AU2002303174A1 - Cytotoxic peptides and peptidomimetics based thereon, and methods for use thereof - Google Patents

Description

CYTOTOXIC PEPTIDES AND PEPTIDOMIMETICS BASED THEREON, AND METHODS FOR USE THEREOF

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 60/280,615, filed March 30, 2001, and U.S. Provisional AppUcation No. 60/281,050, filed April 2, 2000, the contents of both of which are hereby incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to cytotoxic peptides, and the use thereof for developing agents which block undesired apoptosis, and the like. In particular, the present invention relates to methods for using peptides and peptidoinimetics based thereon to induce apoptosis, or to prevent and/ or inhibit undesired apoptosis. In yet another aspect, the present invention relates to methods for identifying and/ or developing agents which induce and/ or inhibit apoptosis.

BACKGROUND OF THE INVENTION

[0003] Cell death in the central nervous system (CNS) occurs extensively in development, during normal aging and in some pathological states associated with degeneration of specific subsets of neurons. The majority of cell deaths in the developing nervous system occur by the activation of programmed cell death, and neural death in at least some disease states may involve components of the apoptotic pathway (Bredesen, Ann. Neurol, 38:839-851 (1995); Sperandio et al., Proc. Natl Acad. Sci, USA 97:14376-14381 (2000); Yuan and Yankner, Nature 407:802-809 (2000). Elucidating the molecular mechanisms that initiate and control pathological cell death in the CNS should help in the development of interventions that may prevent or ameliorate degenerative CNS diseases. [0004] The loss of hippocampal neurons is one of tl e prominent features of Alzheimer's disease (AD). The pathological hallmark of AD is the formation of senile plaques and neuiOfibrillary tangles in brain which is accompanied by substantial neuronal and synaptic loss in the neocortex. β-Amyloid precursor protein (APP) is a ubiquitously expressed membrane-spanning glycoprote n that is cleaved during its normal metabolism to generate the amyloid-β protein (Aβ), a 40 to 42 amino acid peptide that is the main constituent of senile plaques. The deposition of Aβ may account for the enhanced susceptibility of hippocampal and cortical neurons to premature death, since exposure of cultured human neuronal and non-neuronal cells to amyloidogenic Aβ peptide induces the activation of apoptotic cell death pathways (Cotman, Soc.for Neuroscience Satellite Symposium on Neural Apoptosis (1994); Cotman and Anderson, Mol. Neurobiol. 10:19-45 (1995); La Ferla et al., Nature Genet 9:21-30 (1995)).

[0005] In addition to tlie cleavages that result in the formation of Aβ, APP can be cleaved at its C-terminus by caspases, a family of cysteine proteases central to the execution of apoptosis (Lyckman et al., /. Biol. Client. 273:11100-11106 (1998); Gervais et al„ Cell 97:395- 406 (1999); LeBlanc et al., /. Biol. Chan. 274:23426-23436 (1999); Pellegrini et al., /. Biol Oiem. 274:21011-21016 (1999)). In addition, it is possible that this C-terminal caspase cleavage, generating a 31 amino acid fragment (C31), precedes and may favor tlie intramembrane cleaλ'age that leads to the generation of Aβ.

[0006] The formation of Aβ and its subsequent deposition in senile plaques are viewed by many as tl e initiating events that lead to tlie cascade of pathological changes resulting in AD (Selkoe, Trends Cell Biol 8:447-453 (1998)). Aβ is derived from APP by two ox more proteolytic events mediated by β- and δ-secretase activities, and has been shown to be neurotoxic, with pro-apoptotic effects (Cotman, Neurobiol Aging 18:S29-S32 (1998); La Ferla et al, supra; Yankner, Neuron 16:921-932 (1996)). However, whether Aβ cytotoxicity occurs in vivo has not been determined. Indeed, Aβ is not likely to be the only cause of synapse loss and neuronal loss in AD, and may not even prove to be the main cause; several other factors have been proposed as mediators of AD pathogenesis, including oxidative damage, inflammation, mitochondrial dysfunction and apoUpoprotein E, among others. Not only is the cause of the neuronal and syiiaptic loss incompletely understood, but also tl e mode of ceU death that occurs in AD is controversial. Apoptosis has been reported in the brains of patients with AD (Cotman (1998), supra), but this does not seem to be a general process. Thus, both tlie mechanisms and ceUular pathways responsible for neuronal death in AD are still poorly defined.

[0007] Accordingly, there remains a need in the art for compositions and methods to control apoptosis, in particular for application in Alzheimer's disease. The present invention fulfills this need and further provides related advantages.

SUMMARY OF THE INVENTION

[0008] In accordance with one aspect of the present invention, there are provided peptide compositions or peptidomimetics thereof, wherein the peptide is a potent inducer of apoptosis. In specific embodiments, the peptide is derived from β-amyloid precursor protein (APP), APP-like protein 1 (APLP1), or APP-like protein 2 (APLP2).

[0009] In accordance with another aspect of tlie present invention, there are provided methods for inducing apoptosis in a target cell using an effective amount of a peptide or peptidomimetic thereof that is a potent inducer of apoptosis. In a preferred embodiment, tlie target cell is a neural cell, such as a neuron or gUal cell.

[0010] In accordance with yet another aspect of tlie present invention, there are provided methods for reducing or inhibiting apoptosis of cells containing β-amyloid precursor protein (APP) or an APP-like protein by blocking cleavage of the precursor that releases a C-terminal peptide fragment. In specific embodiments of such methods, apoptosis is reduced or inhibited in neural cells, such as neurons or gUal cells. In preferred embodiments, cleavage is blocked by smaU molecule compounds such as peptides, antisense peptides, peptidomimetics, antibodies, antagonists, antisense nucleic acids, and tlie Uke.

[0011] In accordance with another aspect of tlie present invention, there are provided methods for reducing or inhibiting apoptosis of cells containing β-amyloid precursor protein (APP) or an APP-like protein by inactivating the C-terminal peptide fragment formed by cleavage of the precursor protein as it is formed, ϊn specific embodiments of such methods, apoptosis is reduced or inhibited in neural cells, such as neurons or gUal ceUs. In preferred embodiments, the peptide fragment is inactivated by degrading the peptide into inactive fragment(s) or by combining the peptide with a chelator, such as an antibody.

[0012] In accordance with still another aspect of the present invention, there are provided methods of treating a subject in need thereof, comprising administering a therapeutically effective amount of a molecule capable of blocking the cleavage of APP or an APP-Uke protein or capable of inactivating the C-terminal peptide fragment generated by cleavage of the precursor protein. In a preferred embodiment, the subject in need thereof has Alzhekner's disease.

[0013] In accordance with another aspect of the present invention, there are provided methods of identifying s aU molecules that will block cleavage of APP or an APP-like protein, comprising determining which smaU molecules will compete for specific binding to the APP or APP-like protein.

BRIEF DESCRIPTION OF THE FIGURES

[0014] Figure 1 collectively illustrates APP interaction with and cleavage by caspases in cultured cells. In Figure la, APP (a type-1 integral membrane glycoprotein) is illustrated, as are fragments produced by caspase cleavage in the intracellular region of APP; and the antibodies used herein. Cleavage of APP at the caspase consensus site, VEVD/ A, after the aspartic acid, is predicted to yield an N-terminal protein of 664 amino acids (APPΔC31) and a C-terminal peptide of 31 amino acids (C31), which contains the APP intemaUzation signal NPTY (SEQ ID NO:5). 5A3 and 1G7 are monoclonal antibodies against tlie same extracellular region of APP (mixed together to detect tlie full-length APP and APPΔC31); CT15 is a polyclonal rabbit antibody against the C-terminal 15 amino acids of APP; 2όD6 is a monoclonal antibody against and α-1 is a polyclonal antibody against APP amino acids 649-664.

[0015] Figure lb shows that APP interacts with caspases. APP was co- im unoprecipitated with caspases-6, -7, -8 and -9 from 293T cells co-transfected with APP and the respective caspases tested. Catalytic mutant caspases with the active site cysteine mutated to alanine were used so that co-immunoprecipitations could be done without ceU death induction. Monoclonal anti-FLAG M2 was used for the co- immuiioprecipitation of FLAG-tagged caspases. Western blot analysis used monoclonal antibody (5A3/1G7) for APP. Lane 5 shows that caspase-8 does not interact with APPΔC31. Lane 7 shows cells transfected with APP and immunoprecipitated and probed with monoclonal antibody 5A3/1G7 as a positive control. Quantitative densitometry analysis showed that C8 had an intensity about 200% of that of the other caspases tested (Co, 1.2; C7, 1.0; Cδ, 2.2; C9, 1.1).

[0016] Figure lc shows that APP is cleaved in 293T ceUs co-expressing APP and caspase-8. CeU lysate samples were immimodepleted with CT15 (Immunodep CT), then immunoprecipitated with either the mixture of monoclonal mouse antibodies 5A3 and 1G7 (MAb) or CT15 (CT). After immunodepletion, a principal C-terminal truncated species is evident (lane 1); immunodepletion removes most of the fuU-length APP species (lane 2). The faint bands migrating at a higher molecular weight (lanes 1 and 3) represent endogenous APP751 present in 293T cells.

[0017] Figure 2 coUectively iUustrates the results of ceU death and viability assays in cultured cells expressing various APP and C-terminal fragment (CTF) constructs. Figure 2a shows ceU death in N2a ceUs transfected with various constructs. Expression of C31 increases ceU death compared with control (P < 0.001 by one-way ANOVA (P < 0.0001; F = 44.838), post-hoc Tukey-Kramer). Transfection of ceUs with APP (P < 0.001) or V642F (P < 0.001) also causes significant ceU death compared with control.

[0018] Figure 2b shows ceU death in 293T ceUs when various constructs co-expressed with caspase-7 or -8. Caspase-8 is significantly more toxic when co-expressed with APP in 293T ceUs than caspase-8 or APP expressed alone (P < 0.001 by two-way ANOVA (P < 0.00001; F = 186.9), post-hoc Tukey HSD).

[0019] Figure 2c shows tlie viabUity of 293T cells in which apoptosis was induced with tamoxifen in the presence of various constructs. C100 causes more ceU death than APP (P <0.001, one-way ANOVA (P <0.0001; F = 157.58), post-hoc Tukey-Kramer) but sUghtly less ceU death than C31 (P < 0.05). C100-D664A aboUshes aU of the cytotoxic effects of C100, compared with mock transfection with pcDNA3 (P > 0.05). [0020] Figure 3 illustrates in vivo caspase-9 activation in AD and control brains. Crude synaptosomal preparations were immunoprecipitated with a polyclonal antibody against caspase-9, followed by western blot analysis with an activation-specific antibody against caspase-9. Lane 1 shows Hela ceUs transfected with caspase-9 and treated with the pan-caspase inhibitor zVAD.fmk. Lane 2 shows caspase-9 transfected staurosporine- treated Hela cells. Lane 3 shows active recombinant caspase-9. In all five AD patients and one neurologicaHy affected non-AD control patient (with normal-pressure hydrocephalus and dementia) there are activated caspase-9 plO fragments(*).

