The stress response neuropeptide CRF increases amyloid-ß production by regulating γ-secretase activity.

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid-ß (Aß) production. In cells, CRF treatment increases Aß production and triggers CRF receptor 1 (CRFR1) and γ-secretase internalization. Co-immunoprecipitation studies establish that γ-secretase associates with CRFR1; this is mediated by ß-arrestin binding motifs. Additionally, CRFR1 and γ-secretase co-localize in lipid raft fractions, with increased γ-secretase accumulation upon CRF treatment. CRF treatment also increases γ-secretase activity in vitro, revealing a second, receptor-independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ-secretase activity. Unexpectedly, CRFR1 antagonists also increased Aß. These data collectively link CRF to increased Aß through γ-secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aß and in some cases preferentially increase Aß42 via complex effects on γ-secretase.

γ-Secretase processing and effects of γ-secretase inhibitors and modulators on long Aß peptides in cells.

Understanding how different species of Aß are generated by γ-secretase cleavage has broad therapeutic implications, because shifts in γ-secretase processing that increase the relative production of Aßx-42/43 can initiate a pathological cascade, resulting in Alzheimer disease. We have explored the sequential stepwise γ-secretase cleavage model in cells. Eighteen BRI2-Aß fusion protein expression constructs designed to generate peptides from Aß1-38 to Aß1-55 and C99 (CTFß) were transfected into cells, and Aß production was assessed. Secreted and cell-associated Aß were detected using ELISA and immunoprecipitation MALDI-TOF mass spectrometry. Aß peptides from 1-38 to 1-55 were readily detected in the cells and as soluble full-length Aß proteins in the media. Aß peptides longer than Aß1-48 were efficiently cleaved by γ-secretase and produced varying ratios of Aß1-40:Aß1-42. γ-Secretase cleavage of Aß1-51 resulted in much higher levels of Aß1-42 than any other long Aß peptides, but the processing of Aß1-51 was heterogeneous with significant amounts of shorter Aßs, including Aß1-40, produced. Two PSEN1 variants altered Aß1-42 production from Aß1-51 but not Aß1-49. Unexpectedly, long Aß peptide substrates such as Aß1-49 showed reduced sensitivity to inhibition by γ-secretase inhibitors. In contrast, long Aß substrates showed little differential sensitivity to multiple γ-secretase modulators. Although these studies further support the sequential γ-secretase cleavage model, they confirm that in cells the initial γ-secretase cleavage does not precisely define subsequent product lines. These studies also raise interesting issues about the solubility and detection of long Aß, as well as the use of truncated substrates for assessing relative potency of γ-secretase inhibitors.

Reversible pathologic and cognitive phenotypes in an inducible model of Alzheimer-amyloidosis.

Transgenic mice that express mutant amyloid precursor protein (APPsi) using tet-Off vector systems provide an alternative model for assessing short- and long-term effects of Aß-targeting therapies on phenotypes related to the deposition of Alzheimer-type amyloid. Here we use such a model, termed APPsi:tTA, to determine what phenotypes persist in mice with high amyloid burden after new production of APP/Aß has been suppressed. We find that 12- to 13-month-old APPsi:tTA mice are impaired in cognitive tasks that assess short- and long-term memories. Acutely suppressing new APPsi/Aß production produced highly significant improvements in performing short-term spatial memory tasks, which upon continued suppression translated to superior performance in more demanding tasks that assess long-term spatial memory and working memory. Deficits in episodic-like memory and cognitive flexibility, however, were more persistent. Arresting mutant APPsi production caused a rapid decline in the brain levels of soluble APP ectodomains, full-length APP, and APP C-terminal fragments. As expected, amyloid deposits persisted after new APP/Aß production was inhibited, whereas, unexpectedly, we detected persistent pools of solubilizable, relatively mobile, Aß42. Additionally, we observed persistent levels of Aß-immunoreactive entities that were of a size consistent with SDS-resistant oligomeric assemblies. Thus, in this model with significant amyloid pathology, a rapid amelioration of cognitive deficits was observed despite persistent levels of oligomeric Aß assemblies and low, but detectable solubilizable Aß42 peptides. These findings implicate complex relationships between accumulating Aß and activities of APP, soluble APP ectodomains, and/or APP C-terminal fragments in mediating cognitive deficits in this model of amyloidosis.

