WO2018209169A1 - Peptides and methods for treating neurodegenerative disorders - Google Patents

Peptides and methods for treating neurodegenerative disorders Download PDF

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WO2018209169A1
WO2018209169A1 PCT/US2018/032200 US2018032200W WO2018209169A1 WO 2018209169 A1 WO2018209169 A1 WO 2018209169A1 US 2018032200 W US2018032200 W US 2018032200W WO 2018209169 A1 WO2018209169 A1 WO 2018209169A1
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seq
app
ρτρσ
peptide
disease
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PCT/US2018/032200
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French (fr)
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Yingjie Shen
Yuanzheng GU
Kui Xu
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Ohio State Innovation Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1716—Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein

Definitions

  • AD Alzheimer's disease
  • 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_ 1 .
  • ⁇ peptides mainly derive from sequential cleavage of neuronal Amyloid Precursor Protein (APP) by the ⁇ - and ⁇ -secretases.
  • APP 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.
  • Tau is another biomarker that has been intensively studied in AD. Cognitive decline in patients sometimes correlates better with Tau pathology than with ⁇ burden 5 ⁇ 5 . Overwhelming evidence also substantiated that malfunction of Tau contributes to synaptic loss and neuronal deterioration 1 . In addition to AD, many other neurodegenerative diseases also involves ⁇ or Tau pathologies, and there is no disease modifying therapy available for any of these debilitating diseases.
  • peptides, compositions, and methods to treat and prevent neurodegenerative diseases that involve ⁇ -amyloid pathologies and/or Tau pathologies including but not limited to Alzheimer's disease, Lewy body dementia, frontotemporal dementia, cerebral amyloid angiopathy, primary age-related tauopathy, chronic traumatic encephalopathy, Parkinson's disease, postencephalitic parkinsonism, Huntington's disease, amyolateral sclerosis, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, Lytico-Bodig disease, ganglioglioma and gangliocytoma, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and/or Creutzfeldt- Jakob disease.
  • Alzheimer's disease Lewy body dementia, frontotemporal dementia, cerebral amyloid angiopathy, primary age-related tauopathy, chronic traumatic encephalopathy, Parkinson's disease, postencephalitic parkinsonism,
  • neurodegenerative diseases in at-risk subjects such as people with Down syndrome and those who have suffered from brain injuries or cerebral ischemia, as well as the aging population.
  • the disclosed peptides, compositions, and methods disrupt the binding between Protein Tyrosine Phosphatase sigma ( ⁇ ) and APP, preventing ⁇ - amyloidogenic processing of APP as well as Tau aggregation.
  • compositions and methods restore the physiological balance of two classes of ⁇ ligands in the brain microenvironment, namely the chondroitin sulfates (CS) and heparin or its analog heparan sulfates (HS), and thereby prevent abnormally increased ⁇ -amyloidogenic processing of APP.
  • CS chondroitin sulfates
  • HS heparin or its analog heparan sulfates
  • the therapeutic strategy disclosed herein inhibits the process upstream of ⁇ -amyloid production. Unlike the ⁇ - and ⁇ -secretase inhibitors in current clinical trials, the therapeutic strategy disclosed herein inhibits ⁇ -amyloid production without affecting other major substrates of these secretases. Therefore the strategy disclosed herein may be more effective with fewer side effects compared to the most advanced AD drug candidates in clinical trials.
  • the peptide comprising a decoy fragment of APP, a decoy fragment of ⁇ , or a combination thereof.
  • the decoy fragment of APP is a peptide comprising at least 5 consecutive amino acids of SEQ ID NO: l .
  • the decoy fragment of APP is a peptide comprising at least 10 consecutive amino acids of SEQ ID NO: l .
  • the decoy fragment of APP can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO: 101, SEQ ID NO: 112, SEQ ID NO: 139, SEQ ID NO: 151, SEQ ID NO: 157, SEQ ID NO:251, SEQ ID NO:897.
  • the decoy fragment of ⁇ is a peptide comprising at least 4 consecutive amino acids of SEQ ID NO:442.
  • the decoy fragment of ⁇ can comprises the amino acid sequence SEQ ID NO:655, SEQ ID NO:769, SEQ ID NO:898, or SEQ ID NO:899.
  • the peptide further comprises a blood brain barrier penetrating sequence.
  • the blood brain barrier penetrating sequence comprises amino acid sequence SEQ ID NO: 880, SEQ ID NO: 883, SEQ ID NO: 888, SEQ ID NO: 894, SEQ ID NO: 895, SEQ ID NO: 896.
  • administering HS, or its analog heparin, or their mimetics modified to reduce anti -coagulant effect, with a saccharide chain length of 17, 18, 19, 20, 21, 22, 23, 24 units or longer could assist in restoring the CS/HS balance.
  • the physiological molecular CS/HS balance is restored by administering enzymes that digest CS (such as
  • Chondroitinase ABC also known as ChABC
  • HS degradation such as Heparanase inhibitors PI-88, OGT 21 15, or PG545.
  • agents that mimic the HS/heparin effect of ⁇ clustering 8 such as multivalent antibodies, could be administered.
  • the neurodegenerative disease is selected from the group consisting of Alzheimer's Disease, Lewy body dementia, frontotemporal dementia, cerebral amyloid angiopathy, primary age-related tauopathy, chronic traumatic encephalopathy, Parkinson's disease, postencephalitic parkinsonism, Huntington's disease, amyolateral sclerosis, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, Lytico-Bodig disease, ganglioglioma and gangliocytoma, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and/or Creutzfeldt- Jakob disease.
  • subjects are selected from at- risk populations, such as the aging population, people with Down syndrome, and those suffered from brain injuries or cerebral ischemia, to prevent subsequent onset of neurodegenerative diseases.
  • a method of screening for candidate compounds that slow, stop, reverse, or prevent neurodegeneration comprises providing a sample comprising APP and ⁇ in an environment permissive for ⁇ - ⁇ binding, contacting the sample with a candidate compound, and assaying the sample for ⁇ - ⁇ binding, wherein a decrease in ⁇ - ⁇ binding compared to control values is an indication that the candidate agent is effective to slow, stop, reverse, or prevent neurodegeneration.
  • the method comprises contacting/incubating a candidate compound with cell membrane preparations extracted from fresh rodent brain homogenates, wherein a decrease in APP ⁇ - and/or ⁇ -cleavage products is an indication that the candidate agent has the potential to slow, stop, reverse, or prevent neurodegeneration.
  • Figures lA-lL ⁇ is an APP binding partner in the brain, a-f, Colocalization of
  • ⁇ (a, green) and APP (b, red) in hippocampal CA1 neurons of adult rat is shown by confocal imaging. Nuclei of CA1 neurons are stained with DAPI (c, blue), 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
  • Scale bar 20 ⁇ . f
  • 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.
  • ECD extracellular domain
  • ICD transmembrane-intracellular domain
  • APP FL 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.
  • 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 using mice between ages of 1 month to 2 years.
  • FIGS 2A-2C Molecular complex of ⁇ and APP in brains of various rodent species, a, b, Co-immunoprecipitation using an anti-APP antibody specific for amino acid residues 1-16 of mouse ⁇ (clone M3.2).
  • ⁇ and APP binding interaction is detected in forebrains of Balb/c (a) and B6 (b) mice, c, ⁇ co-immunoprecipitates with APP from rat forebrain lysates using an antibody specific for the C-terminus of APP. Images shown are representatives of at least three independent experiments using different animals.
  • APP. a Schematic diagram showing amyloidogenic processing of APP by the ⁇ - and ⁇ - secretases.
  • Full length APP (APP FL) is cleaved by ⁇ -secretase into soluble N-terminal (sAPPP) and C-terminal (CTFP) fragments.
  • APP CTFp 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 CTFP by immunoprecipitation (IP) followed with western blot analysis, using a pair of antibodies as marked in the diagram (a).
  • Antibodies against amino acids 1-16 of ⁇ detect CTFp but not CTF a, as the epitope is absent in CTFa.
  • the mean values from ⁇ deficient samples was normalized to that from the samples with wild type ⁇ . g and h, ⁇ deposition in the hippocampus of 10-month old TgAPP-SwDI mice. Images shown are representatives of 5 pairs of age- and sex -matched mice between 9- to 11 -month old. ⁇ (green) is detected by
  • Figures 4A-4F Genetic depletion of ⁇ reduces ⁇ -amyloidogenic products of APP.
  • a and b Antibody against the C-terminus of APP recognizes full length (FL) and C- terminal fragments (CTFs) of both mouse and human APP.
  • ⁇ deficiency does not affect the expression level of APP FL (a), but reduces the level of an APP CTF at about 15 KD in mouse forebrain lysates (b). Images shown are representatives of at least three independent
  • c Human CTF ⁇ in the forebrains of APP-SwInd transgenic mice is identified using the method as described in Fig.2d.
  • CTF ⁇ is immunoprecipitated by an antibody against the C- terminus of APP and detected by western blot analysis using an antibody against amino acids 1- 16 of human ⁇ (6E10), which reacts with CTF ⁇ but not CTFa (regions of antibody epitopes are shown in Fig. 2a).
  • d Densitometry quantification of experiments as shown in panel c repeated with 5 pairs of mice. For each experiment, the value from ⁇ deficient sample was normalized to the value from the sample with wild type ⁇ .
  • FIGS 5A-5C Lower affinity between BACE1 and APP in ⁇ -deficient brains, a, Co-immunoprecipitation experiments show nearly equal BACEl-APP association in wild type and ⁇ -deficient mouse brains under mild detergent condition (1% NP40). However, in ⁇ -deficient brains, BACEl-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. P40, Nonidet P-40, non-ionic detergent.
  • FIGS 6A-6F ⁇ does not generically modulate b- and g- 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 BACE1 (a) or ⁇ -secretase subunits (b).
  • Presenilinl and 2 (PS 1/2) are the catalytic subunits of ⁇ -secretase, which are processed into N-terminal and C-terminal fragments (NTF and CTF) in their mature forms.
  • NTF and CTF N-terminal fragments
  • Presenilin Enhancer 2 (PEN2), and APHl are other essential subunits of ⁇ -secretase.
  • ⁇ deficiency does not change the level of Neuregulinl (NGR1) CTFp, the C-terminal cleavage product by BACE1.
  • NRG1 FL full length Neuregulinl .
  • 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 using different animals.
  • FIGS 7A-7K ⁇ 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 (red) and DAPI staining of nuclei (blue) in the brains of 9-month old TgAPP-SwDI mice with or without ⁇ , along with their non-transgenic wild type littermate.
  • a-f Dentate gyrus (DG) of the
  • FIGS 8A-8G ⁇ 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; DAP I, 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
  • FIG. 1 Schematic diagram depicting distribution pattern of Tau aggregation (green) detected by immunofluorescent staining using an anti-Tau antibody (Tau-5) against its proline-rich region, in brains of 9 to 11 month-old TgAPP-SwDI transgenic mice. Similar results are seen with Tau- 46, an antibody recognizing the C-terminus of Tau (Extended Data Fig. 6). Aggregated Tau is found most prominently in the molecular layer of piriform and entorhinal cortex, and
  • Bar graph shows quantification of Tau aggregation in coronal brain sections from 4 pairs of age- and sex-matched 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. /? value, Student's t test, 2-tailed.
  • FIGS 10A-10E ⁇ deficiency mitigates Tau pathology in TgAPP-SwInd mice.
  • Tau aggregation green
  • Tau-5 anti-Tau antibody
  • Tau-46 an antibody recognizing the C-terminus of Tau (Extended Data Fig. 6).
  • the mean value of APP-SwInd(+)PTPo(-/-) samples is normalized to that of APP-SwInd(+)PTPo(+/+).
  • j p value Student's t test, 2-tailed. Error bars, SEM. Tau, green; DAPI, blue. Arrows points to Tau aggregates. Scale bars, 50 ⁇ .
  • FIGS 11A-11J Morphology of Tau aggregates found in APP transgenic brains, a- h, Tau aggregation (green) is detected by immunofluorescent staining, using an anti-Tau antibody (Tau-5) against the proline-rich domain of Tau (same as in Fig. 5 and Extended Data Fig. 5).
  • Tau aggregates in TgAPP-SwDI and TgAPP-SwInd brains show similar morphologies, a-f, Many of the Tau aggregates are found in punctate shapes, likely as part of cell debris, in areas that are free of nuclei staining, g, h, Occasionally the aggregates are found in fibrillary structures, probably in degenerated cells before disassembling, i.
  • An additional anti-Tau antibody (Tau-46) which recognizes the C-terminus of Tau, detects Tau aggregation in the same pattern as Tau-5.
  • Tau-46 An additional anti-Tau antibody (Tau-46), which recognizes the C-terminus of Tau, detects Tau aggregation in the same pattern as Tau-5.
  • j Image of staining without primary antibody at the same location of the Tau aggregates in the section adjacent to i. Both these antibodies recognize Tau regardless of its phosphorylation status.
  • Tau green; DAPI, blue. All scale bars, 20 ⁇ .
  • FIG. 12 Tau expression is not affected by ⁇ or human APP transgenes. Upper panel, total Tau level in brain homogenates. Lower panel, Actin as loading control. Tau protein expression level is not changed by genetic depletion of ⁇ or expression of mutated human APP transgenes. All mice are older than 1 year, and mice in each pair are age- and sex matched. Images shown are representatives of three independent experiments.
  • Figures 13A-13C ⁇ 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 short-term 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 APP-SwDI(-)PTPo(+/+) mice. The deficit is reversed by ⁇ depletion in APP-SwDI(+)PTPo(-/-) mice.
  • Figure 14 ⁇ 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 ⁇ .
  • Ages of all genotype groups are similarly distributed between 4 and 11 months. All ⁇ values, Student's t test, 2-tailed. Error bars, SEM.
  • Figures 15A-15D ⁇ deficiency enhances novelty exploration by TgAPP-SwDI mice.
  • NO novel object.
  • FO familiar object, a and b
  • 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 21 .
  • 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. While each has its own advantage and shortcoming, both NO/FO and NO/NO+FO measurements consistently show that the expression of TgAPP-SwDI gene leads to a deficit in attention to the NO, whereas genetic depletion of ⁇ restores novelty exploration to a level close to that of non-transgenic wild type mice, a and c, measurements in terms of exploration time, b and d, measurements in terms of visiting frequency.
  • NO novel object.
  • FO familiar object. NO preference is measured by the ratio of NO exploration time to total object exploration time (b) and the ratio of NO exploration time to FO exploration time (c). ⁇ depletion significantly improves novelty preference in these transgenic mice.
  • APP-SwInd(+)PTPo(+/+), n 43 (21 females and 22 males) ;
  • APP-SwInd(+)PTPo(-/-), n 24 (10 females and 14 males). Ages of both groups are similarly distributed between 5 and 15 months. All p values, Student's t test, 2-tailed. Error bars, SEM.
  • FIG. 17 CS and HS regulate ⁇ -cleavage of APP in opposite manners.
  • Membrane preparations from fresh mouse brain homogenates are incubated with CS18 (chondroitin sulfate of 18 oligosaccharides) or HS17 (heparan sulfate analog, heparin fragment of 17 oligosaccharides) at 37C° for 30 min.
  • CS18 chondroitin sulfate of 18 oligosaccharides
  • HS17 heparan sulfate analog, heparin fragment of 17 oligosaccharides
  • FIGS. 18A and 18B TBI enhances ⁇ - ⁇ binding and ⁇ -cleavage of APP a, Co- immunoprecipitation of ⁇ with APP showed increased ⁇ - ⁇ binding in after TBI in rat.
  • b Level of APP ⁇ -cleavage product (CTFP) is enhanced in correlation with increased ⁇ - ⁇ binding. Similar results are found using in mouse TBI brains.
  • FIG. 19 Heparin fragment of 17 oligosaccharides inhibits ⁇ - ⁇ binding.
  • APP fragment binding to ⁇ is detected by kinetic ELISA assay.
  • Heparin fragment of 17 oligosaccharides (heparan sulfate analog) effectively disrupts ⁇ - ⁇ binding when included in the binding assay.
  • APP fragment used here corresponds to SEQ ID NO: 1, which is the region between El and E2 domains.
  • ⁇ fragment used here includes its IG1 and IG2 domains.
  • FIG. 20 Ligand binding site of ⁇ IG1 domain interacts with APP. Binding of human APP fragment (SEQ ID NO: 1) with various ⁇ fragments is measured by kinetic ELISA assay. APP fragment corresponds to SEQ ID NO: 1, which is a region between El and E2 domains. ⁇ fragments used here include IG1,2 (containing IG1 and IG2 domains), ALysIGl,2
  • Example 1 shows that neuronal receptor ⁇ mediates both ⁇ - amyloid and Tau pathogenesis in two mouse models. In the brain, ⁇ binds to APP.
  • Example 2 shows that two classes of ⁇ ligands in the brain microenvironment, CS and HS, regulate APP amyloidogenic processing in opposite manners. CS increases APP ⁇ -cleavage products, whereas HS decreases APP ⁇ -cleavage products.
  • ⁇ plays a pivotal role in the development of ⁇ -amyloid and Tau pathologies
  • peptides, compositions, and methods disclosed herein may be suitable to treat and prevent neurodegenerative diseases that involve ⁇ -amyloid pathologies and/or Tau pathologies, including but not limited to Alzheimer's disease, Lewy body dementia,
  • frontotemporal dementia cerebral amyloid angiopathy, primary age-related tauopathy, chronic traumatic encephalopathy, Parkinson's disease, postencephalitic parkinsonism, Huntington's disease, amyolateral sclerosis, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, Lytico-Bodig disease, ganglioglioma and gangliocytoma, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and/or Creutzfeldt- Jakob disease.
  • these peptides, compositions, and methods may also be used to prevent these neurodegenerative diseases in at-risk populations, such as subjects with Down syndrome and those suffered from brain injuries or cerebral ischemia, as well as the aging population.
  • a cell includes a plurality of cells, including mixtures thereof.
  • protein protein
  • peptide and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.
  • protein includes amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc., and can contain modified amino acids other than the 20 gene-encoded amino acids.
  • polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art.
  • the term also includes peptidomimetics and cyclic peptides.
  • peptidomimetic means a mimetic of a peptide which includes some alteration of the normal peptide chemistry. Peptidomimetics typically enhance some property of the original peptide, such as increase stability, increased efficacy, enhanced delivery, increased half life, etc. Methods of making peptidomimetics based upon a known polypeptide sequence is described, for example, in U.S. Patent Nos. 5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involve the incorporation of a non-amino acid residue with non-amide linkages at a given position.
  • One embodiment of the present invention is a peptidomimetic wherein the compound has a bond, a peptide backbone or an amino acid component replaced with a suitable mimic.
  • suitable amino acid mimics include ⁇ -alanine, L-a-amino butyric acid, L-y-amino butyric acid, L-a-amino isobutyric acid, L-s-amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L- glutamic acid, ⁇ - ⁇ -Boc-N-a-CBZ-L-lysine, ⁇ - ⁇ -Boc-N-a-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-a-Boc-N-5CBZ-L-ornithine, ⁇ - ⁇ -Boc-N-a
  • a “fusion protein” refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide.
  • the fusion protein can be formed by the chemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein.
  • a single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in frame into a single nucleic acid, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.
  • binding is the binding of one protein to another.
  • the binding may comprise covalent bonds, protein cross-linking, and/or non-covalent interactions such as hydrophobic interactions, ionic interactions, or hydrogen bonds.
  • protein domain refers to a portion of a protein, portions of a protein, or an entire protein showing structural integrity; this determination may be based on amino acid composition of a portion of a protein, portions of a protein, or the entire protein.
  • Amyloid precursor protein (APP) is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. It has been implicated as a regulator of synapse formation, neural plasticity and iron export. APP is cleaved by beta secretase and gamma secretase to yield ⁇ . Amyloid beta ( ⁇ ) denotes peptides of 36-43 amino acids that are involved in Alzheimer's disease as the main component of the amyloid plaques found in the brains of Alzheimer patients. ⁇ molecules cleaved from APP can aggregate to form flexible soluble oligomers which may exist in various forms.
  • seeds can induce other ⁇ molecules to also take the misfolded oligomeric form, leading to a chain reaction and buildup of amyloid plaques.
  • the seeds or the resulting amyloid plaques are toxic to cells in the brain.
  • Protein tyrosine phosphatases or “receptor protein tyrosine phosphatases” (PTPs) are a group of enzymes that remove phosphate groups from phosphorylated tyrosine residues on proteins. Protein tyrosine phosphorylation is a common post-translational modification that can create novel recognition motifs for protein interactions and cellular localization, affect protein stability, and regulate enzyme activity. As a consequence, maintaining an appropriate level of protein tyrosine phosphorylation is essential for many cellular functions. Tyrosine-specific protein phosphatases catalyze the removal of a phosphate group attached to a tyrosine residue. These enzymes are key regulatory components in many signal transduction pathways (such as the MAP kinase pathway) that underlie cellular functions such as cell cycle
  • subject refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • An "at-risk” subject is an individual with a higher likelihood of developing a certain disease or condition.
  • An “at-risk” subject may have, for example, received a medical diagnosis associated with the certain disease or condition.
  • Tau proteins are proteins that stabilize microtubules. They are abundant in neurons of the central nervous system and are less common elsewhere, but are also expressed at very low levels in CNS astrocytes and oligodendrocytes. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and other tauopathies are associated with tau proteins that have become defective, misfolded, tangled, and no longer stabilize microtubules properly.