[0021] Figure 4 demonstrates APP C31 toxicity to both neurons and gUal cells in primary hippocampal cultures. Cultures were treated with various concentrations of the penetration peptide conjugated to C31 (DP-APPC31) or 10 μM of DP, immunostained 24 hours later with antibodies specific for the neuronal marker NeuN or the gUal marker GFAP. Relative area values for NeuN and GFAP immunoreactivity were obtained by image analysis using the Simple PCI software (Compix, Inc., Philadelphia). The broad spectrum caspase inhibitor BAF blocked tlie toxicity of the DP-APPC31 conjugate at 10 μM.

[0022] Figure 5 demonstrates that transduction of APP C31 induces overaU ceU death in hippocampal cultures. Primary hippocampal cultures were transduced with the penetratin peptide (deUvery peptide DP) or the DP-APPC31 conjugate at various concentiations and then assayed for viability 36 hours later by the MTT assay.

[0023] Figure 6 coUectively shows APP C31 induced cell death in primary hippocampal culture. Figure 6a measures viabiUty of the cultures by the trypan blue exclusion method thirty hours after transduction with the peptides. Figure 6b measures condensed, fragmented nuclei by staining cultures with 0.1 mg/ml Hoechst 3334230 hours after transduction with the peptides.

[0024] Figure 7 coUectively shows that the C-terminal cleavage product of the APP homolog, APLP1, induces death in primary hippocampal cultures, in primarUy neurons and not gUal ceUs. Figure 7a shows cultures treated with increasing concentrations of DP-APLP1C31 peptide in the presence or in tlie absence of the caspase inhibitor, BAF. Twenty-four hours later, the cultures were immxinostamed with antibodies specific for NeuN (light) and GFAP (dark). Figure 7b shows primary hippocampal cultures transduced with the indicated concentrations of DP-APLP1C31 or DP alone. Thirty hours later, the viabiUty of the cultures was determined by the trypan blue exclusion method.

DETAILED DESCRIPTION OF THE INVENTION

[0025] In accordance with tlie present invention, it has been discovered that, in addition to Aβ, APP gives rise to a second cytotoxic, proteolyticaUy derived fragment unrelated to Aβ. Furtiiermore, the toxicity of the APP C-terminal fragment, caUed CIOO, is attributable to this pro-apoptotic APP fragment. The mechanism of toxicity appears to be similar to that used by a class of cell death receptors caUed dependence receptors (Bredesen et al., Cell Death Diff. 5:365-371 (1998)); this class includes the common neuiOtrophin receptor p75^NTR, the netrin-1 receptor DCC (deleted in colorectal cancer), and the androgen receptor (Bredesen et al., supra; Mehlen et al., Nature 395:801-804 (1998); Rabizadeh et al., Science 261:345-348 (1993)). This second cytotoxic APP fragment is derived by caspase cleavage of APP at Asp664, mainly by caspase-8 and caspase-9, to generate a C-terminal peptide, called C31, comprising the C-terminal 31 amino acids of APP. C31 is potently pro-apoptotic by a mechanism that involves caspase amplification similar to that induced by DCC (Mehlen et al., supra). The presence of both caspase- cleaved APP fragments and activated caspase-9 species in brains of AD patients indicates that this process occurs in vivo. Thus, this cell death pathway mediated by C31 is also involved in physiological ceU death.

[0026] Also provided herein is evidence that the 31 amino acid peptide released by caspase cleavage of the APP C-terrninus is toxic in neuronal primary culture. These data support the notion that tlie release of the C31 peptide causes neuronal death in AD and plays a pathogenic role in the neurotoxicity associated with AD.

[0027] In accordance with another aspect of the invention, it has also been found that the two APP homologs, APLP1 and APLP2, can also be cleaved by caspases in vitro (the caspase recognition sequences at their C- termini are 100% conserved) and a synthetic peptide of APLP1-C31 deUvered into primary cultures is preferentially toxic to neurons as compared to gUal cells. The LD50 for neurons is around 3 micromolar, compared with an LD50 of 35-40 micromolar for glial ceUs.

[0028] Accordingly, the present invention provides peptides having tlie sequence of the C-terminal peptide of cleaved APP (AAVTPEERHLSKMQQNGYENPTYKFFEQM QN; SEQ ID NO:l) or a peptide having at least 80% sequence identity therewith; tlie sequence of the C-terminal peptide of cleaved APLP1 (PMLTLEEQQLRELQRHGYENP TYRFLEERP; SEQ ID NO:2) or a peptide having at least 80% sequence identity therewith; the sequence of the C-terminal peptide of cleaved APLP2 (PMLTPEERHLNK MQNHGYENPTYKYLEQMQI; SEQ ID NO:3) or a peptide having at least 80% sequence identity therewith, or a peptidomimetic of any of the above peptides, wherein said peptide or peptidomimetic is a potent inducer of apoptosis. Preferably an invention peptide has at least 90% sequence identity with SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3. An invention peptide may also have an amino acid sequence that differs from SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3 by conservative substitutions of one or more residues thereof.

[0029] The term "peptidomimetic" as used herein refers to a non-peptide smaU molecule that exhibits structural similarity to a peptide and has peptide-like properties. Examples include traditional peptides that contain non-amino acid moieties, or alternative linkages. The term "identity" refers to the exact same sequence of amino acids, while the term "sirrύlarity" aUows for conservative amino acid substitutions, for example, a non-polar amino acid substituted for another non-polar amino acid, or a charged for a charged, etc.

[0030] The caspase-generated fragment that comprises the C-terminal 31 amino acids of APP (C31) is a c3^totoxic peptide that sensitizes ceUs to other stressful stimuU in a concentration-dependent manner. The present invention also provides evidence for cleavage of APP at the intraceUular caspase site, D664, in the brains of patients with AD but not control patients (see Figure 3). Taken together, these data strongly suggest that the cleavage of the C-terminal portion of APP plays an important role in the neural toxicity observed in AD pathogenesis, both by increasing the production of the toxic Aβ peptide and by generating a pro-apoptotic C-terminal fragment. Activation of caspases as a result of stress such as that induced by the accumulation of Aβ at neuronal terminals is, therefore, seen to provide the trigger for a 'spiral' of toxicity in which APP is cleaved at its C-terminus and generates an additional toxic fragment. Consistent with this mechanism, mice expressing an APP tiansgene carrying two point mutations linked to autosomal forms of familial AD develop neurological symptoms and extensive neuronal death in the absence of significant Aβ accumulation or amyloid plaque formation (Mucke et al., /. Neurosci. 20:4050-4058 (2000)).

[0031] Accordingly, the present invention provides methods of reducing/ inhibiting apoptosis of a cell containing β-amyloid protein precursor (APP) or an APP-like protein, said method comprising blocking cleavage that releases a C-terminal peptide fragment. In certain embodiments, cleavage is blocked by smaU molecule compounds such as peptides, antisense peptides, peptidomimetics, antibodies, antagonists, antisense nucleic acids, and the like, m preferred embodiments the ceU is a neural cell, such as a neuron or a glial ceU.

[0032] In an alternative embodiment, the invention provides methods of reducmg/ inhibiting apoptosis of a cell containing β-amyloid protein precursor (APP) or an APP-like protein, said method comprising inactivating the C-terminal peptide fragment as it is formed. The peptide fragment is inactivated by degrading the peptide into inactive fragment(s) thereof, or by combining the peptide fragment with a chelator therefor, such as an antibody. In preferred embodiments the ceU is a neural ceU, such as a neuron or a gUal cell.

[0033] APLP1 and APLP2 are members of the APP family of proteins, collectively "APP-like proteins". However, the sites required for γ and β-secretase cleavage of APP are not conserved in either APLP1 or APLP2. These molecules therefore do not have the capacity to generate β-amyloid-Uke peptides. However, the C-terminal caspase cleavage site that aUows for the generation of APP C31 is conserved in both APLP1 and APLP2. For APLP1, the P4-P1' positions would be VEVDP, and for APLP2, the P4-P1' positions would be VEVDP while in APP, the P4-P1' positions are VEVDA. These sequences, like those in APP, fit weU with previously described caspase cleavage sites for the initiator/ apical caspases such as caspase-8 and caspase-9. The predicted APLP1-C31 peptide is 52% identical and 77% sirmlar and the predicted APLP2-C31 is 71% identical and 83% similar to the APP C31 peptide. [0034] Using an in vitro cleavage assay, it has been found that caspases-3, -6 and -8 are capable of cleaving APP, and tlie cleavage by caspase-3 is blocked by mutation of Asp664 to Glu, confirming reports of caspase cleavage at this site (Barnes et al., /. Neurosci. 18:5869-5880 (1996); Weidemann et al., /. Biol Cliem. 274:5823-5829 (1999); Gervais et al., supra; Pellegrini et al., supra; LeBlanc et al., supra). After co-transfection of APP and caspases-6, -7, -8 or -9, complexes of APP and caspases formed, as shown by co- irnmunoprecipitation (see Figure lb). Moreover, in cultured cells, APP was cleaved by both caspase-8 and caspase-9. For full-length APP, caspase cleavage would lead to two fragments: an N-terminal fragment of 664 amino acids (APPΔC31) and a C-terminal fragment (CTF) of 31 amino acids (C31). In 293T cells co-expressing APP and the respective caspase zymogens (that is 'pro-caspase', tlie relatively less-active caspase precursors), a C-terminal-deleted APP fragment consistent with APPΔC31 (see Figure lc) was detected. Expression of a mutant APP, D664A, in which the probable caspase site was mutated to Ala, inliibited die abiUty of caspase-8 to cleave APP. Identical results were obtained when caspase-9 was co-expressed with APP. These results indicate that both caspase-8 and caspase-9 are capable of cleaving APP between residues 664 and 665. In contrast, caspases-3, -6 or -7 did not result in cleavage when tested in tlie same manner.

[0035] In accordance with another aspect of the present invention, there are provided methods for inducing apoptosis in a target ceU, said methods comprising contacting target ceU with an effective amount of a peptide having the sequence of the C- terminal peptide of cleaved APP or a peptide having at least 80% sequence identity therewith, the sequence of the C-terminal peptide of cleaved APLP1 or a peptide having at least 80% sequence identity therewith, the sequence of the C-terminal peptide of cleaved APLP2 or a peptide having at least 80% sequence identity therewith, or a peptidomimetic of any of the above peptides. In preferred embodiments the target cell is a neural cell, such as a neuron or gtial ceU. An "effective amount" as used herein refers to that amount of a peptide which is capable of causing ceU death by apoptosis by any standard test as is known in the art, for example, the MTT assay as provided in the examples below.

