to, the sequence of a recited polypeptide where one or more changes are made and it retains the ability to inhibits or reduces one or more of the activity, signaling, and/or function of the LAR family phosphatase function.
• the therapeutic polypeptide can be in any of a variety of forms of polypeptide derivatives, that include amides, conjugates with proteins, cyclized polypeptides,
• polymerized polypeptides analogs, fragments, chemically modified polypeptides, and the like derivatives.
• conservative substitution can also include the use of a chemically derivatized residue in place of a non-derivatized residue provided that such peptide displays the requisite binding activity.
• “Chemical derivative” refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group.
• Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t- butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
• Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
• Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.
• the imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine.
• chemical derivatives those polypeptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For examples: 4- hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
• Polypeptides described herein also include any polypeptide having one or more additions and/or deletions or residues relative to the sequence of a polypeptide whose sequence is shown herein, so long as the requisite activity is maintained.
• One or more of peptides of the therapeutic peptides described herein can also be modified by natural processes, such as posttranslational processing, and/or by chemical modification techniques, which are known in the art. Modifications may occur in the peptide including the peptide backbone, the amino acid side-chains and the amino or carboxy termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given peptide.
• Modifications comprise for example, without limitation, acetylation, acylation, addition of acetomidomethyl (Acm) group, ADP- ribosylation, amidation, covalent attachment to fiavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation and ubi
• Peptides and/or proteins described herein may also include, for example, biologically active mutants, variants, fragments, chimeras, and analogues; fragments encompass amino acid sequences having truncations of one or more amino acids, wherein the truncation may originate from the amino terminus (N-terminus), carboxy terminus
• Analogues of the invention involve an insertion or a substitution of one or more amino acids.
• Variants, mutants, fragments, chimeras and analogues may function as inhibitors of the LAR family phosphatases (without being restricted to the present examples).
• the therapeutic polypeptides described herein may be prepared by methods known to those skilled in the art.
• the peptides and/or proteins may be prepared using recombinant DNA.
• one preparation can include cultivating a host cell
• the purification of the polypeptides may be done by affinity methods, ion exchange chromatography, size exclusion chromatography, hydrophobicity or other purification technique typically used for protein purification.
• the purification step can be performed under non-denaturating conditions.
• the protein may be renatured using techniques known in the art.
• the therapeutic peptides described herein can include additional residues that may be added at either terminus of a polypeptide for the purpose of providing a "linker" by which the polypeptides can be conveniently linked and/or affixed to other polypeptides, proteins, detectable moieties, labels, solid matrices, or carriers.
• Amino acid residue linkers are usually at least one residue and can be 40 or more residues, more often 1 to 10 residues. Typical amino acid residues used for linking are glycine, tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like.
• a subject polypeptide can differ by the sequence being modified by terminal-NH2 acylation, e.g., acetylation, or thioglycolic acid amidation, by terminal-carboxylamidation, e.g., with ammonia, methylamine, and the like terminal modifications.
• Terminal modifications are useful, as is well known, to reduce susceptibility by proteinase digestion, and therefore serve to prolong half life of the polypeptides in solutions, particularly biological fluids where proteases may be present.
• polypeptide cyclization is also a useful terminal modification, and is particularly preferred also because of the stable structures formed by cyclization and in view of the biological activities observed for such cyclic peptides as described herein.
• the linker can be a flexible peptide linker that links the therapeutic peptide to other polypeptides, proteins, and/or molecules, such as detectable moieties, labels, solid matrices, or carriers.
• a flexible peptide linker can be about 20 or fewer amino acids in length.
• a peptide linker can contain about 12 or fewer amino acid residues, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.
• a peptide linker comprises two or more of the following amino acids: glycine, serine, alanine, and threonine.
• a therapeutic agent comprising the therapeutic peptides described herein provide in the form of a conjugate protein or drug delivery construct includes at least a transport subdomain(s) or moiety(ies) (i.e., transport moieties) that is linked to the therapeutic peptide.
• the transport moieties can facilitate uptake of the therapeutic polypeptides into a mammalian (i.e., human or animal) tissue or cell (e.g., neural cell).
• the transport moieties can be covalently linked to the therapeutic peptides.
• the covalent link can include a peptide bond or a labile bond (e.g., a bond readily cleavable or subject to chemical change in the interior target cell environment).
• the transport moieties can be cross-linked (e.g., chemically cross-linked, UV cross-linked) to the therapeutic polypeptide.
• the transport moieties can also be linked to the therapeutic peptide with linking polypeptide described herein.
• the transport moieties can be repeated more than once in the therapeutic agent.
• the repetition of a transport moiety may affect (e.g., increase) the uptake of the peptides and/or proteins by a desired cell.
• the transport moiety may also be located either at the amino-terminal region of therapeutic peptide or at its carboxy-terminal region or at both regions.
• the transport moiety can include at least one transport peptide sequence that allows the therapeutic peptide once linked to the transport moiety to penetrate into the cell by a receptor-independent mechanism.
