WO2005009374A2

WO2005009374A2 - Ceramide dependent-stabilization of bace1

Info

Publication number: WO2005009374A2
Application number: PCT/US2004/023476
Authority: WO
Inventors: Luigi Puglielli; Dora M. Kovacs
Original assignee: The General Hospital Corporation
Priority date: 2003-07-21
Filing date: 2004-07-21
Publication date: 2005-02-03
Also published as: WO2005009374A3; WO2005009374A8

Abstract

The invention relates to methods and products for inhibiting ceramide associated Aβ accumulation. The invention is useful for screening compounds for such activity, as well as diagnosing, preventing, and treating Alzheimer’s disease and other disorders associated with β-secretase processing of substrates.

Description

CERAMIDE DEPENDENT-STABILIZATION OF BACEl

Related Applications This application claims priority under 35 U.S.C. 119 to U.S. provisional patent application serial number 60/489,955, filed July 21, 2003, which is incorporated by reference in its entirety.

Field of the Invention The invention relates to methods and products for inhibiting ceramide associated Aβ accumulation. The invention is useful for diagnosing, preventing, and treating Alzheimer's disease and other disorders associated with β-secretase processing of substrates.

Background of the Invention Alzheimer's disease (AD) affects approximately 15 million individuals worldwide. The prevalence of the disease doubles every 5 years after age 65 and approaches 50% by age 85. Because of the ongoing increase in life expectancy, the number of people affected by this disease is rapidly increasing. The major risk factor for late-onset AD is aging (1). The molecular events that mediate the effect of aging on AD are a subject of intensive study. The main pathogenic event that occurs in all forms of AD is the abnormal accumulation of amyloid β-peptide (Aβ) into senile (or amyloid) plaques (2). Aβ is a 39-43 amino-acid peptide, proteolytically derived from the amyloid precursor protein (APP). APP is first cleaved by β-site APP cleaving enzyme 1 (BACEl) at the N-terminus of Aβ (β- cleavage), producing a C-terminal fragment (β-APP-CTF) of ~12-kDa, and subsequently in the transmembrane domain (γ-cleavage) by a presenilin-harboring protease complex. The two major sites of γ-cleavage are located at positions 40 and 42 of Aβ, generating Aβ₄o and Aβ₄₂, respectively. Symptoms of AD are progressive and include dementia, which includes characteristics such as loss of memory, problems with reasoning or judgment, disorientation, difficulty in learning, loss of language skills, and decline in the ability to perform routine tasks. Additional AD symptoms may include personality changes, agitation, anxiety, delusions, and hallucinations. The risk of AD in the population increases with age. It is believed that up to 4 million Americans have AD. The onset of AD is generally after age 60, but in rare instances younger individuals may be afflicted. It is generally believed that approximately 3 percent of men and women ages 65 to 74, and almost half of those age 85 and older have AD. Although a pattern of decline in AD patients is generally clinically recognizable as the disease progresses, reliable diagnostic methods are lacking. The only definitive diagnostic test for AD at this time is to determine whether amyloid plaques and tangles are present in a subject's brain tissue, a determination that can only be done after death. Thus, due to the lack of suitable diagnostic methods, health-care professionals are only able to provide a tentative diagnosis of AD in an individual, particularly at the early to mid stages of the disease. Although these diagnoses can indicate that a person "likely" has AD, the absence of a definitive diagnosis reflects a critical need for more accurate and reliable AD diagnostic tests. In addition to the absence of reliable diagnostic methods, the are also very limited treatment options available for patients suspected of having and/or diagnosed as having AD. Several drugs have been approved in the US for treatment of early and mid-stage AD, but they have significant detrimental side effects and limited efficacy. The lack of effective treatments for AD means that even with a diagnosis of probable AD, the therapeutic options are quite limited. Thus, there is a significant need for effective compounds and methods for preventing and/or treating AD.

Summary of the Invention The membrane lipid ceramide is the backbone of all complex sphingolipids and acts as a second messenger in many biological events. In addition, it regulates several biochemical and genetic events that occur during aging/senescence, including inhibition of phospholipase D (PLD) and c-fos dependent signaling pathways, Rb dephosphorylation, arrest of the serum/growth factor mediated activation of protein kinase C (PKC), and arrest of DNA synthesis (3, 4). Endogenous ceramide can be generated by either de novo synthesis or hydrolysis of sphingomyelin (SM) at the cell surface, the latter being the most important source of the active pool of ceramide (5, 6). The intracellular levels of ceramide increase progressively during aging in both cultured cells and the whole organ (7-10). In addition, brains from AD patients contain approximately three times more ceramide when compared to age-matched controls (11). We investigated the role of the second messenger ceramide in the regulation of Aβ generation through several biochemical approaches. Surprisingly, we found that Aβ biogenesis is strictly regulated by the endogenous pool of ceramides, implicating the sphingomyelin, but not the glycosphingolipid, biosynthetic pathway in Aβ generation. We also found, unexpectedly, that the ceramide-dependent regulation of Aβ biogenesis is achieved via control of BACEl steady-state levels, is specific for APP, and is not associated with cell death. According to one aspect of the invention, methods for identifying compounds that inhibit BACE stabilization induced by ceramide are provided. The method include providing a reaction mixture that comprises BACE and ceramide; contacting the reaction mixture with a test compound; determining BACE stability in the absence and in the presence of the test compound, and comparing the BACE stability in the absence and in the presence of the test compound. A test compound that reduces BACE stability below the stability observed in the absence of the test compound is a compound that inhibits BACE stability. In some embodiments, BACE stability is determined by measuring the turnover rate of the BACE protein. BACE stability also can be determined by measuring Aβ production from APP and/or production of APP-C99, the C-terminal product of BACE cleavage. In certain embodiments, reaction mixture is a cell that contains both BACE and ceramide. In such embodiments, the level of ceramide is increased in the cell relative to a control cell. The level of ceramide can be increased by contacting the cell with a compound that increases ceramide, such as ceramide or an active analog or derivative thereof. A preferred active analog or derivative is C6-ceramide. The level of ceramide can be increased by contacting the cell with an enzyme that increases the level of ceramide in the cell, or a nucleic acid that expresses the enzyme. The enzyme is in some embodiments a biosynthetic enzyme, preferably a sphingomyelinase. Preferably the cell used in the assays is a neuronal cell. More preferably still, the neuronal cell manifests phenotypic characteristics of neuronal cell of a subject identified as having Alzheimer's disease. In preferred embodiments, the method is performed simultaneously with a plurality of compounds. The plurality of compounds can be contacted with the same reaction mixture, or each of the plurality of compounds is contacted with a different reaction mixture. The plurality of compounds can be a compound library; preferably the compound library is prepared using combinatorial chemistry. According to another aspect of the invention, methods for reducing Aβ production and/or production of APP-C99 in a cell are provided. The methods include contacting the cell with an amount of a compound effective to decrease the amount of ceramide in the cell, wherein the decrease in the amount of ceramide reduces ceramide-dependent stabilization of BACE. In some embodiments, the compound is a sphingomyelinase inhibitor, preferably manumycin A, scyphostatin, or an analog or derivative thereof that inhibits sphingomyelinase. In other embodiments, the compound is an inhibitor of the de novo biosynthesis of ceramide, preferably fumonisin Bl or an analog or derivative thereof that inhibits ceramide biosynthesis. In a further aspect of the invention, methods for treating Alzheimer's disease ar provided. The method include administering to a subject a therapeutically effective amount of a composition that decreases the amount of ceramide in the brain of the subj ect. In some embodiments, the compound is a sphingomyelinase inhibitor, preferably manumycin A, scyphostatin, or an analog or derivative thereof that inhibits sphingomyelinase. In other embodiments, the compound is an inhibitor of the de novo biosynthesis of ceramide, preferably fumonisin Bl or an analog or derivative thereof that inhibits ceramide biosynthesis. Use of compounds identified in the screening methods, or useful in the foregoing therapeutic methods, in the preparation of a medicament also is provided. The medicament preferably is useful for treating disorders of Aβ and/or APP-C99 production, particularly Alzheimer's disease. These and other objects of the invention will be described in further detail in connection with the detailed description of the invention.

Brief Description of the Figures Fig. 1 shows that C6-ceramide, a cell-permeable analog of ceramide, increases Aβ generation. CHO cells stably transfected with APP₇₅₁ were treated with increasing concentrations of C6-ceramide (C6-cer), a cell-permeable and metabolically active analog of ceramide, for two days. Fig. 1 A, Aβ secretion in the conditioned media was analyzed by sandwich ELISA. C6-cer at 10 μM concentration increased the secretion of both Aβ_totai and Aβ ₂ by ~60 to 70%. Fig. IB, Western blot showing that C6-cer (10 μM) increased the steady- state levels of both β- and α-APP-CTFs without any apparent effect on APP expression or maturation. Fig. 1C, C6-ceramide (10 μM) treatment did not induce CPP32/Mch2α-mediated cleavage of the 116-kDa native form of PARP (f.l. PARP), which follows activation of the apoptotic cascade. Cleavage and generation of the 85-kDa apoptosis-related isoform of PARP was instead induced by staurosporine (Stauro)-mediated activation of the apoptotic cascade. Fig. ID, Inhibition of the apoptotic cascade with ZNAD (100 μM), a specific downstream inhibitor of caspase activity, did not revert the C6-cer (10 μM) effect on Aβ generation. Results are expressed as means ± S.D. of at least three different determinations. Asterisks (*) indicate a significant difference from control at p<0.05. One representative immunoblot (out of at least three) is shown.

Fig. 2 shows that C6-ceramide increases the rate of Aβ biosynthesis by promoting β- but not γ-cleavage of APP. H4 (human neuroglioma) cells were stably transfected with either full length (H4₇₅₁) or the C-terminal 105 amino acids (H4cios) of APP. APPcios is a good substrate for γ- but not α- or β-secretase, and mimics β-APP-CTF (12). Cells were treated with increasing concentrations of C6-ceramide (C6-cer) for two days. Fig. 2A, Western blot showing that 10 μM C6-cer increased the steady-state levels of α- and β-APP-CTFs, but not those of APP_CΪO_S- Fig. 2B, C6-cer treatment did not activate the apoptotic cascade, as assessed by poly (ADP-ribose) polymerase (PARP) activation, f.l. PARP indicates the 116- kDa native form of PARP. The 85-kDa apoptosis-related cleavage product of PARP, which is generated upon activation of the apoptotic cascade (see Fig. lc), could not be detected. C6- cer treatment did not affect "α-like" cleavage of tumor necrosis factor-α (TΝF-α), or full length Notch (f.l. Notch) cleavage by furin. N™ Notch indicates the mature transmembrane domain of Notch produced by furin-cleavage of f.l. Notch, f.l. Notch could only be detected at high exposures and its levels were not affected by ceramide treatment (data not shown). One representative immunoblot (out of at least three) is shown.

Fig. 3 is a schematic view of the sphingomyelin and glycosphingolipid biosynthetic pathways with the inhibitors used in our studies. FB 1 , fumonisin B 1 ; nSMase, neutral sphingomyelinase; NB-DGJ, N-butyldeoxygalactonojirimycin.