  • protein fragment refers to a functional portion of a full-length protein.
  • a fragment of APP or ⁇ may be synthesized chemically or biologically for the purposes of disrupting the binding between APP and ⁇ .
  • Such fragments could be used as "decoy" peptides to prevent or diminish the actual ⁇ - ⁇ binding interaction that results in ⁇ -cleavage of APP and subsequent ⁇ formation.
  • the phrase "functional fragment” or “analog” or mimetic of a protein or other molecule is a compound having qualitative biological activity in common with a full-length protein or other molecule of its entire structure.
  • a functional fragment of a full-length protein may be isolated and attached to a separate peptide sequence.
  • a functional fragment of a blood-brain barrier penetrating protein may be isolated and attached to the decoy peptide that disrupts ⁇ - ⁇ binding, thereby enabling the hybrid peptide to enter the brain and disrupt ⁇ - ⁇ binding.
  • Another example of a functional fragment is a membrane penetrating fragment, or one that relays an ability to pass the lipophilic barrier of a cell's plasma membrane.
  • An analog of heparin for example, may be a compound that binds to a heparin binding site.
  • cyclic peptide or “cyclopeptide” in general refers to a peptide comprising at least one internal bond attaching nonadjacent amino acids of the peptide, such as when the end amino acids of a linear sequence are attached to form a circular peptide.
  • antibody refers to natural or synthetic antibodies that selectively bind a target antigen.
  • the term includes polyclonal and monoclonal antibodies.
  • antibodies are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that selectively bind the target antigen.
  • enzyme refers to a protein specialized to catalyze or promote a specific metabolic reaction.
  • Neurodegenerative disorders or “neurodegenerative diseases” are conditions marked by the progressive loss of structure or function of neural cells, including death of neurons and glia.
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • administering refers to an administration that is intranasal, oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, rectal, vaginal, by inhalation or via an implanted reservoir.
  • parenteral includes
  • pharmaceutically acceptable carrier means a carrier or excipient that is useful in preparing a pharmaceutical composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical use.
  • pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further below.
  • the pharmaceutical compositions also can include preservatives.
  • pharmaceutically acceptable carrier includes both one and more than one such carrier.
  • variant refers to an amino acid or peptide sequence having conservative amino acid substitutions ("conservative variant"), non-conservative amino acid subsitutions (e.g., a degenerate variant), substitutions within the wobble position of each codon (i.e. DNA and RNA) encoding an amino acid, amino acids added to the C-terminus of a peptide, or a peptide having 60%, 70%, 80%, 90%, or 95% homology to a reference sequence.
  • percent (%) sequence identity is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
  • Peptides Disclosed herein are peptides for treating and preventing the aforementioned neurodegenerative diseases, such as Alzheimer's disease.
  • the peptides disrupt the binding between ⁇ and APP, preventing ⁇ -amyloidogenic processing of APP without affecting other major substrates of the ⁇ - and ⁇ -secretases.
  • the peptide may be a decoy fragment of APP, a decoy fragment of ⁇ , or a combination thereof.
  • a decoy peptide could be fabricated from the ⁇ -binding region on APP, which is the fragment between its El and E2 domains (SEQ ID NO: 1). In some embodiments, a decoy peptide could be fabricated from the APP -binding region on ⁇ , which is its IGl domain (SEQ ID NO: 442). In some embodiments, a decoy peptide could be fabricated that corresponds to the entire APP E2 domain or a fragment thereof. In some embodiments, a decoy peptide could be fabricated that corresponds to the entire APP El domain or a fragment thereof. In some embodiments, a ⁇ peptide is used in combination with an APP peptide.
  • the peptide is a fragment of the ⁇ -binding domain of APP. Therefore, in some embodiments, the peptide is a fragment of SEQ ID NO: 1, as listed below, which has at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more amino acids, or a conservative variant thereof.
  • the peptide comprises an amino acid sequence selected from 10 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
  • the peptide comprises an amino acid sequence selected from 11 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
  • the peptide comprises an amino acid sequence selected from 12 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below: SEQ ID NO 181 AEESDNVDSADA
  • the peptide comprises an amino acid sequence selected from 13 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
  • the peptide comprises an amino acid sequence selected from 14 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
  • the peptide comprises an amino acid sequence selected from 24 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
  • the peptide is a fragment of the APP -binding domain of ⁇ . Therefore, in some embodiments, the peptide is a fragment of SEQ ID NO:442, as listed below, which has at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more amino acids, or a conservative variant thereof.
  • the underlined amino acids represent residues in the ligand-binding pocket.
  • the peptide comprises an amino acid sequence selected from 10 consecutive residues of SEQ ID NO: 442, or from the group consisting of the below:
  • the peptide comprises an amino acid sequence selected from 11 consecutive residues of SEQ ID NO: 442, or from the group consisting of the below: SEQ ID NO 531 VHAKLTVLRE
  • the peptide comprises an amino acid sequence selected from 12 consecutive residues of SEQ ID NO: 442, or from the group consisting of the below:
  • the peptide comprises an amino acid sequence selected from 13 consecutive residues of SEQ ID NO: 442, or from the group consisting of the below:
  • the peptide comprises an amino acid sequence selected from 14 consecutive residues of SEQ ID NO: 442, or from the group consisting of the below:
  • the disclosed peptide further comprises a blood brain barrier penetrating sequence.
  • CPPs cell-penetrating peptides
  • BBB blood-brain barrier
  • the cellular internalization sequence can be any cell-penetrating peptide sequence capable of penetrating the BBB.
  • Non-limiting examples of CPPs include Polyarginine (e.g., R9), Antennapedia sequences, TAT, HIV-Tat, Penetratin, Antp-3 A (Antp mutant), Buforin II, Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynBl, Pep-7, HN-1, BGSC (Bis- Guanidinium-Spermidine-Cholesterol, and BGTC (Bis-Guanidinium-Tren-Cholesterol) (see Table 1).
  • Polyarginine e.g., R9
  • Antennapedia sequences e.g., TAT, HIV-Tat, Penetratin, Antp-3 A (Antp mutant), Buforin II, Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynBl, Pep-7
  • Antp-3 A RQIAIWFQNRRMKWAA SEQ ID NO:882
  • Transportan GWTLNSAGYLLGKINKALAALAKKIL SEQ ID NO:885 model KLALKLALKALKAALKLA SEQ ID NO:886 amphipathic
  • the disclosed peptide is a fusion protein, e.g., containing the APP -binding domain of ⁇ , the ⁇ -binding domain of APP, or a combination thereof, and a CPP.
  • Fusion proteins also known as chimeric proteins, are proteins created through the joining of two or more genes, which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with function properties derived from each of the original proteins. Recombinant fusion proteins can be created artificially by recombinant DNA technology for use in biological research or therapeutics.
  • linker (or "spacer") peptides are also added which make it more likely that the proteins fold independently and behave as expected.
  • Linkers in protein or peptide fusions are sometimes engineered with cleavage sites for proteases or chemical agents which enable the liberation of the two separate proteins. This technique is often used for identification and purification of proteins, by fusing a GST protein, FLAG peptide, or a hexa-his peptide (aka: a 6xhis-tag) which can be isolated using nickel or cobalt resins (affinity chromatography).
  • Chimeric proteins can also be manufactured with toxins or antibodies attached to them in order to study disease development.
  • compositions that restore molecular balance of CS and HS in the perineuronal space Compositions that restore molecular balance of CS and HS in the perineuronal space:
  • CS Chondroitin sulfates
  • HS heparin or its analog heparan sulfates
  • GAGs glycosaminoglycans
  • the ratio of CS and HS therefore affects the downstream effects of ⁇ , because CS and HS compete to interact with the receptor yet lead to opposite signaling and neuronal responses (such as neurite regeneration).
  • CS increases but HS decreases APP ⁇ - cleavage products (Example 2). Therefore, methods involving administering to the subject a composition that restore the physiological molecular CS/HS balance may be used to treat and prevent aforementioned neurodegenerative diseases.
  • These therapies could be applied alternatively or in addition to the polypeptides listed above.
  • administering HS, or its analog heparin, or their mimetics modified to reduce anti-coagulant effect, with a saccharide chain length of 17, 18, 19, 20, 21, 22, 23, 24 units or longer could assist in restoring the physiological molecular CS/HS balance.
  • the balance is restored by administering enzymes that digest CS (such as ChABC) or prevent the degradation of HS (such as Heparanase inhibitors PI-88, OGT 2115, or PG545).
  • agents that mimic the HS/heparin effect of ⁇ clustering 8 such as multivalent antibodies, could be administered.
  • the peptides disclosed can be used therapeutically in combination with a
  • compositions suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • the peptides described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art (See, e.g., Ansel,
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
  • a carrier such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.

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Abstract

Disclosed herein are compositions and methods for treating and preventing neurodegenerative diseases, such as Alzheimer's disease. In some embodiments, the composition comprises a peptide that disrupts the binding between PTPs and APP, preventing β-amyloidogenic processing of APP without affecting other major substrates of b- and γ-secretases. Alternatively, in some embodiments, an antibody or a fragment of an antibody against PTPs or APP may be used to disrupt the binding between PTPs and APP. In some embodiments, the composition comprises compounds or enzymes, which restore perineuronal balance of PTPs ligands CS and HS, thereby preventing abnormally increased β-amyloidogenic processing of APP. Compositions and methods disclosed herein can be used in combination to treat and prevent neurodegenerative diseases.

Description

PEPTIDES AND METHODS FOR TREATING
NEURODEGENERATIVE DISORDERS
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application number 62/505,497, filed May 12, 2017, which is hereby incorporated by reference in its entirety for all purposes.
BACKGROUND
Alzheimer's disease (AD) is the most common form of dementia, and its risk accelerates after age 65. With a rapidly expanding aging population, AD is projected to become an overwhelming medical burden to the world.
A definitive pathological hallmark of Alzheimer's disease (AD) is the progressive aggregation of β-amyloid (Αβ) peptides in the brain, a process also known as β-amyloidosis, which is often accompanied by neuroinflammation and formation of neurofibrillary tangles containing Tau, a microtubule binding protein_1.
Evidence from human genetic studies showed that overproduction of Αβ due to gene mutations inevitably inflicts cascades of cytotoxic events, ultimately leading to
neurodegeneration and decay of brain functions. Cerebral accumulation of Αβ peptides, especially in their soluble forms, is therefore recognized as a key culprit in the development of AD 1. In the brain, Αβ peptides mainly derive from sequential cleavage of neuronal Amyloid Precursor Protein (APP) by the β- and γ-secretases. However, despite decades of research, molecular regulation of the amyloidogenic secretase activities remains poorly understood, hindering the design of therapeutics to specifically target the APP amyloidogenic pathway.
Pharmacological inhibition of the β- and γ-secretase activities, although effective in suppressing Αβ production, interferes with physiological function of the secretases on their other substrates. Such intervention strategies therefore are often innately associated with untoward side effects, which have led to several failed clinical trials in the past 2"4. To date, no therapeutic regimen is available to prevent the onset of AD or curtail its progression.
Besides Αβ, Tau is another biomarker that has been intensively studied in AD. Cognitive decline in patients sometimes correlates better with Tau pathology than with Αβ burden 5 <5. Overwhelming evidence also substantiated that malfunction of Tau contributes to synaptic loss and neuronal deterioration 1. In addition to AD, many other neurodegenerative diseases also involves Αβ or Tau pathologies, and there is no disease modifying therapy available for any of these debilitating diseases.
SUMMARY
Disclosed herein are peptides, compositions, and methods to treat and prevent neurodegenerative diseases that involve β-amyloid pathologies and/or Tau pathologies, including but not limited to Alzheimer's disease, Lewy body dementia, frontotemporal dementia, cerebral amyloid angiopathy, primary age-related tauopathy, chronic traumatic encephalopathy, Parkinson's disease, postencephalitic parkinsonism, Huntington's disease, amyolateral sclerosis, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, Lytico-Bodig disease, ganglioglioma and gangliocytoma, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and/or Creutzfeldt- Jakob disease.
These peptides, compositions, and methods may also be used to prevent these
neurodegenerative diseases in at-risk subjects, such as people with Down syndrome and those who have suffered from brain injuries or cerebral ischemia, as well as the aging population.
In some embodiments, the disclosed peptides, compositions, and methods disrupt the binding between Protein Tyrosine Phosphatase sigma (ΡΤΡσ) and APP, preventing β- amyloidogenic processing of APP as well as Tau aggregation.
In some embodiments, the disclosed compositions and methods restore the physiological balance of two classes of ΡΤΡσ ligands in the brain microenvironment, namely the chondroitin sulfates (CS) and heparin or its analog heparan sulfates (HS), and thereby prevent abnormally increased β-amyloidogenic processing of APP.
Unlike the anti-Αβ antibodies in current clinical trials that passively clear β-amyloid, the therapeutic strategy disclosed herein inhibits the process upstream of β-amyloid production. Unlike the β- and γ-secretase inhibitors in current clinical trials, the therapeutic strategy disclosed herein inhibits β-amyloid production without affecting other major substrates of these secretases. Therefore the strategy disclosed herein may be more effective with fewer side effects compared to the most advanced AD drug candidates in clinical trials.
Disclosed herein is a peptide for treating or preventing the aforementioned
neurodegenerative disorders, the peptide comprising a decoy fragment of APP, a decoy fragment of ΡΤΡσ, or a combination thereof. In some embodiments, the decoy fragment of APP is a peptide comprising at least 5 consecutive amino acids of SEQ ID NO: l . In some embodiments, the decoy fragment of APP is a peptide comprising at least 10 consecutive amino acids of SEQ ID NO: l . For example, the decoy fragment of APP can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO: 101, SEQ ID NO: 112, SEQ ID NO: 139, SEQ ID NO: 151, SEQ ID NO: 157, SEQ ID NO:251, SEQ ID NO:897. In some embodiments, the decoy fragment of ΡΤΡσ is a peptide comprising at least 4 consecutive amino acids of SEQ ID NO:442. For example, the decoy fragment of ΡΤΡσ can comprises the amino acid sequence SEQ ID NO:655, SEQ ID NO:769, SEQ ID NO:898, or SEQ ID NO:899. In some embodiments, the peptide further comprises a blood brain barrier penetrating sequence. For example, the blood brain barrier penetrating sequence comprises amino acid sequence SEQ ID NO: 880, SEQ ID NO: 883, SEQ ID NO: 888, SEQ ID NO: 894, SEQ ID NO: 895, SEQ ID NO: 896.
Also disclosed is a method that restores the physiological molecular CS/HS balance that may be used to treat and prevent aforementioned neurodegenerative diseases. In some embodiments, administering HS, or its analog heparin, or their mimetics modified to reduce anti -coagulant effect, with a saccharide chain length of 17, 18, 19, 20, 21, 22, 23, 24 units or longer, could assist in restoring the CS/HS balance. In some embodiments, the physiological molecular CS/HS balance is restored by administering enzymes that digest CS (such as
Chondroitinase ABC, also known as ChABC) or prevent HS degradation (such as Heparanase inhibitors PI-88, OGT 21 15, or PG545). Alternatively or in addition, agents that mimic the HS/heparin effect of ΡΤΡσ clustering 8, such as multivalent antibodies, could be administered.
Also disclosed is a method of treating a neurodegenerative disorder in a subject, the method comprising administering to the subject an aforementioned composition or combination of compositions. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's Disease, Lewy body dementia, frontotemporal dementia, cerebral amyloid angiopathy, primary age-related tauopathy, chronic traumatic encephalopathy, Parkinson's disease, postencephalitic parkinsonism, Huntington's disease, amyolateral sclerosis, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, Lytico-Bodig disease, ganglioglioma and gangliocytoma, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and/or Creutzfeldt- Jakob disease. In some embodiments, subjects are selected from at- risk populations, such as the aging population, people with Down syndrome, and those suffered from brain injuries or cerebral ischemia, to prevent subsequent onset of neurodegenerative diseases. Also disclosed is a method of screening for candidate compounds that slow, stop, reverse, or prevent neurodegeneration. In some embodiments, the method comprises providing a sample comprising APP and ΡΤΡσ in an environment permissive for ΑΡΡ-ΡΤΡσ binding, contacting the sample with a candidate compound, and assaying the sample for ΑΡΡ-ΡΤΡσ binding, wherein a decrease in ΑΡΡ-ΡΤΡσ binding compared to control values is an indication that the candidate agent is effective to slow, stop, reverse, or prevent neurodegeneration. In some embodiments, the method comprises contacting/incubating a candidate compound with cell membrane preparations extracted from fresh rodent brain homogenates, wherein a decrease in APP β- and/or γ-cleavage products is an indication that the candidate agent has the potential to slow, stop, reverse, or prevent neurodegeneration.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
DESCRIPTION OF DRAWINGS
Figures lA-lL ΡΤΡσ is an APP binding partner in the brain, a-f, Colocalization of
ΡΤΡσ (a, green) and APP (b, red) in hippocampal CA1 neurons of adult rat is shown by confocal imaging. Nuclei of CA1 neurons are stained with DAPI (c, blue), d, Merge of three channels. Scale bar, 50 μιη. e, Zoom-in image of the soma layer in d. Arrows, intensive colocalization of ΡΤΡσ and APP in the initial segments of apical dendrites; arrow heads, punctates of
colocalization in the perinuclear regions. Scale bar, 20 μιη. f, Zoom-in image of the very fine grained punctates in the axonal compartment in d. Arrows points to the colocalization of ΡΤΡσ and APP in axons projecting perpendicular to the focal plane. Scale bar, 10 μπι. g, Schematic diagram of ΡΤΡσ expressed on cell surface as a two-subunit complex. ΡΤΡσ 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, i, Co-immunoprecipitation (co-IP) of ΡΤΡσ and APP from mouse forebrain lysates. Left panels, expression of ΡΤΡσ 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, ΡΤΡσ co-IP with APP from forebrain lysates of wild type but not ΡΤΡσ-deficient mice (Balb/c background), detected by an antibody against ΡΤΡσ- ECD. i, ΡΤΡσ co-IP with APP from forebrain lysates of wild type but not APP knockout mice (B6 background), detected by an antibody against ΡΤΡσ-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 using mice between ages of 1 month to 2 years.
Figures 2A-2C. Molecular complex of ΡΤΡσ and APP in brains of various rodent species, a, b, Co-immunoprecipitation using an anti-APP antibody specific for amino acid residues 1-16 of mouse Αβ (clone M3.2). ΡΤΡσ and APP binding interaction is detected in forebrains of Balb/c (a) and B6 (b) mice, c, ΡΤΡσ co-immunoprecipitates with APP from rat forebrain lysates using an antibody specific for the C-terminus of APP. Images shown are representatives of at least three independent experiments using different animals.
Figures 3A-3L Genetic depletion of ΡΤΡσ reduces β-amyloidogenic products of
APP. a, Schematic diagram showing amyloidogenic processing of APP by the β- and γ- secretases. Full length APP (APP FL) is cleaved by β-secretase into soluble N-terminal (sAPPP) and C-terminal (CTFP) fragments. APP CTFp can be further processed by γ-secretase into a C- terminal intracellular domain (AICD) and an Αβ peptide. Aggregation of Αβ is a definitive pathology hallmark of AD. b, ΡΤΡσ 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 CTFP by immunoprecipitation (IP) followed with western blot analysis, using a pair of antibodies as marked in the diagram (a). Antibodies against amino acids 1-16 of Αβ (anti-Αβ 1-16) detect CTFp but not CTF a, as the epitope is absent in CTFa. c,
Mouse endogenous CTFp level is reduced in ΡΤΡσ-deficient mouse brains. 4 repeated experiments were quantified by densitometry, d, Human transgenic CTFP level is reduced in ΡΤΡσ-deficient mouse brains harboring human APP-SwDI transgene. 6 repeated experiments were quantified by densitometry. Within each experiment in both c and d, the value from ΡΤΡσ deficient sample was normalized to that from the sample with wild type ΡΤΡσ. e and f, ΡΤΡσ deficiency reduces the levels of Αβ40 (e) and Αβ42 (f) in TgAPP-SwDI mice as measured by ELISA assays. n=12 for each group. The mean values from ΡΤΡσ deficient samples was normalized to that from the samples with wild type ΡΤΡσ. g and h, Αβ deposition in the hippocampus of 10-month old TgAPP-SwDI mice. Images shown are representatives of 5 pairs of age- and sex -matched mice between 9- to 11 -month old. Αβ (green) is detected by
immunofluorescent staining using anti-Αβ antibodies clone 6E10 (g) and clone 4G8 (h). DAPI staining is shown in blue. ΡΤΡσ deficiency significantly decreases Αβ burden in the brains of TgAPP-SwDI mice, h, Upper panels, the stratum oriens layer between dorsal subiculum (DS) and CA1 (also shown with arrows in g); middle panels, oriens layer between CA1 and CA2; lower panels, the hilus of dentate gyrus (DG, also shown with arrow heads in g). Left column, control staining without primary antibody (no 1° Ab). No Αβ signal is detected in non- transgenic mice (data not shown). Scale bars, 500 μιη in g and 100 μιη in h. i, Genetic depletion of ΡΤΡσ suppresses the progression of Αβ pathology in TgAPP-SwDI mice. ImageJ
quantification of Αβ immunofluorescent staining (with 6E10) in DG hilus from 9- and 16-month old TgAPP-SwDI mice. n=3 for each group. Total integrated density of Αβ 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 ΡΤΡσ. All p values, Student's t test, 2-tailed. Error bars, SEM.