• the transport peptide is a synthetic peptide that contains a Tat-mediated protein delivery sequence and at least one of SEQ ID NOs: 9-13 and 16. These peptides can have, respectively, the amino acid sequences of SEQ ID NOs: 17-22.
• a 16 amino acid region of the third alpha-helix of antennapedia homeodomain has also been shown to enable proteins (made as fusion proteins) to cross cellular membranes (PCT international publication number WO 99/11809 and Canadian application No.:
• HIV Tat protein was shown to be able to cross cellular membranes.
• the transport moiety(ies) can include polypeptides having a basic amino acid rich region covalently linked to an active agent moiety (e.g., intracellular domain- containing fragments inhibitor peptide).
• an active agent moiety e.g., intracellular domain- containing fragments inhibitor peptide.
• the term "basic amino acid rich region” relates to a region of a protein with a high content of the basic amino acids such as arginine, histidine, asparagine, glutamine, lysine.
• a "basic amino acid rich region” may have, for example 15% or more of basic amino acid.
• a “basic amino acid rich region” may have less than 15% of basic amino acids and still function as a transport agent region.
• a basic amino acid region will have 30% or more of basic amino acids.
• the transport moiety(ies) may further include a proline rich region.
• proline rich region refers to a region of a polypeptide with 5% or more (up to 100%) of proline in its sequence. In some instance, a proline rich region may have between 5% and 15% of prolines. Additionally, a proline rich region refers to a region, of a polypeptide containing more prolines than what is generally observed in naturally occurring proteins (e.g., proteins encoded by the human genome). Proline rich regions of this application can function as a transport agent region.
• the therapeutic peptide described herein can be non- covalently linked to a transduction agent.
• a non-covalently linked polypeptide transduction agent is the Chariot protein delivery system (See U.S. Patent No. 6,841,535; / Biol Chem 274(35):24941-24946; and Nature Biotec. 19: 1173-1176, all herein incorporated by reference in their entirety).
• the therapeutic peptides can be expressed in cells being treated using gene therapy to inhibit LAR family signaling.
• the gene therapy can use a vector including a nucleotide encoding the therapeutic peptides.
• a "vector” (sometimes referred to as gene delivery or gene transfer “vehicle”) refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to the cell.
• the polynucleotide to be delivered may comprise a coding sequence of interest in gene therapy.
• Vectors include, for example, viral vectors (such as adenoviruses (Ad), adeno-associated viruses (AAV), and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a target cell.
• viral vectors such as adenoviruses (Ad), adeno-associated viruses (AAV), and retroviruses
• liposomes and other lipid-containing complexes such as adenoviruses (Ad), adeno-associated viruses (AAV), and retroviruses
• liposomes and other lipid-containing complexes such as liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a target cell.
• Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells.
• Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide.
• Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector.
• Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities.
• Selectable markers can be positive, negative or bifunctional. Positive selectable markers allow selection for cells carrying the marker, whereas negative selectable markers allow cells carrying the marker to be selectively eliminated.
• a variety of such marker genes have been described, including bifunctional (i.e. , positive/negative) markers (see, e.g. , Lupton, S., WO 92/08796, published May 29, 1992; and Lupton, S., WO 94/28143, published Dec. 8, 1994).
• Such marker genes can provide an added measure of control that can be advantageous in gene therapy contexts. A large variety of such vectors are known in the art and are generally available.
• Vectors for use herein include viral vectors, lipid based vectors and other non- viral vectors that are capable of delivering a nucleotide encoding the therapeutic peptides described herein to the target cells.
• the vector can be a targeted vector, especially a targeted vector that preferentially binds to neurons and.
• Viral vectors for use in the application can include those that exhibit low toxicity to a target cell and induce production of therapeutically useful quantities of the therapeutic peptide in a cell specific manner.
• viral vectors are those derived from adenovirus (Ad) or adeno- associated virus (AAV). Both human and non-human viral vectors can be used and the recombinant viral vector can be replication-defective in humans.
• Ad adenovirus
• AAV adeno- associated virus
• the vector can comprise a polynucleotide having a promoter operably linked to a gene encoding the therapeutic peptides and is replication-defective in humans.
• HSV vectors deleted of one or more immediate early genes are advantageous because they are generally non-cytotoxic, persist in a state similar to latency in the target cell, and afford efficient target cell transduction.
• Recombinant HSV vectors can incorporate approximately 30 kb of heterologous nucleic acid.
• Retroviruses such as C-type retroviruses and lentiviruses, might also be used in the application.
• retroviral vectors may be based on murine leukemia virus (MLV). See, e.g. , Hu and Pathak, Pharmacol. Rev. 52:493-511, 2000 and Fong et al., Crit. Rev. Ther. Drug Carrier Syst. 17: 1-60, 2000.
• MLV-based vectors may contain up to 8 kb of heterologous (therapeutic) DNA in place of the viral genes.
• the heterologous DNA may include a tissue-specific promoter and a nucleic acid encoding the therapeutic peptide. In methods of delivery to neural cells, it may also encode a ligand to a tissue specific receptor.