Fig. 4 shows that hydrolysis of cell surface sphingomyelin by neutral sphingomyelinase (nSMase) releases active endogenous ceramide and increases Aβ generation. CHO cells stably transfected with APP ₅₁ were treated with 0.25 μM nSMase for three days to release ceramide. Fig. 4A, Cells were labeled with [³H]palmitic acid prior to nSMase treatment. nSMase decreased the levels of sphingomyelin but increased those of ceramide. Results are expressed as percentages of control values. Fig. 4B, Aβ secretion in the conditioned media was analyzed by sandwich ELISA. nSMase increased the secretion of both Aβ_totai and Aβ₄₂ by -80%. Fig. 4C, Western blots showing that nSMase increased the steady- state levels of both β- and α-APP-CTFs, without any apparent effect on APP expression or maturation. Results are expressed as means + S.D. of at least three different determinations. Asterisks (*) indicate a significant difference from control at p<0.05. One representative immunoblot (out of at least three) is shown.

Fig. 5 shows that fumonisin Bl (FBI), an inhibitor of endogenous ceramide biosynthesis, reduces Aβ generation. CHO cells stably transfected with APP₇₅₁ were treated with FB 1 , a general inhibitor of ceramide-synthase, for four days. Fig. 5 A, Cells were labeled with [ Hjpalmitic acid during FBI treatment. FBI reduced the biosynthesis of both ceramide and sphingomyelin by -50 to 65%. Results are expressed as percentage of control values. Fig. 5B, Aβ secretion in the conditioned media was analyzed by sandwich ELISA. FBI treatment reduced the secretion of both Aβ_totai and Aβ ₂ by -50%. Fig. 5C, Western blot showing that FBI reduced the steady-state levels of both β- and α-APP-CTFs, without any apparent effect on APP expression or maturation. Fig. 5D, Cells were first treated with FBI (15 μM) alone, and then with FBI plus C6-ceramide (10 μM). Aβ secretion in the conditioned media was analyzed by sandwich ELISA. C6-ceramide reversed the reduction of Aβ secretion produced by FBI treatment, confirming that ceramide was responsible for such effect. Results are expressed as means + S.D. of at least three different determinations. Asterisks (*) indicate a significant difference from control at_p<0.05. One representative immunoblot (out of at least three) is shown.

Fig. 6 shows that N-butyldeoxygalactonojirimycin (NB-DGJ) does not affect the cellular pool of active ceramide or the rate of Aβ generation. CHO cells stably transfected with APP ₅₁ were treated with increasing concentrations of NB-DGJ for two days. NB-DGJ inhibits the ceramide specific glycosyltransferase without affecting the intracellular pool of active ceramide (16, 17). Fig. 6A, Cells were incubated in the presence of [³H]palmitic acid during NB-DGJ treatment. Results are expressed as percentages of control values. NB-DGJ reduced the incorporation of palmitic acid into the glycosphingolipid GMl but did not affect the biosynthesis of either ceramide or sphingomyelin. Fig. 6B, Following NB-DGJ treatment, media was subjected to sandwich ELISA for Aβ quantitation. No effect was observed on the secretion of either Aβ_totai or Aβ ₂. Fig. 6C, Immunoblot showing that the steady-state levels of β- and α-APP-CTFs were not affected by NB-DGJ treatment. Results are expressed as means + S.D. of at least three different determinations. Asterisks (*) indicate a significant difference from control atp<0.05. One representative immunoblot (out of at least three) is shown. Fig. 7 shows that endogenous ceramide regulates α- and β-, but not γ-cleavage of

APP. H4 (human neuroglioma) cells were stably transfected with either full length APP₇₅₁ (H4₇₅₁) or the C-terminal 105 amino acids of APP (H4cio₅)- APPcios is a good substrate for γ-, but not α- or β-secretase, and mimics β-APP-CTF (12). Cells were treated with nSMase, FBI, or NB-DGJ, as described in Figures 4, 5, and 6, respectively. Fig. 7A and B, Western blots showing that nSMase (at 0.25 μM) increased, while FBI reduced the steady-state levels of β- and α-APP-CTFs. No apparent effect on APP expression or maturation was observed. Neither FBI nor nSMase affected the steady-state levels of APPαos indicating that ceramide levels do not regulate γ-cleavage of APP. Fig. 7C, Immunoblots showing that NB-DGJ, which did not affect endogenous ceramides, did not induce any change in α-, β-, or γ- cleavage of APP. One representative immunoblot (out of at least three) is shown.

