WO2009005783A1 - Peptides, compositions and methods for reducing beta-amyloid-mediated apoptosis - Google Patents

Abstract

The present invention is directed to peptides which bind to proteins and reduce Aβ (39- 42)-mediated apoptosis in neuronal cells, as compared to untreated neuronal cells. The present invention is also directed to pharmaceutical composition comprising such peptides and antibodies to the foregoing peptides.

Description

Peptides, Compositions and Methods for Reducing Beta-Amyloid-Mediated

Apoptosis

FIELD OF THE INVENTION

[0001] The present invention provides peptides, antibodies and anti-idiotypic antibodies that reduce Aβ (39-42)-mediated apoptosis, as compared to untreated neuronal cells.

BACKGROUND OF THE INVENTION

[0002] Alzheimer's Disease (AD) is a chronic and progressive neurodegenerative disorder characterized neuropathologically by the presence of beta-amyloid (Aβ) plaques, neurofibrillary tangles, and gray matter loss. In AD, multiple regions of brain gray matter have a profound neuronal loss, including basal forebrain, hippocampus, entorhinal, and temporal cortices. Neurofibrillary tangles are composed of an abnormally hyperphosphorylated intracellular protein called tau, tightly wound into paired helical filaments and thought to impact microtubule assembly and protein trafficking, resulting in the eventual demise of neuronal viability. The extracellular Aβ plaque deposits are composed of a proteinacious core of insoluble aggregated Aβ peptides, from 39-42 amino acids in length Aβ(l-42). The presence of these aggregates has led to the foundation of the amyloid hypothesis. This hypothesis postulates that Aβ(l-42) is one of the principal causative factors of neuronal death in the brains of Alzheimer's patients.

[0003] Beta-amyloid is a toxic peptide produced by cleavage of amyloid precursor protein

(APP) by β- and γ-secretases. Aβ peptide produces apoptosis in neurons but the mechanism by which Aβ exerts its toxic effect is unknown. Application of Aβ to cultured neuronal cells at micromolar concentrations causes apoptosis, and lower concentrations cause upregulation of apoptosis markers caspase 3 and annexin (White, et al., Neurobiol Dis. 2001 ; 8, 299-316). Biochemical effects of Aβ include activation of calcium channels, production of free radicals, excitotoxicity through activation of NMDA receptors, and glutamate accumulation leading to increased Ca2+ levels. Intracerebroventricular injection of Aβ produces impairment of spatial memory and non-spatial long term memory, reduction of protein kinase C (PKC) activity, induction of apoptosis, and activation of astrocytes and microglia to release excessive amounts of inflammatory cytokines. Transgenic animals expressing human Aβ exhibit many of the pathologies of Alzheimer's disease (AD), including cognitive deficits, age-related formation of amyloid plaques, activation of astrocytes and microglial cells, vascular amyloid pathology, degeneration of cholinergic nerve terminals, and reduced lifespan. However, transgenic mice expressing normal human APP do not exhibit the neurofibrillary tangles and significant neuronal loss characteristic of AD (German, et al., Rev Neurosci, 2004; 15, 353-369).

[0004] Other diseases also are associated with pathologic Aβ. Down syndrome (trisomy 21) patients invariably develop (in their third or fourth decade) cerebral amyloid plaques and neurofibrillary tangles, the characteristic lesions of Alzheimer disease (AD). Some Down Syndrome patients found to have high levels of Aβ deposits in their brains and dementia from an early age have three copies of the APP gene (Robakis N.K., et al., Neurobiol Aging 1994; 15 (Suppl 2), Sl 27-Sl 29). Recent studies have shown that the Aβ(l-42) is the earliest form of this protein deposited in Down syndrome brains, and may be seen in subjects as young as 12 years of age, and that soluble Aβ can be detected in the brains of Down syndrome subjects as early as 21 gestational weeks of age, well preceding the formation of Aβ plaques (Gyure et al., Archives of Pathology and Laboratory Medicine, 2000; 125, 489-492).

[0005] Congophilic angiopathy (CAA), also referred to as amyloid angiopathy, is a form of angiopathy in which Aβ deposits in the walls of the leptomeninges and superficial cerebral cortical blood vessels of the brain. Amyloid deposition predisposes these blood vessel to failure, increasing the risk of a hemorrhagic stroke. Such brain hemorrhages are more common in people who suffer from Alzheimer's, however they can also occur in those who have no history of dementia.

[0006] Formation of Aβ from APP is dependent on the intracellular transport system. APP is transported from the ER and Golgi to the cell surface membrane, where it may be cleaved into the non- amyloidogenic peptide by the enzyme α-secretase. PKC-activated α-secretases also reside in the trans- Golgi network (TON) (Skovronsky, et al., J. Biol. Chem., 2000; 275, 2568-2575), which is a major site for β-secretase activity. Uncleaved APP is then internalized into endocytic compartments, where it is cleaved by β- and γ-secretase to produce Aβ. Aβ can also be produced in the Golgi and ER compartments.

[0007] The normal functions of APP and Aβ are unknown. APP is an integral membrane protein with high affinity for copper (White, et al., J Neurosci, 2002; 19, 9170-9179 and Barnham, et al., J Biol Chem, 2003; 278, 17401-17407). It has been suggested that APP is involved in neurodevelopment (Grilli, et al., Funct Neurol, 2003; 18, 145-148) and is essential for neuronal growth. Mutant mice in which APP has been knocked out develop reactive gliosis, weight loss, cognitive defects, and reduced levels of presynaptic marker proteins (Dawson, et al., Neuroscience, 1999; 90, 1-13), indicating generalized CNS pathology. Downregulation of APP inhibits neurite outgrowth (Allinquant, et al., J. Cell Biol., 1995; 128, 919-927) and anti-APP antibodies block memory formation in chicks (Mileusnic, et al., Eur J Neurosci., 2000; 12, 4487-4495.). After being transported along nerve fibers, APP participates in synaptogenesis (Moya, et al., Dev. Biol., 1994; 161 , 597-603) and cell adhesion (Small, et al., J Alzheimers Dis., 1999; 1, 275-285). Thus, APP may play an important role in normal synaptic plasticity and neuronal growth.

[0008] The Aβ peptide is also found in CSF and blood plasma of normal patients (Seubert, et al., Nature, 1992; 359, 325-327). The production and secretion of Aβ is regulated by neuronal activity (Kamenetz, et al., Neuron, 2003; 37, 925-937). Kamenetz et al., found that APP reversibly depresses synaptic transmission by a mechanism mediated by activation of APP cleavage by NMDA receptors. This suggests that Aβ is normally produced by neurons and has one or more functions in normal cells.

[0009] Levels of LR I 1/SorLA, which is also the receptor for apolipoprotein E, are decreased in sporadic AD (Scherzer, et al., Arch Neurol, 2004; 61 , 1200-1205.). Overexpression of sortilin results in APP localization to the Golgi, which may influence amyloidogenic processing to Aβ (M., A. O., et al., Proc Natl Acad Sci USA, 2005; 102, 13461-13466; Greenfield, et al., Proc Natl Acad Sci USA, 1999; 96, 742-747.; and Spoelgen, et al., J Neurosci, 2006; 26, 418-428). The sortilin receptor SorLA (LRI l ) also interacts with ApoE (45) and regulates Aβ production and APP traffic in endocytic compartments (46). Sortilin is also reported to be a substrate for gamma-secretase and possibly PKC-mediated alpha-secretase (Nyborg, et al., MoI Neurodegener, 2006; 1 , 1-1 1). Its apparent role is to target proteins in the Golgi for transport to late endosomes.