• retroviral vectors that might be used are replication-defective lentivirus-based vectors, including human immunodeficiency (HlV)-based vectors. See, e.g. , Vigna and Naldini, J. Gene Med. 5:308-316, 2000 and Miyoshi et al., J. Virol. 72:8150- 8157, 1998.
• Lenti viral vectors are advantageous in that they are capable of infecting both actively dividing and non-dividing cells.
• Lenti viral vectors for use in the application may be derived from human and non-human (including SIV) lentiviruses.
• lenti viral vectors include nucleic acid sequences required for vector propagation as well as a tissue-specific promoter operably linked to a therapeutic peptide encoding nucleic acid. These former may include the viral LTRs, a primer binding site, a polypurine tract, att sites, and an encapsidation site.
• a lentiviral vector can be employed.
• Lentiviruses have proven capable of transducing different types of CNS neurons (Azzouz et al., (2002) J Neurosci. 22: 10302-12) and may be used in some embodiments because of their large cloning capacity.
• a lentiviral vector may be packaged into any lentiviral capsid.
• the substitution of one particle protein with another from a different virus is referred to as "pseudotyping".
• the vector capsid may contain viral envelope proteins from other viruses, including murine leukemia virus (MLV) or vesicular stomatitis virus (VSV).
• MMV murine leukemia virus
• VSV vesicular stomatitis virus
• the use of the VSV G-protein yields a high vector titer and results in greater stability of the vector virus particles.
• Alphavirus-based vectors such as those made from semliki forest virus (SFV) and Sindbis virus (SIN) might also be used in the application.
• SFV semliki forest virus
• SI Sindbis virus
• Recombinant, replication-defective alphavirus vectors are advantageous because they are capable of high-level heterologous (therapeutic) gene expression, and can infect a wide target cell range.
• Alphavirus replicons may be targeted to specific cell types by displaying on their virion surface a functional heterologous ligand or binding domain that would allow selective binding to target cells expressing a cognate binding partner.
• Alphavirus replicons may establish latency, and therefore long-term heterologous nucleic acid expression in a target cell.
• the replicons may also exhibit transient heterologous nucleic acid expression in the target cell.
• more than one promoter can be included in the vector to allow more than one heterologous gene to be expressed by the vector.
• the vector can comprise a sequence, which encodes a signal peptide or other moiety, which facilitates expression of the therapeutic peptide from the target cell.
• hybrid viral vectors may be used to deliver a nucleic acid encoding a therapeutic peptide to a target neuron, cell, or tissue.
• Standard techniques for the construction of hybrid vectors are well- known to those skilled in the art. Such techniques can be found, for example, in Sambrook, et al., In Molecular Cloning: A laboratory manual. Cold Spring Harbor, N.Y. or any number of laboratory manuals that discuss recombinant DNA technology. Double-stranded AAV genomes in adenoviral capsids containing a combination of AAV and adenoviral ITRs may be used to transduce cells.
• an AAV vector may be placed into a "gutless", “helper-dependent” or "high-capacity” adenoviral vector.
• Adenovirus/ AAV hybrid vectors are discussed in Lieber et al., J. Virol. 73:9314-9324, 1999. Retrovirus/adenovirus hybrid vectors are discussed in Zheng et al., Nature Biotechnol. 18: 176-186, 2000.
• Retroviral genomes contained within an adenovirus may integrate within the target cell genome and effect stable gene expression.
• nucleotide sequence elements which facilitate expression of the therapeutic peptide and cloning of the vector are further contemplated.
• the presence of enhancers upstream of the promoter or terminators downstream of the coding region can facilitate expression.
• tissue-specific promoter can be fused to nucleotides encoding the therapeutic peptides described herein.
• tissue specific promoters By fusing such tissue specific promoter within the adenoviral construct, transgene expression is limited to a particular tissue.
• the efficacy of gene expression and degree of specificity provided by tissue specific promoters can be determined, using the recombinant adenoviral system of the present application.
• Neuron specific promoters such as the platelet-derived growth factor ⁇ -chain (PDGF- ⁇ ) promoter and vectors, are well known in the art.
• non- viral methods may also be used to introduce a nucleic acid encoding a therapeutic peptide into a target cell.
• a review of non- viral methods of gene delivery is provided in Nishikawa and Huang, Human Gene Ther. 12:861-870, 2001.
• An example of a non-viral gene delivery method according to the application employs plasmid DNA to introduce a nucleic acid encoding a therapeutic peptide into a cell. Plasmid-based gene delivery methods are generally known in the art.
• Synthetic gene transfer molecules can be designed to form multimolecular aggregates with plasmid DNA. These aggregates can be designed to bind to a target cell. Cationic amphiphiles, including lipopolyamines and cationic lipids, may be used to provide receptor-independent nucleic acid transfer into target cells.
• preformed cationic liposomes or cationic lipids may be mixed with plasmid DNA to generate cell-transfecting complexes.