Figures 4A-4F. Genetic depletion of ΡΤΡσ reduces β-amyloidogenic products of APP. a and b, Antibody against the C-terminus of APP recognizes full length (FL) and C- terminal fragments (CTFs) of both mouse and human APP. ΡΤΡσ deficiency does not affect the expression level of APP FL (a), but reduces the level of an APP CTF at about 15 KD in mouse forebrain lysates (b). Images shown are representatives of at least three independent
experiments, c, Human CTFβ in the forebrains of APP-SwInd transgenic mice is identified using the method as described in Fig.2d. CTFβ is immunoprecipitated by an antibody against the C- terminus of APP and detected by western blot analysis using an antibody against amino acids 1- 16 of human Αβ (6E10), which reacts with CTFβ but not CTFa (regions of antibody epitopes are shown in Fig. 2a). d, Densitometry quantification of experiments as shown in panel c repeated with 5 pairs of mice. For each experiment, the value from ΡΤΡσ deficient sample was normalized to the value from the sample with wild type ΡΤΡσ. e, Representative images of Αβ immunofluorescent staining (with 6E10) in the hippocampus of 15-month old TgAPP-SwInd mice. Arrows point to Αβ deposits. Scale bars, 50 μπι. f, Αβ immunofluorescent staining in the hippocampus of 15-month old TgAPP-SwInd mice, as shown in panel e, was quantified using ImageJ. APP-SwInd(+)PTPo(+/+), n=7; APP-SwInd(+)PTPo(-/-), n=8. The mean value of APP- SwInd(+)PTPo(-/-) samples was normalized to that of APP-SwInd(+)PTPo(+/+) samples. All error bars, SEM. All p values, Student's t test, 2-tailed.
Figures 5A-5C. Lower affinity between BACE1 and APP in ΡΤΡσ-deficient brains, a, Co-immunoprecipitation experiments show nearly equal BACEl-APP association in wild type and ΡΤΡσ-deficient mouse brains under mild detergent condition (1% NP40). However, in ΡΤΡσ-deficient brains, BACEl-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. P40, Nonidet P-40, non-ionic detergent. SDS, Sodium dodecyl sulfate, ionic detergent, b, Co-immunoprecipitation under buffer condition with 1% NP40 and 0.3% SDS, as shown in the middle panel of a, were repeated with three pair of mice. Each experiment was quantified by densitometry, and the value from ΡΤΡσ-deficient sample was calculated as a percentage of that from the wild type sample (also shown as orange points in c). Error bar, SEM. p value, Student's t test, 2-tailed. c, Co-immunoprecipitation experiments were repeated under each detergent condition. The percentage values shown in dots are derived using the same method as in b. Bars represent means. Increasingly stringent buffer conditions manifest a lower BACEl-APP affinity in ΡΤΡσ-deficient brains, p value and R2, linear regression.
Figures 6A-6F. ΡΤΡσ does not generically modulate b- and g- secretases. Neither expression levels of the secretases or their activities on other major substrates are affected by ΡΤΡσ depletion. Mouse forebrain lysates with or without ΡΤΡσ were analyzed by western blot, a and b, ΡΤΡσ deficiency does not change expression level of BACE1 (a) or γ-secretase subunits (b). Presenilinl and 2 (PS 1/2) are the catalytic subunits of γ-secretase, which are processed into N-terminal and C-terminal fragments (NTF and CTF) in their mature forms. Nicastrin,
Presenilin Enhancer 2 (PEN2), and APHl are other essential subunits of γ-secretase. c, ΡΤΡσ deficiency does not change the level of Neuregulinl (NGR1) CTFp, the C-terminal cleavage product by BACE1. NRG1 FL, full length Neuregulinl . d, The level of Notch cleavage product by γ-secretase is not affected by ΡΤΡσ deficiency. TMIC, Notch transmembrane/intracellular fragment, which can be cleaved by γ-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 γ-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 using different animals.
Figures 7A-7K. ΡΤΡσ 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 ΡΤΡσ depletion. Representative images show GFAP (red) and DAPI staining of nuclei (blue) in the brains of 9-month old TgAPP-SwDI mice with or without ΡΤΡσ, along with their non-transgenic wild type littermate. a-f, Dentate gyrus (DG) of the
hippocampus; scale bars, 100 μπι. g-j, Primary somatosensory cortex; scale bars, 200 μπι. k, ImageJ quantification of GFAP level in DG hilus from TgAPP-SwDI mice aged between 9 to 11 months. APP-SwDI(-)PTPo(+/+), non-transgenic wild type littermates (expressing ΡΤΡσ 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. Mean value of each group was normalized to that of APP-SwDI(-)PTPo(+/+) mice. APP-SwDI(-)PTPo(+/+), n=4; APP-SwDI(+)PTPo(+/+), n=4; APP-SwDI(+)PTPo(-/-), n=6. All p values, Student' s t test, 2- tailed. Error bars, SEM.
Figures 8A-8G. ΡΤΡσ 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; DAP I, blue. Scale bars, 100 μπι. 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. Mean value of each group was normalized to that of wild type APP-SwDI(-)PTPo(+/+) mice. APP-SwDI(-)PTPo(+/+), n=4; APP-
SwDI(+)PTPo(+/+), n=6; APP-SwDI(+)PTPo(-/-), n=6. All p values, Student's t test, 2-tailed. Error bars, SEM.
Figures 9A-9H. ΡΤΡσ deficiency mitigates Tau pathology in TgAPP-SwDI mice, a,
Schematic diagram depicting distribution pattern of Tau aggregation (green) detected by immunofluorescent staining using an anti-Tau antibody (Tau-5) against its proline-rich region, in brains of 9 to 11 month-old TgAPP-SwDI transgenic mice. Similar results are seen with Tau- 46, an antibody recognizing the C-terminus of Tau (Extended Data Fig. 6). Aggregated Tau is found most prominently in the molecular layer of piriform and entorhinal cortex, and
occasionally in hippocampal regions in APP-SwDI(+)PTPo(+/+) mice, b, ΡΤΡσ deficiency diminishes Tau aggregation. Bar graph shows quantification of Tau aggregation in coronal brain sections from 4 pairs of age- and sex-matched 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. /? 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, f, g, Representative images of a few areas with Tau aggregation in age-matched 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-transgenic wild type littermates (expressing ΡΤΡσ but not the human APP transgene). Tau, green; DAPI, blue. Arrows points to Tau aggregates. Scale bars, 50 μιη.
Figures 10A-10E. ΡΤΡσ deficiency mitigates Tau pathology in TgAPP-SwInd mice. Tau aggregation (green) is detected by immunofluorescent staining, using an anti-Tau antibody (Tau-5, as in Fig. 5) in the brains of 15 month-old TgAPP-SwInd transgenic mice. Similar results are seen with Tau-46, an antibody recognizing the C-terminus of Tau (Extended Data Fig. 6). Aggregated Tau is found most prominently in the molecular layer of the entorhrinal (a, b) and piriform cortex (c, d), and occasionally in the hippocampal regions (images not shown), e, ΡΤΡσ deficiency diminishes Tau aggregation as quantified in coronal brain sections from 15 month-old APP-SwInd(+)PTPo(+/+) (n=7) and APP-SwInd(+)PTPo(-/-) mice (n=8). The mean value of APP-SwInd(+)PTPo(-/-) samples is normalized to that of APP-SwInd(+)PTPo(+/+). jp value, Student's t test, 2-tailed. Error bars, SEM. Tau, green; DAPI, blue. Arrows points to Tau aggregates. Scale bars, 50 μπι.
Figures 11A-11J. Morphology of Tau aggregates found in APP transgenic brains, a- h, Tau aggregation (green) is detected by immunofluorescent staining, using an anti-Tau antibody (Tau-5) against the proline-rich domain of Tau (same as in Fig. 5 and Extended Data Fig. 5). Tau aggregates in TgAPP-SwDI and TgAPP-SwInd brains show similar morphologies, a-f, Many of the Tau aggregates are found in punctate shapes, likely as part of cell debris, in areas that are free of nuclei staining, g, h, Occasionally the aggregates are found in fibrillary structures, probably in degenerated cells before disassembling, i, An additional anti-Tau antibody (Tau-46), which recognizes the C-terminus of Tau, detects Tau aggregation in the same pattern as Tau-5. j, Image of staining without primary antibody at the same location of the Tau aggregates in the section adjacent to i. Both these antibodies recognize Tau regardless of its phosphorylation status. Tau, green; DAPI, blue. All scale bars, 20 μπι.
Figure 12. Tau expression is not affected by ΡΤΡσ or human APP transgenes. Upper panel, total Tau level in brain homogenates. Lower panel, Actin as loading control. Tau protein expression level is not changed by genetic depletion of ΡΤΡσ or expression of mutated human APP transgenes. All mice are older than 1 year, and mice in each pair are age- and sex matched. Images shown are representatives of three independent experiments.
Figures 13A-13C. ΡΤΡσ 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 short-term spatial memory, which is rescued by genetic depletion of ΡΤΡσ in APP-SwDI(+)PTPo(-/-) mice. APP- SwDI(-)PTPo(+/+), n=23 (18 females and 5 males); APP-SwDI(+)PTPo(+/+), n=52 (30 females and 22 males); APP-SwDI(+)PTPo(-/-), n=35 (22 females and 13 males). 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 APP-SwDI(-)PTPo(+/+) mice. The deficit is reversed by ΡΤΡσ depletion in APP-SwDI(+)PTPo(-/-) mice. APP-SwDI(-)PTPo(+/+), n=28 (19 females and 9 males); APP-SwDI(+)PTPo(+/+), n=46 (32 females and 14 males); APP- SwDI(+)PTPo(-/-), n=29 (21 females and 8 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.
Figure 14. ΡΤΡσ deficiency restores short-term spatial memory in TgAPP-SwDI mice. In the Y-maze assay, 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. Compared to non-transgenic wild type APP-SwDI(-)PTPo(+/+)mice, APP-SwDI(+)PTPo(+/+) mice show deficit of short-term spatial memory, which is rescued by genetic depletion of ΡΤΡσ. APP-SwDI(-)PTPo(+/+), n=23 (18 females and 5 males); APP- SwDI(+)PTPo(+/+), n=52 (30 females and 22 males); APP-SwDI(+)PTPo(-/-), n=35 (22 females and 13 males). Ages of all genotype groups are similarly distributed between 4 and 11 months. All ^ values, Student's t test, 2-tailed. Error bars, SEM.
Figures 15A-15D. ΡΤΡσ 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. For non-transgenic wild type APP-SwDI(-)PTPo(+/+) mice, the discrimination index is slightly above 0.5 (chance value), similar to what was previously reported for the Balb/c wild type mice 21. Thus, a sole measurement of the discrimination index may not reveal the preference for NO as does the NO/FO ratio. Although not as sensitive in measuring object preference, the NO/(NO+FO) index is most commonly used as it provides a normalization of the NO exploration to total object exploration activity. While each has its own advantage and shortcoming, both NO/FO and NO/NO+FO measurements consistently show that the expression of TgAPP-SwDI gene leads to a deficit in attention to the NO, whereas genetic depletion of ΡΤΡσ restores novelty exploration to a level close to that of non-transgenic wild type mice, a and c, measurements in terms of exploration time, b and d, measurements in terms of visiting frequency. APP-SwDI(-)PTPo(+/+), n=28 (19 females and 9 males); APP-SwDI(+)PTPo(+/+), n=46 (32 females and 14 males); APP-SwDI(+)PTPo(-/-), n=29 (21 females and 8 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.
Figures 16A-16C. ΡΤΡσ deficiency improves behavioral performance of TgAPP- Swlnd mice, a, Performance of spatial navigation is scored by the percentage of spontaneous alternations among total arm entries in the Y-maze assay. Compared to APP- SwInd(+)PTPo(+/+) mice, APP-SwInd(+)PTPo(-/-) mice showed improved short-term spatial memory. APP-SwInd(+)PTPo(+/+), n=40 (20 females and 20 males); APP-SwInd(+)PTPo(-/-), n=18 (9 females and 9 males). Ages of both genotype groups are similarly distributed between 4 and 11 months, b, c, Novel object test. NO, novel object. FO, familiar object. NO preference is measured by the ratio of NO exploration time to total object exploration time (b) and the ratio of NO exploration time to FO exploration time (c). ΡΤΡσ depletion significantly improves novelty preference in these transgenic mice. APP-SwInd(+)PTPo(+/+), n=43 (21 females and 22 males) ; APP-SwInd(+)PTPo(-/-), n=24 (10 females and 14 males). Ages of both groups are similarly distributed between 5 and 15 months. All p values, Student's t test, 2-tailed. Error bars, SEM.
FIG. 17. CS and HS regulate β-cleavage of APP in opposite manners. Membrane preparations from fresh mouse brain homogenates are incubated with CS18 (chondroitin sulfate of 18 oligosaccharides) or HS17 (heparan sulfate analog, heparin fragment of 17 oligosaccharides) at 37C° for 30 min. Levels of APP β-cleavage product (CTFP) as detected by Western blot analysis are enhanced by CS18 treatment but diminished by HS17 treatment. FL APP, full length APP. Control, no treatment.
FIGS. 18A and 18B. TBI enhances ΡΤΡσ-ΑΡΡ binding and β-cleavage of APP a, Co- immunoprecipitation of ΡΤΡσ with APP showed increased ΡΤΡσ-ΑΡΡ binding in after TBI in rat. b, Level of APP β-cleavage product (CTFP) is enhanced in correlation with increased ΡΤΡσ-ΑΡΡ binding. Similar results are found using in mouse TBI brains. FIG. 19 Heparin fragment of 17 oligosaccharides inhibits ΑΡΡ-ΡΤΡσ binding.
Recombinant human APP fragment binding to ΡΤΡσ is detected by kinetic ELISA assay. Heparin fragment of 17 oligosaccharides (heparan sulfate analog) effectively disrupts ΑΡΡ-ΡΤΡσ binding when included in the binding assay. APP fragment used here corresponds to SEQ ID NO: 1, which is the region between El and E2 domains. ΡΤΡσ fragment used here includes its IG1 and IG2 domains.
FIG. 20 Ligand binding site of ΡΤΡσ IG1 domain interacts with APP. Binding of human APP fragment (SEQ ID NO: 1) with various ΡΤΡσ fragments is measured by kinetic ELISA assay. APP fragment corresponds to SEQ ID NO: 1, which is a region between El and E2 domains. ΡΤΡσ fragments used here include IG1,2 (containing IG1 and IG2 domains), ALysIGl,2
(containing IG1 and IG2 domains, with lysine 67, 68, 70, 71 mutated to alanine), IG1-FN1 (containing IG1, IG2, IG3 and FN1 domains), ECD (full extracellular domain of ΡΤΡσ containing all 3 IG domains and 4 FN domains). Value shown are mean±SEM, n=3 for each group. ***, p<0.001, Student t test, comparison with the IG1,2. DETAILED DESCRIPTION
Experimental results in Example 1 show that neuronal receptor ΡΤΡσ mediates both β- amyloid and Tau pathogenesis in two mouse models. In the brain, ΡΤΡσ binds to APP.
Depletion of ΡΤΡσ reduces the affinity between APP and β-secretase, diminishing APP proteolytic products by β- and γ-cleavage without affecting other major substrates of the secretases, suggesting a specificity of β-amyloidogenic regulation. In human APP transgenic mice during aging, the progression of β-amyloidosis, Tau aggregation, neuroinflammation, synaptic loss, as well as behavioral deficits, all show unambiguous dependency on the expression of ΡΤΡσ. Additionally, the aggregates of endogenous Tau are found in a distribution pattern similar to that of early stage neurofibrillary tangles in Alzheimer brains. Together, these findings unveil a gatekeeping role of ΡΤΡσ upstream of the degenerative pathogenesis, indicating a potential for this neuronal receptor as a drug target for Alzheimer's disease.
Experimental results in Example 2 show that two classes of ΡΤΡσ ligands in the brain microenvironment, CS and HS, regulate APP amyloidogenic processing in opposite manners. CS increases APP β-cleavage products, whereas HS decreases APP β-cleavage products.
Because CS and HS compete to interact with receptor ΡΤΡσ yet lead to opposite signaling and neuronal responses, the ratio of perineuronal CS and HS is therefore crucial for the downstream effects of ΡΤΡσ and maintaining the health of the brain. Experimental results in Example 3 further define that the binding between APP and ΡΤΡσ is mediated by a fragment on APP between its El and E2 domain and the IGl domain of ΡΤΡσ.
The findings that ΡΤΡσ plays a pivotal role in the development of β-amyloid and Tau pathologies indicate that peptides, compositions, and methods disclosed herein may be suitable to treat and prevent neurodegenerative diseases that involve β-amyloid pathologies and/or Tau pathologies, including but not limited to Alzheimer's disease, Lewy body dementia,
frontotemporal dementia, cerebral amyloid angiopathy, primary age-related tauopathy, chronic traumatic encephalopathy, Parkinson's disease, postencephalitic parkinsonism, Huntington's disease, amyolateral sclerosis, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, Lytico-Bodig disease, ganglioglioma and gangliocytoma, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and/or Creutzfeldt- Jakob disease.
Additionally, these peptides, compositions, and methods may also be used to prevent these neurodegenerative diseases in at-risk populations, such as subjects with Down syndrome and those suffered from brain injuries or cerebral ischemia, as well as the aging population.
Definitions
As used in the specification and claims, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.
The terms "about" and "approximately" are defined as being "close to" as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.
The terms "protein," "peptide," and "polypeptide" are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another. The term "protein" includes amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc., and can contain modified amino acids other than the 20 gene-encoded amino acids. The
polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. The term also includes peptidomimetics and cyclic peptides.
As used herein, "peptidomimetic" means a mimetic of a peptide which includes some alteration of the normal peptide chemistry. Peptidomimetics typically enhance some property of the original peptide, such as increase stability, increased efficacy, enhanced delivery, increased half life, etc. Methods of making peptidomimetics based upon a known polypeptide sequence is described, for example, in U.S. Patent Nos. 5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involve the incorporation of a non-amino acid residue with non-amide linkages at a given position. One embodiment of the present invention is a peptidomimetic wherein the compound has a bond, a peptide backbone or an amino acid component replaced with a suitable mimic. Some non-limiting examples of unnatural amino acids which may be suitable amino acid mimics include β-alanine, L-a-amino butyric acid, L-y-amino butyric acid, L-a-amino isobutyric acid, L-s-amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L- glutamic acid, Ν-ε-Boc-N-a-CBZ-L-lysine, Ν-ε-Boc-N-a-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-a-Boc-N-5CBZ-L-ornithine, Ν-δ-Boc-N-a-CBZ-L-ornithine, Boc- p-nitro-L-phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.
A "fusion protein" refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein can be formed by the chemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in frame into a single nucleic acid, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.
As used herein, protein "binding" is the binding of one protein to another. The binding may comprise covalent bonds, protein cross-linking, and/or non-covalent interactions such as hydrophobic interactions, ionic interactions, or hydrogen bonds.
The term "protein domain" refers to a portion of a protein, portions of a protein, or an entire protein showing structural integrity; this determination may be based on amino acid composition of a portion of a protein, portions of a protein, or the entire protein.
"Amyloid precursor protein" (APP) is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. It has been implicated as a regulator of synapse formation, neural plasticity and iron export. APP is cleaved by beta secretase and gamma secretase to yield Αβ. Amyloid beta (Αβ) denotes peptides of 36-43 amino acids that are involved in Alzheimer's disease as the main component of the amyloid plaques found in the brains of Alzheimer patients. Αβ molecules cleaved from APP can aggregate to form flexible soluble oligomers which may exist in various forms. Certain misfolded oligomers (known as "seeds") can induce other Αβ molecules to also take the misfolded oligomeric form, leading to a chain reaction and buildup of amyloid plaques. The seeds or the resulting amyloid plaques are toxic to cells in the brain.
"Protein tyrosine phosphatases" or "receptor protein tyrosine phosphatases" (PTPs) are a group of enzymes that remove phosphate groups from phosphorylated tyrosine residues on proteins. Protein tyrosine phosphorylation is a common post-translational modification that can create novel recognition motifs for protein interactions and cellular localization, affect protein stability, and regulate enzyme activity. As a consequence, maintaining an appropriate level of protein tyrosine phosphorylation is essential for many cellular functions. Tyrosine-specific protein phosphatases catalyze the removal of a phosphate group attached to a tyrosine residue. These enzymes are key regulatory components in many signal transduction pathways (such as the MAP kinase pathway) that underlie cellular functions such as cell cycle
control/proliferation, cell death, differentiation, transformation, cell polarity and motility, synaptic plasticity, etc.
The term "subject" refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term "patient" refers to a subject under the treatment of a clinician, e.g., physician. An "at-risk" subject is an individual with a higher likelihood of developing a certain disease or condition. An "at-risk" subject may have, for example, received a medical diagnosis associated with the certain disease or condition.
"Tau proteins" (or τ proteins) are proteins that stabilize microtubules. They are abundant in neurons of the central nervous system and are less common elsewhere, but are also expressed at very low levels in CNS astrocytes and oligodendrocytes. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and other tauopathies are associated with tau proteins that have become defective, misfolded, tangled, and no longer stabilize microtubules properly.