[0010] The 14-3-3 protein, which has many similarities to the Parkinson's disease- associated protein α-synuclein (Ostrerova, et al., J Neurosci., 1991 ; 19, 5782-5791), is found in neurofibrillary tangles (Layfield, et al., Neurosci Lett, 1996; 209, 57-60), binds to tau, and is involved in phosphorylation of tau by GSK-3β (Li, et al., Neurosci Lett, 2007; 414, 203-208 and Hashiguchi, et al., J Biol Chem, 2004; 275, 25247-25254). GSK-3f3, which is the principal enzyme involved in phosphorylating tau, is regulated by 14-3-3 (Yuan, et al., J Biol Chem, 2004; 279, 26105-261 14). Increases in 14-3-3 have been reported in patients with Alzheimer's disease (Fountoulakis, et al., J Neural Transm Suppl, 1999; 57, 323-335). Phosphorylation of Ser-9 in GSK-3P promotes binding of GSK-3p to 14-3-3. 14-3-3 zeta was also one of a small number of proteins found to be significantly oxidized after intracerebral injection of Aβ 1 -42 (Boyd- Kimball, et al., Neuroscience; 2005; 132, 313-324). Thus, binding of Aβ to 14-3-3 could be a missing link that connects two important pathways in AD (Aβ oligomerization and neurofibrillary tangles).

[0011] Orner et al, also recently used phage display to identify proteins that might mediate aggregation of Aβ (Orner, et al., J Am Chem Soc, 20; 128, 1 1882-1 1889.). The predominant peptide motif found by of Orner et al., was a fragment identical to a region of Aβ itself (QKLVFF; SEQ ID NO: 29), containing the α-secretase cleavage site and the "hydrophobic patch" (Fig. 5), suggesting that this region is critical for Aβ aggregation. This supports earlier results indicating that the two phenylalanines are critical for Aβ self-aggregation (Hughes, et al., Proc Natl Acad Sci USA, 1996; 93, 2065-2070). An N-methylated KLVFF (SEQ ID NO: 30) peptide, in which the backbone NH group is replaced by an N-methyl group, was shown to reduce the cytotoxicity of Aβ 1-42 in cultured PC 12 cells (Cruz, et al., J Pept Res, 2004; 63, 324-328).

[0012] Other proteins that have been reported to interact with Aβ peptide include collagenous Alzheimer amyloid plaque component (CLAC) (Kakuyama, et al., Biochemistry, 2005; 44, 15602-15609 and Hashimoto, et al., EMBO J., 2002; 21 , 1524-1534), which is a proteolytic form of collagen type XXV, αl isoform 1 ; α2-macroglobulin, a protease inhibitor that is released in response to inflammatory stimuli (Hughes, et al., Proc Natl Acad Sci USA, 1998; 95, 3275-3280 and Narita, et al., J Neurochem, 1997; 69, 195-204); tau (Prez, et al., J Alzheimers Dis; 2004; 6,461-467); the collagen-like domain of complement CIq A chain (Jiang, et al., J Immunol, 1994; 152, 5050-5059); the p75 neurotrophin receptor (Yaar, et al., J Clin Invest, 1997; 100, 2333-2340); apolipoprotein E (Pillot, et al., J Neurochem, 1999; 72, 1 131-1 137); vascular endothelial growth factor(165) (Yang, et al., J. Neurochem, 2005; 93, 1 18- 127); and the heat- shock protein alphaB-crystallin (Liang, J. J. FEBS Lett, 2000; 484,98-101). Many of these proteins are involved in either inflammation or in Aβ clearance. For example, α2-macroglobulin associates with the low-density lipoprotein receptor related protein (LRP), an established mediator of Aβ clearance. LRP is the major receptor for a 2-macroglobulin and ApoE, both of which bind Aβ.

[0013] Aβ interacts with tubulin, CNPase, and myelin basic protein, which was found by

Verdier et al., who studied synaptosomal proteins that co-precipitated with fibrillar Aβ. Differences between their results and ours may he attributed to their focus on membrane- extracted proteins from the synapse. Since Aβ is created in the endoplasmic reticulum (Aβ 1-42), trans-Golgi network (Aβ 1-40), and endocytic compartments (Aβ 1 -40) (Greenfield, et al., Proc Natl Acad Sci USA, 1999; 96, 742-747 and Soriano, et al., J Biol. Chem, 1999; 274, 32295- 32300), the results of Verdier et al, may relate more specifically to Aβ's possible functions in synaptic function or synaptic vesicle endocytosis.

[0014] Verdier et al. indicate that some form of phosphodiesterase 3 or CNPase interacts with Aβ. However, in this purified system, Aβ had only a minor effect on PDE activity, and co- precipitation experiments from brain cytosol or detergent extracts showed little evidence of a direct interaction. Therefore, it is possible that the interaction between Aβ and PDE is indirect. Phorbol ester-induced phosphorylation of PDE 3A promotes binding to 14-3-3 (Rubio, P. Biochem J, 2005; 392, 163- 172). Phosphorylation of PDE 3B by A-kinase also promotes binding to 14-3-3 (Palmer, et al., J Biol Chem., 2007; 282, 941 1-9419). Thus, the interaction of Aβ with phosphodiesterase and CNPase may be mediated by 14-3-3. Further investigation of the interactions between Aβ and 14-3-3 may shed additional light on the possible involvement of PDE and CNPase.

[0015] Aβ interacts with proteins involved in inflammation. C-reactive protein (CRP), a member of the pentraxin family, is an acute-phase protein normally found in plasma. However, CRP immunoreactivity is also detectable in temporal cortex of AD patients (Wood, et al., Brain Res., 1993; 629, 245-252), more specifically in neurofibrillary tangles (Duong, et al., Brain Res, 1997; 749, 152-156 and McGeer, et al., Neurobiol Aging, 2001 ; 22,843-848). CRP mRNA is also detectable in pyramidal neurons, indicating that it is synthesized in the brain, and CRP is upregulated in AD (Yasojima, et al., Brain Res, 2000; 887, 80-89). [0016] Patients with the pathogenic apolipoprotein APOE4 allele have lower levels of C- reactive protein than normal patients (Haan, et al., Neurobiol Aging, 2007; May 29, 0-0). The question of whether inflammation in AD is a pathogenic event, a response to neurodegeneration, or possibly even a beneficial response, has not been resolved. However, the possibility of new functions for CRP also cannot be ruled out. Other pentraxins, such as the related protein neuronal pentraxin 1 , are involved in synaptic remodeling. Pentraxin 1 can mediate neuronal apoptosis following the loss of neuronal activity (DeGregorio-Rocasolano, et al., J Biol Chem, 2001 ; 276, 796-803). Pentraxin I is increased in dystrophic neurites in patients with sporadic late-onset AD (Abad, et al., J Neurosci., 2006; 26, 12735-12747).

[0017] Aβ also binds to the ER-Golgi intermediate compartment (ERGIC) marker protein

ERGIC-53, which is involved in the calcium-dependent transport of glycoproteins such as APP from the ER to the Golgi intermediate compartment (Itin, et al., MoI Biol Cell, 1996; 7, 483-493), where presenilin is located (Culvenor, et al., J Neurosci Res, 1997; 49, 719-731). Thus, ERGIC- 53, like sortilin, may serve a transport function. Nicastrin, a glycoprotein component of the γ- secretase complex, interacts with ERGIC-53 (Morais, et al., Biochem Biophys Acta, 2006; 1762, 802-810). However, a role of ERGIC-53 in AD has not been established. ERG(C2 (originally named PTX I) is a similar transport protein originally found in prostate (Kwok et al., DNA Cell Biol, 2001 ; 20, 349-357) and may also serve to transport APP or Aβ.

[0018] Apolipoprotein B. Apolipoprotein B (ApoB), like ApoE, is a component of low- density lipoproteins, and is a suspected factor in atherosclerosis. ApoE4 is a genetic predisposing factor in AD. Serum ApoB levels are increased in AD (Caramelli, et al., Acta Neural Scand., 1999; 100, 61 -63 and Kuo, et al., Biochem Biophys Res Commun, 1998; 252, 71 1 -715) and although predominantly a serum protein, ApoB is also found in hippocampus, where it is associated with hippocampal amyloid deposits and neurofibrillary tangles (Namba, et al., Neurosci Lett, 1993; 134, 264-266). Overexpression of ApoB in mice increases APP expression in mice fed a high-cholesterol diet (Bjelik, et al., Neurochem. Int, 2006; 49, 393-400). Apolipoproteins function to transport cholesterol to the cell. The association of Aβ with ApoB, ApoE, steroid reductase, and sortilin, all of which are involved in some way with low-density lipoproteins and cholesterol transport or metabolism, is further evidence of involvement of Aβ with cholesterol uptake or metabolism. There is considerable evidence for a role of cholesterol or its metabolites in AD (Evans, et al., Neurology, 2004; 62, 1869-1871 ; Puglielli, et al., Nat. Neurosci., 2003; 6, 345-351.; Sparks, et al., Proc Natl Acad Sci USA, 2003; 100, 1 1065- 1 1069.; Heverin, et al., J Lipid Res, 2004; 45, 186- 193.; Nelson, et al., J. Biol. Chem., 2005; 280, 7377- 7387.; and Brown, et al., J Biol Chem, 2004; 279, 34674-34681).