[0036] The effects of caspase-mediated cleavage of APP on ceU death were evaluated by expressing wUd-type APP₆₉5, D664A, V642F, APPΔC31 and C31 (the predicted C- terminal APP fragment released after caspase cleavage) in 293T and N2a cells. Expression of wild-type APP and V642F had a pro-apoptotic effect (see Figures 2a and 2b) after staurosporine or tamoxifen induction, although the V642F mutant did not show a significantly greater pro-apoptotic effect than wild-type APP (see Figure 2a). Further analysis showed that expression of C31 but not APPΔC31 produced the pro-apoptotic effects after stimulation by staurosporine that may even exceed those obtained with either APP695 or the APP V642F mutation (see Figures 2a and 2c). Expression of the D664A mutation similarly led to inhibition of the proapoptotic effects (see Figures 2a and 2c).

[0037] The presence of APP potentiated apoptosis initiated by caspases. In 293T ceUs transfected with caspase-8 zymogen, ceU death was significantly greater than in basal conditions or ceUs transfected with caspase-7 (see Figure 2b). However, co- expression of caspase-8 zymogen and APP considerably increased ceU death relative to the conditions described above, indicating a synergistic effect of APP and caspase-8 on cell death (see Figure 2b). This effect was completely dependent on cleavage of APP at Asp664, and thus presumably C31, as the APP mutant D664A faUed to show the additive effects on ceU death (see Figure 2b). Thus, the generation of C31 seemed to amplify the cell death program initiated by caspase-8.

[0038] FinaUy, expression of C31 also induced apoptosis in basal conditions without further ceUular insults. In N2a neuroblastoma cells, expression of C31 alone, without tamoxifen or staurosporine, was strongly correlated with annexin V (Chan et al., /. Neurosci. Res. 57:315-323 (1999) staining (76 ± 10% of C31-positive ceUs were annexin V- positive). This immunoreactivity was indicative of apoptosis, as the ceUs were impermeant to propidium iodide. However, ceUs transfected with APP or with pcDNA3 (mock transfection) were generally negative for annexin V conjugated to fluorescein isothiocyanate (annexin V-FITC) (13 + 5% were positive for both APP and annexin V)(P < 0.001, two-tailed t-test). Treatment with zVAD.fmk decreased annexin V staining of C31 transfected ceUs to control levels. Therefore, these results show that release of a C- terminal caspase-cleaved APP fragment, presumably C31, consistently resulted in a proapoptotic phenotype in cultured ceUs. [0039] C31 can theoreticaUy be generated either from fuU-length APP or APP CTFs, tlie latter derived from α-, β- or δ-secretase cleavages of APP. β-secretase-cleaved APP, the so-caUed CIOO (or C99) CTF, is cytotoxic (Oster-Granite et al, /. Neiirosci 16:6732- 6741 (1996); Yankner et al., Science 245:417-420 (1980); Fukuchi et al, Neiirosci. Lett. 154:145-148 (1992); Sopher et al, Mol. Brain Res. 26:207-217 (1994)) and is increased in neurons expressing disease-associated APP mutations (Oster-Granite et al., supra). Here, CIOO, like APP, was cleaved by caspase-8 and caspase-9. In addition, tlie effects of wild- type and mutant CIOO constructs (D664A-C100) on ceU death were analyzed (see Figure 2c). As expected, CIOO had a pro-apoptotic effect on N2a ceUs (see Figure 2c). However, the CIOO caspase mutant D664A-C100 produced no cytotoxicity, reducing ceU death to the levels obtained with control transfection. These observations therefore indicate that the reported cytotoxic properties of CIOO may be entirely due to tlie generation and release of C31 and its subsequent amplification effect on the ceU death program.

[0040] Having estabUshed that APP can be cleaved by caspase-8 and caspase-9 in cultured cells and that ceU death is potentiated by this cleavage event, it was next sought to determine whether this process occurs in the brains of patients with AD. First, tlie pattern of these two caspases was examined by immunochemistry in mouse brain tissue. Adult mouse brain tissue was immunostained with antibodies raised against the uncleaved forms of caspase-8 and caspase-9. Immunoreactivity for these caspases was readily detectable in almost all neurons in brain, including neocortex, hippocampus and diencephalon. Although the staining was abundant in the perikarya for both caspases, proximal as well as distal neuronal processes were prominently stained by the antibody against caspase-9, indicating transport of caspase-9 to distal sites.

[0041] The studies described above showed that both APP and its CTFs derived from α- or β-secretase are substrates for caspases. Therefore, polypeptides that result from cleavage of APP CTFs (CTFΔC31) rather than from full-length APP were focused on, because these smaU, caspase-generated fragments are resolved much better by SDS- PAGE. As expected, CTFΔC31 fragments derived from caspase cleavage in cultured ceUs were detected. However, using whole-brain homogenates from mid-frontal cortex of both AD and control brain tissue, caspase-derived APP fragments were not detected. Although APP is mainly located in the intermediate compartments in cultured neurons (Caporaso et al., /. Neiirosci. 14:3122-3138 (1994)), it is nonetheless also enriched from synaptosome preparations (Marquez-Sterling et al., /. Neiirosci. 17:140-151 (1997)). Because caspase-9 is apparently distributed into neuronal processes as weU, isolation of synaptosomes may emich for caspase fragments. Indeed, in crude synaptosome samples obtained from mid-frontal regions of AD brains, multiple APP fragments were detected. The immunologic profile was such that it was possible to detect a fragment consistent with caspase cleavage of an α-secretase-derived CTF in AD brain tissue. SpecificaUy, this fragment was recognized by the antibody against intracytoplasmic (I) APP but not by the antibody against tlie APP C terminus (CT15), indicating the absence of tlie C terminus. Moreover, this fragment was present in five of five AD brains examined but was absent in all control brains. Finally, this fragment was also detected in the brain of one adult with Down syndrome.

[0042] In addition to showing the presence of caspase cleavage of APP in brain tissue, tlie present invention provides evidence of caspase activation in the same tissue. The generation of C31 and its proposed downstream cytotoxic effects should be related to caspase activation, otherwise APP would not be cleaved. Effector caspase-3 and effector caspase-6 were not able to cleave APP and are therefore unlikely to initiate the proposed C31-mediated ceU death. Thus, focus was placed on evidence of caspase-8 or caspase-9 activation in brains of AD patients.

[0043] In the crude synaptosomal preparations of AD and control brain tissues, it was not possible to detect caspase-8 by immunoblotting. This was consistent with the predominant perikaryal and sparse neuritic staining of caspase-8 in mouse brains, and therefore no further examination for caspase-8 was carried out. Caspase-9 in its full- length zymogen form was present in crude synaptosome samples, as shown by western blot analysis. Therefore, specific investigation for activated plO fragments of caspase-9 (that is the small subunit resulting from cleavage and activation of caspase-9) was then carried out using an activation-specific antibody against caspase-9, referred to as 315/316. Indeed, there was a fragment about 10 kDa in size, co-migrating with a band from staurosporine-treated Hela ceUs transfected with caspase-9 and a caspase-9 plO recombinant fragment, in the AD brain samples (see Figure 6). Moreover, this activated caspase-9 fragment was present in five of five AD brains examined. However, this activated caspase-9 fragment was not found in four of the five control brains (see Figure 6, control, 1 and 3-5). Tlie one brain with positive results was from a neurologic control subject with dementia (normal-pressure hydrocephalus) but without AD changes (see Figure 6, control, 2).

[0044] A central feature of AD pathology is the profound loss of neurons in cortex, although the mechanisms responsible for neuronal death are unclear. Given recent studies of pro-apoptotic receptors (Bredesen et al., supra; Mehlen et al., supra; Ellerby et al., /. Neurochem. 72:185-195 (1999); Rabizadeh et al., Science 261:345-348 (1993)), it was next determined whether APP is involved in physiological ceU death by using a similar proteolysis-dependent mechanism. According to the present invention, it is shown that APP is a caspase substrate; caspase cleavage of APP at Asp664 generates a cytotoxic C- terminal APP fragment; the toxicity of CIOO is dependent on caspase cleavage; in cultured cells, caspase-8 and caspase-9 were capable of cleaving APP; and both intracytoplasmic cleavage of APP (presumably caspase-mediated) and activation of caspase-9 occurs in tl e brains of AD individuals.

[0045] Consistent with recent reports (Barnes et al., supra; Weidemann et al., supra; Gervais et al., supra; PeUegrini et al., supra; LeBlanc et al., supra) APP was cleaved by caspases at Asp664 both in vitro and in cultured cells. Furthermore, catalytic mutants of caspases-ό, -7, -8 and -9 co-immunoprecipitated with APP. However, only caspase -8 and caspase-9, but not caspases-3, -6 or -7, cleaved APP when co-expressed in cultured cells. Thus, interactions of various caspases with APP did not necessarUy lead to APP proteolysis. This cleavage event produced two predicted fragments: an N-terminal fragment of 664 amino acids and a CTF of 31 amino acids. Consistent with this, an APP C-terminal-deleted fragment (APPΔC31) was present in ceUs co-expressing caspase-8 or caspase-9. Both APP and the α- and β-secretase cleaved CTFs were also substrates for caspase cleavage.

[0046] To determine tlie biological consequences of this cleavage, cell death assays were carried out in cultured cells. Expression of C31 was substantiaUy more proapoptotic (in most ceU death assays) than expression of either APP or V642F in the 293T and N2a cell lines. The smaU differences between experiments are probably due to the assay methods. Mutation of the caspase cleavage site aboUshed most of the pro- apoptotic effects of APP695 and APP with the V642F mutation (see Figure 2a). Furtiiermore, similar results were obtained when CIOO, rather than APP, was tlie (initial) substrate for caspase cleavage. Therefore, caspases may cleave either full-length APP or a C-terminal APP fragment, in both cases generating the cytotoxic C31 peptide. Thus, these findings provide evidence that the cytotoxicity of APP and its fragments, at least in cultured ceUs of a neuronal (N2a) or non-neuronal (293T) phenotype, results mostly from the generation of C31 by caspases.

[0047] In support of this, APP substantiaUy enhanced the cell death induced by expression of caspase-8, but the non-cleavable mutant, APP-D664A, showed no such enhancement. Thus, even though the expression of caspase-8 alone was pro-apoptotic, the ability to generate C31 amplified the effect of caspase-8 in inducing ceU death. The effect was completely dependent on APP cleavage by caspases. As C31 is not Ukely to be a product of constitutive APP processing, the results presented herein indicate that C31 may function by amplifying caspase activation, and thus the ceU death program. As a result, exposure to pro-apoptotic stressors such as Aβ would be more likely to lead to cell death.