The term "protein fragment" refers to a functional portion of a full-length protein. For example, a fragment of APP or ΡΤΡσ may be synthesized chemically or biologically for the purposes of disrupting the binding between APP and ΡΤΡσ. Such fragments could be used as "decoy" peptides to prevent or diminish the actual ΑΡΡ-ΡΤΡσ binding interaction that results in β-cleavage of APP and subsequent Αβ formation.
The phrase "functional fragment" or "analog" or mimetic of a protein or other molecule is a compound having qualitative biological activity in common with a full-length protein or other molecule of its entire structure. A functional fragment of a full-length protein may be isolated and attached to a separate peptide sequence. For example, a functional fragment of a blood-brain barrier penetrating protein may be isolated and attached to the decoy peptide that disrupts ΑΡΡ-ΡΤΡσ binding, thereby enabling the hybrid peptide to enter the brain and disrupt ΑΡΡ-ΡΤΡσ binding. Another example of a functional fragment is a membrane penetrating fragment, or one that relays an ability to pass the lipophilic barrier of a cell's plasma membrane. An analog of heparin, for example, may be a compound that binds to a heparin binding site.
As used herein, "cyclic peptide" or "cyclopeptide" in general refers to a peptide comprising at least one internal bond attaching nonadjacent amino acids of the peptide, such as when the end amino acids of a linear sequence are attached to form a circular peptide.
The term "antibody" refers to natural or synthetic antibodies that selectively bind a target antigen. The term includes polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term "antibodies" are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that selectively bind the target antigen.
As used herein, "enzyme" refers to a protein specialized to catalyze or promote a specific metabolic reaction.
"Neurodegenerative disorders" or "neurodegenerative diseases" are conditions marked by the progressive loss of structure or function of neural cells, including death of neurons and glia.
The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
The term "administering" refers to an administration that is intranasal, oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, rectal, vaginal, by inhalation or via an implanted reservoir. The term "parenteral" includes
subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.
The term "pharmaceutically acceptable carrier" means a carrier or excipient that is useful in preparing a pharmaceutical composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical use. As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. As used herein, the term "carrier" encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further below. The pharmaceutical compositions also can include preservatives. A
"pharmaceutically acceptable carrier" as used in the specification and claims includes both one and more than one such carrier.
The term "variant" refers to an amino acid or peptide sequence having conservative amino acid substitutions ("conservative variant"), non-conservative amino acid subsitutions (e.g., a degenerate variant), substitutions within the wobble position of each codon (i.e. DNA and RNA) encoding an amino acid, amino acids added to the C-terminus of a peptide, or a peptide having 60%, 70%, 80%, 90%, or 95% homology to a reference sequence.
The term "percent (%) sequence identity" or "homology" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
Compositions
Peptides: Disclosed herein are peptides for treating and preventing the aforementioned neurodegenerative diseases, such as Alzheimer's disease. In some embodiments, the peptides disrupt the binding between ΡΤΡσ and APP, preventing β-amyloidogenic processing of APP without affecting other major substrates of the β- and γ-secretases. The peptide may be a decoy fragment of APP, a decoy fragment of ΡΤΡσ, or a combination thereof.
In some embodiments, a decoy peptide could be fabricated from the ΡΤΡσ-binding region on APP, which is the fragment between its El and E2 domains (SEQ ID NO: 1). In some embodiments, a decoy peptide could be fabricated from the APP -binding region on ΡΤΡσ, which is its IGl domain (SEQ ID NO: 442). In some embodiments, a decoy peptide could be fabricated that corresponds to the entire APP E2 domain or a fragment thereof. In some embodiments, a decoy peptide could be fabricated that corresponds to the entire APP El domain or a fragment thereof. In some embodiments, a ΡΤΡσ peptide is used in combination with an APP peptide.
In some embodiments, the peptide is a fragment of the ΡΤΡσ-binding domain of APP. Therefore, in some embodiments, the peptide is a fragment of SEQ ID NO: 1, as listed below, which has at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more amino acids, or a conservative variant thereof.
AEESDNVDSADAEEDDSDVWWGGADTDYADGSEDKVVEVAEEEEVAEVEEEE ADDDEDDEDGDEVEEEAEEPYEEATERTTSIATTTTTTTESVEEVVR (SEQ ID NO: l). Therefore, in some embodiments, the peptide comprises an amino acid sequence selected from 10 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
SEQ ID NO 2 AEESDNVDSA
SEQ ID NO 3 EESDNVDSAD
SEQ ID NO 4 ESDNVDSADA
SEQ ID NO 5 SDNVDSADAE
SEQ ID NO 6 DNVDSADAEE
SEQ ID NO 7 NVDSADAEED
SEQ ID NO 8 VDSADAEEDD
SEQ ID NO 9 DSADAEEDDS
SEQ ID NO 10 SADAEEDDSD
SEQ ID NO 11 ADAEEDDSDV
SEQ ID NO 12 DAEEDDSDVW
SEQ ID NO 13 AEEDDSDVWW
SEQ ID NO 14 EEDDSDVWWG
SEQ ID NO 15 EDDSDVWWGG
SEQ ID NO 16 DDSDVWWGGA SEQ ID NO : 17 DSDVWWGGAD
SEQ ID NO 18 SDVWWGGADT
SEQ ID NO 19 DVWWGGADTD
SEQ ID NO 20 VWWGGADTDY
SEQ ID NO :2l WWGGADTDYA
SEQ ID NO 22 WGGADTDYAD
SEQ ID NO 23 GGADTDYADG
SEQ ID NO 24 GADTDYADGS
SEQ ID NO 25 ADTDYADGSE
SEQ ID NO 26 DTDYADGSED
SEQ ID NO 21 TDYADGSEDK
SEQ ID NO :28 DYADGSEDKV
SEQ ID NO :29 YADGSEDKVV
SEQ ID NO :30 ADGSEDKVVE
SEQ ID NO :31 DGSEDKVVEV
SEQ ID NO :32 GSEDKVVEVA
SEQ ID NO :33 SEDKVVEVAE
SEQ ID NO :34 EDKVVEVAEE
SEQ ID NO :35 DKVVEVAEEE
SEQ ID NO :36 KVVEVAEEEE
SEQ ID NO :37 VVEVAEEEEV
SEQ ID NO :38 VEVAEEEEVA
SEQ ID NO :39 EVAEEEEVAE
SEQ ID NO :40 VAEEEEVAEV
SEQ ID NO A\ AEEEEVAEVE
SEQ ID NO :42 EEEEVAEVEE
SEQ ID NO :43 EEEVAEVEEE
SEQ ID NO :44 EEVAEVEEEE
SEQ ID NO :45 EVAEVEEEEA
SEQ ID NO :46 VAEVEEEEAD
SEQ ID NO :47 AEVEEEEADD
SEQ ID NO :48 EVEEEEADDD
SEQ ID NO :49 VEEEEADDDE
SEQ ID NO :50 EEEEADDDED
SEQ ID NO :51 EEEADDDEDD
SEQ ID NO :52 EEADDDEDDE
SEQ ID NO :53 EADDDEDDED
SEQ ID NO :54 ADDDEDDEDG
SEQ ID NO :55 DDDEDDEDGD
SEQ ID NO :56 DDEDDEDGDE
SEQ ID NO :57 DEDDEDGDEV
SEQ ID NO :58 EDDEDGDEVE
SEQ ID NO :59 DDEDGDEVEE SEQ ID NO :60 DEDGDEVEEE
SEQ ID NO 61 EDGDEVEEEA
SEQ ID NO :62 DGDEVEEEAE
SEQ ID NO :63 GDEVEEEAEE
SEQ ID NO :64 DEVEEEAEEP
SEQ ID NO :65 EVEEEAEEPY
SEQ ID NO 66 VEEEAEEPYE
SEQ ID NO :67 EEEAEEPYEE
SEQ ID NO 68 EEAEEPYEEA
SEQ ID NO 69 EAEEPYEEAT
SEQ ID NO :70 AEEPYEEATE
SEQ ID NO 1\ EEPYEEATER
SEQ ID NO :72 EPYEEATERT
SEQ ID NO :73 PYEEATERTT
SEQ ID NO :74 YEEATERTTS
SEQ ID NO :75 EEATERTTSI
SEQ ID NO :76 EATERTTSIA
SEQ ID NO :77 ATERTTSIAT
SEQ ID NO :78 TERTTSIATT
SEQ ID NO :79 ERTTSIATTT
SEQ ID NO :80 RTTSIATTTT
SEQ ID NO 81 TTSIATTTTT
SEQ ID NO :82 TSIATTTTTT
SEQ ID NO :83 SIATTTTTTT
SEQ ID NO :84 IATTTTTTTE
SEQ ID NO :85 ATTTTTTTES
SEQ ID NO 86 TTTTTTTESV
SEQ ID NO :87 TTTTTTESVE
SEQ ID NO 88 TTTTTESVEE
SEQ ID NO 89 TTTTESVEEV
SEQ ID NO :90 TTTESVEEVV
SEQ ID NO 91 TTESVEEVVR
In some embodiments, the peptide comprises an amino acid sequence selected from 11 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
SEQ ID NO:92 AEESDNVDSAD
SEQ ID NO:93 EESDNVDSADA
SEQ ID NO:94 ESDNVDSADAE
SEQ ID NO:95 SDNVDSADAEE
SEQ ID NO:96 DNVDSADAEED
SEQ ID NO:97 NVDSADAEEDD
SEQ ID NO:98 VDSADAEEDDS SEQ ID NO 99 DSADAEEDDSD
SEQ ID NO 100 SADAEEDDSDV
SEQ ID NO 101 ADAEEDDSDVW
SEQ ID NO 102 DAEEDDSDVWW
SEQ ID NO 103 AEEDDSDVWWG
SEQ ID NO 104 EEDDSDVWWGG
SEQ ID NO 105 EDD SD VWWGGA
SEQ ID NO 106 DDSDVWWGGAD
SEQ ID NO 107 DSDVWWGGADT
SEQ ID NO 108 SDVWWGGADTD
SEQ ID NO 109 DVWWGGADTDY
SEQ ID NO 110 VWWGGADTDYA
SEQ ID NO 111 WWGGADTDYAD
SEQ ID NO 112 WGGADTDYADG
SEQ ID NO 113 GGADTDYADGS
SEQ ID NO 114 GADTDYADGSE
SEQ ID NO 115 ADTDYADGSED
SEQ ID NO 116 DTDYADGSEDK
SEQ ID NO 117 TDYADGSEDKV
SEQ ID NO 118 DYADGSEDKVV
SEQ ID NO 119 YADGSEDKVVE
SEQ ID NO 120 ADGSEDKVVEV
SEQ ID NO 121 DGSEDKVVEVA
SEQ ID NO 122 GSEDKVVEVAE
SEQ ID NO 123 SEDKVVEVAEE
SEQ ID NO 124 EDKVVEVAEEE
SEQ ID NO 125 DKVVEVAEEEE
SEQ ID NO 126 KVVEVAEEEEV
SEQ ID NO 127 VVEVAEEEEVA
SEQ ID NO 128 VEVAEEEEVAE
SEQ ID NO 129 EVAEEEEVAEV
SEQ ID NO 130 VAEEEEVAEVE
SEQ ID NO 131 AEEEEVAEVEE
SEQ ID NO 132 EEEEVAEVEEE
SEQ ID NO 133 EEEVAEVEEEE
SEQ ID NO 134 EEVAEVEEEEA
SEQ ID NO 135 EVAEVEEEEAD
SEQ ID NO 136 VAEVEEEEADD
SEQ ID NO 137 AEVEEEEADDD
SEQ ID NO 138 EVEEEEADDDE
SEQ ID NO 139 VEEEEADDDED
SEQ ID NO 140 EEEEADDDEDD
SEQ ID NO 141 EEEADDDEDDE SEQ ID NO 142 EEADDDEDDED
SEQ ID NO 143 EADDDEDDEDG
SEQ ID NO 144 ADDDEDDEDGD
SEQ ID NO 145 DDDEDDEDGDE
SEQ ID NO 146 DDEDDED GDE V
SEQ ID NO 147 DEDDEDGDEVE
SEQ ID NO 148 EDDEDGDEVEE
SEQ ID NO 149 DDEDGDEVEEE
SEQ ID NO 150 DEDGDEVEEEA
SEQ ID NO 151 EDGDEVEEEAE
SEQ ID NO 152 DGDEVEEEAEE
SEQ ID NO 153 GDEVEEEAEEP
SEQ ID NO 154 DEVEEEAEEPY
SEQ ID NO 155 EVEEEAEEPYE
SEQ ID NO 156 VEEEAEEPYEE
SEQ ID NO 157 EEEAEEPYEEA
SEQ ID NO 158 EEAEEPYEEAT
SEQ ID NO 159 EAEEPYEEATE
SEQ ID NO 160 AEEPYEEATER
SEQ ID NO 161 EEPYEEATERT
SEQ ID NO 162 EPYEEATERTT
SEQ ID NO 163 PYEEATERTTS
SEQ ID NO 164 YEEATERTTSI
SEQ ID NO 165 EEATERTTSIA
SEQ ID NO 166 EATERTTSIAT
SEQ ID NO 167 ATERTTSIATT
SEQ ID NO 168 TERTTSIATTT
SEQ ID NO 169 ERTTSIATTTT
SEQ ID NO 170 RTTSIATTTTT
SEQ ID NO 171 TTSIATTTTTT
SEQ ID NO 172 TSIATTTTTTT
SEQ ID NO 173 SIATTTTTTTE
SEQ ID NO 174 IATTTTTTTES
SEQ ID NO 175 ATTTTTTTESV
SEQ ID NO 176 TTTTTTTESVE
SEQ ID NO 177 TTTTTTESVEE
SEQ ID NO 178 TTTTTESVEEV
SEQ ID NO 179 TTTTESVEEVV
SEQ ID NO 180 TTTESVEEVVR
In some embodiments, the peptide comprises an amino acid sequence selected from 12 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below: SEQ ID NO 181 AEESDNVDSADA
SEQ ID NO 182 EESDNVDSADAE
SEQ ID NO 183 ESDNVDSADAEE
SEQ ID NO 184 SDNVD S AD AEED
SEQ ID NO 185 DNVD S AD AEEDD
SEQ ID NO 186 NVDSADAEEDDS
SEQ ID NO 187 VDS AD AEEDD SD
SEQ ID NO 188 DS AD AEEDD SDV
SEQ ID NO 189 S AD AEEDD SDVW
SEQ ID NO : 190 AD AEEDD SD VW W
SEQ ID NO 191 D AEEDD SD VW WG
SEQ ID NO : 192 AEEDD SD VW WGG
SEQ ID NO 193 EEDDSDVWWGGA
SEQ ID NO : 194 EDD SD VWWGGAD
SEQ ID NO 195 DDSDVWWGGADT
SEQ ID NO 196 DSD VWWGGAD TD
SEQ ID NO : 197 SD VWWGGAD TDY
SEQ ID NO 198 DVWWGGADTDYA
SEQ ID NO 199 VWWGGADTDYAD
SEQ ID NO :200 WWGGADTDYADG
SEQ ID NO :201 WGGADTDYADGS
SEQ ID NO :202 GGADTDYADGSE
SEQ ID NO :203 GADTDYADGSED
SEQ ID NO :204 ADTDYADGSEDK
SEQ ID NO :205 DTDYADGSEDKV
SEQ ID NO :206 TDYADGSEDKVV
SEQ ID NO :207 DYADGSEDKVVE
SEQ ID NO :208 YADGSEDKVVEV
SEQ ID NO :209 ADGSEDKVVEVA
SEQ ID NO :210 DGSEDKVVEVAE
SEQ ID NO 211 GSEDKVVEVAEE
SEQ ID NO :212 SEDKVVEVAEEE
SEQ ID NO 213 EDKVVEVAEEEE
SEQ ID NO 214 DKVVEVAEEEEV
SEQ ID NO 215 KVVEVAEEEEVA
SEQ ID NO :216 VVEVAEEEEVAE
SEQ ID NO :217 VEVAEEEEVAEV
SEQ ID NO 218 EVAEEEEVAEVE
SEQ ID NO :219 VAEEEEVAEVEE
SEQ ID NO :220 AEEEEVAEVEEE
SEQ ID NO :221 EEEEVAEVEEEE
SEQ ID NO :222 EEEVAEVEEEEA
SEQ ID NO :223 EEVAEVEEEEAD SEQ ID NO :224 EVAEVEEEEADD
SEQ ID NO :225 VAEVEEEEADDD
SEQ ID NO :226 AEVEEEEADDDE
SEQ ID NO :227 EVEEEEADDDED
SEQ ID NO :228 VEEEEADDDEDD
SEQ ID NO :229 EEEEADDDEDDE
SEQ ID NO :230 EEEADDDEDDED
SEQ ID NO :231 EEADDDEDDEDG
SEQ ID NO :232 EADDDEDDEDGD
SEQ ID NO :233 ADDDEDDEDGDE
SEQ ID NO :234 DDDEDDEDGDEV
SEQ ID NO :235 DDEDDEDGDEVE
SEQ ID NO :236 DEDDEDGDEVEE
SEQ ID NO :237 EDDEDGDEVEEE
SEQ ID NO :238 DDEDGDEVEEEA
SEQ ID NO :239 DEDGDEVEEEAE
SEQ ID NO :240 EDGDEVEEEAEE
SEQ ID NO :241 DGDEVEEEAEEP
SEQ ID NO :242 GDEVEEEAEEPY
SEQ ID NO :243 DEVEEEAEEPYE
SEQ ID NO :244 EVEEEAEEPYEE
SEQ ID NO :245 VEEEAEEPYEEA
SEQ ID NO :246 EEEAEEPYEEAT
SEQ ID NO :247 EEAEEPYEEATE
SEQ ID NO :248 EAEEPYEEATER
SEQ ID NO :249 AEEPYEEATERT
SEQ ID NO :250 EEPYEEATERTT
SEQ ID NO :251 EPYEEATERTTS
SEQ ID NO :252 PYEEATERTTSI
SEQ ID NO :253 YEE ATERTT SIA
SEQ ID NO :254 EEATERTT SIAT
SEQ ID NO :255 EATERTTSIATT
SEQ ID NO :256 ATERTTSIATTT
SEQ ID NO :257 TERTTSIATTTT
SEQ ID NO :258 ERTTSIATTTTT
SEQ ID NO :259 RTTSIATTTTTT
SEQ ID NO :260 TTSIATTTTTTT
SEQ ID NO :261 TSIATTTTTTTE
SEQ ID NO :262 SIATTTTTTTES
SEQ ID NO :263 IATTTTTTTESV
SEQ ID NO :264 ATTTTTTTESVE
SEQ ID NO :265 TTTTTTTESVEE
SEQ ID NO :266 TTTTTTESVEEV SEQ ID NO:267 TTTTTESVEEVV
SEQ ID NO:268 TTTTESVEEVVR
In some embodiments, the peptide comprises an amino acid sequence selected from 13 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
SEQ ID NO :268 TTTTESVEEVVR
SEQ ID NO :269 AEESDNVDSADAE
SEQ ID NO :270 EESDNVDSADAEE
SEQ ID NO :271 ESDNVDSADAEED
SEQ ID NO :272 SDNVD S AD AEEDD
SEQ ID NO :273 DNVD S AD AEEDD S
SEQ ID NO :274 NVDS AD AEEDD SD
SEQ ID NO :275 VDS AD AEEDD SDV
SEQ ID NO :276 DS AD AEEDD SDVW
SEQ ID NO :277 S AD AEEDD SDVWW
SEQ ID NO :278 AD AEEDD SD VW WG
SEQ ID NO :279 D AEEDD SD VW WGG
SEQ ID NO :280 AEEDD SD VW WGGA
SEQ ID NO :281 EEDD SD VW WGGAD
SEQ ID NO :282 EDD SDVWW GGADT
SEQ ID NO :283 DDSDVWWGGADTD
SEQ ID NO :284 DSDVWWGGADTDY
SEQ ID NO :285 SDVWWGGADTDYA
SEQ ID NO :286 DVWWGGADTDYAD
SEQ ID NO :287 VWWGGADTDYADG
SEQ ID NO :288 WWGGADTDYADGS
SEQ ID NO :289 WGGAD TDYADGSE
SEQ ID NO :290 GGADTDYADGSED
SEQ ID NO :291 GADTDYADGSEDK
SEQ ID NO :292 ADTDYADGSEDKV
SEQ ID NO :293 DTDYADGSEDKVV
SEQ ID NO :294 TDYADGSEDKVVE
SEQ ID NO :295 DYADGSEDKVVEV
SEQ ID NO :296 YADGSEDKVVEVA
SEQ ID NO :297 ADGSEDKVVEVAE