• Methods involving cationic lipid formulations are reviewed in Feigner et al., Ann. N.Y. Acad. Sci. 772: 126-139, 1995 and Lasic and Templeton, Adv. Drug Delivery Rev. 20:221-266, 1996.
• DNA may also be coupled to an amphipathic cationic peptide (Fominaya et al., J. Gene
• EBV Epstein Barr virus
• the nucleic acid encoding the therapeutic peptides can be introduced into the target cell by transfecting the target cells using electroporation techniques. Electroporation techniques are well known and can be used to facilitate transfection of cells using plasmid DNA.
• Vectors that encode the expression of the therapeutic peptides can be delivered in vivo to the target cell in the form of an injectable preparation containing pharmaceutically acceptable carrier, such as saline, as necessary.
• pharmaceutically acceptable carrier such as saline
• Other pharmaceutical carriers, formulations and dosages can also be used in accordance with the present application.
• the vector can be delivered by direct injection at an amount sufficient for the therapeutic peptide to be expressed to a degree, which allows for highly effective therapy.
• the vector By injecting the vector directly into or about the periphery of the neuron, it is possible to target the vector transfection rather effectively, and to minimize loss of the recombinant vectors.
• This type of injection enables local transfection of a desired number of cells, especially at a site of CNS injury, thereby maximizing therapeutic efficacy of gene transfer, and minimizing the possibility of an inflammatory response to viral proteins.
• Other methods of administering the vector to the target cells can be used and will depend on the specific vector employed.
• the therapeutic peptide can be expressed for any suitable length of time within the target cell, including transient expression and stable, long-term expression.
• the nucleic acid encoding the therapeutic peptide will be expressed in therapeutic amounts for a defined length of time effective to induce activity and growth of the transfected cells.
• the nucleic acid encoding the therapeutic peptide will be expressed in therapeutic amounts for a defined length of time effective to in inhibit and/or reduce ⁇ -amyloid accumulation and/or Tau aggregation in a subject in need thereof.
• a therapeutic amount is an amount, which is capable of producing a medically desirable result in a treated animal or human.
• dosage for any one animal or human depends on many factors, including the subject' s size, body surface area, age, the particular composition to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
• Specific dosages of proteins and nucleic acids can be determined readily determined by one skilled in the art using the experimental methods described below.
• therapeutic agents described herein may further be modified
• Such modification may be designed to facilitate manipulation or purification of the molecule, to increase solubility of the molecule, to facilitate
• the therapeutic agents and pharmaceutical compositions comprising the therapeutic agents described herein may be delivered to neurons of the CNS and/or the PNS. Such neurons may be injured or diseased. Such neurons may alternatively be healthy, uninjured neurons. Such neurons may be located at the site of injury, or at a site incident to the injury.
• the neurons to be targeted for therapeutic administration, delivery/contact of the agents and compositions described herein will be neurons from which neuronal outgrowth is thought to prove beneficial to the subject. Such determination is within the ability of the skilled practitioner through no more than routine experimentation.
• the therapeutic agents and therapeutic pharmaceutical compositions described herein may also be delivered to non- neuronal cells of the CNS and/or the PNS, such as to non- neuronal cells that provide support to neural cells.
• non- neuronal cells include, without limitation, glial cells (e.g., astrocytes, oligodendrocytes, ependymal cells, radial glia in the CNS; and Schwann cells, satellite glial cells, enteric glail cells n the PNS).
• the administration is specific for one or more specific locations within the subject's nervous system.
• the preferred mode of administration can vary depending upon the particular agent chosen and the particular target.
• the therapeutic agents When the therapeutic agents are delivered to a subject, they can be administered by any suitable route, including, for example, orally (e.g., in capsules, suspensions or tablets), systemically, or by parenteral administration.
• Parenteral administration can include, for example, intramuscular, intravenous, intraarticular, intraarterial, intrathecal, subcutaneous, or intraperitoneal administration.
• the agent can also be administered orally, transdermally, topically, by inhalation (e.g., intrabronchial, intranasal, oral inhalation or intranasal drops) or rectally. Administration can be local or systemic as indicated.
• the therapeutic agent can be administered by introduction into the cerebrospinal fluid of the subject.
• the therapeutic agent can be introduced into a cerebral ventricle, the lumbar area, or the cistema magna.
• the therapeutic agent can be introduced locally, such as into the site of nerve or cord injury, into a site of pain or neural degeneration, or intraocularly to contact neuroretinal cells.
• the pharmaceutically acceptable formulations can be suspended in aqueous vehicles and introduced through conventional hypodermic needles or using infusion pumps.
• the therapeutic agent can be administered into a subject intrathecally.
• intrathecal administration is intended to include delivering a therapeutic agent directly into the cerebrospinal fluid of a subject, by techniques including lateral cerebroventricular injection through a burrhole or cisternal or lumbar puncture or the like (described in Lazorthes et al., 1991, and Ommaya, 1984, the contents of which are incorporated herein by reference).
• lumbar region is intended to include the area between the third and fourth lumbar (lower back) vertebrae.