Fig. 8 shows that ceramide regulates the molecular stability of the β-secretase, BACEl. Fig. 8 A, H4 (human neuroglioma) cells were treated with 10 μM C6-ceramide for different periods of time and then analyzed for BACEl expression. Fig. 8B, The same experiment was performed with CHO cells stably transfected with BACEl (CHO_BACEI)- Fig. 8C, CHOBA_CEI cells were treated with either FBI (15 μM, for 4 days), nSMase (0.25 μM, for 3 days), or C6-ceramide (C6-cer; 10 μM, for 2 days). FBI reduced, whereas nSMase and C6- cer increased the steady-state levels of BACEl. Fig. 8D, CHO_BACEI cells were grown in the presence or absence of 10 μM C6-cer for 4 days, pulsed with radiolabeled methionine/cysteine, and then chased for different periods of time. C6-ceramide treatment increased the half-life of newly-synthesized BACEl. Fig. 8E, CHO_BA_CEI cells were first grown in the presence or absence of 10 μM C6-ceramide for 4 days and then treated with 0.5 mg/ml of cyclohexamide for increasing periods of time to inhibit protein synthesis. BACEl expression was analyzed by SDS-PAGE followed by immunoblotting. Band intensities were quantitated and expressed as relative densities. C6-ceramide increased the half-life of preformed BACEl. Results are expressed as means + S.D. of at least three different determinations. Asterisks (*) indicate a significant difference from control at p<0.05. One representative immunoblot (out of at least three) is shown. Detailed Description of the Invention The second messenger ceramide was determined to have a major role in the regulation of the generation of amyloid beta (Aβ) fragment of amyloid precursor protein (APP). Surprisingly, we found that Aβ biogenesis is strictly regulated by the endogenous pool of ceramides, and that this ceramide-dependent regulation of Aβ biogenesis is achieved via control of BACEl steady-state levels, is specific for APP, and is not associated with cell death. The invention includes methods for screening for compounds that inhibit the aforementioned ceramide-dependent regulation of BACE stabilization and the resultant increase in Aβ and/or APP-C99 production. As used herein, "BACE" means β-site amyloid precursor protein cleaving enzyme 1, or BACEl. Such methods include cell based (in vitro an in vivo) and non-cell based assays of various kinds. For example, non-cell based assays can involve combining cell extracts from cells that have increased levels of ceramide with compounds that are candidate inhibitors of ceramide-dependent BACE stabilization. Cell based assays can include contacting cells that have increased levels of ceramide (relative to a control cell) with compounds that are candidate inhibitors of ceramide-dependent BACE stabilization. Compounds that inhibit ceramide-dependent BACE stabilization can be selected by determining the stability of BACE protein, or by determining the level of Aβ and/or APP-C99 production, relative to a control cell or cell extract (e.g., a control that does not have increased levels of ceramide and/or that is not contacted with a candidate compound). The compounds used in such assays can be natural or synthetic compounds, such as those in small molecule libraries of compounds (including compounds derived by combinatorial chemistry). Natural product libraries also can be screened using such methods, as can selected libraries of compounds known to exert pharmacological effects, such as libraries of FDA-approved drugs. Compounds identified by the assays can be used in therapeutic methods of the invention described below. A wide variety of assays to identify pharmacological agents that modulate levels of ceramide or ceramide-dependent stability of BACE can be used in accordance with the aspects of the invention, including most particularly assays of the type described in the Exampels, but also labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays, cell-based assays such as two- or three-hybrid screens, expression assays, etc. The assay mixture comprises a candidate pharmacological agent. Typically, a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection. Candidate agents encompass numerous chemical classes, although typically they are organic compounds. In some embodiments, the candidate pharmacological agents are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500. Candidate agents comprise functional chemical groups necessary for structural interactions with proteins and/or nucleic acid molecules, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups. The candidate agents can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Candidate agents also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Where the agent is a nucleic acid molecule, the agent typically is a DNA or RNA molecule, although modified nucleic acid molecules as defined herein are also contemplated. It is contemplated that cell-based assays as described herein can be performed using cell samples and/or cultured cells. Cells include cells that transformed to express a β- secretase protein, or fragment or variant thereof, and cells treated using methods described herein to modulate (e.g. inhibit or enhance) the level of ceramide and/or ceramide-dependent stability of BACE. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means. Further, known pharmacological agents can be tested and further may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the agents. A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used. An assay may be used to identify candidate agents that modulate 1) levels of ceramide, 2) ceramide-dependent stability of BACE and/or 3) production of Aβ and/or APP- C99 resulting from these. In general, the mixture of the foregoing assay materials is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, BACE is stabilized relative to control cells (due to increased ceramide levels) and Aβ and/or APP-C99 production occurs. It will be understood that a candidate pharmacological agent that is identified as a modulating agent may be identified as reducing or eliminating Aβ and/or APP-C99 production. A reduction in ceramide levels need not be the absence of ceramide (which would likely be deleterious to a subject in any case), but may be a lower level of ceramide. Similarly, modulation of BACE stability or activity may be a lowered level, but need not be lowered to the amount of BACE stability or activity observed in control cells. Further, a reduction in Aβ and/or APP-C99 production need not be the absence of Aβ and/or APP-C99 production, but may be a lower level of Aβ and/or APP-C99 production. The order of addition of components, incubation temperature, time of incubation, and other parameters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4°C and 40°C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 1 minute and 10 hours. After incubation, the level of ceramide, ceramide-dependent stability of BACE, the activity of BACE, and/or production of Aβ and/or APP-C99 is detected by any convenient method available to the user. As used herein, the term "modulate" means to change, which in some embodiments means to "enhance" or "increase" and in other embodiments, means to "inhibit" or "reduce". For example, in some embodiments, ceramide-dependent stabilization or activity of BACE is reduced or inhibited. In other embodiments, Aβ and/or APP-C99 production is inhibited. It will be understood that reduction may mean reduction to zero or may mean reduction to a level below a normal level, a previous level, or a control level. The ceramide level, ceramide-dependent BACE stability, BACE activity and Aβ and/or APP-C99 production modulating molecules of the invention may include small molecules, polypeptides, (for example, competitive ligands and antibodies, or antigen- binding fragments thereof), and nucleic acids. For example, compositions of the invention may include nucleic acids that encode a molecule that reduces transcription of ceramide biosynthetic enzymes and/or stability and/or activity of a BACE, fragments and/or complexes thereof, including nucleic acids that bind to other nucleic acids, [e.g. for antisense, RNAi, or small interfering RNA (siRNA) methods], or may be polypeptides that reduce the levels of ceramide, the ceramide-dependent stability and/or activity of BACE or a BACE-containing complex. Such polypeptides include, but are not limited to antibodies or antigen-binding fragments thereof. The invention in other aspects involve the use of compounds that inhibit ceramide- dependent BACE stabilization and resultant increased Aβ and/or APP-C99 production in order to reduce Aβ and/or APP-C99 accumulation. Such methods can be therapeutic methods for treating Alzheimer's disease, or can be in vitro methods useful for evaluating compounds in, e.g., cell-based models of disease. As used herein, the term "ceramide-dependent BACE stabilization" means the posttranslational stabilization of BACE protein turnover resulting from elevated levels of ceramide in cells. As used herein, the term "Aβ and/or APP-C99 production" means the generation of Aβ and/or APP-C99 (the C-terminal product of BACE cleavage of APP) in a cell, tissue, or subject from APP. As used herein, the term "subject" means any mammal that may be in need of treatment with the ceramide lowering compounds of the invention (i.e., compounds identified using the screening methods of the invention) that result in lowered Aβ and/or APP-C99 production via reducing BACE stabilization. Subjects include but are not limited to: humans, non-human primates, cats, dogs, sheep, pigs, horses, cows, rodents such as mice, hamsters, and rats. Certain therapeutic methods of the invention include administering a therapeutically effective amount of a compound that inhibits ceramide-dependent BACE stabilization, such as by reducing ceramide levels in a subject, particularly in neuronal cells. Other therapeutic methods of the invention include administering a therapeutically effective amount of a compound that reduces the BACE stabilization other than by reducing ceramide levels. As used herein, "therapeutically effective amount" is an amount of a compound that is sufficient to reduce ceramide levels in the brain, and/or an amount sufficient to regulate β-secretase activity by stabilization of BACE, and/or to reduce the production of Aβ and/or APP-C99. As used herein, the term "compound that modulates ceramide-dependent BACE stabilization" or "compound that inhibits ceramide-dependent BACE stabilization" means a compound that either modulates or inhibits the level of ceramide in a cell and/or modulates or inhibits the ceramide-dependent stability of a BACE protein in a cell, tissue, or subject. Such compounds are compounds useful for reducing Aβ and/or APP-C99 production in a cell, tissue or subject. The methods of the invention involve the administration of compounds that modulate ceramide-dependent BACE stabilization in neuronal cells and/or tissues and therefore are useful to reduce or prevent Alzheimer's disease, any other diseases or disorders associated with abnormal accumulation of Aβ such as Down's syndrome, cerebro vascular amyloidosis, inclusion body myositis and hereditary inclusion body myopathies and any disease associated with abnormal BACE activity. As used herein, the term "Aβ- accumulation-associated disorder" means Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, inclusion body myositis and hereditary inclusion body myopathies, and any disease associated with abnormal (increased) BACE activity. In addition to mediating processing of APP, β-secretase (BACE) cleaves other substrates, including the sialyltransferase ST6Gal I (Kitazume et al, Proc. Nat 'I. Acad. Set USA 98(24):13554-13559, 2001) and PSGL-1 (P-selectin glycoprotein ligand-1, cleaved by β-secretase in the juxtamembrane domain). For a review of beta-secretase substrates see e.g., Gruninger-Leitch et al., J. Biol Chem. 277(7):4687-4693, 2002. These additional non-APP substrates of β-secretase may be involved in a variety of disease including glycoconjugate metabolism disorders (ST6Gal I), and other diseases (e.g., those involving aberrant leukocyte rolling on the endothelium, transmigration and tissue invasion of leukocytes mediated by PSGL-1). Thus, the stabilization of BACE also provides a target for diseases associated with altered beta-secretase processing of substrates other than APP. Abnormal processing of other substrates by β-secretase (BACE) also can be the result of a lack of secretase stabilization. For example, reduced amounts or activities of β-secretase can be responsible for a reduced level of processing of certain substrates, which also can lead to disease states. Moreover, the reduced amounts or activities of β-secretase can affect other biochemical processes, such as intracellular signaling processes. In such conditions, stabilizing or increasing the amounts of the secretase can provide beneficial effects in the treatment of diseases in which reduced levels are deleterious. One example of this (for γ- secretase) is the effect of a reduced level of presenilin on signaling by β-catenin (see Kang et al, Cell 110:751-762, 2002). Altered β-secretase processing of substrates therefore can result in a variety of disorders that can be diagnosed and/or treated in accordance with the invention. The term "disorder associated with altered β-secretase processing of substrates" as used herein includes Aβ-accumulation-associated disorders such as Alzheimer's disease as well as other disorders correlated with proteins that also are substrates of β-secretase, as described herein. The invention involves a variety of assays based upon detecting the level of ceramide and/or stabilization of BACE in subjects. The assays include (1) characterizing the impact of levels of ceramide and/or stabilization of BACE in a subject; (2) evaluating a treatment for regulating levels of ceramide and/or stabilization of BACE in a subject; (3) selecting a treatment for regulating levels of ceramide and/or stabilization of BACE in a subject; and (4) determining regression, progression or onset of a condition characterized by abnormal levels of ceramide and/or stabilization of BACE in a subject. Thus, subjects can be characterized, treatment regimens can be monitored, treatments can be selected and diseases can be better understood using the assays of the present invention. For example, the invention provides in one aspect a method for measuring the level of ceramide and/or stabilization of BACE in a subject. As provided by the invention, the level of ceramide and/or stabilization of BACE thus correlates with the existence of an Aβ accumulation-associated disorder, e.g. Alzheimer's disease. For example, a level of ceramide and/or stabilization of BACE that is significantly higher in a subject than a control level may indicated a subject has Alzheimer's disease, whereas a relatively normal level of ceramide and/or stabilization of BACE indicates that the subject does not have an Aβ accumulation-associated disorder of the invention, e.g. Alzheimer's disease. The assays described herein are carried out on samples obtained from subjects. As used herein, a subject is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent. In all embodiments, human subjects are preferred. The samples used herein are any cell, body tissue, or body fluid sample obtained from a subject. In some embodiments, the cell or tissue sample includes neuronal cells and/or is a neuronal cell or tissue sample. The biological sample can be located in vivo or in vitro. For example, the biological sample can be a tissue in vivo and an agent specific for levels of ceramide and/or stabilization of BACE can be used to detect the presence of such molecules in the tissue (e.g., by imaging portions of the tissue). Alternatively, the biological sample can be located in vitro (e.g., a biopsy such as a tissue biopsy or tissue extract). In a particularly preferred embodiment, the biological sample can be a cell-containing sample. Samples of tissue and/or cells for use in the various methods described herein can be obtained through standard methods. Samples can be surgical samples of any type of tissue or body fluid. Samples can be used directly or processed to facilitate analysis (e.g., paraffin embedding). Exemplary samples include a cell, a cell scraping, a cell extract, a blood sample, a cerebrospinal fluid sample, a tissue biopsy, including punch biopsy, a tumor biopsy, a bodily fluid, a tissue, or a tissue extract or other methods. Samples also can be cultured cells, tissues, or organs. Particular subjects to which the present invention can be applied are subjects at risk for or known to have an Aβ-accumulation-associated disorder. Such disorders may include, but are not limited to: Alzheimer's disease and any other diseases associated with overproduction of Aβ and/or APP-C99 or reduced clearance of Aβ and/or APP-C99 such as Down's syndrome, cerebrovascular amyloidosis, inclusion body myositis and hereditary inclusion body myopathies, and any disease associated with abnormal BACE activity. The assays described herein (see, e.g., the Examples section) include measuring the level of ceramide and/or stabilization of BACE. Levels of ceramide and/or stabilization of BACE can be measured in a number of ways when carrying out the various methods of the invention. In one type of measurement, the level of ceramide and/or stabilization of BACE is a measurement of absolute level of ceramide and/or stabilization of BACE. This could be expressed, for example, in terms of molecules per cubic millimeter of tissue. Another measurement of the level of ceramide and/or stabilization of BACE is a measurement of the change in the level of ceramide and/or stabilization of BACE over time. This may be expressed in an absolute amount or may be expressed in terms of a percentage increase or decrease over time. Importantly, levels of ceramide and/or stabilization of BACE are advantageously compared to controls according to the invention. The control may be a predetermined value, which can take a variety of forms. It can be a single value, such as a median or mean. It can be established based upon comparative groups, such as in groups having normal amounts of ceramide and/or ceramide-dependent BACE stability (which may be determined by Aβ amounts if correlated to levels of ceramide and/or stabilization of BACE in a particular experimental system) and groups having abnormal amounts of ceramide and/or ceramide- dependent BACE stability. Another example of comparative groups would be groups having a particular disease (e.g., Alzheimer's disease), condition or symptoms, and groups without the disease, condition or symptoms. Another comparative group would be a group with a family history of a condition and a group without such a family history. The predetermined value can be arranged, for example, where a tested population is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group or into quadrants or quintiles, the lowest quadrant or quintile being individuals with the lowest risk or amounts of ceramide and/or ceramide-dependent BACE stability and the highest quadrant or quintile being individuals with the highest risk or amounts of ceramide and/or ceramide-dependent BACE stability. The predetermined value of course, will depend upon the particular population selected. For example, an apparently healthy population will have a different 'normal' range than will a population that is known to have a condition related to Aβ accumulation. Accordingly, the predetermined value selected may take into account the category in which an individual falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art. By abnormally high it is meant high relative to a selected control. Typically the control will be based on apparently healthy normal individuals in an appropriate age bracket. It will also be understood that the controls according to the invention may be, in addition to predetermined values, samples of materials tested in parallel with the experimental materials. Examples include samples from control populations or control samples generated through manufacture to be tested in parallel with the experimental samples. The various assays used to determine the levels of ceramide and/or ceramide- dependent BACE stability include: assays as described in the Examples section herein, and assays such as those using materials that specifically bind to ceramide or BACE; gel electrophoresis; NMR; and the like. Immunoassays may be used according to the invention including sandwich-type assays, competitive binding assays, one-step direct tests and two- step tests such as routinely practiced by those of ordinary skill in the art. As mentioned above, it is also possible to characterize the existence of an Aβ accumulation-associated disorder by monitoring changes in the absolute or relative amounts of ceramide and/or ceramide-dependent BACE stability over time. For example, it is expected that an increase in the amount of ceramide and/or ceramide-dependent BACE stability correlates with increasing severity of an Aβ accumulation-associated disorder. Accordingly one can monitor levels of ceramide and/or ceramide-dependent BACE stability to determine if the status (e.g. severity, existence) of an Aβ accumulation-associated disorder of a subject is changing. Changes in relative or absolute levels of ceramide and/or ceramide- dependent BACE stabilization of greater than 0.1% may indicate an abnormality. Preferably, the change in levels of ceramide and/or ceramide-dependent BACE stabilization, which indicates an abnormality, is greater than 0.2%, greater than 0.5%, greater than 1.0%, 2.0%, 3.0% , 4.0%, 5.0%, 7.0%, 10%,. 15%, 20%, 25%, 30%, 40%, 50%, or more. Other changes, (e.g. increases or reductions) in levels of ceramide and/or ceramide-dependent BACE stability over time may indicate an onset, progression, regression, or remission of the Aβ accumulation-associated disorder in the subject. As described above, in some disorders a decrease in level of ceramide and/or ceramide-dependent BACE stability may mean regression of the disorder. Such a regression may be associated with a clinical treatment of the disorder thus the methods of the invention can be used to determine the efficacy of a therapy for an Aβ-accumulation-associated disorder (e.g. Alzheimer's disease). In some disorders an increase in level of ceramide and/or ceramide-dependent BACE stability may mean progression or onset of the disorder. The invention in another aspect provides a diagnostic method to determine the effectiveness of treatments for abnormal levels of ceramide and/or ceramide-dependent BACE stability. The "evaluation of treatment" as used herein, means the comparison of a subject's levels of ceramide and/or ceramide-dependent BACE stability measured in samples collected from the subject at different sample times, preferably at least one day apart. The preferred time to obtain the second sample from the subject is at least one day after obtaining the first sample, which means the second sample is obtained at any time following the day of the first sample collection, preferably at least 12, 18, 24, 36, 48 or more hours after the time of first sample collection. The comparison of levels of ceramide and/or ceramide-dependent BACE stability in two or more samples, taken on different days, is a measure of level of the subject's diagnostic status for an Aβ accumulation-associated disorder of the invention and allows evaluation of the treatment to regulate levels of ceramide and/or ceramide-dependent BACE stability. Such a comparison provides a measure of the status of the Aβ accumulation-associated disorder to determine the effectiveness of any treatment to regulate levels of ceramide and/or ceramide-dependent BACE stability. As will be appreciated by those of ordinary skill in the art, the evaluation of the treatment also may be based upon an evaluation of the symptoms or clinical end-points of the associated disease. In some instances, the subjects to which the methods of the invention are applied are already diagnosed as having a particular condition or disease. In other instances, the measurement will represent the diagnosis of the condition or disease. In some instances, the subjects will already be undergoing drug therapy for an Aβ accumulation-associated disorder (e.g. Alzheimer's disease), while in other instances the subjects will be without present drug therapy for an Aβ accumulation-associated disorder. Agents, e.g. antibodies and/or antigen-binding fragments thereof, that specifically bind to ceramide or BACE, are useful in screening and diagnostic methods. As described above herein, the antibodies of the present invention thus are prepared by any of a variety of methods, including administering protein, fragments of protein, cells expressing the protein or fragments thereof and the like to an animal to induce polyclonal antibodies. The production of monoclonal antibodies is according to techniques well known in the art. As detailed herein, such antibodies or antigen-binding fragments thereof may be used for example to identify tissues expressing protein or to purify protein.