SEQ ID NO :298 DGSEDKVVEVAEE
SEQ ID NO :299 GSEDKVVEVAEEE
SEQ ID NO :300 SEDKVVEVAEEEE
SEQ ID NO :301 EDKVVEVAEEEEV
SEQ ID NO :302 DKVVEVAEEEEVA
SEQ ID NO :303 KVVEVAEEEEVAE
SEQ ID NO :304 VVEVAEEEEVAEV SEQ ID NO :305 VEVAEEEEVAEVE
SEQ ID NO :306 EVAEEEEVAEVEE
SEQ ID NO :307 VAEEEEVAEVEEE
SEQ ID NO :308 AEEEEVAEVEEEE
SEQ ID NO :309 EEEEVAEVEEEEA
SEQ ID NO 310 EEEVAEVEEEEAD
SEQ ID NO 311 EEVAEVEEEEADD
SEQ ID NO 312 EVAEVEEEEADDD
SEQ ID NO 313 VAEVEEEEADDDE
SEQ ID NO 314 AEVEEEEADDDED
SEQ ID NO 315 EVEEEEADDDEDD
SEQ ID NO 316 VEEEEADDDEDDE
SEQ ID NO 317 EEEEADDDEDDED
SEQ ID NO 318 EEEADDDEDDEDG
SEQ ID NO 319 EEADDDEDDEDGD
SEQ ID NO :320 EADDDEDDEDGDE
SEQ ID NO 321 ADDDEDDEDGDEV
SEQ ID NO :322 DDDEDDEDGDEVE
SEQ ID NO :323 DDEDDED GDE VEE
SEQ ID NO :324 DEDDEDGDEVEEE
SEQ ID NO :325 EDDEDGDEVEEEA
SEQ ID NO :326 DDEDGDEVEEEAE
SEQ ID NO :327 DEDGDEVEEEAEE
SEQ ID NO :328 EDGDEVEEEAEEP
SEQ ID NO :329 DGDEVEEEAEEPY
SEQ ID NO :330 GDEVEEEAEEPYE
SEQ ID NO 331 DEVEEEAEEPYEE
SEQ ID NO :332 EVEEEAEEPYEEA
SEQ ID NO :333 VEEEAEEPYEEAT
SEQ ID NO :334 EEEAEEPYEEATE
SEQ ID NO :335 EEAEEPYEEATER
SEQ ID NO :336 EAEEPYEEATERT
SEQ ID NO :337 AEEPYEEATERTT
SEQ ID NO 338 EEP YEE ATERTT S
SEQ ID NO :339 EPYEEATERTTSI
SEQ ID NO :340 PYEEATERTTSIA
SEQ ID NO 341 YEE ATERTT SIAT
SEQ ID NO :342 EEATERTT SIATT
SEQ ID NO :343 EATERTTSIATTT
SEQ ID NO :344 ATERTTSIATTTT
SEQ ID NO :345 TERTTSIATTTTT
SEQ ID NO :346 ERTTSIATTTTTT
SEQ ID NO :347 RTT SIATTTTTTT SEQ ID NO :348 TTSIATTTTTTTE
SEQ ID NO :349 TSIATTTTTTTES
SEQ ID NO :350 SIATTTTTTTESV
SEQ ID NO 351 IATTTTTTTESVE
SEQ ID NO :352 ATTTTTTTESVEE
SEQ ID NO :353 TTTTTTTESVEEV
SEQ ID NO :354 TTTTTTESVEEVV
SEQ ID NO :355 TTTTTESVEEVVR
In some embodiments, the peptide comprises an amino acid sequence selected from 14 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
SEQ ID NO :356 AEESDNVDSADAEE
SEQ ID NO :357 EESDNVD S AD AEED
SEQ ID NO 358 ESDNVDSADAEEDD
SEQ ID NO :359 SDNVD S AD AEEDD S
SEQ ID NO :360 DNVD S AD AEEDD SD
SEQ ID NO 361 NVDS AD AEEDD SDV
SEQ ID NO :362 VDS AD AEEDD SDVW
SEQ ID NO :363 DS AD AEEDD SDVWW
SEQ ID NO :364 S AD AEEDD SDVWWG
SEQ ID NO :365 AD AEEDD SD VW WGG
SEQ ID NO 366 D AEEDD SD VW WGGA
SEQ ID NO :367 AEEDD SD VW WGGAD
SEQ ID NO 368 EEDD SD VWWGGADT
SEQ ID NO 369 EDD SDVWW GGADTD
SEQ ID NO :370 DDSDVWWGGADTDY
SEQ ID NO 311 DSDVWWGGADTDYA
SEQ ID NO :372 SDVWWGGADTDYAD
SEQ ID NO :373 DVWWGGADTDYADG
SEQ ID NO :374 VWWGGADTDYADGS
SEQ ID NO :375 WWGGADTDYADGSE
SEQ ID NO :376 WGGAD TDYADGSED
SEQ ID NO :377 GGADTD YADGSEDK
SEQ ID NO :378 GADTDYADGSEDKV
SEQ ID NO :379 ADTDYADGSEDKVV
SEQ ID NO :380 DTDYADGSEDKVVE
SEQ ID NO 381 TDYADGSEDKVVEV
SEQ ID NO :382 DYADGSEDKVVEVA
SEQ ID NO 383 YADGSEDKVVEVAE
SEQ ID NO :384 ADGSEDKVVEVAEE
SEQ ID NO 385 DGSEDKVVEVAEEE
SEQ ID NO 386 GSEDKVVEVAEEEE SEQ ID NO :387 SEDKVVEVAEEEEV
SEQ ID NO :388 EDKVVEVAEEEEVA
SEQ ID NO :389 DKVVEVAEEEEVAE
SEQ ID NO :390 KVVEVAEEEEVAEV
SEQ ID NO :39\ VVEVAEEEEVAEVE
SEQ ID NO 392 VEVAEEEEVAEVEE
SEQ ID NO 393 EVAEEEEVAEVEEE
SEQ ID NO 394 VAEEEEVAEVEEEE
SEQ ID NO 395 AEEEEVAEVEEEEA
SEQ ID NO 396 EEEEVAEVEEEEAD
SEQ ID NO 391 EEEVAEVEEEEADD
SEQ ID NO :398 EEVAEVEEEEADDD
SEQ ID NO :399 EVAEVEEEEADDDE
SEQ ID NO :400 VAEVEEEEADDDED
SEQ ID NO :401 AEVEEEEADDDEDD
SEQ ID NO :402 EVEEEEADDDEDDE
SEQ ID NO :403 VEEEEADDDEDDED
SEQ ID NO :404 EEEEADDDEDDEDG
SEQ ID NO :405 EEEADDDEDDEDGD
SEQ ID NO :406 EEADDDEDDEDGDE
SEQ ID NO :407 EADDDEDDEDGDEV
SEQ ID NO :408 ADDDEDDEDGDEVE
SEQ ID NO :409 DDDEDDEDGDEVEE
SEQ ID NO :410 DDEDDED GDE VEEE
SEQ ID NO 411 DEDDEDGDEVEEEA
SEQ ID NO A\2 EDDEDGDEVEEEAE
SEQ ID NO :413 DDEDGDEVEEEAEE
SEQ ID NO :414 DEDGDEVEEEAEEP
SEQ ID NO :415 EDGDEVEEEAEEPY
SEQ ID NO :416 DGDEVEEEAEEPYE
SEQ ID NO :417 GDEVEEEAEEPYEE
SEQ ID NO :418 DEVEEEAEEPYEEA
SEQ ID NO A\9 EVEEEAEEPYEEAT
SEQ ID NO :420 VEEEAEEPYEEATE
SEQ ID NO :421 EEEAEEPYEEATER
SEQ ID NO :422 EEAEEPYEEATERT
SEQ ID NO :423 EAEEPYEEATERTT
SEQ ID NO :424 AEEP YEE ATERTT S
SEQ ID NO :425 EEP YEEATERTT SI
SEQ ID NO :426 EPYEEATERTTSIA
SEQ ID NO :427 P YEEATERTT SIAT
SEQ ID NO :428 YEEATERTT SIATT
SEQ ID NO :429 EEATERTTSIATTT SEQ ID NO :430 EATERTTSIATTTT
SEQ ID NO :431 ATERTTSIATTTTT
SEQ ID NO :432 TERTTSIATTTTTT
SEQ ID NO :433 ERTTSIATTTTTTT
SEQ ID NO :434 RTT SIATTTTTTTE
SEQ ID NO :435 TTSIATTTTTTTES
SEQ ID NO :436 TSIATTTTTTTESV
SEQ ID NO :437 SIATTTTTTTESVE
SEQ ID NO :438 IATTTTTTTESVEE
SEQ ID NO :439 ATTTTTTTESVEEV
SEQ ID NO :440 TTTTTTTESVEEVV
SEQ ID NO :441 TTTTTTESVEEVVR
In some embodiments, the peptide comprises an amino acid sequence selected from 24 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
SEQ ID NO: 900 ATERTTSIATTTTTTTESVEEVVR
In some embodiments, the peptide is a fragment of the APP -binding domain of ΡΤΡσ. Therefore, in some embodiments, the peptide is a fragment of SEQ ID NO:442, as listed below, which has at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more amino acids, or a conservative variant thereof. The underlined amino acids represent residues in the ligand-binding pocket.
EEPPRFIKEPKDQIGVSGGVASF VCQATGDPKPRVTWNKKGKKVNSQRFETIEFD
ESAGAVLRIQPLRTPRDENVYECVAQNSVGEITVHAKLTVLRE (SEQ ID NO:442).
Therefore, in some embodiments, the peptide comprises an amino acid sequence selected from 10 consecutive residues of SEQ ID NO: 442, or from the group consisting of the below:
SEQ ID NO :443 EEPPRFIKEP
SEQ ID NO :444 EPPRFIKEPK
SEQ ID NO :445 PPRFIKEPKD
SEQ ID NO :446 PRFIKEPKDQ
SEQ ID NO :447 RFIKEPKDQI
SEQ ID NO :448 FIKEPKDQIG
SEQ ID NO :449 IKEPKDQIGV
SEQ ID NO :450 KEPKDQIGVS
SEQ ID NO :451 EPKDQIGVSG
SEQ ID NO :452 PKDQIGVSGG
SEQ ID NO :453 KDQIGVSGGV
SEQ ID NO :454 DQIGVSGGVA SEQ ID NO :455 QIGVSGGVAS
SEQ ID NO :456 IGVSGGVASF
SEQ ID NO :457 GVSGGVASFV
SEQ ID NO :458 VSGGVASFVC
SEQ ID NO :459 SGGVASFVCQ
SEQ ID NO :460 GGVASFVCQA
SEQ ID NO :461 GVASFVCQAT
SEQ ID NO :462 VASFVCQATG
SEQ ID NO :463 ASFVCQATGD
SEQ ID NO :464 SFVCQATGDP
SEQ ID NO :465 FVCQATGDPK
SEQ ID NO :466 VCQATGDPKP
SEQ ID NO :467 CQATGDPKPR
SEQ ID NO :468 QATGDPKPRV
SEQ ID NO :469 ATGDPKPRVT
SEQ ID NO :470 TGDPKPRVTW
SEQ ID NO :471 GDPKPRVTWN
SEQ ID NO :472 DPKPRVTWNK
SEQ ID NO :473 PKPRVTWNKK
SEQ ID NO :474 KPRVTWNKKG
SEQ ID NO :475 PRVTWNKKGK
SEQ ID NO :476 RVTWNKKGKK
SEQ ID NO :477 VTWNKKGKKV
SEQ ID NO :478 TWNKKGKKVN
SEQ ID NO :479 WNKKGKKVNS
SEQ ID NO :480 NKKGKKVNSQ
SEQ ID NO :481 KKGKKVNSQR
SEQ ID NO :482 KGKKVNSQRF
SEQ ID NO :483 GKKVNSQRFE
SEQ ID NO :484 KKVNSQRFET
SEQ ID NO :485 KVNSQRFETI
SEQ ID NO :486 VNSQRFETIE
SEQ ID NO :487 NSQRFETIEF
SEQ ID NO :488 SQRFETIEFD
SEQ ID NO :489 QRFETIEFDE
SEQ ID NO :490 RFETIEFDES
SEQ ID NO :491 FETIEFDESA
SEQ ID NO :492 ETIEFDESAG
SEQ ID NO :493 TIEFDESAGA
SEQ ID NO :494 IEFDESAGAV
SEQ ID NO :495 EFDESAGAVL SEQ ID NO :496 FDESAGAVLR
SEQ ID NO :497 DESAGAVLRI
SEQ ID NO :498 ESAGAVLRIQ
SEQ ID NO :499 SAGAVLRIQP
SEQ ID NO :500 AGAVLRIQPL
SEQ ID NO 501 GAVLRIQPLR
SEQ ID NO :502 AVLRIQPLRT
SEQ ID NO :503 VLRIQPLRTP
SEQ ID NO :504 LRIQPLRTPR
SEQ ID NO :505 RIQPLRTPRD
SEQ ID NO :506 IQPLRTPRDE
SEQ ID NO :507 QPLRTPRDEN
SEQ ID NO :508 PLRTPRDENV
SEQ ID NO :509 LRTPRDENVY
SEQ ID NO 510 RTPRDENVYE
SEQ ID NO 511 TPRDENVYEC
SEQ ID NO :512 PRDENVYECV
SEQ ID NO 513 RDENVYECVA
SEQ ID NO 514 DENVYECVAQ
SEQ ID NO :515 ENVYECVAQN
SEQ ID NO 516 NVYECVAQNS
SEQ ID NO :517 VYECVAQNSV
SEQ ID NO 518 YECVAQNSVG
SEQ ID NO 519 ECVAQNSVGE
SEQ ID NO :520 CVAQNSVGEI
SEQ ID NO :521 VAQNSVGEIT
SEQ ID NO :522 AQNSVGEITV
SEQ ID NO :523 QNSVGEITVH
SEQ ID NO :524 NSVGEITVHA
SEQ ID NO :525 SVGEITVHAK
SEQ ID NO :526 VGEITVHAKL
SEQ ID NO :527 GEITVHAKLT
SEQ ID NO :528 EITVHAKLTV
SEQ ID NO :529 ITVHAKLTVL
SEQ ID NO :530 TVHAKLTVLR
SEQ ID NO 531 VHAKLTVLRE
In some embodiments, the peptide comprises an amino acid sequence selected from 11 consecutive residues of SEQ ID NO: 442, or from the group consisting of the below: SEQ ID NO 531 VHAKLTVLRE
SEQ ID NO :532 EEPPRFIKEPK
SEQ ID NO :533 EPPRFIKEPKD
SEQ ID NO :534 PPRFIKEPKDQ
SEQ ID NO :535 PRFIKEPKDQI
SEQ ID NO :536 RFIKEPKDQIG
SEQ ID NO :537 FIKEPKDQIGV
SEQ ID NO 538 IKEPKDQIGVS
SEQ ID NO :539 KEPKDQIGVSG
SEQ ID NO :540 EPKDQIGVSGG
SEQ ID NO 541 PKDQIGVSGGV
SEQ ID NO :542 KDQIGVSGGVA
SEQ ID NO :543 DQIGVSGGVAS
SEQ ID NO :544 QIGVSGGVASF
SEQ ID NO :545 IGVSGGVASFV
SEQ ID NO :546 GVSGGVASFVC
SEQ ID NO :547 VSGGVASFVCQ
SEQ ID NO :548 SGGVASFVCQA
SEQ ID NO :549 GGVASFVCQAT
SEQ ID NO :550 GVASFVCQATG
SEQ ID NO :551 VASFVCQATGD
SEQ ID NO :552 ASFVCQATGDP
SEQ ID NO :553 SFVCQATGDPK
SEQ ID NO :554 FVCQATGDPKP
SEQ ID NO :555 VCQATGDPKPR
SEQ ID NO :556 CQATGDPKPRV
SEQ ID NO :557 QATGDPKPRVT
SEQ ID NO 558 ATGDPKPRVTW
SEQ ID NO :559 TGDPKPRVTWN
SEQ ID NO :560 GDPKPRVTWNK
SEQ ID NO 561 DPKPRVTWNKK
SEQ ID NO :562 PKPRVTWNKKG
SEQ ID NO :563 KPRVTWNKKGK
SEQ ID NO :564 PRVTWNKKGKK
SEQ ID NO :565 RVTWNKKGKKV
SEQ ID NO :566 VTWNKKGKKVN
SEQ ID NO :567 TWNKKGKKVNS
SEQ ID NO :568 WNKKGKKVNSQ
SEQ ID NO :569 NKKGKKVNSQR
SEQ ID NO :570 KKGKKVNSQRF
SEQ ID NO :571 KGKKVNSQRFE
SEQ ID NO :572 GKKVNSQRFET
SEQ ID NO :573 KKVNSQRFETI SEQ ID NO :574 KVNSQRFETIE
SEQ ID NO :575 VNSQRFETIEF
SEQ ID NO :576 NSQRFETIEFD
SEQ ID NO :577 SQRFETIEFDE
SEQ ID NO :578 QRFETIEFDES
SEQ ID NO :579 RFETIEFDESA
SEQ ID NO :580 FETIEFDESAG
SEQ ID NO 581 ETIEFDESAGA
SEQ ID NO :582 TFEFDE S AG A V
SEQ ID NO 583 FEFDESAGAVL
SEQ ID NO :584 EFDESAGAVLR
SEQ ID NO 585 FDESAGAVLRI
SEQ ID NO :586 DESAGAVLRIQ
SEQ ID NO :587 ESAGAVLRIQP
SEQ ID NO :588 SAGAVLRIQPL
SEQ ID NO :589 AGAVLRIQPLR
SEQ ID NO :590 GAVLRIQPLRT
SEQ ID NO 591 AVLRIQPLRTP
SEQ ID NO :592 VLRIQPLRTPR
SEQ ID NO :593 LRIQPLRTPRD
SEQ ID NO :594 RIQPLRTPRDE
SEQ ID NO :595 IQPLRTPRDEN
SEQ ID NO :596 QPLRTPRDENV
SEQ ID NO :597 PLRTPRDENVY
SEQ ID NO :598 LRTPRDENVYE
SEQ ID NO :599 RTPRDENVYEC
SEQ ID NO :600 TPRDENVYECV
SEQ ID NO :601 PRDENVYECVA
SEQ ID NO :602 RDENVYECVAQ
SEQ ID NO :603 DENVYECVAQN
SEQ ID NO :604 ENVYECVAQNS
SEQ ID NO :605 NVYECVAQNSV
SEQ ID NO :606 VYECVAQNSVG
SEQ ID NO :607 YECVAQNSVGE
SEQ ID NO :608 ECVAQNSVGEI
SEQ ID NO :609 CVAQNSVGEIT
SEQ ID NO :610 VAQNSVGEITV
SEQ ID NO 611 AQNSVGEITVH
SEQ ID NO :612 QNSVGEITVHA
SEQ ID NO 613 NSVGEITVHAK
SEQ ID NO :614 SVGEITVHAKL
SEQ ID NO 615 VGEITVHAKLT
SEQ ID NO 616 GEITVHAKLTV SEQ ID NO : 617 EIT VHAKLT VL
SEQ ID NO : 618 IT VHAKLT VLR
SEQ ID NO : 619 TVHAKLT VLRE
In some embodiments, the peptide comprises an amino acid sequence selected from 12 consecutive residues of SEQ ID NO: 442, or from the group consisting of the below:
SEQ ID NO :620 EEPPRFIKEPKD
SEQ ID NO :621 EPPRFIKEPKDQ
SEQ ID NO :622 PPRFIKEPKDQI
SEQ ID NO :623 PRFIKEPKDQIG
SEQ ID NO :624 RFIKEPKDQIGV
SEQ ID NO :625 FIKEPKDQIGVS
SEQ ID NO :626 IKEPKDQIGVSG
SEQ ID NO :627 KEPKDQIGVSGG
SEQ ID NO :628 EPKDQIGVSGGV
SEQ ID NO :629 PKDQIGVSGGVA
SEQ ID NO :630 KDQIGVSGGVAS
SEQ ID NO 631 DQIGVSGGVASF
SEQ ID NO :632 QIGVSGGVASFV
SEQ ID NO :633 IGVSGGVASFVC
SEQ ID NO :634 GVSGGVASFVCQ
SEQ ID NO :635 VSGGVASFVCQA
SEQ ID NO 636 SGGVASFVCQAT
SEQ ID NO :637 GGVASFVCQATG
SEQ ID NO 638 GVASFVCQATGD
SEQ ID NO 639 VASFVCQATGDP
SEQ ID NO :640 ASFVCQATGDPK
SEQ ID NO :641 SFVCQATGDPKP
SEQ ID NO :642 FVCQATGDPKPR
SEQ ID NO :643 VCQATGDPKPRV
SEQ ID NO :644 CQATGDPKPRVT
SEQ ID NO :645 QATGDPKPRVTW
SEQ ID NO :646 ATGDPKPRVTWN
SEQ ID NO :647 TGDPKPRVTWNK
SEQ ID NO :648 GDPKPRVTWNKK
SEQ ID NO :649 DPKPRVTWNKKG
SEQ ID NO :650 PKPRVTWNKKGK
SEQ ID NO 651 KPRVTWNKKGKK
SEQ ID NO :652 PRVTWNKKGKKV
SEQ ID NO :653 RVTWNKKGKKVN
SEQ ID NO :654 VTWNKKGKKVNS
SEQ ID NO :655 TWNKKGKKVNSQ SEQ ID NO :656 WNKKGKKVNSQR
SEQ ID NO :657 NKKGKKVNSQRF
SEQ ID NO :658 KKGKKVNSQRFE
SEQ ID NO :659 KGKKVNSQRFET
SEQ ID NO :660 GKKVNSQRFETI
SEQ ID NO 661 KKVNSQRFETIE
SEQ ID NO :662 KVNSQRFETIEF
SEQ ID NO 663 VNSQRFETIEFD
SEQ ID NO :664 NSQRFETIEFDE
SEQ ID NO :665 SQRFETIEFDES
SEQ ID NO 666 QRFETIEFDESA
SEQ ID NO :667 RFETIEFDE SAG
SEQ ID NO 668 FETIEFDESAGA
SEQ ID NO 669 ETIEFDESAGAV
SEQ ID NO :670 TIEFDESAGAVL
SEQ ID NO :671 IEFDESAGAVLR
SEQ ID NO :672 EFDESAGAVLRI
SEQ ID NO :673 FDESAGAVLRIQ
SEQ ID NO :674 DESAGAVLRIQP
SEQ ID NO :675 ESAGAVLRIQPL
SEQ ID NO :676 SAGAVLRIQPLR
SEQ ID NO :677 AGAVLRIQPLRT
SEQ ID NO :678 GAVLRIQPLRTP
SEQ ID NO :679 AVLRIQPLRTPR
SEQ ID NO :680 VLRIQPLRTPRD
SEQ ID NO 681 LRIQPLRTPRDE
SEQ ID NO :682 RIQPLRTPRDEN
SEQ ID NO 683 IQPLRTPRDENV
SEQ ID NO :684 QPLRTPRDENVY
SEQ ID NO :685 PLRTPRDENVYE
SEQ ID NO 686 LRTPRDENVYEC
SEQ ID NO :687 RTPRDENVYECV
SEQ ID NO 688 TPRDENVYECVA
SEQ ID NO 689 PRDENVYECVAQ
SEQ ID NO :690 RDENVYECVAQN
SEQ ID NO 691 DENVYECVAQNS
SEQ ID NO :692 ENVYECVAQNSV
SEQ ID NO 693 NVYECVAQNSVG
SEQ ID NO :694 VYECVAQNSVGE
SEQ ID NO :695 YECVAQNSVGEI
SEQ ID NO 696 ECVAQNSVGEIT
SEQ ID NO :697 CVAQNSVGEITV
SEQ ID NO 698 VAQNSVGEITVH SEQ ID NO 699 AQNSVGEITVHA
SEQ ID NO :700 QNSVGEITVHAK
SEQ ID NO :701 NSVGEITVHAKL
SEQ ID NO :702 SVGEITVHAKLT
SEQ ID NO :703 VGEITVHAKLTV
SEQ ID NO :704 GEITVHAKLTVL
SEQ ID NO :705 EITVHAKLTVLR
SEQ ID NO :706 ITVHAKLTVLRE
In some embodiments, the peptide comprises an amino acid sequence selected from 13 consecutive residues of SEQ ID NO: 442, or from the group consisting of the below:
SEQ ID NO :707 EEPPRFIKEPKDQ
SEQ ID NO :708 EPPRFIKEPKDQI
SEQ ID NO :709 PPRFIKEPKDQIG
SEQ ID NO :710 PRFIKEPKDQIGV
SEQ ID NO 711 RFIKEPKDQIGVS
SEQ ID NO :712 FIKEPKDQIGVSG
SEQ ID NO 713 IKEPKDQIGVSGG
SEQ ID NO :714 KEPKDQIGVSGGV
SEQ ID NO :715 EPKDQIGVSGGVA
SEQ ID NO :716 PKDQIGVSGGVAS
SEQ ID NO :717 KDQIGVSGGVASF
SEQ ID NO 718 DQIGVSGGVASFV
SEQ ID NO :719 QIGVSGGVASFVC
SEQ ID NO :720 IGVSGGVASFVCQ
SEQ ID NO :721 GVSGGVASFVCQA
SEQ ID NO :722 VSGGVASFVCQAT
SEQ ID NO :723 SGGVASFVCQATG
SEQ ID NO :724 GGVASFVCQATGD
SEQ ID NO :725 GVASFVCQATGDP
SEQ ID NO :726 VASFVCQATGDPK