[0048] The C31 cytotoxic APP fragment disclosed herein may account for the cytotoxicity of the CIOO fragment. CIOO, rather than Aβ, was tlie first cytotoxic fragment to be identified from APP (Yankner et al., supra) Expression of CIOO in cultured cells and in transgenic mice results in significant neuronal death (Oster-Granite et al., supra; Yankner et al., supra). In addition, levels of CIOO are also increased in neurons expressing various APP mutations (McPhie et al, /. Biol Cliem. 272:24743-24746 (1997)). The mechanism of CIOO cytotoxicity has been a matter of debate, but the data presented herein indicate that it is mediated through caspase cleavage of the C-terminal APP fragment and the generation of C31 (or, conceivably, by non-caspase proteolytic cleavage of APP to generate a fragment similar to C31). Thus, in addition to generating increased levels of Aβι-42, APP mutations, through increased levels of CIOO, may provide more substrate for caspase cleavage, thereby enhancing C31 production and apoptosis induction. By this proposed mechanism, this last step may be normaUy relatively quiescent, but leads to a shift in the ceUular 'apostat' (the likelihood that a ceU wUl undergo apoptosis (Salvesen and Dixit, CeU 91:443-446 (1997)) such that any cytotoxic chaUenge would be more likely to result in cell death through C31-mediated amplification of caspase. The mechanism proposed herein does not exclude Aβ toxicity, and in fact complements proposed mechanisms that include Aβ toxicity. Indeed, C31 may function in concert with Aβ to produce the neuronal loss that characterizes AD.

[0049] Accordingly, tlie invention also provides methods of treating a subject in need thereof, said methods comprising administering a therapeuticaUy effective amount of a molecule capable of blocking the cleavage of APP or an APP-like protein, or inactivating the C-terminal peptide fragment generated by cleavage of the precursor. In preferred embodiments, said subject has Alzheimer's disease.

[0050] Evidence of intracytoplasmic APP cleavage in vivo was first reported by detection of the APPΔC31 fragment in a single AD brain by inmiunostairiing with an end-specific antibody (Gervais et al., supra). Biochemical evidence is provided herein of caspase cleavage of APP in five of five AD samples but not in any of tl e control samples. The results presented herein further show that the presence of caspase-cleaved APP fragments coincided with caspase-9 activation in AD brains but not in the four neurologically unaffected control brains. One neurologicaUy affected, non-AD control brain that was also positive for caspase-9 activation but not APP cleavage was from an individual with normal-pressure hydrocephalus and dementia. Nonetheless, the presence of activated caspase-9 along with APPΔC31 fragments from the same synaptosomal preparations in aU the AD brains examined provides compelling evidence that this caspase-mediated cleavage oi APP occurs during the course of AD. The fact that the APP cleavage was detected in crude synaptosome preparations but not in whole-brain homogenates could simply be explained by the paucity of these fragments such that tlie synaptosomes represented a convenient way to emich for APP. Alternatively, the finding may indicate that caspase activation and subsequent cleavage of APP occurs mainly in neurites. The latter interpretation is attractive because it would be consistent with the neuritic and synaptic abnormalities seen in AD brains (Masliah, /. Neural Trans. 53:147-158 (1998); Yang et al., Am. }. Pathol 152:379-389 (1998); Mattson et al, Brain Res. 807:167-176 (1998)). It may also indicate that C31-mediated toxicity is one factor that contributes to synaptic degeneration.

[0051] Evidence of caspase activation in AD remains sparse and conflicting; so far, only activation of effector caspases (3 and 6) has been described (Chan et al, supra; Stadelmann et al., Am. J. Patlιoll55: 59- 66 (1999); Selznick et al., /. Neuropathol. Exp. Neurol 58:1020-1026 (1999); LeBlanc et al., supra). Caspase-6 was shown to be activated in a single AD brain that was examined but not from a single control brain (LeBlanc et al., supra). Whether this result will extend to additional AD brains after further analysis is unclear. The data presented herein on tlie presence of activated caspase-9, an initiator caspase, may be particularly relevant. Although caspase-8 and caspase-9 are able to activate effector caspases (Salvesen and Dixit, supra; Thornberry and Lazebnik, Science 281:1312-1316 (1998); Stermicke et al., /. Biol Cliem. 273:27084-27090 (1998)), it seems that tlie activation of caspase-9 is not necessarily followed by downstream caspase activation in AD. Thus, it may be that the restricted nature of caspase-9 activation (that is, in presynaptic endings) does not lead to widespread caspase activation in perikarya. Alternatively, there may be other cellular mechanisms that limit tlie generalized activation of the caspase cascade. The latter concept would be consistent with evidence that there may be compensatory mechanisms in neurons that respond to the various chronic and perhaps accumulating insults that occur during neurodegenerative disorders (Cotman (1998), supra). Thus, neuronal death in neurodegeneration may represent a form of cell death that is neither classically necrosis nor apoptosis.

[0052] According to the present invention, it is shown that the APP fragment that is generated by caspase cleavage of the APP C-terminus at Asp664 is toxic to hippocampal and cortical neurons in primary culture. This peptide, C31, is relative!)' selectively toxic for the neuronal population, with a LC50 of 1-2 μM for hippocampal neurons, 10-25 μM for astrocytes, and 50-100 μM for 293T human embryonic kidney cells. Moreover, primary cultures that have been exposed to otherwise sublethal concentrations of fibrillar Aβ demonstrate enhanced sensitivity to tlie C31 peptide, decreasing the LC₅₀ to < 500 nM.

[0053] The presence of the C31 peptide in primary neuronal cultures triggers the activation of programmed cell death, as demonstrated by the condensation and fragmentation of nuclei in transduced ceUs and by the abiUty of the general caspase inhibitor BAF to delay the death process. This finding is compatible with the earUer finding that caspase-8 and caspase-9, but not caspase-3, were required for C31-induced ceU death. The biochemical pathway(s) leading from C31 to caspases-8 and -9 and apoptosis activation is not yet known. However, it is compatible with the previous finding of caspase-9 activation in synaptosomal preparations from the brains of patients with AD, but not from control patients.

[0054] The evidence, taken as a whole, suggests that APP is cleaved both in cultured ceUs and in vivo, releasing not only the Aβ peptides, but also APP-C31, a relatively selectively neurotoxic peptide the toxicity of which is enhanced by otherwise sublethal concentrations of Aβ peptide. Thus the C31 peptide is a good candidate to play a role in the death of neurons associated with AD. It should be added that recent work from the d'Adamio Laboratory has shown that the APP-C57 peptide, which results from γ- secretase cleavage, may also be cytotoxic (Passer et al., /. Alzheimer* 's Dis. 2:289- 301 (2000)). However, it is not yet clear whether generation of C31 is required for C57 toxicity, as was previously demonstrated for CIOO. It is also not yet clear whether the toxicity of C57 is relatively selective for neurons.

[0055] In accordance with another aspect of the present invention, there are provided methods of identifying small molecules that wiU block cleavage of APP or an APP-like protein, said method comprising determining which small molecules wiU compete for specific binding to APP or an APP-Uke protein.

[0056] The C-terminal part of APP has been shown to play a critical role in both APP internalization and according to the present invention, in the induction of cell death. This C-terminal fragment of APP harbors a NPTY (SEQ ID NO:5) motif required for the endocytosis of APP and consequent Aβ formation. On the other hand, phosphotyrosine- binding (PTB) domains bind to the NPTY motifs and may play a role in protein endocytosis. For instance, the protein Fe65, containing two PTB domains, has been reported to mediate APP endocytosis. Other proteins harboring PTB domains have been described to bind the NPTY motif. That is the case of Xll, a neuron-specific protein that has been shown to bind in vivo to APP and compete for APP binding with Feό5. The overlapping of APP regions involved in the binding of Fe65 and Xll suggest the existence of competitive mechanisms regulating the binding of the various Ugands to this cytosolic domain and hence represent novel therapeutic targets. [0057] Also, in accordance with tlie present invention, it has been discovered that the

C31 peptide derived from APP binds to tlie PTB domain of Fe65. Bindmg assays can be used to confirm this observation with respect to the C31 peptide derived from APLP1.

[0058] Using X-ray crystallographic information regarding C31, the 3 dimensional conformation of this peptide and its bindmg site was determined. By screening available databases of small molecule compounds, compounds can be identified with the potential to mimic the action of C31, i.e., to induce apoptosis and also conversely to block the binding site for C31, thereby blocking its apoptotic activity. Also candidates can be found that bind directly to C31 and inhibit its activity of caspase amplification and thereby inhibit apoptosis in neuronal cells. Exemplary compounds identified by these screening methods include antibiotics and flavonoids.

[0059] Thus, in accordance with another aspect of the present invention, a rational approach can be used for the development of small molecules that wiU compete for the specific bindmg of Fe65/ APP and Xll/ APP (employing, for example, Catalyst, software from Molecular Simulations Inc.). For this purpose, the avaUable crystallographic structure of Xll/APP complex can be used and tlie Fe65/ APP interaction modeled based on the Xll/APP complex. Applying this approach, 145 potential pharmacophores have been identified from 5 databases containing more than 600,000 compounds. Four of these have a statistically significant docknig score and can be grouped in two chemicaUy distinct groups, i.e., flavonoids and antibiotics. Additional potential compounds contemplated for use in the practice of the present invention include smaU molecules such as, for example, peptides, peptidomimetics, antisense peptides, antibodies, antagonists, antisense nucleic acids, and the Uke.

[0060] The invention will now be described in detail by reference to the foUowing non-limiting examples.

EXAMPLE 1 Plasmid construction and mutagenesis

[0061] Wild-type human APP₆₉5 was subcloned into pcDNA3 (Invitrogen, Carlsbad, California). The mutation of the aspartate residue at codon 664 to glutamate (D664E) or alanine (D664A) and the familial Alzheimer disease mutation of valine to phenlyalanine at codon 642 (V642F, or V717F by APP 770 numbering) was accomplished using the QuikChange method (Stratagene, La JoUa, California). Three constructs encoding different lengtiis of the APP C terminus were made: APP-C125, APP-ClOO and APP-C31. hi APP-C125 and APP-C31, the constructs were generated by PCR from APP₆₉5 to encompass the last 125 and 31 amino-acid residues, respectively. An ATG start codon was introduced before and in-frame with residue 571 (APP-C125) or residue 665 (APP- C31). APP-ClOO comprises the signal peptide sequence of APP fused to tlie C-terminal 99 amino-acid residues beginning at tlie aspartate residue of Aβ. Three C-terminal APP deletion constructs were produced by PCR from the respective full-length cognates: APPΔC31, APP-V642F-ΔC31 and APP-C100-ΔC31. All APP expression constructs were subcloned into pcDNA3 (Invitrogen, Carlsbad, California) and verified by sequencing.

[0062] With the QuikChange Site-DUected Mutagenesis Kit (Stratagene, La JoUa, California), the foUowing catalytic mutant caspases, which disable the catalytic cysteine residue, were generated: caspase-6 (C163A), caspase-7 (C186A), caspase-8 (C360A) and caspase-9 (C287A).