As detailed herein, the foregoing antibodies or antigen-binding fragments thereof and other binding molecules may be used for example to identify ceramide or BACE. Antibodies also may be coupled to specific diagnostic labeling agents for imaging of cells and tissues with abnormal levels of ceramide and/or ceramide-dependent BACE stability; or to therapeutically useful agents according to standard coupling procedures. Diagnostic agents include, but are not limited to, barium sulfate, iocetamic acid, iopanoic acid, ipodate calcium, diatrizoate sodium, diatrizoate meglumine, metrizamide, tyropanoate sodium and radiodiagnostics including positron emitters such as fluorine- 18 and carbon- 11, gamma emitters such as iodine- 123 , technitium-99m, iodine- 131 and indium- 111, nuclides for nuclear magnetic resonance such as fluorine and gadolinium. Other diagnostic agents useful in the invention will be apparent to one of ordinary skill in the art. Thus agents (e.g., antibodies or antigen-binding fragments thereof) can be identified and prepared that bind specifically to ceramide or BACE. As used herein, "binding specifically to" means capable of distinguishing the identified material from other materials sufficient for the purpose to which the invention relates. Thus, "binding specifically to" a ceramide or BACE means the ability to bind to and distinguish these molecules from other lipids or proteins, respectively. The invention also provides agents (e.g. antibodies) for use in methods to stabilize β- secretase to inhibit the ceramide-dependent stabilization of BACE. In such methods, the antibodies recognize and bind specifically to BACE or another component of a cell that affects ceramide-dependent BACE stability. Such antibodies can be identified using the screening methods of the invention described herein. Methods to stabilize BACE may be used to treat Aβ accumulation-associated disorders such as, for example, Alzheimer's disease. The agents mentioned above include polypeptides. Such polypeptides include polyclonal and monoclonal antibodies, prepared according to conventional methodology, and include antibodies that bind to ceramide or BACE. Certain antibodies useful in the methods of the invention already are known in the art and include for example, the antibodies provided in the Examples section herein. Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F9(ab')2 fragment, retains both of the antigen binding sites of an intact antibody.

Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd Fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation. Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (Frs), which maintain the tertiary structure of the paratope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity. It is now well-established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. See, e.g., U.S. patents 4,816,567, 5,225,539, 5,585,089, 5,693,762 and 5,859,205. Thus, for example, PCT International Publication Number WO 92/04381 teaches the production and use of murine RSN antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin. Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as "chimeric" antibodies.

Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (HAMA) responses when administered to humans. Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab')2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or Fr and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or nonhuman sequences. The present invention also includes so-called single chain antibodies. Thus, the invention involves polypeptides of numerous size and type that bind specifically to a ceramide biosynthetic enzyme, a secretase pathway associated protein, or fragments thereof. These polypeptides may be derived also from sources other than antibody technology. For example, such polypeptide-binding agents can be provided by degenerate peptide libraries, which can be readily prepared in solution, in immobilized form or as phage display libraries. Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptoids and non-peptide synthetic moieties. Various methods may be used to decrease Aβ accumulation. Aβ accumulation may be decreased,e.g., for treatment of Alzheimer's disease, using methods to decrease the level of ceramide and/or ceramide-dependent stabilization of BACE. For example, methods of the invention include 1) the administration of molecules that are antisense of the nucleic acids that encode a ceramide biosynthetic enzyme, 2) RNAi and/or siRNA inhibition methods or such enzymes, and/or 3) administration of antibodies that block the functional activity of the proteins in the production of ceramide and/or ceramide-dependent stabilization of BACE.

The methods of reducing activity of the proteins may also include administering polypeptides or nucleic acids that encode polypeptides that are variants of the ceramide biosynthetic enzymes and are not fully functional. Such dominant negative variants may compete with the functional endogenous versions in a cell, tissue, or subject, and thereby reduce the ceramide content of cells or the ceramide-dependent BACE stabilization. The ceramide-, BACE stabilization and Aβ and/or APP-C99 production-modulating compounds of the invention, which include for example, antisense oligonucleotides, RNAi and/or siRNA oligonucleotides, antibodies, nucleic acids, an/or polypeptides may be administered as part of a pharmaceutical composition. In some embodiments of the invention, the stability and/or activity of BACE may be increased, for example, to produce cell or animal models of Alzheimer's disease or other neurological disorders. In these embodiments, the functional activity of BACE may be increased using methods such as administration of ceramide analogs (e.g., C6-ceramide) nucleic acids that encode the ceramide biosynthetic pathway enzymes, or other methods that enhance the level of ceramide in a cell. One set of embodiments of the aforementioned compositions and methods include the use of antisense molecules or nucleic acid molecules that reduce expression of genes via RNA interference (RNAi or siRNA). One example of the use of antisense, RNAi or siRNA in the methods of the invention is their use to decrease the level of expression of one or more ceramide biosynthetic pathway enzymes. The antisense oligonucleotides, RNAi, or siRNA nucleic acid molecules used for this purpose may be composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5' end of one native nucleotide and the 3' end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester intemucleoside linkage. These oligonucleotides may be prepared by art-recognized methods, which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors. In some embodiments of the invention, the antisense or siRNA oligonucleotides also may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a number of ways, which do not prevent them from hybridizing to their target but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness. The term "modified oligonucleotide" as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic intemucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide. Preferred synthetic intemucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides. The term "modified oligonucleotide" also encompasses oligonucleotides with a covalently modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having backbone sugars that are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus, modified oligonucleotides may include a 2'-O- alkylated ribose group. In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose. The present invention, thus, contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acid molecules encoding proteins of the invention, together with pharmaceutically acceptable carriers. The methods to modulate ceramide levels and to treat Alzheimer's disease also include methods to increase expression of fragments or variants of a ceramide biosynthetic pathway enzyme that has reduced function (e.g., dominant negative molecules). Additionally, the invention includes methods that include cells or models of Alzheimer's disease. Thus, it will be recognized that the invention embraces the use of sequences that encode dominant negative ceramide biosynthetic pathway enzymes or fragments or variants thereof, in expression vectors, as well their use to transfect host cells and cell lines, be these prokaryotic (e.g., E. coli), or eukaryotic (e.g., CHO cells, COS cells, yeast expression systems and recombinant baculovirus expression in insect cells). Especially useful are mammalian cells such as human, mouse, hamster, pig, goat, primate, etc. They may be of a wide variety of tissue types, including mast cells, fϊbroblasts, oocytes, monocytes, lymphocytes, and leukocytes, and they may be primary cells or cell lines. Specific examples include neuronal cells and embryonic stem cells. The expression vectors require that the pertinent sequence, i.e., those nucleic acids described supra, be operably linked to a promoter. The invention also permits the construction of gene "knock-outs", "knock-downs" or "knock-ins" in cells and in animals, providing materials for studying certain aspects of disorders associated with a increased ceramide levels and ceramide-dependent BACE stabilization. For example, a knock-in mouse may be constructed and examined for clinical parallels between the model and characteristics and symptoms found in subjects with Alzheimer's disease. Thus, animal or cell models may be constructed in which the level of ceramide and/or the stability of a BACE protein is increased. Such a cellular or animal model may be useful for assessing treatment strategies for Aβ accumulation-associated disorders, e.g. Alzheimer's disease. This type of "knock-in" model provides a model with which to evaluate the effects of candidate pharmacological agents (e.g. inhibitory effects) on a living animal that has an abnormal level of Aβ and/or APP-C99 production resulting from increased ceramide levels and/or stabilization of BACE. According to still a further aspect of the invention, a transgenic non-human animal comprising an expression vector of the invention is provided, including a transgenic non- human animal which has altered expression of molecule that modulates the level of ceramide and/or the ceramide-dependent stability of BACE. As used herein, "transgenic non-human animals" includes non-human animals having one or more exogenous nucleic acid molecules incorporated in germ line cells and/or somatic cells. Thus the transgenic animal include "knock-out" animals having a homozygous or heterozygous gene disruption by homologous recombination, animals having episomal or chromosomally inco orated expression vectors, etc. Knock-out animals can be prepared by homologous recombination using embryonic stem cells as is well known in the art. The recombination can be facilitated by the cre/lox system or other recombinase systems known to one of ordinary skill in the art. In certain embodiments, the recombinase system itself is expressed conditionally, for example, in certain tissues or cell types, at certain embryonic or post-embryonic developmental stages, inducibly by the addition of a compound which increases or decreases expression, and the like. In general, the conditional expression vectors used in such systems use a variety of promoters which confer the desired gene expression pattern (e.g., temporal or spatial). Conditional promoters also can be operably linked to nucleic acid molecules of the invention to increase or decrease expression of the encoded polypeptide molecule in a regulated or conditional manner. Trans-acting negative or positive regulators of polypeptide activity or expression also can be operably linked to a conditional promoter as described above. Such tn s-acting regulators include antisense nucleic acid molecules, nucleic acid molecules that encode dominant negative molecules, ribozyme molecules specific for nucleic acid molecules, and the like. Knock-down animals can be prepared using RNAi approaches (including siRNA) to reduce the levels of gene expression, as is known in the art. The transgenic non-human animals are useful in experiments directed toward testing biochemical or physiological effects of diagnostics or therapeutics for conditions characterized by increased or decreased levels of ceramide and/or increased or decreased ceramide-dependent stability of a BACE protein. Other uses will be apparent to one of ordinary skill in the art. Thus, the invention also permits the construction of gene "knockouts" and "knock-downs" in cells and in animals, providing materials for studying certain aspects of Aβ accumulation-associated disorders. Proteins useful in accordance with the invention, and fragments thereof, can be isolated from biological samples including tissue or cell homogenates, and can also be expressed recombinantly in a variety of prokaryotic and eukaryotic expression systems by constructing an expression vector appropriate to the expression system, introducing the expression vector into the expression system, and isolating the recombinantly expressed protein. Short polypeptides, including antigenic peptides (such as those presented by MHC molecules on the surface of a cell for immune recognition) also can be synthesized chemically using well-established methods of peptide synthesis. Thus, as used herein with respect to proteins, "isolated" means separated from its native environment and present in sufficient quantity to permit its identification or use. Isolated, when referring to a protein or polypeptide, means, for example: (i) selectively produced by expression of a recombinant nucleic acid or (ii) purified as by chromatography or electrophoresis. Isolated proteins or polypeptides may, but need not be, substantially pure. The term "substantially pure" means that the proteins or polypeptides are essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use. Substantially pure proteins may be produced by techniques well known in the art. Because an isolated protein may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the protein may comprise only a small percentage by weight of the preparation. The protein is nonetheless isolated in that it has been separated from the substances with which it may be associated in living systems, e.g. isolated from other proteins. The prevention and treatment methods of the invention include administration of ceramide-modulating compounds, to decrease (or increase) the level of ceramide in cells or tissues, and thereby to decrease (or increase) ceramide-dependent BACE stabilization and Aβ and/or APP-C99 production in the cells or tissues. Various techniques may be employed for introducing ceramide-modulating compounds of the invention to cells or tissues, depending on whether the compounds are introduced in vitro or in vivo in a host. In some embodiments, the ceramide-modulating compounds target neuronal cells and/or tissues. Thus, the ceramide-modulating compounds can be specifically targeted to neuronal tissue (e.g. neuronal cells) using various delivery methods, including, but not limited to: administration to neuronal tissue, the addition of targeting molecules to direct the compounds of the invention to neuronal cells and/or tissues. Additional methods to specifically target molecules and compositions of the invention to brain tissue and/or neuronal tissues are known to those of ordinary skill in the art. In some embodiments of the invention, a ceramide-modulating compound of the invention may be delivered in the form of a delivery complex. The delivery complex may deliver the ceramide-modulating compound into any cell type, or may be associated with a molecule for targeting a specific cell type. Examples of delivery complexes include a ceramide-modulating compound of the invention associated with: a sterol (e.g., cholesterol), a lipid (e.g., a cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g., an antibody, including but not limited to monoclonal antibodies, or a ligand recognized by target cell specific receptor). Some delivery complexes may be sufficiently stable in vivo to prevent significant uncoupling prior to intemalization by the target cell. However, the delivery complex can be cleavable under appropriate conditions within the cell so that the ceramide-modulating compound is released in a functional form. An example of a targeting method, although not intended to be limiting, is the use of liposomes to deliver a ceramide-modulating compound of the invention into a cell.