SEQ ID NO :727 ASFVCQATGDPKP
SEQ ID NO :728 SFVCQATGDPKPR
SEQ ID NO :729 FVCQATGDPKPRV
SEQ ID NO :730 VCQATGDPKPRVT
SEQ ID NO 731 CQATGDPKPRVTW
SEQ ID NO :732 QATGDPKPRVTWN
SEQ ID NO :733 ATGDPKPRVTWNK
SEQ ID NO :734 TGDPKPRVTWNKK
SEQ ID NO :735 GDPKPRVTWNKKG
SEQ ID NO :736 DPKPRVTWNKKGK
SEQ ID NO :737 PKPRVTWNKKGKK SEQ ID NO :738 KPRVTWNKKGKKV
SEQ ID NO :739 PRVTWNKKGKKVN
SEQ ID NO :740 RVTWNKKGKKVNS
SEQ ID NO :741 VTWNKKGKKVNSQ
SEQ ID NO :742 TWNKKGKKVNSQR
SEQ ID NO :743 WNKKGKKVNSQRF
SEQ ID NO :744 NKKGKKVNSQRFE
SEQ ID NO :745 KGKKVNSQRFET
SEQ ID NO :746 KGKKVNSQRFETI
SEQ ID NO :747 GKKVNSQRFETIE
SEQ ID NO :748 KKVNSQRFETIEF
SEQ ID NO :749 KVNSQRFETIEFD
SEQ ID NO :750 VNSQRFETIEFDE
SEQ ID NO :751 NSQRFETIEFDES
SEQ ID NO :752 SQRFETIEFDESA
SEQ ID NO :753 QRFETIEFDESAG
SEQ ID NO :754 RFETIEFDESAGA
SEQ ID NO :755 FETIEFDESAGAV
SEQ ID NO :756 ETIEFDESAGAVL
SEQ ID NO :757 TFEFDESAGAVLR
SEQ ID NO :758 FEFDESAGAVLRI
SEQ ID NO :759 EFDESAGAVLRIQ
SEQ ID NO :760 FDESAGAVLRIQP
SEQ ID NO :761 DESAGAVLRIQPL
SEQ ID NO :762 ESAGAVLRIQPLR
SEQ ID NO :763 SAGAVLRIQPLRT
SEQ ID NO :764 AGAVLRIQPLRTP
SEQ ID NO :765 GAVLRIQPLRTPR
SEQ ID NO :766 AVLRIQPLRTPRD
SEQ ID NO :767 VLRIQPLRTPRDE
SEQ ID NO :768 LRIQPLRTPRDEN
SEQ ID NO :769 RIQPLRTPRDENV
SEQ ID NO :770 IQPLRTPRDENVY
SEQ ID NO :771 QPLRTPRDENVYE
SEQ ID NO :772 PLRTPRDENVYEC
SEQ ID NO :773 LRTPRDENVYECV
SEQ ID NO :774 RTPRDENVYECVA
SEQ ID NO :775 TPRDENVYECVAQ
SEQ ID NO :776 PRDENVYECVAQN
SEQ ID NO :777 RDENVYECVAQNS
SEQ ID NO :778 DENVYECVAQNSV
SEQ ID NO :779 ENVYECVAQNSVG
SEQ ID NO :780 NVYECVAQNSVGE SEQ ID NO 781 VYECVAQNSVGEI
SEQ ID NO :782 YECVAQNSVGEIT
SEQ ID NO :783 ECVAQNSVGEITV
SEQ ID NO :784 CVAQNSVGEITVH
SEQ ID NO :785 VAQNSVGEITVHA
SEQ ID NO :786 AQNSVGEITVHAK
SEQ ID NO :787 QNSVGEITVHAKL
SEQ ID NO :788 NSVGEITVHAKLT
SEQ ID NO :789 SVGEITVHAKLTV
SEQ ID NO :790 VGEITVHAKLTVL
SEQ ID NO :791 GEITVHAKLTVLR
SEQ ID NO :792 EITVHAKLTVLRE
In some embodiments, the peptide comprises an amino acid sequence selected from 14 consecutive residues of SEQ ID NO: 442, or from the group consisting of the below:
SEQ ID NO: 793 EEPPRFIKEPKDQI
SEQ ID NO: 794 EPPRFIKEPKDQIG
SEQ ID NO: 795 PPRFIKEPKDQIGV
SEQ ID NO: 796 PRFIKEPKDQIGVS
SEQ ID NO: 797 RFIKEPKDQIGVSG
SEQ ID NO: 798 FIKEPKDQIGVSGG
SEQ ID NO: 799 IKEPKDQIGVSGGV
SEQ ID NO: 800 KEPKDQIGVSGGVA
SEQ ID NO: 801 EPKDQIGVSGGVAS
SEQ ID NO: 802 PKDQIGVSGGVASF
SEQ ID NO: 803 KDQIGVSGGVASFV
SEQ ID NO: 804 DQIGVSGGVASFVC
SEQ ID NO: 805 QIGVSGGVASFVCQ
SEQ ID NO: 806 IGVSGGVASFVCQA
SEQ ID NO: 807 GVSGGVASFVCQAT
SEQ ID NO: 808 VSGGVASFVCQATG
SEQ ID NO: 809 SGGVASFVCQATGD
SEQ ID NO: 810 GGVASFVCQATGDP
SEQ ID NO: 811 GVASFVCQATGDPK
SEQ ID NO: 812 VASFVCQATGDPKP
SEQ ID NO: 813 ASFVCQATGDPKPR
SEQ ID NO: 814 SF VC Q ATGDPKPR V
SEQ ID NO: 815 FVCQATGDPKPRVT
SEQ ID NO: 816 VCQATGDPKPRVTW
SEQ ID NO: 817 CQATGDPKPRVTWN
SEQ ID NO: 818 QATGDPKPRVTWNK
SEQ ID NO: 819 ATGDPKPRVTWNKK SEQ ID NO :820 TGDPKPRVTWNKKG
SEQ ID NO 821 GDPKPRVTWNKKGK
SEQ ID NO :822 DPKPRVTWNKKGKK
SEQ ID NO :823 PKPRVTWNKKGKKV
SEQ ID NO :824 KPRVTWNKKGKKVN
SEQ ID NO :825 PRVTWNKKGKKVNS
SEQ ID NO :826 RVTWNKKGKKVNSQ
SEQ ID NO :827 VTWNKKGKKVNSQR
SEQ ID NO :828 TWNKKGKKVNSQRF
SEQ ID NO :829 WNKKGKKVNSQRFE
SEQ ID NO :830 NKKGKKVNSQRFET
SEQ ID NO 831 KKGKKVNSQRFETI
SEQ ID NO :832 KGKKVNSQRFETIE
SEQ ID NO 833 GKKVNSQRFETIEF
SEQ ID NO :834 KKVNSQRFETIEFD
SEQ ID NO 835 KVNSQRFETIEFDE
SEQ ID NO 836 VNSQRFETIEFDES
SEQ ID NO :837 NSQRFETIEFDESA
SEQ ID NO :838 SQRFETIEFDESAG
SEQ ID NO :839 QRFETIEFDESAGA
SEQ ID NO :840 RFETIEFDESAGAV
SEQ ID NO 841 FETIEFDESAGAVL
SEQ ID NO :842 ETIEFDESAGAVLR
SEQ ID NO :843 TIEFDESAGAVLRI
SEQ ID NO :844 IEFDESAGAVLRIQ
SEQ ID NO :845 EFDESAGAVLRIQP
SEQ ID NO :846 FDESAGAVLRIQPL
SEQ ID NO :847 DESAGAVLRIQPLR
SEQ ID NO :848 ESAGAVLRIQPLRT
SEQ ID NO :849 SAGAVLRIQPLRTP
SEQ ID NO :850 AGAVLRIQPLRTPR
SEQ ID NO 851 GAVLRIQPLRTPRD
SEQ ID NO :852 AVLRIQPLRTPRDE
SEQ ID NO 853 VLRIQPLRTPRDEN
SEQ ID NO :854 LRIQPLRTPRDENV
SEQ ID NO 855 RIQPLRTPRDENVY
SEQ ID NO :856 IQPLRTPRDENVYE
SEQ ID NO :857 QPLRTPRDENVYEC
SEQ ID NO :858 PLRTPRDENVYECV
SEQ ID NO :859 LRTPRDENVYECVA
SEQ ID NO :860 RTPRDENVYECVAQ
SEQ ID NO 861 TPRDENVYECVAQN
SEQ ID NO :862 PRDENVYECVAQNS SEQ ID NO 863 RDENVYECVAQNSV
SEQ ID NO :864 DENVYECVAQNSVG
SEQ ID NO :865 ENVYECVAQNSVGE
SEQ ID NO 866 NVYECVAQNSVGEI
SEQ ID NO :867 VYECVAQNSVGEIT
SEQ ID NO 868 YECVAQNSVGEITV
SEQ ID NO 869 ECVAQNSVGEITVH
SEQ ID NO :870 CVAQNSVGEITVHA
SEQ ID NO 871 VAQNSVGEITVHAK
SEQ ID NO :872 AQNSVGEITVHAKL
SEQ ID NO :873 QNSVGEITVHAKLT
SEQ ID NO :874 NSVGEITVHAKLTV
SEQ ID NO :875 SVGEITVHAKLTVL
SEQ ID NO :876 VGEITVHAKLTVLR
SEQ ID NO :877 GEITVHAKLTVLRE
In some embodiments, the disclosed peptide further comprises a blood brain barrier penetrating sequence. For example, cell-penetrating peptides (CPPs) are a group of peptides, which have the ability to cross cell membrane bilayers. CPPs themselves can exert biological activity and can be formed endogenously. Fragmentary studies demonstrate their ability to enhance transport of different cargoes across the blood-brain barrier (BBB). The cellular internalization sequence can be any cell-penetrating peptide sequence capable of penetrating the BBB. Non-limiting examples of CPPs include Polyarginine (e.g., R9), Antennapedia sequences, TAT, HIV-Tat, Penetratin, Antp-3 A (Antp mutant), Buforin II, Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynBl, Pep-7, HN-1, BGSC (Bis- Guanidinium-Spermidine-Cholesterol, and BGTC (Bis-Guanidinium-Tren-Cholesterol) (see Table 1).
Table 1 : Cell Internalization Transporters
Name Sequence SEQ ID NO
Polyarginine RRRRRRRRR SEQ ID NO: 878
Antp RQPKIWFPNRRKPWKK SEQ ID NO: 879
HIV-Tat GRKKRRQRPPQ SEQ ID NO:880
Penetratin RQIKT F QNRRMKWKK SEQ ID NO:881
Antp-3 A RQIAIWFQNRRMKWAA SEQ ID NO:882
Tat RKKRRQRRR SEQ ID NO:883
Buforin II TRS SRAGLQFP VGRVHRLLRK SEQ ID NO:884
Transportan GWTLNSAGYLLGKINKALAALAKKIL SEQ ID NO:885 model KLALKLALKALKAALKLA SEQ ID NO:886 amphipathic
peptide (MAP)
K-FGF AAVALLPAVLLALLAP SEQ ID NO:887
Ku70 VPMLK- PMLKE SEQ ID NO:888
Prion MA LGYWLLALFVTMWTDVGLCKKRPKP SEQ ID NO:889 pVEC LLIILRRRIRKQAHAHSK SEQ ID NO:890
Pep-1 KET W WET W W TEW S QPKKKRK V SEQ ID NO:891
SynBl RGGRLSYSRRRFSTSTGR SEQ ID NO:892
Pep-7 SDLWEMMMVSLACQY SEQ ID NO:893
HN-1 T SPLNIHNGQKL SEQ ID NO:894
Tat GRKKRRQRRRPQ SEQ ID NO:895
Tat RKKRRQRRRC SEQ ID NO:896
Therefore, in some embodiments, the disclosed peptide is a fusion protein, e.g., containing the APP -binding domain of ΡΤΡσ, the ΡΤΡσ-binding domain of APP, or a combination thereof, and a CPP. Fusion proteins, also known as chimeric proteins, are proteins created through the joining of two or more genes, which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with function properties derived from each of the original proteins. Recombinant fusion proteins can be created artificially by recombinant DNA technology for use in biological research or therapeutics.
In some embodiments, linker (or "spacer") peptides are also added which make it more likely that the proteins fold independently and behave as expected. Linkers in protein or peptide fusions are sometimes engineered with cleavage sites for proteases or chemical agents which enable the liberation of the two separate proteins. This technique is often used for identification and purification of proteins, by fusing a GST protein, FLAG peptide, or a hexa-his peptide (aka: a 6xhis-tag) which can be isolated using nickel or cobalt resins (affinity chromatography).
Chimeric proteins can also be manufactured with toxins or antibodies attached to them in order to study disease development.
Compositions that restore molecular balance of CS and HS in the perineuronal space:
Chondroitin sulfates (CS) and heparin or its analog heparan sulfates (HS) are two main classes of glycosaminoglycans (GAGs) in the brain that are sensed by neurons via Receptor Protein Tyrosine 8. The ratio of CS and HS therefore affects the downstream effects of ΡΤΡσ, because CS and HS compete to interact with the receptor yet lead to opposite signaling and neuronal responses (such as neurite regeneration). CS increases but HS decreases APP β- cleavage products (Example 2). Therefore, methods involving administering to the subject a composition that restore the physiological molecular CS/HS balance may be used to treat and prevent aforementioned neurodegenerative diseases. These therapies could be applied alternatively or in addition to the polypeptides listed above. In some embodiments,
administering HS, or its analog heparin, or their mimetics modified to reduce anti-coagulant effect, with a saccharide chain length of 17, 18, 19, 20, 21, 22, 23, 24 units or longer, could assist in restoring the physiological molecular CS/HS balance. In some embodiments, the balance is restored by administering enzymes that digest CS (such as ChABC) or prevent the degradation of HS (such as Heparanase inhibitors PI-88, OGT 2115, or PG545). Alternatively or in addition, agents that mimic the HS/heparin effect of ΡΤΡσ clustering 8, such as multivalent antibodies, could be administered.
Pharmaceutical Compositions
The peptides disclosed can be used therapeutically in combination with a
pharmaceutically acceptable carrier. Pharmaceutical carriers suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
In some embodiments, the peptides described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art (See, e.g., Ansel,
Introduction to Pharmaceutical Dosage Forms, 4th Edition, 1985, 126).
Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
Dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared. Methods for preparation of these compositions are known to those skilled in the art. The contemplated compositions may contain 0.001%- 100% active ingredient, or in one embodiment 0.1-95%.
Methods of Screening
Also disclosed are methods of screening for candidate compounds that slow, stop, reverse, or prevent neurodegeneration. Methods of screening based on ΑΡΡ-ΡΤΡσ binding:
In some embodiments, the method comprising providing a sample comprising APP and ΡΤΡσ in an environment permissive for ΑΡΡ-ΡΤΡσ binding, contacting the sample with a candidate compound, and assaying the sample for ΑΡΡ-ΡΤΡσ binding, wherein a decrease in ΑΡΡ-ΡΤΡσ binding compared to control values is an indication that the candidate agent is effective to slow, stop, reverse, or prevent neurodegeneration.
The binding of ΡΤΡσ to APP can be detected using routine methods that do not disturb protein binding.
In some embodiments, the binding of ΡΤΡσ to APP can be detected using
immunodetection methods. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Maggio et al., Enzyme-Immunoassay, (1987) and Nakamura, et al., Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Handbook of Experimental Immunology, Vol. 1 : Immunochemistry, 27.1-27.20 (1986), each of which is incorporated herein by reference in its entirety and specifically for its teaching regarding immunodetection methods. Immunoassays, in their most simple and direct sense, are binding assays involving binding between antibodies and antigen. Examples of immunoassays are enzyme linked immunosorbent assays (ELISAs), radioimmunoassays (RIA), radioimmune precipitation assays (RIP A), immunobead capture assays, Western blotting, dot blotting, gel- shift assays, Flow cytometry, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery/localization after photobleaching (FRAP/ FLAP).
The methods can be cell-based or cell-free assays.
In some embodiments, the binding between ΡΤΡσ and APP can be detected using fluorescence activated cell sorting (FACS). For example, disclosed are cell lines transfected with of ΡΤΡσ and APP fused to fluorescent proteins. These cell lines can facilitate high-throughput screens for biologically expressed and chemically synthesized molecules that disrupt the binding between ΡΤΡσ and APP.
In some embodiments, the binding between ΡΤΡσ and APP can be detected in a cell-free setting where one of these two binding partners is purified and immobilized/captured through covalent or non-covalent bond to a solid surface or beads, while the other binding partner is allowed to bind in the presence of biologically expressed and chemically synthesized molecules to screen candidate agents for their efficacies in dissociating ΑΡΡ-ΡΤΡσ interaction. In some embodiments, the binding between ΡΤΡσ and APP can be detected in a setting where cell membrane preparations extracted from fresh rodent brain homogenates (containing both APP and ΡΤΡσ) are contacted with biologically expressed and chemically synthesized molecules. Subsequently, one of the binding partners is immunoprecipitated and the binding or co-immunoprecipitation of the other binding partner is detected using its specific antibody.
A candidate agent that decreases or abolishes ΑΡΡ-ΡΤΡσ binding in a disclosed method herein has the potential to slow, stop, reverse, or prevent neurodegeneration.
Methods of screening based on APP amyloidogenic processing:
In some embodiments, the method comprising contacting/incubating a candidate compound with cell membrane preparations extracted from fresh rodent brain homogenates, wherein a decrease in APP β- and/or γ-cleavage products is an indication that the candidate agent has the potential to slow, stop, reverse, or prevent neurodegeneration. APP β- and/or γ- cleavage products can be detected by routine biochemical methods such as Western blot analysis, ELISA, and immnuopurification.