• cistema magna is intended to include the area where the skull ends and the spinal cord begins at the back of the head.
• the ten-n “cerebral ventricle” is intended to include the cavities in the brain that are continuous with the central canal of the spinal cord.
• Administration of therapeutic agent to any of the above mentioned sites can be achieved by direct injection of the therapeutic agent or by the use of infusion pumps. Implantable or external pumps and catheter may be used.
• therapeutic agent can be formulated in liquid solutions, typically in physiologically compatible buffers such as Hank's solution or Ringer's solution.
• the therapeutic agent may be formulated in solid form and re-dissolved or suspended immediately prior to use. Lyophilized forms are also included.
• the injection can be, for example, in the form of a bolus injection or continuous infusion (such as using infusion pumps) of the therapeutic agent.
• the therapeutic agent can be administered by lateral cerebroventricular injection into the brain of a subject.
• the injection can be made, for example, through a burr hole made in the subject's skull.
• the therapeutic agent can be administered through a surgically inserted shunt into the cerebral ventricle of a subject.
• the injection can be made into the lateral ventricles, which are larger, even though injection into the third and fourth smaller ventricles can also be made.
• the therapeutic agent can be administered by injection into the cistema magna, or lumbar area of a subject.
• An additional means of administration to intracranial tissue involves application to the olfactory epithelium, with subsequent transmission to the olfactory bulb and transport to more proximal portions of the brain.
• Such administration can be by nebulized or aerosolized preparations.
• the therapeutic agent can be administered to a subject at the site of injury or systemically to the subject.
• the therapeutic agent can administered to a subject for an extended period of time. Sustained contact with the active compound can be achieved, for example, by repeated administration of the active compound(s) over a period of time, such as one week, several weeks, one month or longer.
• the pharmaceutically acceptable formulation used to administer the therapeutic agent(s) can also be formulated to provide sustained delivery of the active compound to a subject.
• the formulation may deliver the active compound for at least one, two, three, or four weeks, inclusive, following initial administration to the subject.
• a subject to be treated in accordance with the present invention is treated with the active compound for at least 30 days (either by repeated administration or by use of a sustained delivery system, or both).
• Sustained delivery of the therapeutic agent can be demonstrated by, for example, the continued therapeutic effect of the therapeutic agent over time.
• sustained delivery of the therapeutic agent may be demonstrated by detecting the presence of the therapeutic agents in vivo over time.
• Approaches for sustained delivery include use of a polymeric capsule, a minipump to deliver the formulation, a biodegradable implant, or implanted transgenic autologous cells (see U.S. Patent No. 6,214,622).
• Implantable infusion pump systems e.g., INFUSAID pumps (Towanda, PA)
• osmotic pumps are available commercially and otherwise known in the art.
• Another mode of administration is via an implantable, externally programmable infusion pump.
• Infusion pump systems and reservoir systems are also described in, e.g., U.S. Patents No. 5,368,562 and No. 4,731,058.
• Vectors encoding the therapeutic peptides can often be administered less frequently than other types of therapeutics.
• an effective amount of such a vector can range from about 0.01 mg/kg to about 5 or 10 mg/kg, inclusive; administered daily, weekly, biweekly, monthly or less frequently.
• the ability to deliver or express the therapeutic peptides allows for cell activity modulation in a number of different cell types.
• the therapeutic peptides can be expressed, for example, in neural cells or brain areas affected by degenerative diseases, such as
• the therapeutic agents can be used to treat diseases, disorders, or condition associated with ⁇ -amyloid accumulation and/or Tau aggregation in a subject in need thereof.
• the disease, disorder, and/or condition includes at least one of a disease, disorder, and/or condition of the nervous system.
• the disease, disorder, and/or condition of the nervous system includes at least one of a neurological disorder, neuropsychiatric disorder, neural injury, neural toxicity disorder, a neuropathic pain, and neural degenerative disorders.
• the neurological disorder can include at least one of Alzheimer's disease or dementias related to Alzheimer's disease.
• the therapeutic agent described herein can be any therapeutic agent described herein.
• anti- Alzheimer's agent or “anti- Alzheimer agent”, as employed herein refers to any compound that can be employed for the treatment of Alzheimer's disease and other dementias; such as, but not limited to, N-methyl-D-aspartate receptor (NMDA) receptor antagonists, acetyl
• NMDA N-methyl-D-aspartate receptor
• cholinesterase inhibitor acetylcholine synthesis modulators, acetylcholine storage modulators, acetylcholine release modulators, ⁇ inhibitors, ⁇ plaque removal agents, inhibitors of ⁇ plaque formation, inhibitors of amyloid precursor protein processing enzymes, ⁇ -amyloid converting enzyme inhibitors, ⁇ -secretase inhibitors, ⁇ -secretase modulators, nerve growth factor agonists, hormone receptor blockade agents,
• the anti- Alzheimer's agent is an NMDA receptor antagonist.