Liposomes may be targeted to a particular tissue, such as neuronal cells, by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein.

Such proteins include proteins or fragments thereof specific for a particular cell type, antibodies for proteins that undergo intemalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like. Liposomes are commercially available from Life Technologies, Inc., for example, as

LIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids such as N-[l-

(2,3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods for making liposomes are well known in the art and have been described in many publications. Liposomes also have been reviewed by Gregoriadis, G. in Trends in Biotechnology, 3:235-241 (1985). When administered, the ceramide-modulating compounds (also referred to herein as therapeutic compounds and/or pharmaceutical compounds) of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The characteristics of the carrier will depend on the route of administration. The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intranasal, intracavity, subcutaneous, intradermal, or transdermal. The therapeutic compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the therapeutic agent, which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile iηjectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in lJ-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of iηjectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Compositions suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the therapeutic agent. Other compositions include suspensions in aqueous liquors or non-aqueous liquids such as a syrup, an elixir, or an emulsion. The invention provides a composition of the above-described agents for use as a medicament, methods for preparing the medicament and methods for the sustained release of the medicament in vivo. Delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the therapeutic agent of the invention, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer-based systems such as polylactic and polyglycolic acid, poly(lactide-glycolide), copolyoxalates, polyanhydrides, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polycaprolactone. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di- and tri-glycerides; phospholipids; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like. Specific examples include, but are not limited to: (a) erosional systems in which the polysaccharide is contained in a form within a matrix, found in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation. In one particular embodiment, the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. PCT/US95/03307 (Publication No. WO 95/24929, entitled

"Polymeric Gene Delivery System". PCT/US95/03307 describes a biocompatible, preferably biodegradable polymeric matrix for containing an exogenous gene under the control of an appropriate promoter. The polymeric matrix is used to achieve sustained release of the exogenous gene in the patient. In accordance with the instant invention, the compound(s) of the invention is encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US95/03307. The polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the compound is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the compound is stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the compounds of the invention include films, coatings, gels, implants, and stents. The size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted. The size of the polymeric matrix device further is selected according to the method of delivery that is to be used. The polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material that is bioadhesive, to further increase the effectiveness of transfer when the devise is administered to a vascular surface. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time. Both non-biodegradable and biodegradable polymeric matrices can be used to deliver agents of the invention of the invention to the subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers. In general, the agents of the invention are delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix. Exemplary synthetic polymers that can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone. Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof. Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. Bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein by reference, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate). Use of a long-term sustained release implant may be particularly suitable for treatment of established neurological disorder conditions as well as subjects at risk of developing a neurological disorder. "Long-term" release, as used herein, means that the implant is constmcted and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days. The implant may be positioned at or near the site of the neurological damage or the area of the brain or nervous system affected by or involved in the neurological disorder. Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above. Some embodiments of the invention include methods for treating a subject to reduce the risk of a disorder associated with abnormal levels of ceramide and/or ceramide-dependent stabilization of BACE. The methods involve selecting and administering to a subject who is known to have, is suspected of having, or is at risk of having an abnormal level of ceramide and/or stabilization of BACE, an Aβ accumulation-modulating compound for treating the disorder. Preferably, the an Aβ accumulation-modulating compound is a compound for modulating (e.g. inhibiting) levels of ceramide and/or ceramide-dependent stabilization of BACE and is administered in an amount effective to modulate (reduce) levels of ceramide and/or ceramide-dependent stabilization of BACE. Another aspect of the invention involves reducing the risk of a disorder associated with abnormal levels of ceramide and/or ceramide-dependent stabilization of BACE, by the use of treatments and/or medications to modulate levels of ceramide and/or ceramide- dependent stabilization of BACE, therein reducing, for example, the subject's risk of an Aβ accumulation-associated disorder of the invention. In a subject determined to have an Aβ-accumulation-associated disorder, an effective amount of an Aβ accumulation-modulating compound is that amount effective to modulate (e.g. increase of decrease) levels of Aβ accumulation in the subject. For example, in the case of Alzheimer's disease an effective amount may be an amount that inhibits (reduces) the abnormally high levels of ceramide and/or ceramide-dependent stabilization of BACE, in the subject. A response to a prophylatic and/or treatment method of the invention can, for example, also be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. For example, the behavioral and neurological diagnostic methods that are used to ascertain the likelihood that a subject has Alzheimer's disease, and to determine the putative stage of the disease can be used to ascertain the level of response to a prophylactic and/or treatment method of the invention. The amount of a treatment may be varied for example by increasing or decreasing the amount of a therapeutic composition, by changing the therapeutic composition administered, by changing the route of administration, by changing the dosage timing and so on. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration, and the like factors within the knowledge and expertise of the health practitioner. For example, an effective amount can depend upon the degree to which an individual has abnormal levels of ceramide and/or ceramide-dependent stabilization of BACE. The factors involved in determining an effective amount are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons. The therapeutically effective amount of a pharmacological agent of the invention is that amount effective to modulate Aβ accumulation, and/or the levels of ceramide and/or ceramide-dependent stabilization of BACE and reduce, prevent, or eliminate the Aβ accumulation-associated disorder. For example, testing can be performed to determine the levels of ceramide and/or ceramide-dependent stabilization of BACE in a subject's tissue and/or cells. Additional tests useful for monitoring the onset, progression, and/or remission, of Aβ accumulation-associated disorders such as those described above herein, are well known to those of ordinary skill in the art. As would be understood by one of ordinary skill, for some disorders (e.g. Alzheimer's disease) an effective amount would be the amount of a pharmacological agent of the invention that decreases the levels of ceramide and/or ceramide- dependent stabilization of BACE to a level and/or activity that diminishes the disorder, as determined by the aforementioned tests. In the case of treating a particular disease or condition the desired response is inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition. The pharmaceutical compositions used in the foregoing methods preferably are sterile and contain an effective amount of a pharmacological agent for producing the desired response in a unit of weight or volume suitable for administration to a patient. The doses of pharmacological agents administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. The dosage of a pharmacological agent of the invention may be adjusted by the individual physician or veterinarian, particularly in the event of any complication. A therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0J mg/kg to about 200 mg/kg, and most preferably from about 0J mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days. Various modes of administration will be known to one of ordinary skill in the art which effectively deliver the pharmacological agents of the invention to a desired tissue, cell, or bodily fluid. The administration methods include: topical, intravenous, oral, inhalation, intracavity, intrathecal, intrasynovial, buccal, sublingual, intranasal, transdermal, intravitreal, subcutaneous, intramuscular and intradermal administration. The invention is not limited by the particular modes of administration disclosed herein. Standard references in the art (e.g., Remington's Pharmaceutical Sciences, 18th edition, 1990) provide modes of administration and formulations for delivery of various pharmaceutical preparations and formulations in pharmaceutical carriers. Other protocols which are useful for the administration of pharmacological agents of the invention will be known to one of ordinary skill in the art, in which the dose amount, schedule of administration, sites of administration, mode of administration (e.g., intra-organ) and the like vary from those presented herein. Administration of pharmacological agents of the invention to mammals other than humans, e.g. for testing purposes or veterinary therapeutic purposes, is carried out under substantially the same conditions as described above. It will be understood by one of ordinary skill in the art that this invention is applicable to both human and animal diseases including Aβ accumulation-associated disorders of the invention. Thus, this invention is intended to be used in husbandry and veterinary medicine as well as in human therapeutics. When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts. Preferred components of the composition are described above in conjunction with the description of the pharmacological agents and/or compositions of the invention. A pharmacological agent or composition may be combined, if desired, with a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the pharmacological agents of the invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. The pharmaceutical compositions may contain suitable buffering agents, as described above, including: acetate, phosphate, citrate, glycine, borate, carbonate, bicarbonate, hydroxide (and other bases) and pharmaceutically acceptable salts of the foregoing compounds. The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal. In general, the treatment methods involve administering an agent to modulate the level and/or activity of ceramide. Such agents can include enzymes that are involved in catabolism of ceramide, or enzymes that modify ceramide in a manner that renders is unable to stabilize BACE. The agents also can include molecules (such as RNAi or siRNA molecules) that reduce the level of enzymes involved in ceramide synthetic pathways. Thus, these methods include gene therapy applications. The procedure for performing ex vivo gene therapy is outlined in U.S. Patent 5,399,346 and in exhibits submitted in the file history of that patent, all of which are publicly available documents. In general, it involves introduction in vitro of a functional copy of a gene into a cell(s) of a subject which contains a defective copy of the gene, and returning the genetically engineered cell(s) to the subject. The functional copy of the gene is under operable control of regulatory elements, which permit expression of the gene in the genetically engineered cell(s). Numerous transfection and transduction techniques as well as appropriate expression vectors are well known to those of ordinary skill in the art, some of which are described in PCT application WO95/00654. In vivo gene therapy using vectors such as adenovirus, retroviruses, herpes virus, and targeted liposomes also is contemplated according to the invention. In certain embodiments, the method for treating a subject with a disorder characterized by abnormal levels of ceramide and/or ceramide-dependent stabilization of BACE of involves administering to the subject an effective amount of a nucleic acid molecule to treat the disorder. In certain of these embodiments, the method for treatment involves administering to the subject an effective amount of an antisense, RNAi, or siRNA oligonucleotide to reduce the level of a ceramide biosynthetic pathway associated protein (e.g., an enzyme such as neutral sphingomyelinase) and thereby, treat the disorder. An exemplary molecule for modulating the levels of ceramide and/or ceramide-dependent stabilization of BACE is a siRNA molecule that is selective for the nucleic acid encoding a neutral sphingomyelinase or other ceramide synthetic pathway associated protein. Alternatively, the method for treating a subject with a disorder characterized by abnormal levels of ceramide involves administering to the subject an effective amount of a ceramide synthetic pathway associated protein (or the nucleic acid that encodes such a protein) that has a reduced ability to increase ceramide levels, in order to treat the disorder. In yet another embodiment, the treatment method involves administering to the subject an effective amount of a binding polypeptide (e.g., antibody, or antigen-binding fragment thereof) to modulate binding between one or more proteins of the invention and, thereby, treat the disorder. In some embodiments, the treatment method involves administering to the subject an effective amount of a binding polypeptide to reduce the levels of ceramide and/or ceramide-dependent stabilization of BACE to decrease Aβ and/or APP- C99 production activity. In certain preferred embodiments, the binding polypeptide is an antibody or an antigen-binding fragment thereof; more preferably, the antibodies or antigen- binding fragments are labeled with one or more cytotoxic agents. According to yet another aspect of the invention, expression vectors comprising any of the isolated nucleic acid molecules of the invention described above, preferably operably linked to a promoter, are provided. In a related aspect, host cells transformed or transfected with such expression vectors also are provided. Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second

Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA) encoding a protein of the invention, fragment, or variant thereof. The heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell. As used herein, a "vector" may be any of a number of nucleic acid molecules into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids and virus genomes. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells that have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins that increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes that encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase, luciferase or alkaline phosphatase), and genes that visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein). Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined. As used herein, a coding sequence and regulatory sequences are said to be "operably" joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably j oined if induction of a promoter in the 5 ' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide. The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5' non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art. In some embodiments, a viras vector for delivering a nucleic acid molecule encoding a sphingomyelinase (e.g., dominant negative), fragment thereof, sphingomyelinase antisense molecule, RNAi, or siRNA molecule of the invention, is selected from the group consisting of adenoviruses, adeno-associated viruses, poxviruses including vaccinia viruses and attenuated poxviruses, Semliki Forest virus, Venezuelan equine encephalitis viras, retrovirases, Sindbis viras, and Ty virus-like particle. Examples of viruses and virus-like particles which have been used to deliver exogenous nucleic acids include: replication- defective adenoviruses (e.g., Xiang et al, Virology 219:220-227, 1996; Eloit et al., J. Virol. 7:5375-5381, 1997; Chengalvala et al., Vaccine 15:335-339, 1997), a modified retroviras (Townsend et al, J. Virol. 71 J365-3374, 1997), a nonreplicating retroviras (Irwin et al., J. Virol. 68:5036-5044, 1994), a replication defective Semliki Forest viras (Zhao et al., Proc. Natl Acad. Sci. USA 92:3009-3013, 1995), canarypox viras and highly attenuated vaccinia viras derivative (Paoletti, Proc. Natl. Acad. Sci. USA 93:11349-11353, 1996), non-replicative vaccinia viras (Moss, Proc. Natl. Acad. Sci. USA 93:11341-11348, 1996), replicative vaccinia viras (Moss, Dev. Biol. Stand. 82:55-63, 1994), Venzuelan equine encephalitis viras (Davis et al., Virol. 70:3781-3787, 1996), Sindbis viras (Pugachev et al, Virology 212:587-594, 1995), and Ty virus-like particle (Allsopp et al., Eur. J. Immunol 26: 1951-1959, 1996). In preferred embodiments, the viras vector is an adenoviras. Another preferred viras for certain applications is the adeno-associated viras, a double-stranded DNA virus. The adeno-associated viras is capable of infecting a wide range of cell types and species and can be engineered to be replication-deficient. It further has advantages, such as heat and lipid solvent stability, high transduction frequencies in cells of diverse lineages, including hematopoietic cells, and lack of superinfection inhibition thus allowing multiple series of transductions. The adeno-associated viras can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression. In addition, wild-type adeno-associated viras infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated viras genomic integration is a relatively stable event. The adeno-associated viras can also function in an extrachromosomal fashion. In general, other preferred viral vectors are based on non-cytopathic eukaryotic virases in which non-essential genes have been replaced with the gene of interest. Non- cytopathic virases include retrovirases, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Adenoviruses and retrovirases have been approved for human gene therapy trials. In general, the retrovirases are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retrovirases (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retrovirases by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, M., "Gene Transfer and Expression, A Laboratory Manual," W.H. Freeman Co., New York (1990) and Murry, E.J. Ed. "Methods in Molecular Biology," vol. 7, Humana Press, Inc., Cliffton, New Jersey (1991). Preferably the foregoing nucleic acid delivery vectors: (1) contain exogenous genetic material that can be transcribed and translated in a mammalian cell and that reduce levels of ceramide and/or ceramide-dependent stabilization of BACE to treat Aβ accumulation- associated disorders, and preferably (2) contain on a surface a ligand that selectively binds to a receptor on the surface of a target cell, such as a mammalian cell, and thereby gains entry to the target cell. Various techniques may be employed for introducing nucleic acid molecules of the invention into cells, depending on whether the nucleic acid molecules are introduced in vitro or in vivo in a host. Such techniques include transfection of nucleic acid molecule-calcium phosphate precipitates, transfection of nucleic acid molecules associated with DEAE, transfection or infection with the foregoing virases including the nucleic acid molecule of interest, liposome-mediated transfection, and the like. For certain uses, it is preferred to target the nucleic acid molecule to particular cells. In such instances, a vehicle used for delivering a nucleic acid molecule of the invention into a cell (e.g., a retroviras, or other viras; a liposome) can have a targeting molecule attached thereto. For example, a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incorporated within the nucleic acid molecule delivery vehicle. Especially preferred are monoclonal antibodies. Where liposomes are employed to deliver the nucleic acid molecules of the invention, proteins that bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake. Such proteins include capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo intemalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like. Polymeric delivery systems also have been used successfully to deliver nucleic acid molecules into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acid molecules. In addition to delivery through the use of vectors, nucleic acids of the invention may be delivered to cells without vectors, e.g. as "naked" nucleic acid delivery using methods known to those of skill in the art. The invention will be more fully understood by reference to the following examples. These examples, however, are merely intended to illustrate the embodiments of the invention and are not to be construed to limit the scope of the invention.

Examples The lipid second messenger ceramide regulates several biochemical events that occur during aging. In addition, its level is highly elevated in the amyloid-burdened brains of Alzheimer's disease (AD) patients. Here, we analyzed the impact of aberrant ceramide levels on amyloid β-peptide (Aβ) generation by using a cell permeable analog of ceramide, C6- ceramide, and several biochemical inhibitors of the sphingomyelin/glycosphingolipid biosynthetic pathway. We found that C6-ceramide increased the biogenesis of Aβ by affecting β- but not γ-cleavage of the amyloid precursor protein (APP). Similarly to C6- ceramide, increased levels of endogenous ceramide induced by neutral sphingomyelinase treatment, also promoted the biogenesis of Aβ. Conversely, fumonisin Bl, which inhibits the biosynthesis of endogenous ceramide, reduced Aβ production. Exogenous C6-ceramide restored both intracellular ceramide levels and Aβ generation in fumonisin Bl -treated cells. These events were specific for APP and were not associated with apoptotic cell death. Pulse- chase and time-course degradation experiments showed that ceramide post-translationally stabilizes the β-secretase BACEl . Taken together, these data indicate that the lipid second messenger ceramide, which is elevated in the brains of AD patients, increases the half-life of BACEl and thereby promotes Aβ biogenesis.

Cell culture and treatments Chinese Hamster Ovary (CHO) and H4 (human neuroglioma) cell lines were grown as described previously (12). Cells were grown in either 6-well plates or 100 mm tissue culture dishes (Becton Dickinson Labware, Franklin Lakes, New Jersey). nSMase, FBI, NB- DGJ, and C6-ceramide (C6-cer) were obtained from either Sigma Chemicals Co. (St. Louis, Missouri) or Calbiochem (La Jolla, California). Pharmacological treatment was for three (nSMase), four (FB 1 ), or two (C6-cer and NB-DGJ) days.

Lipid labeling and extraction Labeling of glycosphingolipids was performed using [9,10-³H(N)]palmitic acid (60 Ci/mmol) (NEN Life Science, Boston, Massachusetts). Cells were incubated in the presence of [³H]palmitic acid for at least three days (ad equilibrium) to allow steady-state labeling of palmitic-containing lipids. At the end of each treatment (see above), cells were washed twice in PBS, scraped and extracted in chloroform:methanol (2:1, v/v). After "Folch" extraction, the lipid phase was dried, resuspended in chloroform and applied, together with standards, to a Silica Gel-G (EM Science, Gibbstown, New Jersey) thin layer chromatography (TLC) plate. Plates were developed in either chloroform:methanol:acetic acid:formic acid:water (70:30:12:4:2, v/v/v/v/v) (13), or chloroform:methanol:9.8mM CaCl₂ (60:35:8, v/v/v) (14). Spots were scraped and counted in a liquid-scintillation counter.

Antibodies and Western blot analysis Polyclonal antibodies C7 and C8 against the C-terminus of APP were a generous gift from Dr. Dennis J. Selkoe (Harvard Medical School, Boston, Massachusetts). The polyclonal antibodies against caveolin-1 came from Santa Cruz Biotechnology, Inc. (Santa Cruz, California) and against BACEl from Abeam (Cambridge, United Kingdom). Monoclonal antibodies against PARP (C-2-10) were obtained from Clontech Laboratories, Inc. (Palo Alto, California) and against Tumor Necrosis Factor-α (TNF-α, M32255a) from Fitzgerald Industries International, Inc. (Concord, Massachusetts). The hybridoma antibody against Notch developed by S. Artavanis-Tsakonas was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences (Iowa City, Iowa). For Western blot analysis, total proteins (50 to 100 μg/lane) were separated on NuPage 4-12% BisTris-polyacrylamide gel electrophoresis (Invitrogen, Carlsbad, California) using MES running buffer (Invitrogen), and then blotted on Immun-Blot™ PVDF membranes (BIO-RAD, Hercules, California). Proteins were visualized using the LumiGLO™ protein detection kit (KPL, Gaithersburg, Maryland) as described by the manufacturer.

Aβ concentration determinations For Aβ determination, APP ₅₁ stably transfected CHO cell lines were grown in 6-well plates (Becton Dickinson Labware, Franklin Lakes, New Jersey). When -80-90% confluent, cells were washed in PBS and incubated in 1 ml of fresh medium for 24 hours (12). Secreted Aβ_totai and Aβ ₂ were quantitated by standard sandwich ELISA (Aβ ELISA Core Facility, Center for Neurological Diseases, Harvard Institutes of Medicine, Harvard Medical School).