Complex relationships between substrate sequence and sensitivity to alterations in γ-secretase processivity induced by γ-secretase modulators.

γ-Secretase catalyzes the final cleavage of the amyloid precursor protein (APP), resulting in the production of amyloid-ß (Aß) peptides with different carboxyl termini. Presenilin (PSEN) and amyloid precursor protein (APP) mutations linked to early onset familial Alzheimer's disease modify the profile of Aß isoforms generated, by altering both the initial γ-secretase cleavage site and subsequent processivity in a manner that leads to increased levels of the more amyloidogenic Aß42 and in some circumstances Aß43. Compounds termed γ-secretase modulators (GSMs) and inverse GSMs (iGSMs) can decrease and increase levels of Aß42, respectively. As GSMs lower the level of production of pathogenic forms of long Aß isoforms, they are of great interest as potential Alzheimer's disease therapeutics. The factors that regulate GSM modulation are not fully understood; however, there is a growing body of evidence that supports the hypothesis that GSM activity is influenced by the amino acid sequence of the γ-secretase substrate. We have evaluated whether mutations near the luminal border of the transmembrane domain (TMD) of APP alter the ability of both acidic, nonsteroidal anti-inflammatory drug-derived carboxylate and nonacidic, phenylimidazole-derived classes of GSMs and iGSMs to modulate γ-secretase cleavage. Our data show that point mutations can dramatically reduce the sensitivity to modulation of cleavage by GSMs but have weaker effects on iGSM activity. These studies support the concept that the effect of GSMs may be substrate selective; for APP, it is dependent on the amino acid sequence of the substrate near the junction of the extracellular domain and luminal segment of the TMD.

γ-Secretase inhibitors and modulators.

γ-Secretase is a fascinating, multi-subunit, intramembrane cleaving protease that is now being considered as a therapeutic target for a number of diseases. Potent, orally bioavailable γ-secretase inhibitors (GSIs) have been developed and tested in humans with Alzheimer's disease (AD) and cancer. Preclinical studies also suggest the therapeutic potential for GSIs in other disease conditions. However, due to inherent mechanism based-toxicity of non-selective inhibition of γ-secretase, clinical development of GSIs will require empirical testing with careful evaluation of benefit versus risk. In addition to GSIs, compounds referred to as γ-secretase modulators (GSMs) remain in development as AD therapeutics. GSMs do not inhibit γ-secretase, but modulate γ-secretase processivity and thereby shift the profile of the secreted amyloid ß peptides (Aß) peptides produced. Although GSMs are thought to have an inherently safe mechanism of action, their effects on substrates other than the amyloid ß protein precursor (APP) have not been extensively investigated. Herein, we will review the current state of development of GSIs and GSMs and explore pertinent biological and pharmacological questions pertaining to the use of these agents for select indications. This article is part of a Special Issue entitled: Intramembrane Proteases.

Steroids as γ-secretase modulators.

Aggregation and accumulation of Aß42 play an initiating role in Alzheimer's disease (AD); thus, selective lowering of Aß42 by γ-secretase modulators (GSMs) remains a promising approach to AD therapy. Based on evidence suggesting that steroids may influence Aß production, we screened 170 steroids at 10 µM for effects on Aß42 secreted from human APP-overexpressing Chinese hamster ovary cells. Many acidic steroids lowered Aß42, whereas many nonacidic steroids actually raised Aß42. Studies on the more potent compounds showed that Aß42-lowering steroids were bonafide GSMs and Aß42-raising steroids were inverse GSMs. The most potent steroid GSM identified was 5ß-cholanic acid (EC50=5.7 µM; its endogenous analog lithocholic acid was virtually equipotent), and the most potent inverse GSM identified was 4-androsten-3-one-17ß-carboxylic acid ethyl ester (EC50=6.25 µM). In addition, we found that both estrogen and progesterone are weak inverse GSMs with further complex effects on APP processing. These data suggest that certain endogenous steroids may have the potential to act as GSMs and add to the evidence that cholesterol, cholesterol metabolites, and other steroids may play a role in modulating Aß production and thus risk for AD. They also indicate that acidic steroids might serve as potential therapeutic leads for drug optimization/development.