EXAMPLE 2 Cell culture and antibodies

[0063] Human embryonic kidney 293T ceUs were grown and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C and 5% CO₂. 293T cells were transiently transfected with plasmids using the calcium phosphate method.

[0064] Mouse N2a neuroblastoma ceUs were grown at 37 °C and 5 % CO₂ in 45 % Dulbecco's modified Eagle's medium and 45% OptiMEM I (Life Technologies) supplemented with 10% fetal bovine serum and 2 mM glutamine. Plasmid constructs were introduced into the N2a ceUs with the LipofectAMINE plus transfection reagent (Life Technologies) according to the manufacturer's instructions.

[0065] APP antibodies included the foUowing: CT15, a polyclonal rabbit antibody recognizing the C-terminal 15 amino acids of APP (Sisodia et al., /. Neiirosci 13:3136-3142 (1993)); a mixture of two monoclonal mouse antibodies, 5A3 and 1G7, which recognize non-overlapping epitopes in tlie extracellular region of APP (Koo and Squazzo, /. Biol Chem. 269:17386-17389 (1994)); (the two monoclonal antibodies were used together to increase sensitivity); and a monoclonal antibody 26D6 recognizing tlie Aβ peptide sequence of amino acids 1-12 (provided by M. Kounnas and S. Wagner of Merck Research Labs, San Diego, California); rabbit polyclonal antiserum, α-1 (provided by D. Selkoe, Brigham and Women's Hospital, Boston Massachusetts), raised agamst a synthetic peptide of APP arniiio acids 649-664. Monoclonal ANTI-FLAG M2 was obtained from Sigma.

[0066] Rabbit antiserum Bur49, raised against human caspase-9, was generated as described (Krajewski et al, Proc. Natl. Acad. Set USA 96:5752-5757 (1999)). Rabbit antiserum 1890, raised against human caspase-8, was produced using the same methods as for Bur49. The specificity and affinity of antibodies 1890 and Bur49 to caspase-8 and caspase-9, respectively, were confirmed as described (Krajewski et al., supra). For subsequent immunohistochemistiy, Bur49 was used at a dilution of 1:25,000 and 1890, at a dilution of 1:15,000.

[0067] Polyclonal antibody against caspase-9, directed against the entire caspase-9 zymogen, was used for immunoprecipitatioii (Stennicke et al., supra; Wotf et al., Blood 94:1683-1692 (1999)). Rabbit antiserum 315/316 (Biosource, CamarUlo, California) was used for subsequent western blot analysis. This antibody is specific for the N terminus of the cleavage site 315/316 of human caspase-9 and consequently detects the plO fragment of active caspase-9. For caspase-8 immunoblotting, the monoclonal antibody B9-2 (PharMingen, San Diego, California), recognizing amino acids 335-469 of caspase-8 fragment, was used.

EXAMPLE 3 Induction of apoptosis and assessment of viability

[0068] After transfection, apoptosis was induced in 293T cells as described (Ellerby et al., supra). After incubation of 293T ceUs (plated in six-weU plates at a density of 5 x 10⁵ ceUs per weU) in the calcium-phosphate-DNA solution for 20-24 h, the apoptosis- inducing agent tamoxifen was added at a final concentiation of 50 μM. After 3 h more of incubation, ceUs undergoing cell death were quantified by the trypan blue method (EUerby et al., supra).

[0069] Apoptosis of N2a ceUs was assessed by Hoechst staining and the MTS (3-(4,5- dimethyltliiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-suUophenyl)-2H-tetrazolium, inner salt) assay according to manufacturers' instructions (Promega, Madison, Wisconsin). MTS is a ceU proliferation assay that measures the number of viable cells for mitochondria activity (dye reduction), and therefore it indirectly measures cell viabUity. Apoptosis was induced with tamoxifen using the protocol described above for 293T cells. CeU viability was calculated by normaUzing the absorbance value of each respective well to the absorbance value of the control well without transfection and expressed as percent viability of control cells.

[0070] For Hoechst staining, N2a ceUs were treated with 0.5 μM staurospor e for three hours, foUowed by a 5-minute incubation with 5 μg/ml bis-benzimide (Hoechst 33258; Molecular Probes, Eugene, Oregon) as described (Shindler et al., /. Neiirosci. 17:3112-3119 (1997)). Apoptotic cells, defined by abnormal morphology under ultraviolet visualization, were assessed in photomicrographs of transfected ceUs. Cells were counted in four random fields from each weU of cultured ceUs (about 300-500 ceUs), in tripUcate for each condition. The results are expressed as a percentage of apoptotic nuclei divided by the total number of ceUs. hi some experiments, the ceUs were co- transfected with a green fluorescent protein control vector to monitor transfection efficiency, which was typically approximately 70%. SimUar results were obtained when the results are expressed as a percentage of total ceUs or total transfected ceUs determined by the use of green fluorescent protein.

EXAMPLE 4 In vitro protein synthesis and caspase cleavage

[0071] In vitro transcription and translation used the Promega Coupled kit (Promega, Madison, Wisconsin). The constructs pcDNA3-APPC125 (C-terminal 125 amino acids of APP) and ρcDNA3-APPC125-D664E (D664E mutation in APP₆₉₅) were translated, and the protein products were used to assess caspase cleavage. Cleavage with caspases-3, -6, -7, -8, -9 and -10 was done and assessed as described (EUerby et al., supra).

EXAMPLE 5

Caspase interaction assay in cultured cells

[0072] Cells were co-transfected with catalytic mutant caspases-6, -7, -8 or -9 and APP or the deletion construct APPΔC31. CeU lysates of co-transfected 293T cells were prepared by incubation of cells for 30 min on ice, with occasional vortexing, in Nonidet- P40 lysis buffer (0.1% Nonidet-P40, 50 M HEPES, pH 7.4, 250 mM NaCl and 5 mM EDTA). For immunoprecipitation, samples were incubated for 12 h with a monoclonal ANTI-FLAG M2-Agarose affinity gel (Sigma) or with the mixture of monoclonal anitbodies 5A3 and 1G7) and Sepharose A beads to bind FLAG-tagged mutant caspases or APP, respectively. The beads were washed three times by centrifugation and resuspension in Nonidet-P40 lysis buffer and were resuspended in Laernrnli sample buffer. The immunoprecipitated proteins were resolved by 10% SDS-PAGE and were transferred to PVDF membranes for western blot analysis with monoclonal ANTI-FLAG M2 (Sigma) or monoclonal antibody against APP to detect mutant caspase or APP, respectively. The immunoblots were developed with peroxidase-conjugated secondary antibody and enhanced chem uminescence. Quantitative densitometry used NIH Image (Version 1.61).

EXAMPLE 6 Caspase cleavage of APP in cultured cells

[0073] 293T ceUs were co-transfected with constructs encoding wild-type APP or APP D664A mutant, and caspase-3, -6, -7,-B or -9 zymogens. In some experiments, 40 μM zVAD.fmk (benzoxycarbonyl-Val-Ala-Asp-CH2F) was added to the cells during cotransfection of APP and caspase-8 or caspase-9. At 24 h after transfection, the ceUs were lysed in 1% Nonidet-P40. CT15 or a mixture of two monoclonal mouse antibodies, 5A3 and 1G7, was added to the lysate along with protein A-Sepharose beads (Zymed, San Francisco, California) or antibody against mouse IgG-agarose beads (American Qualex, San Clemente, California), respectively, for overnight immunoprecipitation. The samples of protein-Ig-bead complexes were washed twice in lysis buffer, mixed 1:1 with 2x sample buffer, and boiled for 5 min. The protein samples were separated by 5% PAGE and transferred to PVDF membranes. Imniuiioblotting used the mixture of monoclonal mouse antibodies 5A3 and 1G7. Secondary antibody against mouse was used for chemiluminescence to detect bound primary antibody as described before (Koo and Squazzo, supra).

EXAMPLE 7 Irnmunocytochemistry

[0074] N2a ceUs were plated on glass cover sUps treated with polylysine and were transfected as described above. At 24 h after transfection, cells were directly stained with annexin V-FITC for apoptosis in tissue culture media, according to the manufacturer's instructions (annexin V-FITC Apoptosis Detection Kit; Calbiochem, La JoUa, California). After being stained with annexin V-FITC, ceUs were fixed for 20 min in 4% paraformaldehyde in phosphate-buffered saline and permeabUized in 0.5% Triton X- 100 in phosphate-buffered saline for 5 min. After being blocked in 5% BSA in phosphate-buffered saline, the ceUs were incubated for 1 h at room temperature with CT15, dUuted 1:2,000. In paraUel, ceUs were stained with propidium iodide after annexin V staining and paraformaldehyde fixation but without permeabiUzation to verify membrane integrity; the combination of annexin V staining and membrane integrity indicated apoptosis without secondary necrosis. The primary antibody was detected with Texas Red^®-conjugated secondary antibody against rabbit (Molecular Probes, Eugene, Oregon). Negative controls, which included preimmune serum and ceUs transfected with pcDNA3 (mock transfection), were assayed in parallel using the protocol described above. The ceUs were analyzed by confocal microscopy using a BioRad MRC-1024 system. CeUs in five random fields were counted for each sUde, in dupUcate for each condition. The number of ceUs positive and negative for APP or C31 and apoptosis (annexin V-FITC) were determined, and relative risk was calculated (RR = pi /p2) = concordant staining [(+/+) times (-/-)] divided by discordant staining [(+/-) times (-/+)].

[0075] Double-labeling of APP and annexin V in N2a ceUs after transfection was performed. N2a ceUs were transfected with either APP or C31 foUowed by staining with annexin V-FITC to visualize apoptotic ceUs. Transfected ceUs were immunolocaUzed with CT15 followed by Texas Red conjugated secondary antibody to visuaUze APP or C31 expression. When expressed in N2a ceUs, C31 but not APP is associated with apoptosis. A high proportion of C31 transfected ceUs are positive for annexin V-FITC (green outline of the cell; RR = 6.3), whereas in APP-expressing cells, annexin V-FITC staining is sparse (RR = 0.77). In control cells transfected with pcDNA3 (mock transfection; control), there is no staining with annexin V-FITC and essentiaUy no staining of endogenous APP at this antibody dilution. Images are representations of four different fields of view of each of three separate experiments.

[0076] For immunostaining of caspase-8 and caspase-9, free-floating tissue sections of mouse cerebral cortex and hippocampus were incubated with the appropriate polyclonal antibodies as described using a peroxidase system (Krajewski et al., supra). Brightfield images were obtained with Nikon Inverted E-300 Microscope.

[0077] Caspase-8 and caspase-9 are expressed in the frontal cortex and hippocampus of mice. Mouse frontal cortex and CA1 of hippocampus show neurons irnmunoreactive to antibodies against caspase-8 or caspase-9. Tlie immunoreactivity is most abundant in the neuronal perikarya with antibodies against caspase-8 or caspase-9, whereas proximal neuronal processes are also imniunostaiiied by antibody against caspase-9. Control staining with pre-immune serum showed no background staining in the cerebral cortex.