• the NMDA receptor antagonist includes, but not limited to, memantine, amantadine, neramexane (1, 3, 3, 5, 5- pentamethylcyclohexan-1- amine), ketamine, rimantidine, eliprodil, ifenprodil, dizocilpine, remacemide, riluzole, aptiganel, phencyclidine, flupirtine, celfotel, felbamate, spermine, spermidine, levemopamil, and/or combinations thereof.
• NMDA receptor antagonist employed in the present invention is an Anti- Alzheimer agent.
• the anti- Alzheimer's agent is an inhibitor of cholinesterase.
• the acetylcholinesterase inhibitor includes, but is not limited to, donepezil, tacrine, rivastigmine, galantamine, physostigmine, neostigmine, Huperzine A, icopezil (CP- 118954, 5,7-dihydro-3-[2-[l-(phenylmethyl)-4-piperidinyl]ethyl]-6H-pyrrolo-[4,5-f- ]-l,2- benzisoxazol-6-one maleate), ER-127528 (4-[(5,6-dimethoxy-2-fluoro-l-indanon)-2- yl] methyl- 1- (3 -fluorobenzyl) piperidine hydrochloride), zanapezil (TAK-147; 3-[l- (TAK-147; 3-[
• the anti- Alzheimer's agent is an ⁇ inhibitor, ⁇ plaque removal agents, inhibitors of ⁇ plaque formation, inhibitors of amyloid precursor protein processing enzymes, ⁇ -amyloid converting enzyme inhibitors, ⁇ -secretase inhibitors, ⁇ - secretase modulators.
• the ⁇ inhibitor includes, but is not limited to, tarenflurbil, tramiprosate, clioquinol, PBT-2 and other 8-hydroxyquinilone derivatives, ⁇ plaque removal agents, inhibitors of ⁇ plaque formation, inhibitors of amyloid precursor protein processing enzymes, ⁇ -amyloid converting enzyme inhibitors, ⁇ -secretase inhibitors, a-secretase modulators (LY450139; N-[N-(3,5-difluorophenacetyl)-L-alanyl)-S- phenylglycine t-butyl ester), and combinations thereof.
• the anti- Alzheimer's agent is a nerve growth factor agonist.
• the nerve growth factor agonist is, but not limited to, xaliproden or brain derived neurotrophic factor or nerve growth factor.
• the anti- Alzheimer's agent is a hormone receptor blockade agent.
• the hormone receptor blockade agent is, but not limited to, leuproelide or a derivative thereof.
• the anti- Alzheimer's agent is a neurotransmission modulator.
• the neurotransmission modulator is, but not limited to, ispronicline.
• the Leukocyte-common Antigen-Related (LAR) family of phosphatases consists of three members: LAR itself, receptor protein tyrosine phosphatase Sigma (RPTPo) and receptor protein tyrosine phosphatase delta (RPTP5). Structural and sequence analysis has revealed that all members of the LAR family contain a wedge-shaped helix-loop-helix motif in the first intracellular catalytic domain that mediates homo/heterophilic receptor interaction. Using peptide mimetics of this wedge domain tagged to a cytosolic localizing TAT sequence, LAR activity was successfully abolished in neurotrophin signaling paradigms.
• ILP Intraceullar LAR blocking peptide
• ISP intracellular Sigma blocking peptide
• IDP intracellular delta blocking peptide
• This example shows that inhibition of neuronal receptor ⁇ (protein tyrosine phosphatase sigma) curbs ⁇ -amyloid ( ⁇ ) pathogenesis and aggregation of Tau.
• ⁇ protein tyrosine phosphatase sigma
• Genetic depletion of ⁇ lowers ⁇ -secretase affinity to APP and suppresses ⁇ accumulation in a specific manner that does not generically inhibit ⁇ - and ⁇ -secretase activities.
• Genetic depletion of ⁇ also inhibits the aggregation of Tau.
• mice were maintained under standard conditions approved by the Institutional Animal Care and Use Committee. Wild type and ⁇ -deficient mice of Balb/c background were provided by Dr. Michel L. Tremblay. Homozygous TgAPP-SwDI mice, C57BL/6- Tg(Thyl-APPSwDutIowa)BWevn/Mmjax, stock number 007027, were from the Jackson Laboratory.
• mice express human APP transgene harboring Swedish, Dutch, and Iowa mutations, and were bred with Balb/c mice heterozygous for the ⁇ gene to generate bigenic mice heterozygous for both TgAPP-SwDI and ⁇ genes, which are hybrids of 50% C57BL/6J and 50% Balb/c genetic background. These mice were further bred with Balb/c mice heterozygous for the ⁇ gene.
• mice heterozygous for TgAPP-SwDI transgene with wild type ⁇ mice heterozygous for TgAPP-SwDI transgene with wild type ⁇
• TgAPP- SwDI(+/-)PTPo(-/-) mice heterozygous for TgAPP-SwDI transgene with genetic depletion of ⁇
• TgAPP-SwDI(-/-)PTPo(+/+) mice free of TgAPP-SwDI transgene with wild type ⁇ .