[0019] P2X2. Purinergic receptors are upregulated in AD. Recent studies have shown that caffeine and adenosine receptor antagonists prevent Aβ-induced cognitive deficits in mice (Dall'Igna, et al., Exp Neural, 2007; 203, 241 -245) and reduces Aβ production (Arendash, et al., Neuroscience, 2006; 142, 941 -952). P2X2 has been shown to interact with Fe65 (Masin, et al., J Biol Chem, 2006; 281 , 4100-4108), an adaptor protein for APP that associates with tau in vivo (Barbato, et al., Neurobiol Dis, 2005; 18, 399-408).

SUMMARY OF THE INVENTION

[0020] The present invention provides isolated peptides having amino acid sequences

VSVGMLWC (SEQ ID NO: 4); SVLDRQRC (SEQ ID NO: 5); LGSYKPSC (SEQ ID NO: 6); NDRGLLAC (SEQ ID NO: 11); and YQDSAKTC (SEQ ID NO: 10).

[0021] The present invention also provides nucleic acids, both RNA and DNA, encoding the peptides of SEQ ID NO: 4, 5, 6, 10 and 1 1. The present invention also discloses cloning and expression vectors comprising the nucleic acids of the present invention operably linked to a promoter. The present invention also provides host cells, including prokaryotic and eukaryotic cells, such as nonpathogenic yeast, transformed with the vectors of the present invention. The present invention also provides methods of producing the peptides of SEQ ID NO: 4, 5, 6, 10 and 11 comprising the steps of culturing the transformed host cells and isolating the expressed peptides.

[0022] Further provided are pharmaceutical compositions comprising an isolated peptides having amino acid sequences VSVGMLWC (SEQ ID NO: 4); SVLDRQRC (SEQ ID NO: 5); LGSYKPSC (SEQ ID NO: 6); NDRGLLAC (SEQ ID NO: 1 1); and YQDSAKTC (SEQ ID NO: 10) in a pharmaceutically acceptable carrier.

[00231 Antibodies to the foregoing peptides also are provided (AbI), along with anti-idiotypic antibodies or fragments thereof derived from the anti-peptide antibodies (Ab2). [0024] The present invention also provides a method of reducing Aβ (39-42)-mediated apoptosis in a neuronal cell, which method comprises contacting the neuronal cell with an effective amount of at least one of the following peptides: VSVGMLWC (SEQ ID NO: 4); SVLDRQRC (SEQ ID NO: 5); LGSYKPSC (SEQ ID NO: 6); NDRGLLAC (SEQ ID NO: 1 1); or YQDSAKTC (SEQ ID NO: 10), as compared to untreated neuronal cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025 J Figure IA-B. Fig. 1 depicts results of co-precipitation experiments of Aβ 1-42 and Aβ- binding proteins in rabbit brain extracts. Fig. IA shows co-precipitation with C-reactive pre-cursor protein; GDNF receptor; steroid 5α-reductase; 14-3-3ε; and 14-3-3γ. Fig. IB shows co-precipitation with ERGlC-52; ERGIC-2; sortilin; P2XP and ApoB.

[0026] Figure 2. Fig. 2 depicts Aβ-binding proteins identified in mice by results of mass spectrometry. A= pyruvate kinase, B= 14-3-3ε, C= 14-3-3η, D= peroxiredoxin I, E= neurofilament triplet L protein, F= tubulin, G= phosphodiesterase (CNPase), H= spectrin, 1= C-reactive protein precursor, J= C-reactive protein precursor, K= pyruvate carboxylase, L= syntaxin-binding protein, M= tubulin βl , N= phosphodiesterase (CNPase), O= 14-3-3 ζ / δ, P= histone H4.

[0027] Figure 3A-C. Fig. 3A-C shows results of peptides derived from Aβ-binding proteins on the activity of purified phosphodiesterase 3B or phosphodiesterase 3B purified from rabbit brain homogenates. Fig. 3A shows total phosphodiesterase activity. Fig. 3B shows the effect on purified phosphodiesterase 3A. Fig. 3C shows effect on purified phosphodiesterase 3B.

[0028] Figure 4A-C. Fig. 4 shows the effects of peptides derived from Aβ 1-42 on apoptosis of cultured neuronal cells. Fig. 4A shows the effects of peptides on pBad. Fig. 4B shows the effects on caspase. Fig. 4C shows the effects on β-galactosidase, an indicator of cell senescence.

[0029] Figure 5. Fig. 5 depicts the regions of Aβ 1-42 which bind to the Aβ-binding proteins and peptides derived from such proteins. DETAILED DESCRIPTION

[0030] To further elucidate the biochemical pathways in which Aβ participates, phage display screenings, co-precipitation experiments, and mass spectrometry were performed to identify the binding partners of Aβ in normal rabbit brain. The ability of peptides based on these binding partners to block the toxic effects of Aβ also was examined. Aβ was found to interact with apolipoprotein B and the C- reactive protein precursor, the cholesterol transporter proteins sortilin, ERGIC2, and ERGIC53, and the regulatory proteins 14-3-3 epsilon and 14-3-3 gamma. These peptides bound to the hydrophobic region (residues 17-21) or to the nearby PKC pseudo-phosphorylation site (residues 26-30) of Aβ, regions involved in Aβ's effector action and aggregation. As described in more detail in the Examples, peptides based on these binding regions were effective blockers of Aβ toxicity.

[0031] The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them.

[0032] As used herein, the term "isolated" means that the referenced material is removed from the environment in which it is normally found. Thus, an isolated biological material can be free of cellular components, i.e., components of the cells in which the material is found or produced. In the case of peptides molecules, an isolated peptide is free from other proteins or nucleic acids, or both, in which it is comprised or with which it associates in the cell. An isolated material may be, but need not be, purified.

[0033] The term "purified" as used herein refers to material, such as a peptide of the present invention, that has been isolated under conditions that reduce or eliminate unrelated materials, i.e., contaminants. For example, a purified peptide is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell. As used herein, the term "substantially free" is used operationally, in the context of analytical testing of the material. Preferably, purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by conventional means, e.g., chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.

[0034] As used here in "β-amyloidoses" or a "disorder associated with Aβ" refers to amyloid diseases which involve the formation, deposition, accumulation and/or persistence of Aβ (i.e., beta- amyloid protein), including but not limited to Aβ containing 39-43 amino acids in length, but more preferably, Aβ 1-40, or Aβ 1-42, and mixtures or fragments thereof. Such disorders include but are not limited to but are not limited to Alzheimer's disease, Down's syndrome, forms of familial amyloidosis, cerebrovascular amyloidosis and cerebral hemorrhage; cystatin C amyloid angiopathy; hereditary cerebral hemorrhage with amyloidosis (Dutch type); hereditary cerebral hemorrhage with amyloidosis (Icelandic type); and inclusion body myositis.

[0035] The terms "patient" or "patient population" or "individual in need thereof refer to individual(s) diagnosed as having a β-amyloidosis, or at risk of developing a β-amyloidosis, such as but not limited to AD or Down Syndrome. Methods of and criteria for diagnosis of such disorders is described further below.

[0036] The terms "therapeutically effective dose" and "effective amount" refer to an amount sufficient to elicit a therapeutic response. A therapeutic response may be any response that a user (e.g., a clinician) will recognize as an effective response to the therapy. Thus, a therapeutic response will generally be an amelioration or inhibition of one or more symptoms of a disease or disorder. As one example, a therapeutic response to AD may include amelioration of the foregoing symptoms and surrogate clinical markers of β-amyloidoses, such as inhibition of neuronal apoptosis or reduction in Aβ deposits or aggregates. Exemplary methods for evaluating a therapeutic response also are described further below.