C6-ceramide promotes Aβ generation The role of ceramide in APP processing and Aβ generation was initially analyzed using C6-ceramide, a cell permeable and active analog of ceramide. Chinese Hamster Ovary (CHO) cells, stably transfected with APP ₅₁, were treated with 10 μM C6-ceramide for two days. C6-ceramide increased the secretion of both Aβ_totai and Aβ₄₂ by -60% (Figure 1 A). This increase was accompanied by elevated steady-state levels of both α- and β-APP-CTFs (Figure IB), produced by α- and β-cleavage of APP, respectively. In addition, C6-ceramide increased the release of the secreted form of APP (sAPP) into the conditioned media (data not shown). C6-ceramide treatment did not affect CHO cell viability, as assessed by trypan blue uptake and by lactate dehydrogenase (LDH) release into the media (Table I). It also did not activate the apoptotic cascade, as indicated by the absence of the 85-kDa apoptosis-related isoform of the poly (ADP-ribose) polymerase (PARP) (Figure 1C). PARP-85 is an early indicator of apoptosis; it is produced by CPP32/Mch2α-mediated cleavage of the 116-kDa native form of PARP (f.l. PARP), which follows activation of the apoptotic cascade (Figure 1C). Finally, to assess whether the C6-ceramide-dependent increase in Aβ secretion was due to caspase activation, we used ZVAD, a downstream inhibitor of caspase activity, in conjunction with C6-ceramide treatment. The increase in Aβ generation induced by C6- ceramide was not reversed by ZVAD (Figure ID), indicating that C6-ceramide upregulates Aβ generation in CHO cells in the absence of apoptosis.

Table I. C6-cer amide did not affect the release of the cytosolic enzyme lactate dehydrogenase (LDH) into the conditioned media. Stable transfected CHO cell lines were treated with 10 μM C6-ceramide up to 6 days. LDH release in the conditioned media was analyzed using the enzymatic assay from Sigma, as described by the manufacturer. Days 0 2 6

LDH (U/L) 29.3±1J 31J±2.0 29J±2.6

To confirm the above results in a different cell type and to assess whether the changes in the rate of Aβ generation were determined by changes in only β- or in both β- and γ- cleavage of APP, we used H4 (human neuroglioma) cells stably transfected with either full length APP ₅₁ or the C-terminal 105 amino acids of APP (APPc.-ios), which mimic β-APP- CTF (12). C6-ceramide increased the steady-state levels of both α- and β-APP-CTFs in H4 cells expressing full length APP₇₅₁, without any evident effect on APP expression or maturation (Figure 2A). Significantly, no effect was observed on the steady-state levels of APPcιo₅ (Figure 2 A), indicating that the changes in Aβ secretion were due to changes in β- but not γ-cleavage of APP. Again, we did not detect any effect on cell viability (data not shown), or caspase-mediated cleavage of full length PARP (Figure 2B). Finally, C6-ceramide did not stimulate the "α-like" cleavage of tumor necrosis factor-α (TNF-α) by TNF-α- converting enzyme (TACE), or the furin-dependent cleavage of full-length Notch (Figure 2B). Taken together, the above results suggest that the second messenger ceramide regulates the rate of Aβ generation by affecting β-, but not γ-cleavage of APP. Reduced α- cleavage of APP is unlikely to lower Aβ levels. Instead, most reports indicate that it would elevate Aβ under normal cellular conditions (15). Finally, low levels of C6-ceramide did not induce apoptotic cell death of CHO and H4 cell lines under our experimental conditions. Endogenous ceramide levels regulate Aβ generation In order to confirm that endogenous ceramide, and not only cell-permeable analogues, can modulate Aβ production, we used additional biochemical approaches known to regulate the endogenous pool of active ceramide. The different approaches used in these studies are schematically described in Figure 3. nSMase increases the intracellular pool of ceramide by hydrolysis of cell surface SM. In contrast, FBI inhibits ceramide-synthase, preventing the biosynthesis of ceramide and all the other glycosphingolipids beyond the ceramide moiety. In addition to nSMase and FBI, we also used NB-DGJ, a biochemical inhibitor of the ceramide- specific glycosyltransferase. NB-DGJ blocks the glycosphingolipid, but not the SM, biosynthetic pathway and does not affect the levels of the signaling-active ceramide (16, 17). Neither nSMase nor FBI affected cell viability under the conditions used in our studies, as assessed by the uptake of trypan blue and by the release of the cytosolic enzyme LDH into the media (data not shown). When used at 0.25 μM, nSMase produced a -60% decrease of SM levels (Figure 4A). Similar to C6-ceramide, nSMase increased both ceramide levels (Figure 4A) and Aβ secretion (Figure 4B) by -2-fold. The increase in Aβ secretion was accompanied by increased steady-state levels of both α- and β-APP-CTFs, in the absence of any evident effect on APP expression or maturation (Figure 4C). As seen with nSMase, FBI treatment reduced SM by -50% (Figure 5A). However, in contrast with C6-ceramide and nSMase, FBI reduced ceramide levels by -60-70% (Figure 5A). Decreased endogenous ceramide levels were paralleled by a corresponding reduction in Aβ_totai and Aβ₄ secretion into the media by -50% (Figure 5B). Steady-state levels of α- and β-APP-CTF also decreased, with no apparent effect on APP expression or maturation (Figure 5C). Most importantly, exogenous C6-ceramide could recover Aβ levels reduced by FBI (Figure 5D), confirming that decreased ceramide levels were responsible for lowering Aβ in the first place. In contrast to nSMase and FB 1 , NB-DGJ, which did not affect the endogenous pool of the signaling active ceramide (Figure 6A), was not able to produce any effect on Aβ secretion (Figure 6B) or APP processing (Figure 6C). Finally, we assessed the effect of nSMase, FBI, and NB-DGJ on β- and γ-secretase cleavage of APP by using H4 (human neuroglioma) cells stably transfected with either full length APP₇₅₁ or with APPcios- As seen in CHO cells, both nSMase and FB 1 reduced SM levels, while producing opposite effects on ceramide levels. nSMase increased, whereas FBI reduced ceramide levels by -30 and -50%, respectively (data not shown). Again, the changes in ceramide production were paralleled by similar changes in the steady-state levels of both α- and β-APP-CTFs: nSMase increased, whereas FBI reduced APP CTFs (Figures 7A, B). Significantly, no effect was observed on the steady-state levels of APPcios (Figures 7 A and B) indicating that the changes in Aβ secretion were only due to changes in β- and not γ- cleavage of APP. As already observed in CHO cells, NB-DGJ did not affect ceramide levels or APP processing (Figure 7C). Overall, our results indicate that both cell-permeable analogues and endogenous ceramides regulate Aβ generation by affecting β-, but not γ-cleavage of APP. Although ceramide levels also modulate α-cleavage of APP, this clip does not directly contribute to Aβ generation and could be regulated by separate cellular events. Moreover, our data argue for a direct effect of ceramide on BACEl activity instead of APP trafficking, because γ-cleavage of APP is not affected by ceramide in APPcios-expressing H4 cells. Finally, biotinylation of cell surface proteins, subcellular fractionation studies, and analysis of APP mature/immature ratios revealed that ceramide levels do not significantly affect APP trafficking or localization (data not shown).

C6-ceramide reduces the turn-over rate of BACEl To assess the effect of altered ceramide levels on BACEl, we asked whether C6- ceramide regulates either the subcellular/membrane distribution or the steady-state levels of this enzyme. Endogenous BACEl was detected in H4 cells as a double band at around 65-70- kDa (Figure 8A). H4 cells were grown in the presence or absence of C-6 ceramide for up to six days and then analyzed for subcellular/membrane distribution or steady-state levels of BACEl. C6-ceramide did not affect the overall distribution of BACEl among intracellular membranes or membrane microdomains (data not shown). However, C6-ceramide progressively increased BACEl levels reaching a plateau after approximately four days of treatment (Figure 8 A). No effect was observed on the steady-state levels of BACE2, a BACEl homologue, or TACE, a regulated form of α-secretase (data not shown). To assess whether post-transcriptional events lead to increased BACEl protein levels, we used a CHO cell line stably expressing C-terminally myc-tagged human BACEl under the control of a cytomegaloviras promoter. When these cells were treated with C6-ceramide, the steady-state levels of both native and epitope-tagged BACEl increased progressively, reaching a plateau after four days, at which point they were -3 -fold higher than control (Figure 8B). Most importantly, as observed for the rate of Aβ secretion (see Figures 4 and 5), steady-state levels of BACEl also paralleled changes in endogenous ceramide induced by FB1 and nSMase. Figure 8C shows that FBI reduced, whereas nSMase increased, the steady- state levels of BACEl, strongly suggesting that ceramide levels regulate Aβ generation by modulating the amount of enzyme available for β-secretase cleavage of APP. Finally, we performed pulse-chase analysis and cycloheximide degradation assays to directly study changes in the turnover rate of BACEl . CHO cells stably expressing BACEl were treated with C-6 ceramide, followed by pulse-chase with radio-labeled methionine/cysteine in order to calculate the half-life of BACEl . Ceramide treatment increased the half-life of BACEl from -16-20 hours to -30 hours (Figure 8D). It is worth noting that the levels of newly-synthesized BACEl found in ceramide-treated cells after 56 hours of chase were very similar to those found in control cells after only 24 hours of chase (Figure 8D). Very similar results were obtained when the same cells were treated with cycloheximide in order to inhibit protein synthesis. Ceramide treatment significantly reduced the turnover rate of BACEl and increased the half-life of pre-formed BACEl from -20 hours to -56 hours (Figure 8E). Taken together, the above data indicate that ceramide regulates Aβ generation by affecting the steady-state levels of BACEl, the rate-limiting enzyme in the biogenesis of Aβ. They also suggest that the increase in BACEl levels is, at least in part, the result of post- transcriptional stabilization of BACEl. Our results show for the first time that intracellular levels of ceramide regulate Aβ generation by modulating β-secretase cleavage of APP. C6-ceramide, a cell-permeable and active analog of ceramide, increased the rate of Aβ biosynthesis by affecting β-, but not γ-, cleavage of APP. nSMase, FBI, and NB-DGJ, general inhibitors of the SM/glycosphingolipid metabolic pathway, caused changes in ceramide levels, which were consistently paralleled by changes in Aβ generation. Additionally, we found that ceramide controls the processing of APP by affecting the molecular stability of the β-secretase, BACEl . These effects occur under physiological conditions that do not perturb cell viability and do not activate apoptosis. Finally, we found that ceramide levels do not affect γ-cleavage of APP, as shown by the absence of changes in Aβ₄₂/Aβ_totai ratios and in the steady-state levels of APPcios, a short C-terminal fragment of APP that mimics β-APP-CTF. Ceramide is a lipid second messenger involved in many biological events that regulate terminal differentiation of neurons, cellular senescence, proliferation, and death (7, 14, 18, 19). Depending on the cell type and the doses used, exogenously-added ceramide has been shown to either activate or inhibit apoptosis (3, 18). Intracellular levels of ceramide increase during aging in both cultured cells and the whole organ (7-10). In addition, senescent-like doses (-10 μM) of ceramide are able to induce a senescent phenotype in young cultured cells (7, 9, 10). Under those conditions, ceramide has been shown to promote outgrowth and survival of cultured neurons (14, 18, 20). In apparent contrast to the above studies, a chronic increase in intracellular ceramide can inhibit axonal elongation, receptor-mediated intemalization of NGF, and activate cell death (13, 21). In addition, it also reduces receptor-mediated intemalization of lipoprotein- associated cholesterol (13), which is involved in the regulation of synaptogenesis (22). These effects may be part of a delicate set of events that occur during aging. Very recently, Han et al. (11) also reported that ceramide levels are increased more than 3 -fold in the brains of AD patients, when compared to age-matched controls. In this study, we showed that ceramide levels control Aβ biosynthesis, the first pathogenic event in the generation of senile (or amyloid) plaques. It is worth noting that our results with exogenous C6-ceramide (Figures 1, 2, and 8) were observed at 10 μM concentration, which is known to produce a cellular concentration of active ceramide very close to that observed in senescent cells (7). Additionally, the signaling function of ceramide is likely to be required for its effect on Aβ generation, as the signaling inactive analog dehydroceramide did not produce any effect on Aβ production in our CHO cells (data not shown). Dehydroceramide is a naturally occurring ceramide that lacks the 4-5 trans double bond, which is required for the signaling activity, but retains the stereochemical configuration of ceramides and is metabolized very similarly to ceramides (13, 18). Cell surface SM is mostly found in cholesterol-rich domains (CRD), which are specialized membrane microdomains highly enriched in SM, cholesterol, and the glycosphingolipid GMl (23). Hydrolysis of SM has been reported to reduce the clustering of cholesterol into CRD and to induce retro-transport of cholesterol from the plasma membrane to the endoplasmic reticulum (ER) (23). In the ER excess cholesterol activates the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT), which we have recently implicated with Aβ generation (12). However, ACAT activation follows retro-transport of cholesterol, which occurs only in the presence substantial SM hydrolysis and massive sterol mobilization from CRD (23). In our study, both nSMase and FBI reduced the clustering of cholesterol into CRD (data not shown) but they only induced a modest mobilization of sterols, which was not accompanied by ACAT activation (data not shown). In addition, although FBI and nSMase had similar effects on SM levels and on cholesterol distribution between membrane microdomains, they produced opposite effects on Aβ generation. Those effects paralleled the changes in ceramide levels and could be reproduced by C6-ceramide. Finally, the reduction in Aβ biosynthesis produced by FBI treatment was reversed by C6-ceramide, confirming that indeed ceramide was responsible for the changes observed in Aβ generation. BACEl is a type I integral membrane protein with an aspartyl protease motif in its lumenal domain that fulfills most of the requirements expected for a candidate β-secretase (24). It is highly expressed in brain and neurons, and colocalizes with Golgi and endosomal markers. BACEl is the primary brain β-secretase and is highly increased, in both protein levels and enzymatic activity, in the neocortex of AD patients (25, 26). Disraption of BACEl in AD transgenic mice almost completely abolished β-cleavage of APP, together with the ability to generate Aβ (27), further confirming that BACEl is indeed the long-searched APP β-secretase. Even if much is known about intracellular trafficking and post-translational modifications of BACEl, very little or nothing is known about regulation of BACE expression/activity. Very recently Tamagno et al. have shown that oxidative stress is able to increase the expression of BACEl, together with β-cleavage of APP (28). In this previous work, BACEl activation involved cell damage and most likely required transcriptional activation of BACEl. In contrast, our results indicate that the ceramide-dependent regulation of BACEl expression occurs, at least in part, at the level of protein degradation. Whether ceramide also affects transcription and/or translation of BACEl remains to be further analyzed. However, it is worth mentioning that for the stable transfection of CHO_BA_CEI cells we only used the coding region of BACEl, eliminating potential 5'- and 3 '-regulatory elements. In conclusion, our study implicates for the first time the lipid second messenger ceramide in the generation of Aβ and proposes ceramide as a potential novel link between AD and aging. It also shows that ceramide regulates β-secretase activity at the level of BACEl stabilization. Identification of the downstream molecules that mediate the ceramide- dependent regulation of BACEl turnover may provide novel targets for the therapeutic treatment of AD patients.