EXAMPLE 8 In vivo caspase cleavage of APP in AD and control brains

[0078] For the in vivo study, brains of AD and control patients were obtained from the Alzheimer Disease Research Center at Johns Hopkins University. Diagnosis of AD was estabUshed by both CERAD (Consortium to Establish a Registry for Alzheimer's Disease) and NINDS/ ADRDA (National Institute of Neurological Disorders and Stroke/ Alzheimer's Disease and Related Disorders Association) criteria. The AD patients ranged in age from 72 to 86 years; the unaffected control subjects were 19, 40, 66 and 85 years old. Tlie individual with normal-pressure hydrocephalus was 63 years old at death. Crude synaptosomes were prepared from the mid frontal cortex of the frozen tissue (Hui et al., J. Biol Client. 273:31053-31060 (1998)). Approximately 2.5 g of gray matter was homogenized in 10% sucrose containing 1 mM dithiothreitol. The homogenate was centrifuged at 700g for 20 min and the peUet was rehomogenized and ceiitrifuged again at 700g for 20 min. The two supernatants were combined and centrifuged at 10,000g for 30 min to obtain the crude synaptosome pellet. The synaptosome peUet was subsequently lysed in the extraction buffer (50 mM HEPES, pH7.ό, 250 mM NaCl, 0.1% Nonidet-P40, 50 mM EDTA and 0.5 mM dithiothreitol) with protease inhibitors. The lysate was precleared with protein A-Sephorose beads and incubated for 48 h with antibody against intracytoplamic (I) and protein A-Sepharose beads. The sepharose beads were coUected and washed four to five times. Proteins immunoprecipitated were separated by 15% tiicine gel electiophoresis and visuaUzed by western blot analysis with CT15 or intracytoplasmic (I) APP.

[0079] To verify the identity of APP fragments generated by caspases, CTFs of APP were first assessed by western blot analysis for cleavage in cultured ceUs. In C100- transfected ceUs, there are two species of CTFs, one generated by β-secretase; the other, by α-secretase. As expected, both species are immunoreactive to CT15 and antibody against intracytoplasmic APP (αl), whereas only the fragment generated by β-secretase is positive for 26D6. In APP-transfected cells, the CTFs are mostly generated by α- secretase, as shown by positive immunoreactivity to CT15 and αl but negative reactivity to 26D6. After co-transfection of CIOO with caspase-8, CTFs of β- and α-secretase missing the epitopes of CT15 are generated. The immunoreactive profUes of β-CTFΔC31 fragment and α-CTFΔC31 are as expected: the β-CTFΔC31 fragment is positive for 26D6 and αl, but negative for CT15; and the α-CTFΔC31 fragment is positive for only αl. Co- transfection of APP with caspase-8 generates mau ly α-CTFΔC31 and some α-CTF.

[0080] Crude synaptosomal preparations were immunoprecipitated with αl foUowed by western blot analysis for APP CTFΔC31 fragments. Immunoprecipitated products of 293T cell lysates co-transfected with CIOO and caspase-8 were assayed next to the AD and control samples. In aU five AD patients, α-CTFΔC31 is present and its identity is consistent with the irnmunologic profile of caspase-cleaved fragments: positive imrnunoreactivity for α-1 and negative immunreactivity for CT15. In control tissues, this fragmentation is absent. Synaptosome samples from 10 subjects were assayed for synaptophysin ύnmunoreactivity to verify equal loading of protein. EXAMPLE 9 In vivo detection of caspase-9 activation in AD and control brains

[0081] Crude synaptosomal preparations were analyzed for the presence of caspase- 9 activation. The same crude synaptosome samples prepared from AD and control patients were subjected to immunoprecipitation (1:100 dilution) with the polyclonal antibody against caspase-9. The immunoprecipitates were separated by 15% Tris- glycine SDS-PAGE and immunoblotted with the activation-specific antibody against caspase-9, 315/316 (Biosource, CamarUlo, California). As a negative control, He La ceUs, transfected with caspase-9 zymogen, were treated with 40 μM zVAD.fmk (pan-caspase mhibitor). HeLa cells transfected with caspase-9 zymogen and treated with 1 μM staurosporine for 5 h served as a positive control for caspase-9 activation. For caspase-8 imrnunoblotting, the monoclonal antibody B9-2 (PharMingen, San Diego, California), recognizing amino acids 335-469 of caspase-8 fragment, was used.

EXAMPLE 10 Hippocampal cultures

[0082] Hippocampal or cortical neurons derived from 17-day old rat embryos were plated in modified rriinimum essential media (MEM-PAK) supplemented with 5% horse serum. Three days later, the cultures were treated with 10 μM cytosine arabinoside (AraC). Twenty-four h later the ceUs were treated with the peptide conjugates and incubated for an additional 24 or 48 h. CeUs were incubated in tlie presence of 50 μM of the general caspase inhibitor BOC-Asp(Ome)-FMK (BAF) for 30 min prior to addition of peptides or with an equivalent volume of dimethylsulfoxide (DMSO) and then maintained in the presence of the same concentiation of the inhibitor for the duration of the experiment. In aU experiments involving Aβ, a 1 mM Aβ42 stock solution was used that had been incubated at 37 °C for 48 h and then stored at 4 °C to aUow for the formation of Aβ fibrils. Aβ42 was purchased from AnaSpec (San Jose, CA).

EXAMPLE 11 Peptide delivery into cells

[0083] Peptides were synthesized and purified at the Stanford University Protein and Nucleic Acid (PAN) FacUity. AU peptide stocks were solubiUzed in water at 1 or 10 mM concentration. The deUvery peptide derived from the Drosophila Antennapedia homeodomain (RQIKIWFQNRRMKWKK; SEQ ID NO:4) (Dorn et al., Proc. Natl. Acad. Sci. USA 96:12798-12803 (1999) also called penetratin (Nakagawa et al., Nature 403:98-103 (2000)), was cross-linked via an N-terminal Cys-Cys bond to the 31 amino acid peptide generated by caspase cleavage of APP (DP-APPC31) or APLP1(DP-APLP1C31), to an irrelevant peptide (C-N), or to itseU (DP-DP) at tlie Stanford University PAN facility. Cargo peptides are released from tlie carrier by reduction of the disulfide bond in the intraceUular environment. No toxicity has been observed in transduction experiments using the conjugate of penetratin to itseU at the concentrations assayed. A control peptide used was a fusion of tlie human immunodeficieiicy vUus-1 TAT protein (HIV- TAT) deUvery peptide sequence (HUeman et al, FEBS Lett. 415:145-154 (1997) and the first heUx of the p75 receptor intracellular domain.

EXAMPLE 12 Cell death assessment

[0084] Primary hippocampal or cortical neuronal cultures or 293 ceUs transduced with the different penetratin-peptide conjugates were assayed for viabiUty at 24 and 48 h after transduction by trypan blue exclusion or by conversion of 3-[4,5-Dimethylfhiazol-2- yl]-2,5-diphenylteti."azolium bromide; Thiazolyl blue (MTT, Sigma, St. Louis) to formazan by dehydrogenase enzymes (MTT, Sigma, St. Louis) and by the LIVE/ DEAD assay (Molecular Probes, Eugene, OR), which distinguishes live cells by the presence of traceUular esterase activity, which results in the conversion of the non-fluorescent ceU permeant calcein-AM to the intensely green fluorescent calcein. Calcein is retained within Uve ceUs. Etiiidium homodimer-1 (EthD-1) enters cells with damaged membranes and becomes mtensely fluorescent when bonding to nucleic acids. EthD-1 is excluded by the intact plasma membrane of live cells. Media were removed and replaced by 4μM EthD-1 and 2μM calcein in PBS. Images were taken 30 in after treatment. The morphology of nuclei in the cultures was examined by staining with 0.1 μg/ml Hoechst 33342. EXAMPLE 13 Antibodies, immunostaining and image analysis of hippocampal cultures

[0085] Hippocampal cultures were fixed in 4% paraformaldehyde in IX phosphate- buffered saline (PBS) for 20 min at room temperature (RT). CeUs were then rinsed in IX PBS and then washed once in IX Tris-buffered saline (TBS) foUowed by blocking in 10% donkey serum (Jackson ImniunoResearch Labs, West Grove, PA) with 0.1% Triton X-100 in IXTBS for 1 h at RT. Cultures were incubated overnight in the presence of rabbit anti- GFAP (Sigma, St. Louis) at 1:800 dUution and mouse anti-NeuN (Chemicon, Temecula, CA) at 1:100 at 4 °C. Negative controls were incubated hi 2 mg/ml rabbit and mouse preimmune IgGs (Sigma, St. Louis). AU primary antibodies were dUuted in IXTBS containing 10% donkey serum. Cultures were washed for 90 min in 4 changes of IXTBS and incubated in the presence of donkey anti-rabbit IgG conjugated to Cy3 and donkey anti-mouse IgG conjugated to FITC (Jackson ImmunoResearch Laboratories, hie, West Grove, PA), at 1:250 and 1:400, respectively, in lxTBS containing 1% donkey serum for 1 h at RT. CeUs were washed for 90 min in 4 changes of lxTBS and mounted in VectaShield-DAPI mounting medium (Vector Laboratories, Burlingame, CA). Images were acquired using Nikon EcUpse-800 microscope and Optronics MagnaFire camera and software, and analyzed using Compix Simple PCI software. The total surface area corresponding to red and green fluorescence in each confocal image was determined by image analysis using Simple PCI software (Compix, Inc., PhUadelphia).

[0086] These experiments showed that C31 induces death of rat hippocampal neurons in primary culture. In order to determine whether C31 is also toxic in primary neuronal cultures, in which transfection efficiencies are relatively low, protein transduction was used. This approach allows for the introduction of polypeptides into ceUs with an efficiency close to 100%, and utilizes relatively stress-free conditions (Schwarze et al., Trends Cell Biol 10:290-295 (2000)). The D. melanogaster Antennapedia homeodomain-derived deUvery peptide (penetratin) linked by an N-terrninal disulfide bond to the APP-derived C31 peptide or to control peptides was used. Disulfide-linkage was chosen over other types of covalent bond in order to allow tlie C31 peptide to be released intraceUularly, in association with reduction of the S-S bond in the intiaceUular environment. [0087] Hippocampal neuronal cultures derived from 17-day old rat embryos were transduced with 10 μM DP-C31 peptide or the DP control, and a marked decrease in viabiUty observed in the DP-C31-transduced cultures, but not the control cultures, 24 h after transduction. The cells treated with DP-C31 peptide showed prominent cytoplasmic shrinkage and an almost complete disaggregation of the neuritic network. Fluorescence microscopic examination of tlie same cultures showed a profound reduction in the number of viable ceUs (cells capable of calcein retention in their cytoplasm), and a proportional increase in the number of cells with damaged membranes permeable to EthD-1. EssentiaUy identical results were obtained for cortical neuronal cultures. To assess the efficiency of transduction, primary hippocampal cultures were transduced with penetratin conjugated to FITC (DP-FITC) and analyzed by confocal microscopy. The DP-FITC peptide was internalized in >95% of the ceUs in the culture, incubation of primary hippocampal neurons in the presence of increasing concentrations of DP-C31, but not DP, reduced the number of viable ceUs capable of converting MTT into msoluble formazan.