Retention in endoplasmic reticulum 1 (RER1) modulates amyloid-ß (Aß) production by altering trafficking of γ-secretase and amyloid precursor protein (APP).

BACKGROUND: Aß production is influenced by intracellular trafficking of secretases and amyloid precursor protein (APP). RESULTS: Retention in endoplasmic reticulum 1 (RER1) regulates the trafficking of γ-secretase and APP, thereby influences Aß production. CONCLUSION: RER1, an ER retention/retrieval factor for γ-secretase and APP, modulates Aß production. SIGNIFICANCE: RER1 and its influence on γ-secretase and APP may be implicated for a safe strategy to target Aß production. The presence of neuritic plaques containing aggregated amyloid-ß (Aß) peptides in the brain parenchyma is a pathological hallmark of Alzheimer disease (AD). Aß is generated by sequential cleavage of the amyloid ß precursor protein (APP) by ß- and γ-secretase, respectively. As APP processing to Aß requires transport through the secretory pathway, trafficking of the substrate and access to the secretases are key factors that can influence Aß production (Thinakaran, G., and Koo, E. H. (2008) Amyloid precursor protein trafficking, processing, and function. J. Biol. Chem. 283, 29615-29619). Here, we report that retention in endoplasmic reticulum 1 (RER1) associates with γ-secretase in early secretory compartments and regulates the intracellular trafficking of γ-secretase. RER1 overexpression decreases both γ-secretase localization on the cell surface and Aß secretion and conversely RER1 knockdown increases the level of cell surface γ-secretase and increases Aß secretion. Furthermore, we find that increased RER1 levels decrease mature APP and increase immature APP, resulting in less surface accumulation of APP. These data show that RER1 influences the trafficking and localization of both γ-secretase and APP, thereby regulating the production and secretion of Aß peptides.

Cathepsins B and L differentially regulate amyloid precursor protein processing.

Previous studies have shown that cathepsins control amyloid beta (Abeta) levels in chromaffin cells via a regulated secretory pathway. In the present study, this concept was extended to investigations in primary hippocampal neurons to test whether Abeta release was coregulated by cathepsins and electrical activity, proposed components of a regulated secretory pathway. Inhibition of cathepsin B (catB) activity with CA074Me or attenuation of catB expression through small interfering RNA produced decreases in Abeta release, similar to levels produced with suppression of beta-site APP-cleaving enzyme 1 (BACE1) expression. To test whether the catB-dependent release of Abeta was linked to ongoing electrical activity, neurons were treated with tetrodotoxin (TTX) and CA074Me. These comparisons demonstrated no additivity between decreases in Abeta release produced by TTX and CA074Me. In contrast, pharmacological inhibition of cathepsin L (catL) selectively elevated Abeta42 levels but not Abeta40 or total Abeta. Mechanistic studies measuring C-terminal fragments of amyloid precursor protein (APP) suggested that catL elevated alpha-secretase activity, thereby suppressing Abeta42 levels. The mechanism of catB-mediated regulation of Abeta release remains unclear but may involve elevation of beta-secretase. In summary, these studies provide evidence for a significant alternative pathway for APP processing that involves catB and activity-dependent release of Abeta in a regulated secretory pathway for primary neurons.

Carbamate-appended N-alkylsulfonamides as inhibitors of gamma-secretase.