REFERENCES

1. Morris, J. C. (1999) J Clin Invest 104, 1171-1173.

2. Selkoe, D. J. (1999) Nature 399, A23-31. 3. Venable, M. E., and Obeid, L. M. (1999) Biochim Biophys Acta 1439, 291-298.

4. Cutler, R. G., and Mattson, M. P. (2001) Mech Ageing Dev 111, 895-908.

5. Merrill, A. H. Jr (1994) In: Current Topics in Membranes. Cell Lipids (Hoekstra, D., Ed.), Academic Press, New York, NY. 6. Venkataraman, K., and Futerman, A. H. (2000) Trends Cell Biol 10, 408-412.

7. Venable, M. E., Lee, J. Y., Smyth, M. J., Bielawska, A., and Obeid, L. M. (1995) J Biol Chem 270, 30701-30708.

8. Lightle, S. A., Oakley, J. I., and Nikolova-Karakashian, M. N. (2000) Mech Ageing De 120, 111-125. 9. Mouton, R. E., and Venable, M. E. (2000) Mech Ageing Dev 113, 169-181.

10. Miller, C. J., and Stein, G. H. (2001) JGerontolA Biol Sci MedSci 56, B8-19.

11. Han, X., Holtzman, D. M., McKeel, D. W. Jr., and Morris, J. C. (2002) JNeurochem 82, 809-818.

12. Puglielli, L., Konopka, G., Pack-Chung, E., Ingano, L. A., Berezovska, O., Hyman, B. T., Chang, T. Y., Tanzi, R. E., and Kovacs, D. M. (2001) Nat Cell Biol 3, 905-912.

13. de Chaves, E. P., Bussiere, M., Maclnnis, B., Vance, D. E., Campenot, R. B., and Vance, J. E. (2001) JBiol Chem 276, 36207-36214.

14. Brann, A. B., Scott, R., Νeuberger, Y., Abulafia, D., Boldin, S., Fainzilber, M., and Futerman, A. H. (1999) JNeurosci 19, 8199-8206. 15. Νuna, J., and Small, D. H. (2000) FEBS Lett 483, 6-10.

16. Tepper, A. D., Diks, S. H., van Blitterswijk, W. J., and Borst, J. (2000) JBiol Chem 275, 34810-34817.

17. Platt, F. M., Reinkensmeier, G., Dwek, R. A., and Butters, T. D. (1997) JBiol Chem 272, 19365-19372. 18. Irie, F., and Hirabayashi, Y. (1998) JNeurosci Res 54, 475-485.

19. Ito, A., and Horigome, K. (1995) JNeurochem 65, 463-466.

20. DeFreitas, M. F., McQuillen, P. S., and Shatz, C. J. (2001) JNeurosci 21, 5121-5129.

21. Brann, A. B., Tcherpakov, M., Williams, I. M., Futerman, A. H., and Fainzilber, M. (2002) JBiol Chem 111, 9812-9818. 22. Mauch, D. H., Νagler, K., Schumacher, S., Goritz, C, Muller, E. C, Otto, A., and Pfrieger, F. W. (2001) Science 294, 1354-1357. 23. Slotte, J. P, Pom, M. S., and Harmala, A-S. (1994) In: Current Topics in Membranes. Cell Lipids (Hoekstra, D., Ed.), Academic Press, New York, NY. 24. Vassar, R., and Citron, M. (2000) Neuron 27, 419-422.

25. Fukumoto, H., Cheung, B.S., Hyman, B.T., and Irizarry, M.C. (2002) Arch Neurol 59, 1381-1389.

26. Holsinger, R.M.D.m McLean, C.A., Beyreuther, K., Masters, C.L., and Evin, G. (2002) Ann Neurol 51, 783-786.

27. Luo, Y., Bolon, B., Kahn, S., Bennett, B.D., Babu-Khan, S., Denis, P., Fan, W., Kha, H., Zhang, J., Gong, Y., Martin, L., Louis, J-C, Yan, Q., Richards, W.G., Citron, M., and Vassar, R. (2001) Nat Neurosci 4, 231-232.

28. Tamagno, E., Bardini, P., Obbili, A., Vitali, A., Borghi, R., Zaccheo, D., Pronzato, M.A., Danni, O., Smith, M.A., Perry, G., and Tabaton, M. (2002) Neurobiol Dis 10, 279-288.

EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. All references, including patent documents, disclosed herein are incorporated by reference in their entirety.

We claim:

Claims

1. A method for identifying compounds that inhibits BACE stabilization induced by ceramide, comprising providing a reaction mixture that comprises BACE and ceramide, contacting the reaction mixture with a test compound, determining BACE stability in the absence and in the presence of the test compound, and comparing the BACE stability in the absence and in the presence of the test compound, wherein a test compound that reduces BACE stability below the stability in the absence of the test compound is a compound that inhibits BACE stability.

2. The method of claim 1, wherein BACE stability is determined by measuring the turnover rate of the BACE protein.

3. The method of claim 1 , wherein BACE stability is determined by measuring Aβ production from APP and/or production of APP-C99, the C-terminal product of BACE cleavage.

4. The method of claim 1, wherein the reaction mixture is a cell that contains both BACE and ceramide.

5. The method of claim 4, wherein the level of ceramide is increased in the cell relative to a control cell.

6. The method of claim 5, wherein the level of ceramide is increased by contacting the cell with a compound that increases ceramide.

7. The method of claim 6, wherein the compound is ceramide or an active analog or derivative thereof.

8. The method of claim 7, wherein the active analog or derivative is C6-ceramide.

9. The method of claim 6, wherein the compound is an enzyme that increases the level of ceramide in the cell, or a nucleic acid that expresses the enzyme.

10. The method of claim 9, wherein the enzyme is a biosynthetic enzyme.

11. The method of claim 9, wherein the enzyme is a sphingomyelinase.

12. The method of claim 4, wherein the cell is a neuronal cell.

13. The method of claim 11 , wherein the neuronal cell manifests phenotypic characteristics of neuronal cell of a subject identified as having Alzheimer's disease.

14. The method of claim 1 , wherein the method is performed simultaneously with a plurality of compounds.

15. The method of claim 14, wherein the plurality of compounds is contacted with the same reaction mixture.

16. The method of claim 14, wherein each of the plurality of compounds is contacted with a different reaction mixture.

17. The method of claim 14, wherein the plurality of compounds is a compound library.

18. The method of claim 17, wherein the compound library is prepared using combinatorial chemistry.

19. A method for reducing Aβ production and/or production of APP-C99 in a cell, comprising contacting the cell with an amount of a compound effective to decrease the amount of ceramide in the cell, wherein the decrease in the amount of ceramide reduces ceramide- dependent stabilization of BACE.

20. The method of claim 19, wherein the compound is a sphingomyelinase inhibitor.

21. The method of claim 20 wherein the sphingomyelinase inhibitor is manumycin A, scyphostatin, or an analog or derivative thereof that inhibits sphingomyelinase.

22. The method of claim 19, wherein the compound is an inhibitor of the de novo biosynthesis of ceramide.

23. The method of claim 22, wherein the compound is fumonisin Bl or an analog or derivative thereof that inhibits ceramide biosynthesis.

24. A method for treating Alzheimer's disease comprising administering to a subject a therapeutically effective amount of a composition that decreases the amount of ceramide in the brain of the subject.

25. The method of claim 24, wherein the compound is a sphingomyelinase inhibitor.

26. The method of claim 25 wherein the sphingomyelinase inhibitor is manumycin A, scyphostatin, or an analog or derivative thereof that inhibits sphingomyelinase.

27. The method of claim 24, wherein the compound is an inhibitor of the de novo biosynthesis of ceramide.

28. The method of claim 27, wherein the compound is fumonisin B 1 or an analog or derivative thereof that inhibits ceramide biosynthesis.