[0037] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. [0038] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, 18th Edition, or other editions.

[0039] The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies

(mAbs), chimeric antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments, regions or derivatives thereof, provided by any known technique, such as, but not limited to enzymatic cleavage, peptide synthesis or recombinant techniques.

[0040] As used herein, the term Aβ (1-42) refers to a human Aβ polypeptide having an amino acid sequence set forth in SEQ ID NO: 1 and Figure 5.

[0041] "Function-conservative variants" are those in which a given amino acid residue in a protein has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Such changes are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide. Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70 % to 99 % as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm.

[0042] A "function-conservative variant" also includes a polypeptide which has at least at least

75%, preferably at least 85%, and even more preferably at least 90%, and which has the same or substantially similar properties or functions as the native or parent protein to which it is compared. [0043] The terms "about" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

Neuroprotective Peptides

[0044] The present invention provides novel isolated peptides that competitively bind to Aβ, thereby blocking pathological Aβ effector functions. Also provided are antibodies raised against such peptides, and anti-idiotypic antibodies raised against the anti-peptide antibodies. Methods of using the peptides and anti-idiotypic antibodies for the treatment or prevention of β-amyloidoses associated with Aβ also are provided.

[0045] One embodiment of the invention provides novel peptides which bind to amino acid residues 17-21 of human Aβ (LVFFA; SEQ ID NO: 2).

[0046| In another embodiment, provided are novel peptides which bind to amino acid residues

26-30 of human Aβ (SNKGA; SEQ ID NO: 3). This region is the PKC pseudo-phosphorylation site.

[0047] In one specific embodiment, the peptide comprises the sequence VSVGMLWC (SEQ

ID NO: 4).

[0048] In a second specific embodiment, the peptide comprises the sequence SVLDRQRC

(SEQ ID NO: 5).

[0049] In a third specific embodiment, the peptide comprises the sequence LGSYKPSC (SEQ

ID NO: 6). [0050] In addition to the foregoing, provided are peptides having conservative amino acid substitutions which do not adversely impact binding of the peptides to Aβ (but which may increase the binding affinity of the peptides to Aβ). Such peptides are referred to as "function-conserved variants."

[0051] In one specific embodiment, provided are function-conserved variants of the peptides.

In a preferred embodiment, the peptides contain no more than two amino acid substitutions with function-conserved variants, preferably no more than one function-conserved variant.

[0052] As one example, the amino acids of peptide VSVGMLWC (SEQ ID NO: 4) can have the following amino acid function-conserved substitutions: i) valine is replaced with other aliphatic, hydrophobic amino acids such as isoleucine or leucine; ii) serine is replaced with other polar, small amino acids such as threonine; iii) glycine is preferably not replaced; iv) methionine is replaced with other hydrophobic amino acids such as leucine, isoleucine or valine; v) leucine is replaced with other aliphatic hydrophobic amino acids such as methionine, isoleucine, or valine; vi) tryptophan is replaced with other aromatic, hydrophobic amino acids such as tyrosine or phenylalanine; and vii) cysteine is replaced with small amino acids but is preferably not replaced.

[0053] As another example, the amino acids of peptide SVLDRQRC (SEQ ID NO: 5) can have the following amino acid function-conserved substitutions: i) serine is replaced with other polar, small amino acids such as threonine; ii) valine is replaced with other aliphatic, hydrophobic amino acids such as isoleucine or leucine; iii) leucine is replaced with other aliphatic hydrophobic amino acids such as methionine, isoleucine, or valine; iv) aspartic acid is replaced with other negatively charged, polar amino acids such as glutamic acid or asparagine; v) arginine is replaced with other positively charged, polar amino acids such as lysine or glutamine; vi) glutamine is replaced with other polar amino acids such as glutamic acid; and vii) cysteine is preferably not substituted.

[0054] As a third example, the amino acids of peptide LGSYKPSC (SEQ ID NO: 6) can have the following amino acid function-conserved substitutions: i) leucine is replaced with other aliphatic hydrophobic amino acids such as methionine, isoleucine, or valine; ii) glycine is preferably not substituted; iii) serine is replaced with other polar, small amino acids such as threonine; iv) tyrosine is replaced with other aromatic, more hydrophobic amino acids such as phenylalanine or tryptophan, or sometimes histidine; v) lysine is replaced with other positively charged amino acids such as arginine, glutamic acid, or glutamine; vi) proline is preferably not substituted; vii) serine is replaced with other polar, small amino acids such as threonine; and viii) cysteine is preferably not substituted.

[0055] The peptides of the present invention may contain chemical modifications to increase physical or chemical stability ex vivo during synthesis or in formulations or in vivo following administration to an individual. Such modifications include capping and pegylation. In one embodiment, the peptides are modified, or "capped," at the N or C termini. Exemplary modifications include acetylation at the N terminus (N-acetylation) and amidation at the C terminus (C-amidation). Such modifications protect the peptide from degradation and make the peptide more closely mimic the charge state of the alpha amino and carboxyl groups in the native protein.

[0056] Other modifications include the conjugation of polymers to peptides to improve their stability, solubility, and circulating half-life, and protect peptides from degradation. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine. The N-terminal amino group and the C-terminal carboxylic acid can also be used. Exemplary polymers include linear or branched polyethylene glycols (PEG) and derivatives and other polyvinylalcohols (PVA). Polymer conjugation techniques, commonly referred to a PEGylation, are well known in the art (Harris and Chess, Nature, 2003; 2: 214-221).

[0057] Polysialylation, namely, conjugation of peptides and proteins to the naturally occurring, biodegradable alpha-(2— >8) linked polysialic acid has recently been developed as an alternative to PEGylation (Gregoriadis et al, Int J Pharm., 2005;300(l-2): 125-30). Still other modifications include replacing some "L" amino acids with "D" amino acids.

Antibodies

[0058] Also provided are antibodies which specifically bind to the peptides of the present invention. In on embodiment, the anti-peptide antibodies are monoclonal (mAb). The mAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature 256:495-497 (1975); U.S. Pat. No. 4,376, 1 10: Ausubel et al, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1987, 1992); and Harlow and Lane ANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Laboratory (1988); Colligan et al, eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N. Y., (1992, 1993), the contents of which references are incoporated entirely herein by reference. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. A hybridoma producing a mAb of the present invention may be cultivated in vitro, in situ or in vivo.

[0059] Further provided are anti-idiotypic antibodies to the anti-peptide antibodies described above. Hypervariable regions of an antibody (AbI) can themselves act as antigens. The antibodies produced in this way are known as anti-idiotype antibodies (Ab2) since they bind to the idiotypic region (binding site) of the first antibody. Such anti-idiotypic antibodies therefore represent an "internal image" of the original antigen, i.e., a structural mimic of the antigen to which the first antibody (AbI) was raised. Thus, anti-idiotypic antibodies raised against antibodies which specifically bind to the Aβ-derived peptides of the present invention will structurally mimic the peptides. Accordingly, such anti-idiotypic antibodies and fragments thereof are contemplated for use in the method of the present invention since they will prevent Aβ binding to its effector proteins, thereby inhibiting pathologic Aβ-mediated effects.

[0060] The anti-idiotypic antibodies also may be chimeric. Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having variable region derived from a murine mAb and a human immunoglobulin constant region (Fc), which are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (Cabilly et al, Proc. Natl. Acad. Sci. USA. 1984; 81 :3273-3277). Chimeric antibodies containing a human Fc region are referred to as "humanized" antibodies.

[0061] In another embodiment, to mitigate the production of an untoward anaphylactic response in patients, provided are antibody fragments derived from naturally-occurring anti-idiotypic antibodies. Such fragments will comprise either both heavy and light chains (e.g., Fab fragments), or single heavy or light chains (e.g., light chain dimers), together with their constant region component stretches. In a further embodiment, the antibody fragment may be a single-chain variable region (scFv), which is a fusion of the variable regions of the heavy and light chains of immunoglobulin, linked together with a short (usually serine, glycine) linker. [0062] The Fab or scFv antibody fragments can be generated using phage display. In this way, antibody fragments that are entirely human in origin can be obtained. According to the phage display technique, gene segments encoding the antigen-binding variable domains of antibodies are fused to genes encoding the coat protein of a bacteriophage. Bacteriophage containing such gene fusions are used to infect bacteria, and the resulting phage particles have coats that express the antibody-like fusion protein, with the antigen-binding domain displayed on the outside of the bacteriophage.