Libraries of molecules and compounds:
In general, candidate agents can be identified from large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) used.
Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts,
fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi- synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available, e.g., from purveyors of chemical libraries including but not limited to ChemBridge Corporation (16981 Via Tazon, Suite G, San Diego, CA, 92127, USA, www.chembridge.com); ChemDiv (6605 Nancy Ridge Drive, San Diego, CA 92121, USA); Life Chemicals (1103 Orange Center Road, Orange, CT 06477); Maybridge (Trevillett, Tintagel, Cornwall PL34 0HW, UK). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including 02H,
(Cambridge, UK), MerLion Pharmaceuticals Pte Ltd (Singapore Science Park II, Singapore 117528) and Galapagos NV (Generaal De Wittelaan LI 1 A3, B-2800 Mechelen, Belgium).
In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods or by standard synthetic methods in combination with solid phase organic synthesis, micro-wave synthesis and other rapid throughput methods known in the art to be amenable to making large numbers of compounds for screening purposes. Furthermore, if desired, any library or compound, including sample format and dissolution is readily modified and adjusted using standard chemical, physical, or biochemical methods.
Candidate agents encompass numerous chemical classes, but are most often organic molecules, e.g., small organic compounds having a molecular weight of more than 100 and less than about 2,500 Daltons, or, in some embodiments, having a molecular weight of more than 100 and less than about 5,000 Daltons. Candidate agents can include functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, for example, at least two of the functional chemical groups. The candidate agents often contain cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
In some embodiments, the candidate agents are proteins. In some aspects, the candidate agents are naturally occurring proteins or fragments of naturally occurring proteins. Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, can be used. In this way libraries of procaryotic and eucaryotic proteins can be made for screening using the methods herein. The libraries can be bacterial, fungal, viral, and vertebrate proteins, and human proteins.
Methods of Treatment
Disclosed herein are methods for treating neurodegenerative diseases that involve β- amyloid pathologies and/or Tau pathologies, including but not limited to Alzheimer's disease, Lewy body dementia, frontotemporal dementia, cerebral amyloid angiopathy, primary age- related tauopathy, chronic traumatic encephalopathy, Parkinson's disease, postencephalitic parkinsonism, Huntington's disease, amyolateral sclerosis, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, Lytico-Bodig disease, ganglioglioma and gangliocytoma, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and/or Creutzfeldt-Jakob disease.
These peptides, compositions, and methods may also be used to prevent these neurodegenerative diseases in populations at risk, such as people with Down syndrome and those suffered from brain injuries or cerebral ischemia, as well as the aging population.
In some embodiments, these methods involve disrupting the binding between ΡΤΡσ and APP, preventing β-amyloidogenic processing of APP without affecting other major substrates of β- and γ-secretases. For example, the methods can involve administering to a subject a peptide disclosed herein. In other embodiments, monoclonal antibodies could be formed against the IGl domain of ΡΤΡσ or a fragment thereof, a fragment between the El and E2 domain of the
APP695 isoform, or both, and these antibodies, or fragments thereof, could be administered to the subject.
Chondroitin sulfates (CS) and heparin or its analog heparan sulfates (HS) are two main classes of glycosaminoglycans (GAGs) in the brain that are "sensed" by neurons via Receptor Protein Tyrosine 8. The ratio of CS and HS therefore affects the downstream effects of ΡΤΡσ, because CS and HS compete to interact with the receptor yet lead to opposite signaling and neuronal responses (such as neurite regeneration). CS increases but HS decreases APP β- cleavage products (Example 2). Therefore, in some embodiments, the methods involve administering to the subject a composition, which restores the physiological molecular CS/HS balance, may be used to treat and prevent aforementioned neurodegenerative diseases. These therapies could be applied alternatively or in addition to the polypeptides listed above. In some embodiments, administering HS, or its analog heparin, or their mimetics modified to reduce anti -coagulant effects, with a saccharide chain length of 17, 18, 19, 20, 21, 22, 23, 24 units or longer, could assist in restoring the physiological molecular CS/HS balance. In some embodiments, the balance is restored by administering enzymes that digest CS (such as
Chondroitinase ABC) or prevent the degradation of HS (such as Heparanase inhibitors PI-88, OGT 2115, or PG545). Alternatively or in addition, agents that mimic the HS/heparin effect of ΡΤΡσ clustering 8, such as multivalent antibodies, could be administered.
In some embodiments, the method involves administering a composition described herein in a dose equivalent to parenteral administration of about 0.1 ng to about 100 g per kg of body weight, about 10 ng to about 50 g per kg of body weight, about 100 ng to about 1 g per kg of body weight, from about ^g to about 100 mg per kg of body weight, from about 1 μg to about 50 mg per kg of body weight, from about 1 mg to about 500 mg per kg of body weight; and from about 1 mg to about 50 mg per kg of body weight. Alternatively, the amount of composition administered to achieve a therapeutic effective dose is about 0.1 ng, 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 500 mg per kg of body weight or greater.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. EXAMPLES
Example 1: Alzheimer's disease pathogenesis is dependent on neuronal receptor
ΡΤΡσ
Methods and Materials
Mouse lines: Mice were maintained under standard conditions approved by the
Institutional Animal Care and Use Committee. Wild type and ΡΤΡσ-deficient mice of Balb/c background were provided by Dr. Michel L. Tremblay 9. Homozygous TgAPP-SwDI mice, C57BL/6-Tg(Thyl-APPSwDutIowa)BWevn/Mmjax, stock number 007027, were from the Jackson Laboratory. These mice express human APP transgene harboring Swedish, Dutch, and Iowa mutations, and were bred with Balb/c mice heterozygous for the ΡΤΡσ gene to generate bigenic mice heterozygous for both TgAPP-SwDI and ΡΤΡσ 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 ΡΤΡσ gene. The offspring from this mating are used in experiments, which include littermates of the following genotypes: TgAPP-SwDI(+/-)PTPa(+/+), mice
heterozygous for TgAPP-SwDI transgene with wild type ΡΤΡσ; TgAPP-SwDI(+/-)PTPa(-/-), mice heterozygous for TgAPP-SwDI transgene with genetic depletion of ΡΤΡσ; TgAPP-SwDI(- /-)ΡΤΡσ(+/+), mice free of TgAPP-SwDI transgene with wild type ΡΤΡσ. Both TgAPP-SwDI(-/- )ΡΤΡσ(+/+) and Balb/c ΡΤΡσ(+/+) are wild type mice but with different genetic background. Heterozygous TgAPP-SwInd (J20) mice, 6.Cg-Tg(PDGFB-APPSwInd)20Lms/2Mmjax, were provided by Dr. Lennart Mucke. These mice express human APP transgene harboring Swedish and Indiana mutations, and were bred with the same strategy as described above to obtain mice with genotypes of TgAPP-SwInd (+/-)ΡΤΡσ(+/+) and TgAPP-SwInd (+/-)ΡΤΡσ(-/-). Antibodies:
Figure imgf000049_0001
Immunohistochemistry: Adult rat and mice were perfused intracardially with fresh made 4% paraformaldehyde in cold phosphate-buffered saline (PBS). The brains were collected and post-fixed for 2 days at 4 °C. Paraffin embedded sections of 10 μΜ thickness were collected for immunostaining. The sections were deparaffinized and sequentially rehydrated. Antigen retrieval was performed at 100 °C in Tris-EDTA buffer (pH 9.0) for 50 min. Sections were subsequently washed with distilled water and PBS, incubated at room temperature for 1 hour in blocking buffer (PBS, with 5% normal donkey serum, 5% normal goat serum, and 0.2% Triton X-100). Primary antibody incubation was performed in a humidified chamber at 4°C overnight. After 3 washes in PBS with 0.2% Triton X-100, the sections were then incubated with a mixture of secondary and tertiary antibodies at room temperature for 2 hours. All antibodies were diluted in blocking buffer with concentrations recommended by the manufacturers. Mouse primary antibodies were detected by goat anti-mouse Alexa488 together with donkey anti-goat Alexa488 antibodies; rabbit primary antibodies were detected by chicken anti-rabbit CF568 and donkey anti-chicken Cy3 antibodies; chicken antibody was detected with donkey anti-chicken Cy3 antibody. Sections stained with only secondary and tertiary antibodies (without primary antibodies) were used as negative controls. At last, DAPI (Invitrogen, 300 nM) was applied on sections for nuclear staining. Sections were washed 5 times before mounted in Fluoromount (SouthernBiotech).
Wide field and confocal images were captured using Zeiss Axio Imager M2 and
LSM780, respectively. Images are quantified using the Zen 2 Pro software and ImageJ.
Protein extraction, immunoprecipitation, and western blot analysis: For the co- immunoprecipitation of APP and ΡΤΡσ, RIPA buffer was used (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% P40, 0.1% SDS, 0.5% sodium deoxycholate). For the co- immunoprecipitation of APP and BACE1, P40 buffer was used (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% P40) without or with SDS at concentration of 0.1%, 0.3%, and 0.4%). For total protein extraction and immunopurification of CTF , SDS concentration in RIPA buffer was adjusted to 1%> to ensure protein extraction from the lipid rafts. Mouse or rat forebrains were homogenized thoroughly on ice in homogenization buffers (as mention above) containing protease and phosphatase inhibitors (Thermo Scientific). For each half of forebrain, buffer volume of at least 5 ml for mouse and 8 ml for rat was used to ensure sufficient detergent/tissue ratio. 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.
For co-immunoprecipitation and immunopurification, the homogenates were then centrifuged at 85,000 x g for 1 hour at 4°C and the supernatants were collected. Protein concentration was measured using BCA Protein Assay Kit (Thermo Scientific). 0.5 mg total proteins of brain homogenates were incubated with 5 μg of designated antibody and 30 μΐ of Protein-A sepharose beads (50%> slurry, Roche), in a total volume of 1 ml adjusted with RIPA buffer. Samples were gently mixed at 4°C overnight. Subsequently, the beads were washed 5 times with cold immunoprecipitation buffer. Samples were then incubated in Laemmli buffer with 100 mM of DTT at 75°C for 20 minutes and subjected to western blot analysis.
For analysis of protein expression level, the homogenates were centrifuged at 23,000 x g for 30 min at 4°C and the supernatants were collected. Protein concentration was measured using BCA Protein Assay Kit (Thermo Scientific). 30 μg of total proteins were subjected to western blot analysis.
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 μιη 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).
Western blot band intensity was quantified by densitometry.
Αβ ELISA assays: 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. The supernatants were collected and diluted 1 :20 in neutralization buffer (1 M Tris base, 0.5 M Na2HP04, 0.05% NaN3) 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 Αβ peptides. Serial dilutions of synthesized human Αβ 1-40 or 1-42 (American Peptide) were loaded to determine a standard curve. Αβ was detected using an HRP labeled antibody for either Αβ 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 Αβ in samples were determined using the standard curve.
Behavior assays: The Y-maze assay: 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. Cages and objects were cleaned with 70% ethanol between each animal. Subsequently, 2 hours after the familiarization session on day 4, mice were put back to the same cage where one of the familiar objects (randomly chosen) was replaced with a novel object, and allowed to explore for 5 minutes. Mice were scored using Observer software (Noldus) on their time duration and visiting frequency exploring either object. Object exploration was defined as facing the object and actively sniffing or touching the object, whereas any climbing behavior was not scored. The discrimination indexes reflecting interest in the novel object is denoted as either the ratio of novel object exploration to total object exploration (NO/NO+FO) or the ratio of novel object exploration to familiar object exploration (NO/FO). All tests and data analyses were conducted in a double-blinded manner.
Statistics: 2-tailed Student' s t test was used for two-group comparison. Relationship between two variables was analyzed using linear regression. All error bars show standard error of the means (SEM).
Results
ΡΤΡσ is an APP binding partner in the brain.
Previously identified as a neuronal receptor of extracellular proteoglycans 8 10 11, ΡΤΡσ is expressed throughout the adult nervous system, most predominantly in the hippocampus 12 13, one of earliest affected brain regions in AD. Using immunohistochemistry and confocal imaging, it was found that ΡΤΡσ and APP (the precursor of Αβ) colocalize in hippocampal pyramidal neurons of adult rat brains, most intensively in the initial segments of apical dendrites, and in the perinuclear and axonal regions with a punctate pattern (Fig. la-f). To assess whether this colocalization reflects a binding interaction between these two molecules, co- immunoprecipitation experiments were run from brain homogenates. In brains of rats and mice with different genetic background, using various antibodies of APP and ΡΤΡσ, a fraction of ΡΤΡσ that co-immunoprecipitates with APP was consistently detected, providing evidence of a molecular complex between these two transmembrane proteins (Fig. lh, i; Fig. 2).
Genetic depletion of ΡΤΡσ reduces β-amyloidogenic products of APP.
The molecular interaction between ΡΤΡσ and APP prompted an investigation on whether ΡΤΡσ plays a role in amyloidogenic processing of APP. In neurons, APP is mainly processed through alternative cleavage by either a- or β-secretase. These secretases release the N-terminal portion of APP from its membrane-tethering C-terminal fragment (CTFa or CTFP,
respectively), which can be further processed by the γ-secretase 14 15. Sequential cleavage of
APP by the β- and γ-secretases is regarded as amyloidogenic processing since it produces Αβ peptides 16. When overproduced, the Αβ peptides can form soluble oligomers that trigger ramification of cytotoxic cascades, whereas progressive aggregation of Αβ eventually results in the formation of senile plaques in the brains of AD patients (Fig. 3a). To test the effect of ΡΤΡσ in this amyloidogenic processing, the levels of APP β- and γ-cleavage products in mouse brains were analyzed, with or without ΡΤΡσ.
Western blot analysis with protein extracts from mouse brains showed that genetic depletion of ΡΤΡσ does not affect the expression level of full length APP (Fig. 3b; Fig 4a). However, an antibody against the C-terminus of APP detects a band at a molecular weight consistent with CTFp, which is reduced in ΡΤΡσ-deficient mice as compared to their age- sex- matched wild type littermates (Fig. 3b). Additionally, in two AD mouse models expressing human APP genes with amyloidogenic mutations 17 18, a similar decrease of an APP CTF upon ΡΤΡσ depletion was observed (Fig. 3b; Fig. 4b). The TgAPP-SwDI and TgAPP-SwInd mice, each expressing a human APP transgene harboring the Swedish mutation near the β-cleavage site, were crossed with the ΡΤΡσ line to generate offsprings that are heterozygous for their respective APP transgene, with or without ΡΤΡσ. Because the Swedish mutation carried by these APP transgenes is prone to β-cleavage, the predominant form of APP CTF in these transgenic mice is predicted to be CTFp. Thus, the reduction of APP CTF in ΡΤΡσ-deficient APP transgenic mice may indicate a regulatory role of ΡΤΡσ on CTFP level. However, since the APP C-terminal antibody used in these experiments can recognize both CTFa and CTFp, as well as the phosphorylated species of these CTFs (longer exposure of western blots showed multiple CTF bands), judging the identity of the reduced CTF simply by its molecular weight may be inadequate. CTFP immunopurification was therefore performed with subsequent western blot detection, using an antibody that recognizes CTFP but not CTFa (Fig. 3c, d; Fig. 4c, d). With this method, we confirmed that ΡΤΡσ depletion decreases the level of CTFP originated from both mouse endogenous and human transgenic APP.
Because CTFP is an intermediate proteolytic product between β- and γ-cleavage, its decreased steady state level could result from either reduced production by β-cleavage or increased degradation by subsequent γ-secretase cleavage (Fig. 3a). To distinguish between these two possibilities, the level of Αβ peptides was measured, because they are downstream products from CTFβ degradation by γ-cleavage. Using ELISA assays with brain homogenates from the TgAPP-SwDI mice, it was found that ΡΤΡσ depletion decreases the levels of Αβ peptides to a similar degree as that of CTFβ (Fig. 3e, f). Consistently, as Αβ peptides gradually aggregate into plaques during aging of the transgenic mice, a substantial decrease of cerebral Αβ deposition was observed in APP transgenic ΡΤΡσ-deficient mice as compared to the age- matched APP transgenic littermates expressing wild type ΡΤΡσ (Fig. 3g, h; Fig. 4e, f). Thus, the concurrent decrease of β- and γ-cleavage products argues against an increased γ-secretase activity, but instead suggests a reduced β-secretase cleavage of APP, which suppresses not only the level of CTFp but also downstream Αβ production in ΡΤΡσ-deficient brains.
Curtailed progression of β-amyloidosis in the absence of ΡΤΡσ.
Progressive cerebral Αβ aggregation (β-amyloidosis) is regarded as a benchmark of AD progression. To investigate the effects of ΡΤΡσ on this pathological development, Αβ deposits in the brains of 9-month old (mid-aged) and 16-month old (aged) TgAPP-SwDI mice were monitored. At age of 9 to 11 months, Αβ deposits are found predominantly in the hippocampus, especially in the hilus of the dentate gyrus (DG) (Fig. 3g, h). By 16 months, the pathology spreads massively throughout the entire brain. The propagation of Αβ deposition, however, is curbed by genetic depletion of ΡΤΡσ, as quantified using the DG hilus as a representative area (Fig. 3i). Between the ages of 9 and 16 months, the Αβ burden is more than doubled in TgAPP- SwDI mice expressing wild type ΡΤΡσ [APP-SwDI(+)PTPo(+/+)], but only shows marginal increase in the transgenic mice lacking functional ΡΤΡσ [APP-SwDI(+)PTPo(-/-)]. Meanwhile, the Αβ loads measured in 9-month old APP-SwDI(+)PTPo(+/+) mice are similar to those of 16- month old APP-SwDI(+)PTPo(-/-) mice (p=0.95), indicating a restraint of disease progression by ΡΤΡσ depletion (Fig. 3i).
Decreased BACE1-APP affinity in ΡΤΡσ-deficient brains.
Consistent with these observations that suggest a facilitating role of ΡΤΡσ in APP β- cleavage, the data further reveal that ΡΤΡσ depletion weakens the interaction of APP with BACEl, the β-secretase in the brain. To test the in vivo affinity between BACEl and APP, co- immunoprecipitation were performed of the enzyme and substrate from mouse brain
homogenates in buffers with serially increased detergent stringency. Whereas BACEl -APP association is nearly equal in wild type and ΡΤΡσ-deficient brains under mild buffer conditions, increasing detergent stringency in the buffer unveils that the molecular complex is more vulnerable to dissociation in brains without ΡΤΡσ (Fig. 5). Thus a lower BACEl-APP affinity in ΡΤΡσ-deficient brains may likely be an underlying mechanism for the decreased levels of CTFβ and its derivative Αβ.
Although it cannot be ruled out that some alternative uncharacterized pathway may contribute to the parallel decrease of CTFβ and Αβ in ΡΤΡσ-deficient brains, these data consistently support the notion that ΡΤΡσ regulates APP amyloidogenic processing, likely via facilitation of BACE1 activity on APP, the initial process of Αβ production.
The specificity of β-amyloidogenic regulation by ΡΤΡσ.
The constraining effect of ΡΤΡσ on APP amyloidogenic products led to further questions regarding whether this observation reflects a specific regulation of APP metabolism, or alternatively, a general modulation on the β- and γ-secretases. First, the expression level of these secretases in mouse brains were assessed with or without ΡΤΡσ. No change was found for BACE1 or the essential subunits of γ-secretase (Fig. 6a, b). Additionally, the question of whether ΡΤΡσ broadly modulates β- and γ-secretase activities was tested by examining the proteolytic processing of their other substrates. Besides APP, Neuregulinl (NRG1) 19-21 and Notch 22-24 are the major in vivo substrates of BACE1 and γ-secretase, respectively. Neither BACE1 cleavage of NRG1 nor γ-secretase cleavage of Notch is affected by ΡΤΡσ deficiency (Fig. 6c, d). Taken together, these data rule out a generic modulation of β- and γ-secretases, but rather suggest a specificity of APP amyloidogenic regulation by ΡΤΡσ.
ΡΤΡσ depletion relieves neuroinflammation and synaptic impairment in APP transgenic mice.
Substantial evidence from earlier studies has established that overproduction of Αβ in the brain elicits multiplex downstream pathological events, including chronic inflammatory responses of the glia, such as persistent astrogliosis. The reactive (inflammatory) glia would then crosstalk with neurons, evoking a vicious feedback loop that amplifies neurodegeneration during disease progression 25-2V.
The TgAPP-SwDI model is one of the earliest to develop neurodegenerative pathologies and behavioral deficits among many existing AD mouse models 11. These mice were therefore chosen to further examine the role of ΡΤΡσ in AD pathologies downstream of neurotoxic Αβ.
The APP-SwDI(+)PTPo(+/+) mice, which express the TgAPP-SwDI transgene and wild type ΡΤΡσ, 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. 7). In the DG hilus, for example, GFAP expression level in the APP-SwDI(+)PTPo(+/+) mice is more than tenfold compared to that in age-matched non-transgenic littermates [APP-SwDI(- )ΡΤΡσ(+/+)]. ΡΤΡσ deficiency, however, effectively attenuates astrogliosis induced by the amyloidogenic transgene. In the APP-SwDI(+)PTPo(-/-) brains, depletion of ΡΤΡσ restores GFAP expression in DG hilus back to a level close to that of non-transgenic wild type littermates (Fig. 7k).