[0088] Immunocytochemical examination of hippocampal cultures using antibodies specific for a neuronal marker, neuron-specific nuclear protein (NeuN), and a glial marker, glial fibrUlary acidic protein (GFAP), revealed a marked decrease in the number of NeuN-immunoreactive cells present in the cultures that had been treated with 10 μM C-C31. No difference in the number of NeuN-reactive ceUs was found in cultures treated with vehicle, with a control peptide, or with 10 μM C-C31 in the presence of the broad-spectrum caspase inliibitor BAF.

[0089] These experiments showed that C31 induces programmed ceU death in both neuronal and gUal ceUs. The morphology of tlie ceUs that survived transduction with DP was suggestive of glial origin. To investigate whether this was due to a greater sensitivity of neurons than glial ceUs to DP-APPC31 -induced death, 3-day-old rat hippocampal cultures were exposed to increasing concentrations of DP-APPC31 or control DP peptide and fixed 48 h after transduction. The fixed cultures were then immunostained with antibodies specific for GFAP (red) and NeuN (green). A quantitative assessment of the total area of red and green fluorescence present hi low- magnification confocal images of representative fields obtained from three mdependent experhnents was performed using a digital image analysis system (SimplePCl, Compix, Inc, Philadelphia). Both the neuronal and the gUal populations were significantly reduced in cultures treated with 10 μM DP-APPC3I when compared to untreated or control peptide-tieated cultures (see Figure 4). Transduction with higher concentrations of DP-APPC31 (25 μM) were required to reduce tlie viabUity of the neuronal population further, whUe no further toxicity was observed for gUal cells at the concentrations assayed. Incubation in the presence of tlie broad caspase inhibitor, Boc-aspartyl- fluoromethylketone (BAF), delayed tlie toxicity resulting from transduction with DP- APPC31, confhmiiig that C31 -induced neuronal death in primary cultures is caspase mediated.

[0090] To resolve the discrepancy in the LC50 values obtained by the MTT assay for cell viabUity (see Figure 5, 5 μM) and by quantitative image analysis (see Figure 3, 3.75 μM), the extent of cell death induced by transduction of DP-APPC31 in neuronal cultures was further examined by the trypan blue exclusion method. The LC50 value obtained by trypan blue exclusion for neuronal cultures transduced with DP- APPC31 was 3.75 μM, in agreement with the value obtained by quantitative image analysis (see Figure όa). Given that 3-day old cultures of primary neurons were used in aU experiments, it is conceivable that the higher LC50 value obtained using the MTT assay was due to variabiUty in the proportion of gUal cells present in different batches of primary neurons at the time of plating.

[0091] FinaUy, the number of apoptotic nuclei in cultures transduced with different concentrations of DP-APPC31 was quantitated by Hoechst 33342 staining. An increase in the percentage of condensed, fragmented nuclei present in neuronal cultures was observed when increasmgly high concentrations of DP-APPC31 were used for transduction (see Figure 6b). This observation, together with the finding that APPC31 toxicity was delayed by caspase inhibitors, is consistent with the conclusion that the cellular death induced by the C31 peptide was apoptotic in nature.

[0092] Experiments further indicated that exposure to Aβ increases the sensitivity of neurons to C31. It has been shown that exposure of cultured human neuronal and non- neuronal cells to amyloidogenic Aβ peptide induces the activation of apoptotic ceU death pathways (Cotman, supra; Cotman and Anderson, supra; La Ferla et al., supra; Nakagawa et al., supra). The concentiations of Aβ used in most of these experiments, however, are likely to be greater than those that may be found in tlie vicmity of axonal terminals, particularly at early stages in the pathogenesis of AD. It was found that Aβ toxicity in ceU lines is augmented by C31. Tlie effect of exposing hippocampal neurons to both Aβ and C31 was therefore assessed. No measurable toxicity was found in hippocampal cultures exposed to concentrations of Aβ alone up to 25 μM. Treatment with 50 μM Aβ, however, was sufficient to decrease the number of viable ceUs in the culture substantially with a concomitant increase in the number of inviable cells showmg permeabiUty for EthD-1.

[0093] In the next set of experiments, hippocampal cultures were preincubated in the presence of sublethal concentiations (5 μM) of fibrUlar Aβ and these cultures transduced with different peptides 24 h later. Transduction was performed in the absence of Aβ. Preincubation of the primary hippocampal cultures in the presence of 5 μM Aβ for 24 h exacerbated the sensitivity of hippocampal neuronal cultures to C-C31 induced death (LC50 < 500 nM). The presence of Aβ itseU could not account for the toxicity observed, since incubation of ceUs in 5 μM Aβ alone did not increase the number of dead cells present in the culture above background. There was toxicity observed when sublethal concentrations of Aβ and control peptide were added; however, the toxicity observed in cultures treated with Aβ and transduced with C-C31 was Ukely not due to additive toxic effect (P < 0.01 by two-way ANOVA) [[P = 0.0059]]. Examination by Hoechst 33342 staining revealed a sharp increase in the percentage of apoptotic nuclei in cultures that had been incubated in the presence of Aβ and then exposed to C-C31 (72%), but not in cultures that had been mock-treated or treated with Aβ (approximately 6% and 9%, respectively).

[0094] Aβ exacerbates the sensitivity of hippocampal cultures to C31 toxicity. Primary hippocampal cultures were left untreated or exposed to varying concentrations of Aβ. Thirty-six hours later, the cultures were evaluated by the LIVE/ DEAD assay. Primary hippocampal cultures were also tested by preincubation for 24 hours in the presence of a sublethal concentration of Aβ and exposed to varying concentrations of C- C31 or C-C control peptide for an additional 24 hours. Cultures were then assayed by the trypan blue exclusion method. Cells were stained with Hoechst 33342 and scored for nuclear condensation.

EXAMPLE 14 Generation of an APP-Neo antibody [0095] An antibody that recognizes specifically the epitope generated by cleavage of APP at D664 by caspases was generated at ResGen (Invitrogen Corp., Alabama). Briefly, rabbits were immunized with the peptide 657CIHHGWEVD664, (SEQ ID NO:ό) which includes the nine amino acids immediately preceding the caspase cleavage site at position 664 in APP695, coupled to KLH. Antisera from three bleeds over a 10-week period were pooled and affinity purified in three successive steps. (1) Peptide antigen was irrimobilized on an activated support. Antisera was passed through the co lumn and then washed. After washing, the bound antibodies were eluted by a pH gradient. (2) The eluate from (1) was depleted of immunoglobulins that recognize the intact APP molecule by adsorption to a bridging peptide that encompasses the caspase cleavage site (TSIHHGWEVDAAVTPEE; SEQ ID NO:7). (3) The flowthrough from (2) was affinity purified on tlie immobilized immunogenic peptide. After washing, specific antibodies were eluted by a pH gradient, coUected and stored in borate buffer. The ELISA titer for this preparation was <1:142,000 (<5 ng/ml) against the immunizing peptide (corresponding to the "novel" C-terminus of APP, an epitope that is generated only after caspase cleavage) versus >1:70 (>10 mg/ml) against the bridging peptide that corresponds to tlie intact APP sequence across the caspase cleavage site at D664.

EXAMPLE 15 Human tissue immunohistochemistry

[0096] Human hippocampi obtained from AD or age-matched control patients (Harvard Brain Tissue Resource Center, Belmont, MA) fixed with 4% paraformaldehyde were embedded in paraffin. Seven μm microtome sections were deparaff inized in xylene, rehydrated in 100, 95, 80 and 70% ethanol, and washed in 1 XTBS for 15 mm at room temperature. A 3% H₂O solution in methanol was used to neutraUze endogenous peroxidase-Iike activity. Microwave antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) for 5 min at 440 watts. SUdes were aUowed to cool to room temperature and were washed in 1 XTBS for 15 min. Samples were blocked in 10% normal horse serum in IXTBS for 1 hour at room temperature. Primary rabbit IgG to APP-Neo was applied at a dilution of 1:10,000 in 1%BSA in IXTBS; sections were incubated overnight at 4 °C. Rabbit preimmune IgG (Sigma, St. Louis) dUuted to 1 μg/mL in the 1%BSA in IXTBS was used as a negative control. Sections were washed for 30 min in 3 changes of IXTBS; biotinylated horse anti-rabbit IgG (Vector Laboratories, Burlingame, CA) was applied at a dilution of 1:250 for 1 hour at room temperature. Peroxidase-based ABC Elite kit (Vector Laboratories, Burlingame, CA) was used according to the manufacturer's instructions followed by a 30 min wash in 3 changes of IX TBS. A Uquid DAB kit (Vector Laboratories, Burlingame, CA) was used for the detection; color development was monitored under the microscope. Sections were washed in IXTBS, briefly counterstained in aqueous hematoxylin, dehydrated, cleared, and mounted in Permount (Fisher Scientific, Pittsburgh, PA). Images were acquired using Nikon Eclipse- 800 microscope and the Optronics MagnaFire camera and software. Low magnification images were acquired using Nikon SMZ-U dissecting microscope and the CoolSnap camera and software.

[0097] The APP-neo antibody of the present invention was used to show caspase- cleaved APP in the brains of patients with AD and control, non-AD patients. To document the generation of C31 peptides in cultured ceUs and tissues, an antibody was generated that is capable of recognizing exclusively tlie novel epitope that arises by caspase cleavage of APP at its C terminus (APP-Neo), as described in Example 14. Hippocampi obtained from AD or age-matched control subjects were examined by immunohistocheinisti-y using the APP-Neo antibody. Hippocampal sections from AD brains showed that APP-Neo immunoreactivity, indicative of cleavage of APP at its C- terminus, is intense anteriorly in tlie polymorphic layer, reduced in the stratum granulosum, decreased in CA4-CA2 and absent from the stratum moleculare. APP-Neo staining was less intense at more posterior levels, but could be detected as dense deposits and in efferent fibers near CA3. Staining was aboUshed U the primary antibody was preadsorbed with the irrununizing peptide, but not if it was preadsorbed with a peptide that encompasses the immunizing peptide sequences and the first 5 N-terminal amino acids of the C31 peptide, past the caspase cleavage site (bridge peptide). Specific APP-Neo irrununostaniing occurred in the hippocampus of a 90 y.o. without AD as well (i.e., control brain), but to a lesser degree, staining was low to moderate in ceUs and fibers of the polymorphic layer and stratum granulosum, declining in CA4-CA2 and absent from the stratum moleculare. hi contrast to tlie AD brains, no APP-Neo staining could be detected at more posterior levels in the hippocampus. Staining was aboUshed by preadsorption with the immunogenic peptide, but not by preadsorption with bridge peptide.