[0063] A collection of recombinant phage, each displaying a different antigen-binding domain on its surface, is known as a phage display library. Phage expressing antigen-binding domains specific for a particular antigen can be isolated by selecting the phage in the library for binding to that antigen. The phage particles that bind are recovered and used to infect fresh bacteria. Each phage isolated in this way produces a monoclonal antigen-binding particle analogous to a monoclonal antibody. The genes encoding the antigen-binding site, which are unique to each phage, can then be recovered from the phage DNA. When the genes encoding antibody fragment are introduced into a suitable host cell line, such as bacteria, the transfected cells can secrete antibody fragments.

Combination Therapy

[0064] The peptides and antibodies described herein can be used in a method to reduce Aβ (39-

42) mediated apoptosis in a neuronal cell, comprising the step of contacting the neuronal cell with an effective amount of a composition comprising a combination of one or more of the peptides or antibodies of the present invention and a therapeutic agent selected from the group consisting of Cognex^® (tacrine), Aricept^® (donepezil), Exelon^® (rivastigmine), Reminyl^® (galantamine), and NMDA receptor antagonists such as Namenda^® (memantine). Other suitable therapeutic agents include protease inhibitors (see e.g., U.S. Patent Nos. 5,863,902; 5,872,101 , each incorporated herein by reference in their entireties); inhibitors of Aβ production (see e.g., U.S. Patent Nos. 7,01 1,901 ; 6,495,540; 6,610,734; 6,632,812; 6,713,476; and 6,737,420, each incorporated herein by reference in their entireties); modulators of Aβ (see e.g., U.S. Patent Nos: 6,303,567; 6,689,752, each incorporated by reference in their entireties); and inhibitors of β-secretase (see e.g., U.S. Patent Nos. 6,982,264; 7,034,182; 7,030,239, each incorporated by reference in their entireties).

[0065] All references, patents and printed publications mentioned in the present application are hereby incorporated by reference in their entirety into this application. [0066] The following Examples serve to illustrate further the present invention and are not to be construed as limiting the scope of the invention in any way.

EXAMPLES EXAMPLE 1: Screening for Aβ-Interacting Target Proteins

[0067] Phage display screenings were performed to identify the proteins that interact with Aβ, using Aβ 1-42, Aβ 25-35, Aβ 12-28, and Aβ 1-20. To account for the possibility that the binding site of the peptides might be hindered by binding of the peptide to the polystyrene plate, a solution screening with all four peptides was also performed. BLAST searching and co-immunoprecipitation studies were then performed to identify the proteins to which the Aβ peptides bound.

Materials and Methods

[0068] Phage display screening. Phage display screening was performed using a Ph.D. 7 peptide display system (New England Biolabs). Aβ 1-42, Aβ 1-20, Aβ 25-35, or Aβ 12-28 (37.5/cg) were coated onto an Immulon 4 HBX flat-bottom microtiter plate (Thermo Labsystems, Waltham, MA) and panned with 4 μl phage display peptide library according to the manufacturer's instructions. Bound proteins were eluted with 40 μl 0.2 M glycine pH 2.2, followed by 20 μl Tris-HCl, pH 9 and 20 μl Tris base. Four rounds of panning were performed on the plate.

[0069] Because phage display screening using low MW peptides can be inefficient, we also performed a solution panning using mouse anti-Aβ 18-30 antibody (Calbiochem 171587), which was determined to give the broadest reactivity for all forms of Aβ of the four antibodies we tested. Phage were purified using Ultralink Protein A Agarose alternating with Ultralink Protein G Agarose to avoid purifying phage that had affinity for protein A or protein G. Three rounds of solution panning were performed.

[0070] One vial of Pro-Ject protein transduction reagent (Pierce) was then dissolved in 250 μl

CHCl₃. Four μl of Pro-Ject solution were transferred to 1.5-ml polypropylene centrifuge tubes and evaporated with nitrogen. Peptide was diluted in PBS to 9.1 μg/ml and 0.22 μg of peptide was added. The vial was incubated for 5 min, then vortexed, and the volume was brought to 0.5 ml with serum-free DMEM. [0071] Solution screening. The peptides were allowed to interact in solution with phage and then recovered with a specific Aβ antibody, and the phage was isolated using alternating pannings with protein A-agarose and protein G-agarose.

[0072] Co-precipitation. One rabbit brain was homogenized by sonication in 3 vol. 10 mM

Tris-HCl pH 7.4 containing 50 mM NaF and 1 mM PMSF. The homogenate was centrifuged for 20 min at 100,000 x g. The supernatant was then re-centrifuged to produce the cytosolic fraction, and the original pellet was sonicated in the original volume of 10% N-lauroyl sarcosine or 10% CHAPS and centrifuged for 20 min at 100,000 x g. The detergent extracts and cytosol were divided into 4-ml aliquots and incubated in 17 x 100 mm polypropylene tubes (Falcon 2059) with 10 μg biotin-LC-13-β- amyloid 1-42 (Anaspec) for 1 h at room temperature. Avidin-agarose (50 μl) was added and the incubation continued on an orbital shaker (800 rpm) for an additional 60 min. The mixture was cooled on ice, transferred to a Bio-Rad Poly-Prep column, and rapidly washed with 2 ml ice-cold 10 mM Tris- HCl + 100 mM NaCl using pressure. The co-precipitated proteins were eluted with 2 ml 4M NaCl and 2 ml 0.2M glycine-HCl pH 2.2. The eluted fractions were concentrated and desalted in Centricon-3 ultrafiltration units, then mixed with SDS sample buffer for Western blotting. The non-eluted proteins were eluted from the Agarose beads by boiling with SOS sample buffer. Samples were applied to a 4- 20% SDS polyacrylamide gel and stained with Coomassie blue.

[0073] Co-precipitation using protein A-Agarose. Rabbit brain or N-lauroyl sarcosine extracts

(3 ml) were incubated with 1 μg Aβ 1-42 for 1 1 1 at room temperature. Anti-Aβ antibody (Calbiochem 171604, 9.09 /eg) was added. After 45 min of shaking, 30 μg protein A-agarose was added and the samples were incubated with shaking for 30 min at room temperature. Samples were then transferred to disposable plastic columns and rapidly washed with 1 ml of 10 mM Tris-HCl (pH 7.4) containing 150 mM NaCl and 1 mM PMSF. The agarose beads were transferred to a centrifuge tube, proteins were eluted with SDS sample buffer, and the proteins were identified by electrophoresis and Western blotting.

[0074) Western blotting. Protein was measured using the Bradford dye-binding technique

(Bradford, M. M. Anal Biochem., 1976; 72, 248-254). Samples were boiled in SDS sample buffer and loaded on a 4-20% polyacrylamide gel. Identical amounts of protein were applied to each gel. The samples were subjected to SDS polyacrylamide gel electrophoresis, nitrocellulose blotting, and antibody staining using commercial antibodies and alkaline phosphatase conjugated secondary antibody as described previously (Nelson, et al., Hippocampus., 2004; 14, 46-57).

[0075] Mass spectrometry. One rabbit brain was sonicated in 3 volumes of buffer (10 mM

Tris-HCI pH 7.4, 50 mM NaF, I mM PMSF, 0.3 mM leupeptin, 0.125 mM pepstatin, and 5% CHAPS) and centrifuged at 100,000 x g. The supernatant was aliquoted into four 17 x 100 mm Falcon 2059 tubes and incubated for 1 h at room temperature in the presence of 10 μl CuC12 or 1 μM EDTA, with 100 pg fresh biotin-LC-β-amyloid 1-42 or biotin-LC-β-amyloid 1-42 that had been preincubated at 37° for 4 days. After incubation, 0.5 ml of avidin-Agarose was added and incubation continued with shaking for 30 min. The sample was cooled to 4°, transferred to a disposable column, and rapidly washed with 4 ml ice-cold homogenization buffer. The proteins with affinity for Aβ were then sequentially eluted with 2 μl of 0.5M NaCl, 1.5M NaCl, 4M NaCl, IM Tris-HCI pH 9, or 0.2M glycine HCl pH 2.2. All buffers contained 0.1% CHAPS + 1 mM PMSF. All NaCl solutions also contained 10 mM Tris-HCI pH 7.4. The eluates were concentrated and desalted in Centricon-3 ultrafiltration units. The proteins were then separated by SDS-polyacrylamide gel electrophoresis followed by Coomassie blue staining.