• TgAPP-SwDI(-/-)PTPo(+/+) and Balb/c ⁇ (+/+) are wild type mice but with different genetic background.
• RIPA buffer 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate.
• NP40 buffer 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% NP40 without or with SDS at concentration of 0.1%, 0.3%, and 0.4%.
• the homogenates were incubated at 4°C for 1 hour with gentle mixing, sonicated on ice for 2 minutes in a sonic dismembrator (Fisher Scientific Model 120, with pulses of 50% output, 1 second on and 1 second off), followed with another hour of gentle mixing at 4°C. All samples were used fresh without freezing and thawing.
• Electrophoresis of protein samples was conducted using 4-12% Bis-Tris Bolt Plus Gels, with either MOPS or MES buffer and Novex Sharp Pre-stained Protein Standard (all from Invitrogen). Proteins were transferred to nitrocellulose membrane (0.2 ⁇ pore size, Bio-Rad) and blotted with selected antibodies (see table above) at concentrations suggested by the manufacturers. Primary antibodies were diluted in SuperBlock TBS Blocking Buffer (Thermo Scientific) and incubated with the nitrocellulose membranes at 4°C overnight; secondary antibodies were diluted in PBS with 5% nonfat milk and 0.2% Tween20 and incubated at room temperature for 2 hours. Membranes were washes 4 times in PBS with 0.2% Tween20 between primary and secondary antibodies and before chemiluminescent detection with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).
• Mouse forebrains were thoroughly homogenized in tissue homogenization buffer (2 mM Tris pH 7.4, 250 mM sucrose, 0.5 mM EDTA, 0.5 mM EGTA) containing protease inhibitor cocktail (Roche), followed by centrifugation at 135,000 x g (33,500 RPM with SW50.1 rotor) for 1 hour at 4°C. Proteins in the pellets were extracted with formic acid (FA) and centrifuged at 109,000 x g (30,100 RPM with SW50.1 rotor) for 1 hour at 4°C.
• tissue homogenization buffer (2 mM Tris pH 7.4, 250 mM sucrose, 0.5 mM EDTA, 0.5 mM EGTA) containing protease inhibitor cocktail (Roche), followed by centrifugation at 135,000 x g (33,500 RPM with SW50.1 rotor) for 1 hour at 4°C. Proteins in the pellets were extracted with formic acid (FA) and centrifuged
• the supernatants were collected and diluted 1:20 in neutralization buffer (1 M Tris base, 0.5 M Na 2 HP0 4 , 0.05% NaN 3 ) and subsequently 1:3 in ELISA buffer (PBS with 0.05% Tween-20, 1% BSA, and 1 mM AEBSF). Diluted samples were loaded onto ELISA plates pre-coated with 6E10 antibody (Biolegend) to capture ⁇ peptides. Serial dilutions of synthesized human ⁇ 1-40 or 1-42 (American Peptide) were loaded to determine a standard curve. ⁇ was detected using an HRP labeled antibody for either ⁇ 1-40 or 1-42 (see table above). ELISA was developed using TMB substrate (Thermo Scientific) and reaction was stopped with IN HC1. Plates were read at 450nm and concentrations of ⁇ in samples were determined using the standard curve.
• mice were placed in the center of the Y-maze and allowed to move freely through each arm. Their exploratory activities were recorded for 5 minutes. An arm entry is defined as when all four limbs are within the arm. For each mouse, the number of triads is counted as "spontaneous alternation", which was then divided by the number of total arm entries, yielding a percentage score.
• the novel object test On day 1, mice were exposed to empty cages (45 cm x 24 cm x 22 cm) with blackened walls to allow exploration and habituation to the arena. During day 2 to day 4, mice were returned to the same cage with two identical objects placed at an equal distance. On each day mice were returned to the cage at approximately the same time during the day and allowed to explore for 10 minutes.
• ⁇ is an APP binding partner in the brain
• ⁇ is expressed throughout the adult nervous system, most predominantly in the hippocampus, one of earliest affected brain regions in AD.
• ⁇ and APP the precursor of ⁇
• Fig. 1A- F the perinuclear and axonal regions with a punctate pattern
• APP is mainly processed through alternative cleavage by either a- or ⁇ -secretase. These secretases release the N-terminal portion of APP from its membrane-tethering C-terminal fragment (CTFoc or CTF , respectively), which can be further processed by the ⁇ -secretase. Sequential cleavage of APP by the ⁇ - and ⁇ -secretases is regarded as amyloidogenic processing since it produces ⁇ peptides.
• CTFoc membrane-tethering C-terminal fragment
• ⁇ peptides When overproduced, the ⁇ peptides can form soluble oligomers that trigger ramification of cytotoxic cascades, whereas progressive aggregation of ⁇ eventually results in the formation of senile plaques in the brains of AD patients (Fig. 2a).