The synthesis and gamma-secretase inhibition data for a series of carbamate-appended N-alkylsulfonamides are described. Carbamate 54 was found to significantly reduce brain Abeta in transgenic mice. 54 was also found to possess markedly improved brain levels in transgenic mice compared to previously disclosed 1 and 2.

Nitrogen-appended N-alkylsulfonamides as inhibitors of gamma-secretase.

The synthesis and gamma-secretase inhibition data for a series of nitrogen-appended N-alkylsulfonamides (11-47) are described. Inhibition of brain Abeta in transgenic mice was demonstrated by two of these compounds (23 and 44).

A comparative analysis of brain and plasma Abeta levels in eight common non-transgenic mouse strains: validation of a specific immunoassay for total rodent Abeta.

Transgenic mouse models of Alzheimer's disease (AD) are being utilized as models for elucidating AD etiology and potential therapeutic approaches. However, two major drawbacks of these models are: (1) transgenic animals often over-express amyloid beta (Abeta) to high levels compared to that seen in sporadic human AD and (2) the current intellectual property issues surrounding a number of these models make them difficult to utilize in a commercial setting. Our goal was to identify an appropriate non-transgenic mouse strain, devoid of these patent restrictions and test whether amyloid-modulating compounds will lower total brain and plasma Abeta. Plasma and brain samples were collected from eight commonly used mouse strains (C57BL/6, SJL, CF-1, DBA/2, CD-1, 129, FVB and B6D2F1; Charles River Labs) and total Abetalevels were validated and quantified with a rodent-specific monoclonal Abetaantibody. Plasma Abeta in SJL mice was the highest of the eight strains tested (213 pM +/- 21 pM), but was not significantly different than the seven other strains. Total brain Abeta in SJL mice was also the greatest of the mouse strains tested (356 pM +/- 73 pM). SJL, C57BL/6 and CF-1 mice had total brain Abeta levels that were significantly greater than Abeta levels in B6D2F1 mice (242 +/- 20 pM). In vivo efficacy of an Abeta lowering agent was observed in CF-1 mice upon oral administration of the gamma-secretase inhibitors, DAPT and LY-411575. The absolute levels of rodent brain Abeta detected and the efficacy of the gamma-secretase treatment were dependent upon the antibodies used, as well as the extraction methodology. The measurement of total brain Abeta lowering in a common mouse strain could help accelerate drug discovery programs for Alzheimer's disease without relying on costly transgenic animals that overexpress APP in a manner that may not be predictive of the effects of these compounds in human AD.

Differential Inhibition of Signal Peptide Peptidase Family Members by Established γ-Secretase Inhibitors.

The signal peptide peptidases (SPPs) are biomedically important proteases implicated as therapeutic targets for hepatitis C (human SPP, (hSPP)), plasmodium (Plasmodium SPP (pSPP)), and B-cell immunomodulation and neoplasia (signal peptide peptidase like 2a, (SPPL2a)). To date, no drug-like, selective inhibitors have been reported. We use a recombinant substrate based on the amino-terminus of BRI2 fused to amyloid ß 1-25 (Aß1-25) (FBA) to develop facile, cost-effective SPP/SPPL protease assays. Co-transfection of expression plasmids expressing the FBA substrate with SPP/SPPLs were conducted to evaluate cleavage, which was monitored by ELISA, Western Blot and immunoprecipitation/MALDI-TOF Mass spectrometry (IP/MS). No cleavage is detected in the absence of SPP/SPPL overexpression. Multiple γ-secretase inhibitors (GSIs) and (Z-LL)2 ketone differentially inhibited SPP/SPPL activity; for example, IC50 of LY-411,575 varied from 51±79 nM (on SPPL2a) to 5499±122 nM (on SPPL2b), while Compound E showed inhibition only on hSPP with IC50 of 1465±93 nM. Data generated were predictive of effects observed for endogenous SPPL2a cleavage of CD74 in a murine B-Cell line. Thus, it is possible to differentially inhibit SPP family members. These SPP/SPPL cleavage assays will expedite the search for selective inhibitors. The data also reinforce similarities between SPP family member cleavage and cleavage catalyzed by γ-secretase.