[0076] After excision, Coomassie-stained protein hands were destained by washing with acetonitrile/acetic acid/water (1 : 1 :1), reduced, alkylated, and digested with 120 ng trypsin using the in- gel method (Rosenfeld, et al., Anal Biochem., 1992; 203, 173-179 and Hellman, et al., Anal Biochem., 1995; 224, 451-455). Digestion with trypsin was carried out overnight at 37° C. Peptides were twice extracted from the gel into 50% acetonitrile/5% formic acid. The extracts were pooled, the volume reduced by vacuum centrifugation, and the final volume was brought up to 6 microliters with 25 mM ammonium bicarbonate. The peptides from the tryptic digests were analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Liquid chromatography was performed using an LC Packings UltiMate NanoLC at 250 nl/min using a PepMap CI8 reverse-phase IOOA pore column. Peptides were separated using a linear gradient from 95%A+567013 to 5%A+95%B (A=H20 + 0.1% formic acid; B=80% ACN + 0.1% formic acid) over 38 min, followed by 95% B for 17 min. The LC effluent was electrosprayed directly into the sampling orifice of an LTQ mass spectrometer (Thermo Finnigan) using a nanospray interface. The LTQ was operated to collect MS/MS spectra in a data dependent manner, with up to five of the most intense ions that exceeded a pre-set threshold being subjected to fragmentation and analysis. The MS/MS data generated were analyzed and matches to protein sequences in the NCBI non-redundant nr database (human/trypsin subset) were determined using SEQUEST (Eng, et al., J. Amer. Soc. Mass Spec. 1994; 5, 976-989) program.

[0077] Sequence identification was based on the cross-correlation normalized for peptide length (XConl) and delta correlation ΔCn) scores. SEQUEST-derived peptide identifications and protein identifications were evaluated for statistical significance and filtered with the Peptide Prophet and Protein Prophet software tools (Nesvizhskii, et al., Anal. Chem., 2003; 75, 4646-4658). In each case, the predicted Mr and pi of the identification matched the Mr and pi values on the gel within ± 5%.

Results

[0078] Phage display. A total of 160 clones were sequenced, yielding 61 different peptides.

The peptides are shown in Table 1. Although an unambiguous identification cannot be made from a seven-amino acid sequence, in most cases a mammalian-constrained BLAST search identified only a single protein, or in some cases two candidate proteins, as having a close match (Table 1). The peptides found by phage display contained sequences found in phosphodiesterase 3A and 3B, and a number of proteins involved in cholesterol transport and metabolism (sortilin and steroid 5α- reductase type 2). The strongest binding (17 clones) was to a peptide found in the P2X2 purinergic receptor and the protein Suppressor of Ty 3 homolog (SUPT3H). (Yu, et al , Genomics, 1998; 53, 90-96).

Table 1. Aβ-binding peptides identified by phage display. Only peptides found in two or more clones are shown.

For peptides for which BLAST search yielded two or more results, the ambiguities were resolved by co- precipitation studies and the correct protein is listed first.

[0079] Co-precipitation. To confirm the interactions between Aβ and the candidate proteins, and to resolve the ambiguities in the phage display results, co-precipitation studies were performed. Rabbit brain cytosol and brain N-lauroyl sarcosine extract or CHAPS extract were incubated with biotin-LC-β-amyloid 1-42. Aβ-binding proteins were collected using avidin-agarose and analyzed by Western blotting. Since the protein interactions of some candidate proteins (such as 14-3-3) depend on Ca2+, the co-precipitation experiments were carried out in the presence and absence of added calcium. Since copper also binds to Aβ (Atwood, et al., J Neurochem, 2000; 75, 1219-1233 and Atwood, et al., J Biol. Chem., 1998; 273, 12817-12826), copper (10 μM) was also added to some samples.

[0080] These co-precipitation experiments confirmed that Aβ interacts with the following proteins in rabbit brain extracts: C-reactive protein precursor, 14-3-3ε, 14-3-3γ, GDNF receptor, ERGIC-53, P2X2, ERGIC2, apolipoprotein B, and sortilin (Fig. 1, Tables 2 and 3). No co- precipitation could be found for phosphodiesterase 3A, phosphodiesterase 3B, CNPase, tubulin, SorLa, KIDINS-220, SUPT3H, or CD34. The interaction between Aβ and 14-3-3 and ERGIC-53 was calcium-dependent. The co-precipitation signal with C-reactive protein precursor was very weak, because of the low levels of expression of C-reactive protein precursor in brain. Aβ co-precipitated with the P2X2 purinergic receptor but not with CD34 or SUPT3H, indicating that peptide YQDSAKT (SEQ ID NO: 7) represented P2X2. Similarly, co-precipitation experiments indicated that the peptide SVLDRQR (SEQ ID NO: 8) corresponded to sortilin, and not KIDINS-220, because Aβ co- precipitated only with sortilin (Fig. 1); however, the sortilin band was relatively weak, suggesting low levels of sortilin in the rabbit brain. No co-precipitation was observed with a SorLA antibody. Interestingly, co-precipitation was observed for both ERGIC2 and ERGIC-53 (Fig. T), despite the fact that a peptide matching LGSYKPS (SEQ ID NO: 9) is only found in ERGIC2.

[0081] The inability to detect tubulin by co-precipitation in either cytosol or N-lauroyl sarcosine extract, despite the strong signal observed by mass spectrometry, is not surprising since binding of Aβ may be specific to particular oligomeric or fibrillar forms of Aβ. Tubulin binding to Aβ (Verdier, et al., J Neurochem, 2005; 94, 617-628) and carboxyl-terminal fragments of Aβ precursor protein (Islam, et al., Am J Pathol, 1997; 151 , 265-271) has been observed by previous researchers. Antibodies were not available for tumor differentially expressed protein or TMS membrane protein; thus it was not possible to determine which of these two proteins bound Aβ. The proteins that were confirmed by co-precipitation to interact with Aβ are summarized in Table 2.

Table 2. Aβ-binding proteins identified by co-precipitation

[0082] Mass spectrometry. To identify Aβ-interacting proteins by mass spectrometry, biotin-

LC-β-amyloid 1-42 was incubated with CHAPS extract from rabbit or mouse brain, collected with avidin-agarose, washed, and eluted with increasing salt and buffer concentrations. In one experiment, fresh and oligomerized biotin-LC-β-amyloid were also compared. Since Cu2+ has been shown to bind to 4 (Atwood, et al., J Neurochem, 2000; 75, 1219-1233 and Atwood, et al., J Biol. Chem., 1998; 273, 12817-12826), 10 μM CuC12 was also added to some samples. Rabbit brain was used because the sequence of rabbit Aβ is identical to that of humans. The proteins were analyzed by SDS polyacrylamide gel electrophoresis, eluted from the gel and identified by liquid chromatography / ion trap tandem mass spectrometry. The proteins are shown in Table 3.

Table 3: Aβ -binding peptides identified by mass spectrometry

Fractions not listed did not contain any identifiable proteins. Pyruvate carboxylase is a biotin-binding protein and was not investigated further. [0083] The predominant Aβ-binding proteins found by mass spectrometry were

14-3-3 and 2',3'-cyclic nucleotide 2'3'-cyclic nucleotide 3 '-phosphodiesterase (CNPase). Similar patterns were observed for rabbit and mouse, although more mouse proteins than rabbit proteins were identified, presumably because the rabbit nr database is relatively incomplete. Addition of Ca2+ had no effect on the binding of Aβ for any of the proteins (Fig. 2). No differences were observed between fresh and oligomerized biotin-Aβ (not shown). Oligomerization of Aβ is essential for toxicity, however, oligomerization of Aβ 1-42 occurs rapidly (within minutes) in solution (El-Agnaf, et al., Biochem. Biophys Res Commun., 2000; 273,1003-1007); thus, the fresh biotin-Aβ used in these experiments may also have been partially oligomerized.