• ⁇ amyloidogenic processing
• mice each expressing a human APP transgene harboring the Swedish mutation near the ⁇ -cleavage site, were crossed with the ⁇ line to generate offsprings that are heterozygous for their respective APP transgene, with or without ⁇ . Because the Swedish mutation carried by these APP transgenes is prone to ⁇ -cleavage, the predominant form of APP CTF in these transgenic mice is predicted to be CTF . Thus, the reduction of APP CTF in ⁇ -deficient APP transgenic mice may indicate a regulatory role of ⁇ on CTF level.
• CTFP is an intermediate proteolytic product between ⁇ - and ⁇ -cleavage
• its decreased steady state level could result from either reduced production by ⁇ -cleavage or increased degradation by subsequent ⁇ -secretase cleavage (Fig. 2A).
• Fig. 2A we measured the level of ⁇ peptides, which are downstream products from CTFP degradation by ⁇ -cleavage.
• TgAPP-SwDI model is one of the earliest to develop neurodegenerative pathologies and behavioral deficits among many existing AD mouse models. We therefore chose these mice to further examine the role of ⁇ in AD pathologies downstream of neurotoxic ⁇ .
• the APP-SwDI(+)PTPo(+/+) mice which express the TgAPP-SwDI transgene and wild type ⁇ , have developed severe neuroinflammation in the brain by the age of 9 months, as measured by the level of GFAP (glial fibrillary acidic protein), a marker of astrogliosis (Fig. 5).
• GFAP glial fibrillary acidic protein
• Fig. 5 a marker of astrogliosis
• GFAP expression level in the APP- SwDI(+)PTPo(+/+) mice is more than tenfold compared to that in age-matched non- transgenic littermates [APP-SwDI(-)PTPo(+/+)].
• Neurofibrillary tangles composed of hyperphosphorylated and aggregated Tau are commonly found in AD brains. These tangles tend to develop in a hierarchical pattern, appearing first in the entorhinal cortex before spreading to other brain regions. The precise mechanism of tangle formation, however, is poorly understood. The fact that Tau tangles and ⁇ deposits can be found in separate locations in postmortem brains has led to the question of whether Tau pathology in AD is independent of ⁇ accumulation. Additionally, despite severe cerebral ⁇ -amyloidosis in many APP transgenic mouse models, Tau tangles have not been reported, further questioning the relationship between ⁇ and Tau pathologies in vivo.
• TgAPP-SwDI or TgAPP-SwInd transgene which apparently causes Tau aggregation, does not enhance the phosphorylation of Tau residues including Serinel91, Therioninel94, and Therionine220 (data not shown), whose homologues in human Tau (Serine202, Therionine205, and Therionine231) are typically hyperphosphorylated in neurofibrillary tangles.
• Malfunction of Tau is broadly recognized as a neurodegenerative marker since it indicates microtubule deterioration.
• the constraining effect on Tau aggregation by genetic depletion of ⁇ thus provides additional evidence for the role of this receptor as a pivotal regulator of neuronal integrity.
• the Y-maze assay which allows mice to freely explore three identical arms, measures their short-term spatial memory. It is based on the natural tendency of mice to alternate arm exploration without repetitions. The performance is scored by the percentage of spontaneous alternations among total arm entries, and a higher score indicates better spatial navigation. Compared to the non-transgenic wild type mice within the colony, the APP- SwDI(+)PTPo(+/+) mice show a clear deficit in their performance. Genetic depletion of ⁇ in the APP-SwDI(+)PTPo(-/-) mice, however, unequivocally restores the cognitive performance back to the level of non-transgenic wild type mice (Fig. 8A, Fig. 9).
• Apathy the most common neuropsychiatric symptom reported among individuals with AD, is characterized by a loss of motivation and diminished attention to novelty, and has been increasingly adopted into early diagnosis of preclinical and early prodromal AD. Many patients in early stage AD lose attention to novel aspects of their environment despite their ability to identify novel stimuli, suggesting an underlying defect in the circuitry responsible for further processing of the novel information. As a key feature of apathy, such deficits in attention to novelty can be accessed by the "curiosity figures task" or the "oddball task” in patients.
• the expression of APP-SwDI transgene in the APP-SwDI(+)PTPo(+/+) mice leads to a substantial decrease in NO exploration as compared to non-transgenic wild type mice (Fig. 8B, C; Fig. 10). Judging by their NO/FO ratios, it is evident that both the transgenic and non-transgenic groups are able to recognize and differentiate between the two objects (Fig. 10A, B). Thus, the reduced NO exploration by the APP-SwDI(+)PTPo(+/+) mice may reflect a lack of interest in the NO or an inability to shift attention to the NO.
• Figs. 12 illustrates an immunoblot showing the effects ISP in combination with a ⁇ -secretase inhibitor on APP processing compared to a ⁇ -secretase inhibitor administered alone or a BACE1 inhibitor in combination with a ⁇ -secretase inhibitor.
• ISP in combination with a ⁇ -secretase inhibitor substantially inhibited APP processing compared to a ⁇ -secretase inhibitor administered alone or a BACEl inhibitor in combination with a ⁇ -secretase inhibitor.

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