Cholestenoic acid, an endogenous cholesterol metabolite, is a potent γ-secretase modulator.

BACKGROUND: Amyloid-ß (Aß) 42 has been implicated as the initiating molecule in the pathogenesis of Alzheimer's disease (AD); thus, therapeutic strategies that target Aß42 are of great interest. γ-Secretase modulators (GSMs) are small molecules that selectively decrease Aß42. We have previously reported that many acidic steroids are GSMs with potencies ranging in the low to mid micromolar concentration with 5ß-cholanic acid being the most potent steroid identified GSM with half maximal effective concentration (EC50) of 5.7 µM. RESULTS: We find that the endogenous cholesterol metabolite, 3ß-hydroxy-5-cholestenoic acid (CA), is a steroid GSM with enhanced potency (EC50 of 250 nM) relative to 5ß-cholanic acid. CA i) is found in human plasma at ~100-300 nM concentrations ii) has the typical acidic GSM signature of decreasing Aß42 and increasing Aß38 levels iii) is active in in vitro γ-secretase assay iv) is made in the brain. To test if CA acts as an endogenous GSM, we used Cyp27a1 knockout (Cyp27a1-/-) and Cyp7b1 knockout (Cyp7b1-/-) mice to investigate if manipulation of cholesterol metabolism pathways relevant to CA formation would affect brain Aß42 levels. Our data show that Cyp27a1-/- had increased brain Aß42, whereas Cyp7b1-/- mice had decreased brain Aß42 levels; however, peripheral dosing of up to 100 mg/kg CA did not affect brain Aß levels. Structure-activity relationship (SAR) studies with multiple known and novel CA analogs studies failed to reveal CA analogs with increased potency. CONCLUSION: These data suggest that CA may act as an endogenous GSM within the brain. Although it is conceptually attractive to try and increase the levels of CA in the brain for prevention of AD, our data suggest that this will not be easily accomplished.

Emerging beta-amyloid therapies for the treatment of Alzheimer's disease.

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder marked by loss of memory, cognition, and behavioral stability. AD is defined pathologically by extracellular neuritic plaques comprised of fibrillar deposits of beta-amyloid peptide (Abeta) and neurofibrillary tangles comprised of paired helical filaments of hyperphosphorylated tau. Current therapies for AD, such as cholinesterase inhibitors, treat the symptoms but do not modify the progression of the disease. The etiology of AD is unclear. However, data from familial AD mutations (FAD) strongly support the "amyloid cascade hypothesis" of AD, i.e. that neurodegeneration in AD is initiated by the formation of neurotoxic beta-amyloid (Abeta) aggregates; all FAD mutations increase levels of Abeta peptide or density of Abeta deposits. The likely link between Abeta aggregation and AD pathology emphasizes the need for a better understanding of the mechanisms of Abeta production. This review summarizes current therapeutic strategies directed at lowering Abeta levels and decreasing levels of toxic Abeta aggregates through (1) inhibition of the processing of amyloid precursor protein (APP) to Abeta peptide, (2) inhibition, reversal or clearance of Abeta aggregation, (3) cholesterol reduction and (4) Abeta immunization.

Regenerative medicine in Alzheimer's disease.

Identifying novel, effective therapeutics for Alzheimer's disease (AD) is one of the major unmet medical needs for the coming decade. Because the current paradigm for developing and testing disease-modifying AD therapies is protracted and likely to be even longer, with the shift toward earlier intervention in preclinical AD, it is an open issue whether we can develop, test, and widely deploy a novel therapy in time to help the current at-risk generation if we continue to follow the standard paradigms of discovery and drug development. There is an imperative need to find safe and effective preventive measures that can be distributed rapidly to stem the coming wave of AD that will potentially engulf the next generation. We can define regenerative medicine broadly as approaches that use stem cell-based therapies or approaches that seek to modulate inherent neurogenesis. Neurogenesis, although most active during prenatal development, has been shown to continue in several small parts of the brain, including the hippocampus and the subventricular zone, suggesting its potential to reverse cognitive deficits. If AD pathology affects neurogenesis, then it follows that conditions that stimulate endogenous neurogenesis (eg, environmental stimuli, physical activity, trophic factors, cytokines, and drugs) may help to promote the regenerative and recovery process. Herein, we review the complex logistics of potentially implementing neurogenesis-based therapeutic strategies for the treatment of AD.