EXAMPLE 2: Evaluation of Target Peptides on Neuroprotection

[0084] Synthetic peptides. Aβ-binding peptides were synthesized by Genscript

(Piscataway, NJ). All peptides were dissolved in phosphate-buffered saline (PBS) at a concentration of I mM, except for peptide #3 (VSVGML WC-SEQ ID NO: 4), which was dissolved at 0.1 mM. Peptides were then sterilized by filtration before use. For cell culture experiments, one vial of Pro-Ject protein transduction reagent (Pierce) was then dissolved in 250 μl CHCl₃. Four μl of Pro-Ject solution were transferred to 1.5-ml polypropylene centrifuge tubes and evaporated with nitrogen. Peptide was diluted in PBS to 9.1 μg/ml and 0.22 μl of peptide was added. The vial was incubated for 5 min, then vortexed, and the volume was brought to 0.5 mi with serum-free DMEM.

[0085] β-amyloid oligomerization. Human Aβ (Anaspec 20276, 1 mg) was dissolved in 3 ml deionized water by the addition of a minimum volume of 1% ammonium hydroxide and was incubated for 3 days at 37°. The solution was then reduced in volume to 0.886 ml by lyophilization and incubated for an additional 48h at 37°. The oligomerized Aβ was then stored at -80°.

[0086] Cells and cell culture. Rat hippocampal H 19-7/IGF-IR cells (ATCC,

Manassas, VA) were plated onto poly-L-lysine coated plates and grown at 35° in DMEM/10%FCS for several days until coverage was obtained. The cells were then induced to differentiate into a neuronal phenotype by replacing the medium with 5 ml N2 medium containing 10 ng/ml basic fibroblast growth factor at 39° C and grown in T-75 flasks at 37° C.

[0087] For neuroprotection experiments, differentiated cells were grown in 12- well plates containing 1 ml N2 culture medium. When the cells reached 75-80% confluence, they were washed with serum-free DMEM and peptide/Pro-Ject mixture dissolved in 1/2 ml serum-free DMEM was added. After 4h incubation at 37°C, 1/2 ml of DMEM containing 20% fetal bovine serum was added. After 18h, oligomerized Aβ was added. Cells were monitored daily. After 7d, the medium was removed and the cells were washed twice with 1 x PBS. A 100 μl aliquot of PBS was added and the cells were removed by gentle scraping. The cells were then homogenized by sonication and stored at -80°.

[0088] β-Galactosidase. Cell senescence was tested using the hydrolysis by β- galactosidase at pH 6 of 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal), a commonly-used β-galactosidase substrate. Under these conditions, β-galactosidase is easily detectable in senescent cells, but undetectable in quiescent, immortal, or tumor cells (Dimri, et al., Proc Natl Acad Sci USA, 1995; 92, 9363-9367). To measure β- galactosidase, cell homogenate (20 μl) was incubated in 100 id of 0.5M Tris-HCl, pH 6.8 containing 0.1 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-(β-D-galactopyranoside). After 24h at 37°C, the samples were diluted to 1 ml and absorbance at 610 nm was measured.

[0089] pBad. Bad is a member of the Bcl-2 family, and regulates the survival signal (of Bc 12, P. and regulation of apoptosis, Leukemia, 2001 ; 15, 515-522). Unphosphorylated Bad dimerizes with Bcl-2 and BcI-XL, which neutralizes their anti- apoptotic activity. Activation of the phosphoinositol 3-kinase pathway ultimately leads to activation of Akt, which phosphorylates Bad on serine 136. Activation of MAP kinase pathways results in phosphorylation of Bad on serine 1 12. Phosphorylated Bad is sequestered from its proapoptotic role by binding with 14-3-3 protein (Masters, et al., Biochem Soc Trans., 2002; 30, 360-36516). Thus, a decrease in Bad phosphorylation indicates apoptosis. pBad was measured by densitometry of Western blots stained with phospho-Bad antibody. Image quantitation and molecular weight estimation were done on 16-bit images using the Unix-based image analysis program imal (http://brneurosci.org/imal.html). The background value for each band was calculated by fuzzy k-means clustering analysis of the appropriate region of the image (Kaufman, et al., Finding groups in data: an introduction to cluster analysis, Wiley-Interscience, New York, (1990); Bezdek, J. C, Pattern recognition with fuzzy objective function algorithms, Plenum, New York, (1981); Bezdek, J. C, J. Cybern., 1971 ; 3, 58; and Dunn, J. C, J. Cybern., 1974; 3, 32). Densitometry results are expressed as units of relative staining.

[0090] Caspase 3 assay. Members of the caspase family of cysteine aspartyl proteases are related to the C. elegans CED-3 death protein. Caspases 8, 9, and 10 are activated by receptor clustering and are known as "initiator caspases" (Riedl, et al., Nat Rev MoI Cell Biol, 2004; 5, 897-907). Caspases 3, 6, and 8 are activated by changes in mitochondrial permeability that are associated with apoptosis, and are known as "effector caspases". Effector caspase 3 proteolyzes a number of substrates, including DFF45/ICAD, PARP, gelsolin, and nuclear lamins 1221. Proteolysis of DFF45/ICAD liberates the DNAse subunit of DFF to cause chromatin degradation (Wolf, et al., J Biol Chem., 1999; 274, 30651-30656). Proteolysis of PARP has been used by many researchers as a marker for apoptosis. Caspase 3 activity was measured fluorometrically using the commercially-available substrate Ac-Asp-Glu-Val-Asp-NH2-methylcoumarin, which contains the PARP cleavage site (Fernandes-Alnemri, et al., Cancer Res., 1995; 55, 6045-6052). Hydrolysis of this peptide yields a species that fluoresces at 440-460 nm.

[0091] To measure caspase 3, samples (20 μl) were incubated with 20 Al Caspase

Buffer (40 mM Tris-HCI pH 7.4, 150 mM NaCl, 5 raM EDTA, 0.2% CHAPS, and 15% sucrose) containing 0.3% dithiothreitol and 0.54 μg AcDEVD-AMC peptide in 1.5-ml polypropylene centrifuge tubes. The samples were incubated for 1 hr at 37° C diluted to 1 ml, and fluorescence was measured (ex=354 nm, em=442 nm) in a Spex Fluorolog 2 spectrofluorometer. [0092] Phosphodiesterase assay. Phosphodiesterase was measured by the method of Alvarez and Daniels (Alvarez, et al., Anal Biochem., 1992; 203, 76-82) with slight modifications. Fines were removed from acidic alumina (Brockmann grade 1) by resuspending several times in water. Bio-Rad Poly-Prep columns were packed with a slurry containing 1.3 g alumina to a height of 3 cm. Columns were precycled with 0.1 M ammonium acetate to remove any cAMP, then equilibrated with water. In an 0.5-ml polypropylene centrifuge tube, 0.15 μl rat brain homogenate was diluted to 100 μl with water and 10 μl buffer (I M Tris-HCl pH 7.5 2 mM MgSO4) and 0.5 μl 3H-cAMP were added. After 10 min incubation at 30° C, the samples were boiled and cooled and 0.1 Al 5'-nucleotidase (Biomol, 500 kU/ml). The samples were incubated for 30 min at 30° C and applied to the column. The adenosine was eluted with 4 ml 5 mM HCl, mixed with scintillation fluid, and counted in a scintillation counter.

Results

[0093] Effects on phosphodiesterase activity. Since both mass spectrometry and phage display both implicated some form of phosphodiesterase, we measured the effects of Aβ on cGMP-sensitive PDE 3A and 3B. Aβ had no effect on commercially-available purified phosphodiesterase 3A but produced a slight inhibition of phosphodiesterase activity in rabbit brain homogenate. Aβ peptides also produced a slight (about 20%) inhibition of purified PDE 3B (Fig. 3). Aβ had no effect on the inhibition of PDE 3 A or PDE 3B by cGMP (not shown).