EXAMPLE 16 Analysis of APP-like proteins

[0098] APLPl and APLP2 are cleaved by caspases. Three members of the APP family of proteins exist: APP, APLPl and APLP2. Even though the overaU sirrularity of tlie APP family C-termini is not high, the caspase cleavage site that is required for the generation of APPC31 is completely conserved in aU three members. If the DEVD sequences in APLPl and APLP2 can function as caspase cleavage sites, both proteins could potentially generate C-teririinal peptides. It should be noted, however, that the PI' position in APP is Ala (VEVDA; SEQ ID NO:8), whereas the PI' position in APLPl is Pro (VEVDP; SEQ ID NO:9) as it is in APLP2. Caspases tend to prefer less bulky residues such as Gly, Ala, or Ser, in tlie PI' position, rather than more bulky residues such as Pro. Therefore, at least in theory, the VEVD site in APP should be more readUy cleaved by caspases than the sites in APLPl and APLP2. To determine whether APLPl and APLP2 can be cleaved by caspases, a panel of recombinant caspases were assayed for their abiUties to cleave ³⁵S-labeUed, in vitro transcribed/ translated APP, APLPl and APLP2. The results show that APP can be cleaved by caspases -3 and -6, but not by caspase-8. APLPl, on the other hand, was cleaved in vitro only by caspase-3, not by caspase -6 or - 8. Like APLPl, APLP2 may be cleaved in vitro by caspase-3 only, but with very low efficiency, if at aU. The ³⁵S-Met-labeUed C31 peptide product of the cleavage of APP by caspases -3 and -6 was detected as a ~4 kDa band. However, the homologous peptide generated by caspase-3 cleavage of APLPl was not detectable, likely due to the fact that only one methionine (of a total of two in APPC31) is conserved in APLP1C31.

[0099] To determine whether cleavage of APLPl and APLP2 can occur in cultured ceUs, CMV-driven constructs expressing N-terminally FLAG-tagged APLPl and APLP2 or a fuU-length APP construct were transfected in 293 ceUs and activated the caspase cascade by treatment with staurosporine. Both APP and APLPl were cleaved in staurosporine-treated 293 ceUs and in both cases, cleavage was prevented by incubation of the cells in the presence of BAF. Both the fuU-length and tlie truncated forms of APP were detected. Full-length APLPl appeared to be completely degraded in 293 cells treated with staurosporine, but not when BAF was present. No cleavage products of FLAG-APLP1 could be detected in these cultures. Both in vitro and in transfected 293 cells, caspases could cleave APP and APLPl at more than one site. No evidence was found for the cleavage of FLAG-APLP2 in transfected 293 cells.

[0100] To determine whether APLPl is effectively cleaved at position 664, the selective reactivity of the APP-Neo antibody was used. Given that the 5 amino acids that constitute the novel C-termini in cleaved APLPl and APLP2 are relatively conserved, epitopes could be generated that might be recognized selectively by APP-Neo after caspase cleavage. To determine whether APP-Neo immunoreactive epitopes are generated by caspases in APP, APLPl and APLP2, unlabelled, in vitro transcribed/ translated fuU length APP (APP_bP₅), APPD₀6₄A (a mutant of APP in which tlie D residue at position 664 has been replaced by A), and full-length APLPl and APLP2 were incubated in the presence of recombinant caspases. The products of the reactions were separated on polyacrylamide gels and immunoblotted with APP-Neo antibody. Control hnmunoblots were performed using lysates from 293 ceUs transfected with full length APP605, with an APP construct lacking the APP C-terminal 31 amino acids (APPdeltaC31), or with APP₆95 and treated with 10 μM staurosporine, in the presence or absence of 50 ιiM BAF. APP-Neo immunoreactive bands were detected only in lysates from 293 ceUs expressing APPdeltaC31 and in lysates from ceUs expressing APPOQS and treated with staurosporine in the absence of BAF. Also, an APP-Neo-immunoreactive epitope was detected in immunoblots of in vitro transcribed/ translated fuU length APP that had been incubated in the presence of recombinant caspase-3, but not caspase-7 or - 8. Likewise, in vitro transcribed/ translated APLPl and APLP2 yielded APP-Neo immunoreactive cleavage products only when incubated in the presence of recombinant caspase-3. Providnig a control for the specificity of the reaction, a mutant form of APP that cannot be cleaved by caspases, APPD664A, did not yield detectable APP-Neo immunoreactive products after incubation with recombinant caspase-3, -7 or -8.

[0101] APLP1C31 induces death in primary hippocampal cultures. The results presented suggest that APLPl can be cleaved by caspase-3 at the aspartic acid residue at position 620. If this event occurs in vivo, APLPl would have the potential to generate a pro-apoptotic C-terminal peptide homologous to APPC31. To determine whether the peptide generated by caspase cleavage of APLPl is toxic, a fusion of APLP1C31 to the Antennapedia delivery peptide (DP- APLPl C31) was generated and assayed hi protein transduction experiments. Three-day-old rat hippocampal cultures were exposed to increasing concentrations of DP- APLPl C31 or control peptide, fixed 36 h after transduction and irnniunostained with antibodies specific for GFAP and NeuN. A quantitative assessment of the relative areas of red (GFAP) and green (NeuN) fluorescence present in low-magnification confocal images was performed using a digital image analysis system (SimplePCl, Compix, Inc, Philadelphia). The NeuN- immunoreactive population was markedly reduced in cultures treated with 10 μM DP- APPC31 (see Figure 7a). At higher concentrations of DP-APPC31 (25 μM), the viabiUty of tlie neuronal population was reduced further. A modest decline in the viabiUty of glial cellswas observed, which may have been due to a relatively higher sensitivity of neurons to APLPC31 toxicity. Incubation in tlie presence of the broad caspase inhibitor, Boc-aspartyl-fluoromethylketone (BAF), delayed DP-APLP1C31 toxicity, consistent with the suggestion that cell death induced by APLPl C31 depends on caspase activity.

[0102] The extent of cell death induced by transduction of DP-APPC31 in neuronal cultures was further examined by the trypan blue exclusion method. As shown in Figure 7b, a dose-dependent reduction in the viabiUty of the cultures was observed at increasmg concentrations of transduced DP- APLPl C31 but not of control DP peptide. The LC50 value obtained for neuronal cultures transduced with DP-APPC31 was 4 μM, whUe the value obtained by image analysis was approximately 5 μM.

EXAMPLE 17

Identification of non-peptide small molecule compounds competing for the specific binding of the PTB domain of Xll and the C-terminal domain of APP

[0103] Since tlie C-terminal part of APP plays a critical role in both APP internalization and in the induction of ceU death, and it has been shown herein that C31 (peptide sequence: AAVTPEERHLSKMQQNGYENPTYKFFEQMQN; SEQ ID NO:l) is capable of inducing ceU death in ceUs expressing APP (Lu et al, supra), a rational approach has been employed for the identification of peptide and non-peptide smaU molecules that wiU compete for the specific binding of Fe65/ APP and Xll/APP (employing, for example, Catalyst, software from Molecular Summations Inc.). For this purpose, the avaUable crystallographic structure of Xll/APP complex are used and the Feό5/APP interaction modeled based on the Xll/APP complex. Applying this approach, 145 potential pharmacophores have been identified from 5 databases containing more than 600,000 compounds. Four of these have a statisticaUy significant dockmg score and can be grouped in two chemicaUy distinct groups, i.e., flavonoids and antibiotics. Additional potential compounds contemplated for use in the practice of the present invention include small molecules such as, for example, peptides, peptidomimetics, antisense peptides, antibodies, antagonists, antisense nucleic acids, and the like.

[0104] While tlie invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

[0105] AU references cited herein are hereby incorporated by reference in their entirety.

Claims (22)

WHAT IS CLAIMED IS:

1. A peptide having the sequence set forth in

(a) SEQ ID NO:l or a peptide having at least 80% sequence identity therewith,

(b) SEQ ID NO:2 or a peptide having at least 80% sequence identity therewith,

(c) SEQ ID NO:3 or a peptide having at least 80% sequence identity therewith, or

(d) a peptidomimetic of (a), (b), or (c); wherein said peptide or peptidomimetic is a potent inducer of apoptosis.

2. A peptide according to claim 1, wherein said peptide has at least 90% sequence identity with SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3.

3. A peptide according to claim 1, wherein the amino acid sequence of said peptide differs from SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3 by conservative substitutions of one or more residues thereof.

4. A method for inducing apoptosis in a target cell, said method comprising contacting said cell with an effective amount of a peptide according to claim 1.

5. A method according to claim 4, wherein said target ceU is a neural ceU.

6. A method according to claim 5, wherein said neural ceU is a neuron.

7. A method according to claim 5, wherein said neural ceU is a glial cell.

8. A method of reducing/ inhibiting apoptosis of a ceU containing β-amyloid protein precursor (APP) or an APP-like protein, said method comprising blocking cleavage that releases a C-terminal peptide fragment.

9. A method according to claim 8, wherein said cleavage is blocked by smaU molecule compounds such as peptides, antisense peptides, peptidomimetics, antibodies, antagonists, antisense nucleic acids, and the Uke.

10. A method according to claim 8, wherein said cell is a neural ceU.

11. A method according to claim 10, wherein said neural cell is a neuron.

12. A method accordhig to claim 10, wherein said neural cell is a glial ceU.

13. A method of reducing/ inhibiting apoptosis of a ceU containing β-amyloid protem precursor (APP) or an APP-like protem, said method comprismg inactivating the C-terminal peptide fragment as it is formed.

14. A method accordmg to claim 13, wherein said peptide fragment is inactivated by degrading the peptide into mactive fragment(s) thereof.

15. A method according to claim 13, wherein said peptide fragment is inactivated by combining with a chelator therefor.

16. A method according to claim 15, wherein said chelator is an antibody.

17. A method according to claim 13, wherein said target ceU is a neural cell.

18. A method according to claim 17, wherein said neural cell is a neuron.

19. A method according to claim 17, wherein said neural cell is a gUal cell.

20. A method of treating a subject in need thereof, said method comprising administeriiig a therapeuticaUy effective amount of a molecule capable of:

(a) blocknig the cleavage of APP or an APP-like protein, or

(b) inactivating the C-terminal peptide fragment generated by cleavage of the precursor.

21. A method according to claim 20, wherein said subject has Alzheimer's disease.

22. A method of identifying smaU molecules that wUl block cleavage of APP or an APP-Uke protein, said method comprising determining wliich small molecules wiU compete for specific binding to APP or an APP-Uke protein.