Among all brain regions, the most affected by the expression of TgAPP-SwDI transgene appears to be the hilus of the DG, where Αβ deposition and astrogliosis are both found to be the most severe (Fig. 3g, h; Fig. 7). The question was therefore raised whether the pathologies in this area have an impact on the mossy fiber axons of DG pyramidal neurons, which project through the hilus into the CA3 region, where they synapse with the CA3 dendrites. Upon examining the presynaptic markers in CA3 mossy fiber terminal zone, decreased levels of Synaptophysin and Synapsin-1 were found in the APP-SwDI(+)PTPo(+/+) mice, comparing to their age-matched non-transgenic littermates (Fig. 8, data not shown for Synapsin-1). Such synaptic impairment, evidently resulting from the expression of the APP transgene and possibly the overproduction of Αβ, is reversed by genetic depletion of ΡΤΡσ in the APP-SwDI(+)PTPo(- /-) mice (Fig. 8).
Interestingly, the APP-SwDI(+)PTPo(-/-) mice sometimes express higher levels of presynaptic markers in the C A3 terminal zone than their age-matched non-transgenic wild type littermates (Fig. 8g). This observation, although not statistically significant, may suggest an additional synaptic effect of ΡΤΡσ that is independent of the APP transgene, as observed in previous studies 28.
Tau pathology in aging AD mouse brains is dependent on ΡΤΡσ.
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 5 <5. The precise mechanism of tangle formation, however, is poorly understood. The fact that Tau tangles and Αβ deposits can be found in separate locations in postmortem brains has led to the question of whether Tau pathology in AD is independent of Αβ accumulation 5 <5. Additionally, despite severe cerebral β-amyloidosis in many APP transgenic mouse models, Tau tangles have not been reported, further questioning the relationship between Αβ and Tau pathologies in vivo.
Nonetheless, a few studies did show non-tangle like assemblies of Tau in dystrophic neurites surrounding Αβ plaques in APP transgenic mouse lines 29"31, arguing that Αβ can be a causal factor for Tau dysregulation, despite that the precise nature of Tau pathologies may be different between human and mouse. In the histological analysis using an antibody against the proline-rich domain of Tau, Tau aggregation was observed in the brains of both TgAPP-SwDI and TgAPP-SwInd mice during the course of aging (around 9 months for the APP- SwDI(+)PTPo(+/+) mice and 15 months for the APP-SwInd(+)PTPo(+/+) mice) (Fig. 9; Fig. 10). Such aggregation is not seen in aged-matched non-transgenic littermates (Fig. 9h), suggesting that it is a pathological event downstream from the expression of amyloidogenic APP transgenes, possibly a result of Αβ cytotoxicity. Genetic depletion of ΡΤΡσ, which diminishes Αβ levels, suppresses Tau aggregation in both TgAPP-SwDI and TgAPP-SwInd mice (Fig. 9; Fig. 10).
In both TgAPP-SwDI and TgAPP-SwInd mice, the Tau aggregates are found
predominantly in the molecular layer of the piriform and entorhinal cortices, and occasionally in the hippocampal region (Fig. 9; Fig. 10), reminiscent of the early stage tangle locations in AD brains 32. Upon closer examination, the Tau aggregates are often found in punctate shapes, likely in debris from degenerated cell bodies and neurites, scattered in areas free of nuclear staining (Fig. 1 la-f). Rarely, a few are in fibrillary structures, probably in degenerated cells before disassembling (Fig. 1 lg, h). To confirm these findings, an additional antibody was used to recognize the C-terminus of Tau. The same morphologies (Fig. Hi) and distribution pattern (Fig. 9a) were detected.
Consistent with the findings in postmortem AD brains, the distribution pattern of Tau aggregates in the TgAPP-SwDI brain does not correlate with that of Αβ deposition, which is pronounced in the hippocampus yet only sporadic in the piriform or entorhinal cortex at the age of 9 months (Fig. 3g, h). Given that the causation of Tau pathology in these mice is possibly related to the overproduced Αβ, the segregation of predominant areas for Αβ and Tau depositions may indicate that the cytotoxicity originates from soluble Αβ instead of the deposited amyloid. It is also evident that neurons in different brain regions are not equally vulnerable to developing Tau pathology.
Next, the question of whether the expression of APP transgenes or genetic depletion of ΡΤΡσ regulates Tau aggregation by changing its expression level and/or phosphorylation status was examined. Western blot analysis of brain homogenates showed that Tau protein expression is not affected by the APP transgenes or ΡΤΡσ (Fig. 12), suggesting that the aggregation may result from local misfolding of Tau rather than an overexpression of this protein. These experiments with brain homogenates also revealed that 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. These findings are consistent with a recent quantitative study showing similar post-translational modifications of Tau in wild type and TgAPP-SwInd mice 33. Furthermore, unlike previously reported 29'30, we could not detect these phosphorylated residues in the Tau aggregates, suggesting that the epitopes are either missing (residues not phosphorylated or cleaved off) or embedded inside the misfolding. Given the complexity of Tau post-translational modification, one cannot rule out that the aggregation may be mediated by some unidentified modification(s) of Tau. It is also possible that other factors, such as molecules that bind to Tau, may precipitate the aggregation.
Although the underlying mechanism is still unclear, the finding of Tau pathology in these mice establishes a causal link between the expression of amyloidogenic APP transgenes and a dysregulation of Tau assembly. The data also suggest a possibility that ΡΤΡσ depletion may suppress Tau aggregation by reducing amyloidogenic products of APP.
Malfunction of Tau is broadly recognized as a neurodegenerative marker since it indicates microtubule deterioration 1. The constraining effect on Tau aggregation by genetic depletion of ΡΤΡσ thus provides additional evidence for the role of this receptor as a pivotal regulator of neuronal integrity.
ΡΤΡσ deficiency rescues behavioral deficits in AD mouse models.
Next, the question was assessed of whether the alleviation of neuropathologies by ΡΤΡσ depletion is accompanied with a rescue from AD relevant behavioral deficits. The most common symptoms of AD include short-term memory loss and apathy among the earliest, followed by spatial disorientation amid impairment of many cognitive functions as the dementia progresses. Using Y maze and novel object assays as surrogate models, these cognitive and psychiatric features were evaluated in the TgAPP-SwDI and TgAPP-SwInd mice.
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 ΡΤΡσ in the APP- SwDI(+)PTPo(-/-) mice, however, unequivocally restores the cognitive performance back to the level of non-transgenic wild type mice (Fig. 13a, Fig. 14).
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 34"36. 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 34,35. 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 34>35>37. These visual -based novelty encoding tasks are very similar to the novel object assay for rodents, which measures the interest of animals in a novel object (NO) when they are exposed simultaneously to a prefamiliarized object (FO). This assay was therefore used to test the attention to novelty in the APP transgenic mice. When mice are pre-trained to recognize the FO, their attention to novelty is then measured by the discrimination index denoted as the ratio of NO exploration to total object exploration (NO+FO), or alternatively, by the ratio of NO exploration to FO exploration. Whereas both ratios are commonly used, a combination of these assessments provides a more comprehensive evaluation of animal behavior. In this test, as indicated by both measurements, 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. l ib, c; Fig. 15). 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. 15a, 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. Once again, this behavioral deficit is largely reversed by ΡΤΡσ deficiency in the APP-SwDI(+)PTPo(- /-) mice (Fig. 13b, c; Fig. 15), consistent with previous observation of increased NO preference in the absence of ΡΤΡσ 28.
To further verify the effects of ΡΤΡσ on these behavioral aspects, the TgAPP-SwInd mice were also tested using both assays, and similar results were observed. This confirms an improvement on both short-term spatial memory and attention to novelty upon genetic depletion of ΡΤΡσ (Fig. 16).
Discussion
The above data showed that β-amyloidosis and several downstream disease features are dependent on ΡΤΡσ in two mouse models of genetically inherited AD. This form of AD develops inevitably in people who carry gene mutations that promote amyloidogenic processing of APP and overproduction of Αβ. The data presented herein suggest that targeting ΡΤΡσ is a potential therapeutic approach that could overcome such dominant genetic driving forces to curtail AD progression. The advantage of this targeting strategy is that it suppresses Αβ accumulation without broadly affecting other major substrates of the β- and γ-secretases, thus predicting a more promising translational potential as compared to those in clinical trials that genetically inhibit the secretases.
ΡΤΡσ was previously characterized as a neuronal receptor of the chondroitin sulfate- and heparan sulfate-proteoglycans (CSPGs and HSPGs) 10 U. In response to these two classes of extracellular ligands, ΡΤΡσ functions as a "molecular switch" by regulating neuronal behavior in opposite manners 8. The finding presented herein of a pivotal role for the proteoglycan sensor ΡΤΡσ in AD pathogenesis may therefore implicate an involvement of the perineuronal matrix in AD etiology.
More than 95% of AD cases are sporadic, which are not genetically inherited but likely result from insults to the brain that occurred earlier in life. AD risk factors, such as traumatic brain injury and cerebral ischemia 38"41, have been shown to induce overproduction of Αβ in both human and rodents 42"46, and speed up progression of this dementia in animal models 47"49. However, what promotes the amyloidogenic processing of APP in these cases is still a missing piece of the puzzle in understanding the AD-causing effects of these notorious risk factors.
Coincidently, both traumatic brain injury and cerebral ischemia cause pronounced remodeling of the perineuronal microenvironment at lesion sites, marked by increased expression of CSPGs 50"53, a major component of the perineuronal net that is upregulated during neuroinflammation and glial scar formation 54"56. In the brains of AD patients, CSPGs were found associated with Αβ depositions, further suggesting an uncanny involvement of these proteoglycans in AD development 51. On the other hand, analogues of heparan sulfate (HS, carbohydrate side chains of HSPGs that bind to ΡΤΡσ) were shown to inhibit BACEl activity, suggesting their function in preventing Αβ overproduction 58. After cerebral ischemia, however, the expression of Heparanase, an enzyme that degrades HS, was found markedly increased 59. Collectively, these findings suggest a disrupted molecular balance between CSPGs and HSPGs in brains after lesion, which may ignite insidious signaling cascades preceding the onset of AD.
Further study could include investigation of a potential mechanism, whereby chronic CSPG upregulation or HSPG degradation in lesioned brains may sustain aberrant signaling through their neuronal sensor ΡΤΡσ, leading to biased processing of APP and a neurotoxic "Αβ cascade". As such, altered signaling from ΡΤΡσ after traumatic brain injury and ischemic stroke may explain how these risk factors can trigger subsequent onset of AD. Restoring the integrity of brain microenvironment therefore could be essential in preventing AD for the population at risk. Example 2: CS and HS regulates APP amyloidogenic processing in opposite manners
CS and HS/heparin are two classes of ΡΤΡσ ligands in the perineuronal space that compete for binding to the same site on receptor ΡΤΡσ with similar affinities 8. Increased CS/HS ratio is often found after brain injuries or ischemic stroke 50_53>59 3 both of which are prominent risk factors for AD and alike neurodegenerative diseases.
These two classes of ligands were shown previously to oppositely regulate neuronal responses, such as neurite outgrowth, through their common receptor ΡΤΡσ. Whereas CS inhibits neurite outgrowth, HS/heparin promotes neurite outgrowth.
When tested in an in vitro assay for their effects on APP amyloidogenic processing, these ΡΤΡσ ligands again showed opposite effects. As in Figure 17, incubation of cell membrane preparations extracted from fresh mouse brain homogenates with these ΡΤΡσ ligands results in an increased level of APP β-cleavage by CS, but a decreased level of APP β-cleavage by HS/heparin. Whereas CS levels are well documented to be upregulated after traumatic brain injury (TBI) in rats and mice, this study found increased ΑΡΡ-ΡΤΡσ binding accompanied with significantly enhanced level of APP β-cleavage product (CTF ) in injured brains (Fig. 18). On the contrary, HS/heparin, which inhibits APP β-cleavage, effectively disrupts ΑΡΡ-ΡΤΡσ binding (Fig. 19). These data thus suggest that the molecular balance of ΡΤΡσ ligands CS and HS in the brain is important in regulating APP amyloidogenic processing, and that the promoting and suppressing effects on APP β-cleavage by CS and HS, respectively, are mediated by their control on ΑΡΡ-ΡΤΡσ binding.
Example 3: Defining binding regions on human APP and ΡΤΡσ
Domain regions were subcloned from human APP695 (construct by Denis Selkoe and Tracy Yang labs purchased through Addgene.com) and ΡΤΡσ (constructs from Radu Aricescu lab). Recombinant APP and ΡΤΡσ proteins were tested in solid phase ELISA binding assays to define the binding regions on each partner. Neither El or E2 domain of APP interacts with
ΡΤΡσ (data not shown), however the region in between these two APP domains (SEQ ID NO: 1) appears to have high affinity with ΡΤΡσ IG1 domain (Fig. 20). The lysine residues (K67, 68, 70, 71) in ΡΤΡσ IG1 ligand binding site, which was shown to be responsible for CS and HS binding 8 11>60 3 are also important for its interaction with APP, as mutation of these residues abolishes ΑΡΡ-ΡΤΡσ binding. Comparing APP binding strength of difference ΡΤΡσ fragments, it appears that inclusion of the fibronectin (FN) domains of ΡΤΡσ weakens the interaction with APP, likely due to folding of ΡΤΡσ that covers up the ligand binding site in its IG1 domain 61. Full ΡΤΡσ extracellular domain nearly lost binding with APP SEQ ID NO: 1, suggesting that factors triggering the unfold ΡΤΡσ are required for ΑΡΡ-ΡΤΡσ binding.
Sequences:
Sequences for the peptides used in Example 3 are provided in Tables 3, 4, and 5.
Figure imgf000062_0001
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
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Claims

WHAT IS CLAIMED IS:
1. A peptide for treating or preventing a neurodegenerative disorder, the peptide
comprising;
5 a decoy fragment of Amyloid Precursor Protein (APP), a decoy fragment of Receptor
Protein Tyrosine Phosphatase Sigma (ΡΤΡσ), or a combination thereof, and
a blood brain barrier penetrating sequence.
2. The peptide of claim 1, wherein the decoy fragment of APP is a peptide comprising at o least 5 consecutive amino acids of SEQ ID NO: 1.
3. The peptide of claim 2, wherein the decoy fragment of APP is a peptide comprising at least 10 consecutive amino acids of SEQ ID NO: l . 5 4. The peptide of any one of claims 1 to 3, wherein the decoy fragment of APP comprises an amino acid sequence selected from the group consisting of SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO: 101, SEQ ID NO: 112, SEQ ID NO: 139, SEQ ID NO: 151, SEQ ID NO: 157, SEQ ID NO:251, SEQ ID NO:897, SEQ ID NO: 900. 0 5. The peptide of claim 1, wherein the decoy fragment of ΡΤΡσ is a peptide comprising at least 4 consecutive amino acids of SEQ ID NO:442.
6. The peptide of claim 5, wherein the decoy fragment of ΡΤΡσ is a peptide comprising at least 10 consecutive amino acids of SEQ ID NO:442.
5
7. The peptide of claim 5, wherein the decoy fragment of ΡΤΡσ comprises the amino acid sequence SEQ ID NO:898, SEQ ID NO:899. SEQ ID NO:655, or SEQ ID NO:769. 0 8. The peptide of any one of claims 1 to 7, wherein the blood brain barrier penetrating
sequence comprises amino acid sequence SEQ ID NO: 880, SEQ ID NO: 883, SEQ ID NO: 888, SEQ ID NO: 894, SEQ ID NO: 895, SEQ ID NO: 896.
9. The peptide of any one of claims 1 to 6, wherein the peptide is cyclic.
10. A composition, comprising the peptide of any one of claims 1 to 9 and further comprising a pharmaceutically acceptable excipient.
5
11. An antibody or an antibody fragment against APP or ΡΤΡσ for treating or preventing a neurodegenerative disorder, wherein the antibody or antibody fragment binds an epitope on APP or an epitope on ΡΤΡσ. o 12. The antibody or antibody fragment of claim 11, wherein the epitope on APP is a peptide sequence between the El and E2 domains of APP.
13. The antibody or antibody fragment of claim 11, wherein the epitope on ΡΤΡσ is a peptide sequence on the ΡΤΡσ IG1 domain.
5
14. The antibody or antibody fragment of claim 11, wherein the epitope on ΡΤΡσ is the entire ΡΤΡσ IG1 domain or SEQ ID NO:442.
15. The antibody or antibody fragment of any one of claims 11 to 14, further comprising a0 pharmaceutically acceptable excipient.
16. One or more compounds or enzymes for treating or preventing a neurodegenerative disorder, wherein the compound or enzyme restores the physiological molecular balance of chondroitin sulfate (CS) and heparan sulfate (HS) in the brain.
5
17. The one or more compounds or enzymes of claim 16, wherein the compound or enzyme is an analog of heparin, an analog of HS, a mimetic of heparin, a mimetic of HS, an inhibitor of heparanase, chondroitinase ABC (ChABC), or a combination thereof. 0 18. The one or more compounds or enzymes of claim 16, wherein the compound is an
inhibitor of heparanase.
19. The one or more compounds or enzymes of claim 16, wherein the compound is an analog or mimetic of heparin or HS.
20. The compound or enzyme of claim 16, wherein the compound or enzyme is ChABC.
21. The compound or enzyme of any one of claims 17 to 20, further comprising a pharmaceutically acceptable excipient.
22. A method of treating a neurodegenerative disorder in a subject, the method comprising administering to the subject a composition that interferes with the binding of Amyloid Precursor Protein (APP) to Receptor Protein Tyrosine Phosphatase Sigma (ΡΤΡσ).
23. The method of claims 22, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's Disease, Lewy body dementia, frontotemporal dementia, cerebral amyloid angiopathy, primary age-related tauopathy, chronic traumatic encephalopathy, Parkinson's disease, postencephalitic parkinsonism, Huntington's disease, amyolateral sclerosis, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, Lytico-Bodig disease, ganglioglioma and gangliocytoma, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and/or Creutzfeldt- Jakob disease.
24. The method of claim 22, wherein the composition comprises the composition of any one of claims 10, 15, 21, or a combination thereof.
25. A method of preventing a neurodegenerative disorder in an at-risk subject, the method comprising administering to the subject a composition that interferes with the binding of Amyloid Precursor Protein (APP) to Receptor Protein Tyrosine Phosphatase Sigma (ΡΤΡσ), wherein the at-risk subject is at age older than 60 years or has received a medical diagnosis associated with Down syndrome, brain injury, or cerebral ischemia.
26. The method of claim 25, wherein the composition comprises the composition of any one of claims 10, 15, 21, or a combination thereof.
27. A method of treating a neurodegenerative disorder in a subject, the method comprising administering to the subject a composition that restores the physiological molecular balance of CS and HS in the brain.
28. The method of claims 27, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's Disease, Lewy body dementia, frontotemporal dementia, cerebral amyloid angiopathy, primary age-related tauopathy, chronic traumatic encephalopathy, Parkinson's disease, postencephalitic parkinsonism, Huntington's disease, amyolateral sclerosis, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, Lytico-Bodig disease, ganglioglioma and gangliocytoma, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and/or Creutzfeldt- Jakob disease.
29. The method of claim 27, wherein the composition comprises the composition of any one of claims 10, 15, 21, or a combination thereof.
30. A method of preventing a neurodegenerative disorder in an at-risk subject, the method comprising administering to the at-risk subject a composition that restores the physiological molecular balance of CS and HS in the brain, wherein the at-risk subject has received a medical diagnosis associated with Down syndrome, brain injury, or cerebral ischemia.
31. The method of claim 30, wherein the composition comprises the composition of any one of claims 10, 15, 21, or a combination thereof.
32. A method of screening for candidate compounds that slow, stop, reverse, or prevent neurodegeneration, the method comprising;
providing a sample comprising Amyloid Precursor Protein (APP) and Receptor Protein Tyrosine Phosphatase Sigma (ΡΤΡσ) in an environment permissive for ΑΡΡ-ΡΤΡσ binding, contacting the sample with a candidate compound, and
assaying the sample for ΑΡΡ-ΡΤΡσ binding, wherein a decrease in ΑΡΡ-ΡΤΡσ binding compared to control values is an indication that the candidate agent is effective to slow, reverse, or prevent neurodegeneration.
33. A method of screening for candidate compounds that slow, stop, reverse, or prevent neurodegeneration, the method comprising;
providing a sample comprising cell membrane extracted from rodent brain homogenates, contacting the sample with a candidate compound, and
5 assaying the sample for APP amyloidogenic processing, wherein a decrease in APP
amyloidogenic product level compared to control values is an indication that the candidate agent has a potential to slow, reverse, or prevent neurodegeneration.
34. The method of claim 33, wherein the rodent brain homogenate is fresh rodent brain o homogenate.
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US20030069198A1 (en) * 1998-08-28 2003-04-10 Barger Steven W. Materials and methods related to the inflammatory effects of secreted amyloid precursor proteins
US20090215665A1 (en) * 2004-12-21 2009-08-27 Robert Gourdie Compositions and methods for promoting wound healing and tissue regeneration

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US20030069198A1 (en) * 1998-08-28 2003-04-10 Barger Steven W. Materials and methods related to the inflammatory effects of secreted amyloid precursor proteins
US20090215665A1 (en) * 2004-12-21 2009-08-27 Robert Gourdie Compositions and methods for promoting wound healing and tissue regeneration

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