[0094] Protection against β-amyloid. To test whether the Aβ-binding peptides could protect against the toxic effects of Aβ, we applied eight synthetic peptides mixed with Pro-Ject, a cationic lipid protein transduction reagent, to cultured H19-7/IGF-IR cells. These cells are hippocampal neurons from R. norvegicus that have been immortalized by retroviral transduction of temperature sensitive tsA58 SV40 large T antigen (Eves, et al., Proc Natl Acad Sci U S A., 1992; 89,4373-4377). After 18h, oligomerized Aβ 1-42 was applied to the cells. The cells were collected after 7 days in culture and analyzed for markers of apoptosis (caspase 3 and pBad) and senescence (β- galactosidase). The peptide sequences are shown in Table 4. Table 4: Sequences of Aβ-binding peptides tested in cultured cells for their ability to protect against Aβ. The binding region on A13 found by phage display is also shown. The probable identity of each peptide was determined by BLAST, using co-precipitation studies to discriminate between high-scoring proteins.

[0095] Phosphorylated Bad was significantly decreased in H19-7/IGF-IR cells after Aβ 1 -42 treatment (Fig. 4A), indicating early stages of apoptosis. Peptides #3 (VSVGMLWC; SEQ ID NO. 4), representing steroid 5-α reductase) and #4 (SVLDRQRC; SEQ ID NO: 5), representing sortilin-related receptor) prevented the decrease, with #3 almost completely blocking the decrease. Similarly, peptides #3, #4, and #5 (LGSYKPSC; SEQ ID NO: 6), representing ERGIC2) also prevented the increase in caspase activity produced by Aβ 1-42 treatment (Fig. 4B), with peptides #3 and #5 being equally potent at blocking caspase activation. Peptides #4 and #5 also reduced the increase in β-galactosidase produced by Aβ 1 -42 treatment (Fig. 4C), indicating that they were partially protective against Aβ-induced senescence. In contrast, peptides #1 (YQDSAKTC; SEQ ID NO: 10, P2X2 purinergic receptor) and #2 (NDRGLLAC; SEQ ID NO: 1 1, TMS Membrane protein) had either no effect or a slight worsening effect. The three peptides with the strongest beneficial effect (#3, #4, and #5) corresponded to sortilin, steroid-5α reductase, and ERGIC2, Aβ-interacting proteins that are involved in cholesterol, LDL, or ER membrane transport.

[0096] Based on the overlapping peptides used in the phage display experiment, the binding region for phosphodiesterase and steroid reductase can be narrowed down to amino acids 12-20 of Aβ (VHHQKLVFF; SEQ ID NO: 28). Likewise the binding sites for sortilin and ApoB are within the region of amino acids 25-25, and the binding region for P2X2 is within the region of amino acids 12-28 (Fig. 5). This region includes a PKC pseudo-phosphorylation site (amino acids 26- 30, SNKGA; SEQ ID NO: 3). Although no phosphorylation of this site by PKC has been reported, it binds PKC and is critical for the inhibition of PKC by low micromolar concentrations of Aβ 1-42 and Aβ 26-36 (Lee, et al., MoI Cell Neurosci, 2004; 26, 222-231). This region also contains a hydrophobic region or "patch" (LVFFA; SEQ ID NO: 2), which may participate in binding of cholesterol, facilitating contact with cholesterol-metabolizing proteins such as steroid reductase and LDL-associated proteins such as sortilin. Aβ 1-42 competitively inhibits cholesterol binding to ApoE and low-density lipoprotein (Yao, et al., FASEB J., 2002; 16, 1677-1679).

[0097] The preceeding Examples identified that Aβ peptide binds to 14-3-3,

ERGIC-53, ERGIC2, sortilin, P2X2, ApoB, steroid reductase, and the C-reactive protein precursor. The interactions with 14-3-3 and sortilin are of particular relevance to Alzheimer's Disease. Sortilin expression has been shown to correlate inversely with AD neuropathology. Inherited variants in the sortilin SORL l (LR II) lipoprotein receptor, which mediates a variety of cellular sorting and trafficking functions, are genetically associated with late-onset AD (Rogaeva, et al. , Nat Genet., 2000; 39,68-77).

[0098] Research with a similar goal was carried out by Blanchard et al.,

(Blanchard, et al., Brain Res., 1997; 776, 40-50 and Blanchard, et al., J Alzheimers Dis., 2000; 2, 137-149), who used a combinatorial library of hexapeptides containing random combinations of Ala, He, VaI, Ser, Thr, and GIy, selected on the basis of their ability to complex with Aβ, to identify potential therapeutic peptides. Additional peptides were engineered by adding prolines and N- or C-terminal modifications. These peptides were intended to act as "decoy peptides" that would mix with endogenous Aβ and prevent the formation of fibrils.

[0099] A BLAST search was performed using the longer peptides reported in their papers. Although their peptides contained a restricted set of common amino acids, resulting in a large number of possible matches, two matches were of potential interest: DP(25) (TPIRTPAPA; SEQ ID NO: 31), which matched radical fringe homolog protein (a Golgi protein involved in signaling of the γ-secretase substrate Notch), and DP3 (TVIGTIGGG; SEQ ID NO: 32), which showed some similarity to protocadherin. However, none of the peptides in either Blanchard et al., or Orner et al., matched any proteins found in the present study. This was not unexpected, considering that Blanchard et al., used rational design rather than library screening to create their peptides. A similar approach was taken by Chalifour et al., who used rational peptide design to create inhibitors of Aβ fibrillogenesis using D-amino acids (Chalifour, et al., J Biol Chem, 2003; 278, 34874-34881).

Claims

WHAT IS CLAIMED:

1. An isolated peptide consisting essentially of the amino acid sequence VSVGMLWC (SEQ ID NO: 4).

2. An isolated peptide consisting essentially of the amino acid sequence SVLDRQRC (SEQ ID NO: 5).

3. An isolated peptide consisting essentially of the amino acid sequence LGSYKPSC (SEQ ID NO: 6).

4. An isolated peptide consisting essentially of the amino acid sequence NDRGLLAC (SEQ ID NO: 1 1).

5. An isolated peptide consisting essentially of the amino acid sequence YQDSAKTC (SEQ ID NO: 10).

6. A pharmaceutical composition comprising an isolated, purified peptide having an amino acid sequence VSVGMLWC (SEQ ID NO: 4), or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.

7. A pharmaceutical composition comprising an isolated, purified peptide having an amino acid sequence SVLDRQRC (SEQ ID NO: 5), or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.

8. A pharmaceutical composition comprising an isolated, purified peptide having an amino acid sequence LGSYKPSC (SEQ ID NO: 6), or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.

9. A pharmaceutical composition comprising an isolated, purified peptide having an amino acid sequence NDRGLLAC (SEQ ID NO: 1 1), or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.

10. A pharmaceutical composition comprising an isolated, purified peptide having an amino acid sequence YQDSAKTC (SEQ ID NO: 10), or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.

1 1. An antibody which specifically binds to a peptide having the amino acid sequence VSVGMLWC (SEQ ID NO: 4).

12. An antibody which specifically binds to a peptide having the amino acid sequence SVLDRQRC (SEQ ID NO: 5).

13. An antibody which specifically binds to a peptide having the amino acid sequence LGSYKPSC (SEQ ID NO: 6).

14. An antibody which specifically binds to a peptide having the amino acid sequence NDRGLLAC (SEQ ID NO: 1 1).

15. An antibody which specifically binds to a peptide having the amino acid sequence YQDSAKTC (SEQ ID NO: 10).

16. A method of preventing Aβ (39-42)-mediated apoptosis in a neuronal cell, which method comprises contacting the neuronal cell with an effective amount of a peptide selected from the group consisting of amino acid sequences VSVGMLWC (SEQ ID NO: 4); SVLDRQRC (SEQ ID NO: 5); and LGSYKPSC (SEQ ID NO: 6).

17. The method of claim 16, wherein the peptide is VSVGMLWC (SEQ ID NO: 4).

18. The method of claim 16, wherein the peptide is SVLDRQRC (SEQ ID NO: 5).

19. The method of claim 16, wherein the peptide is LGSYKPSC (SEQ ID NO: 6).

20. The method of claim 16, wherein the peptide binds to Aβ.