Independent relationship between amyloid precursor protein (APP) dimerization and γ-secretase processivity.

Altered production of ß-amyloid (Aß) from the amyloid precursor protein (APP) is closely associated with Alzheimer's disease (AD). APP has a number of homo- and hetero-dimerizing domains, and studies have suggested that dimerization of ß-secretase derived APP carboxyl terminal fragment (CTFß, C99) impairs processive cleavage by γ-secretase increasing production of long Aßs (e.g., Aß1-42, 43). Other studies report that APP CTFß dimers are not γ-secretase substrates. We revisited this issue due to observations made with an artificial APP mutant referred to as 3xK-APP, which contains three lysine residues at the border of the APP ectodomain and transmembrane domain (TMD). This mutant, which dramatically increases production of long Aß, was found to form SDS-stable APP dimers, once again suggesting a mechanistic link between dimerization and increased production of long Aß. To further evaluate how multimerization of substrate affects both initial γ-secretase cleavage and subsequent processivity, we generated recombinant wild type- (WT) and 3xK-C100 substrates, isolated monomeric, dimeric and trimeric forms of these proteins, and evaluated both ε-cleavage site utilization and Aß production. These show that multimerization significantly impedes γ-secretase cleavage, irrespective of substrate sequence. Further, the monomeric form of the 3xK-C100 mutant increased long Aß production without altering the initial ε-cleavage utilization. These data confirm and extend previous studies showing that dimeric substrates are not efficient γ-secretase substrates, and demonstrate that primary sequence determinants within APP substrate alter γ-secretase processivity.

Comment on "ApoE-directed therapeutics rapidly clear ß-amyloid and reverse deficits in AD mouse models".

Cramer et al. (Reports, 23 March 2012, p. 1503; published online 9 February 2012) demonstrates short-term bexarotene treatment clearing preexisting ß-amyloid deposits from the brains of APP/PS1ΔE9 mice with low amyloid burden, providing a rationale for repurposing this anticancer agent as an Alzheimer's disease (AD) therapeutic. Using a nearly identical treatment regimen, we were unable to detect any evidence of drug efficacy despite demonstration of target engagement.

Cyanobacterial peptides as a prototype for the design of potent ß-secretase inhibitors and the development of selective chemical probes for other aspartic proteases.

Inspired by marine cyanobacterial natural products, we synthesized modified peptides with a central statine-core unit, characteristic for aspartic protease inhibition. A series of tasiamide B analogues inhibited BACE1, a therapeutic target in Alzheimer's disease. We probed the stereospecificity of target engagement and determined additional structure-activity relationships with respect to BACE1 and related aspartic proteases, cathepsins D and E. We cocrystallized selected inhibitors with BACE1 to reveal the structural basis for the activity. Hybrid molecules that combine features of tasiamide B and an isophthalic acid moiety-containing sulfonamide showed nanomolar cellular activity. Compounds were screened in a series of rigorous complementary cell-based assays. We measured secreted APP ectodomain (sAPPß), membrane bound carboxyl terminal fragment (CTF), levels of ß-amyloid (Aß) peptides and selectivity for ß-secretase (BACE1) over γ-secretase. Prioritized compounds showed reasonable stability in vitro and in vivo, and our most potent inhibitor showed efficacy in reducing Aß levels in the rodent brain.

Modulation of γ-secretase by EVP-0015962 reduces amyloid deposition and behavioral deficits in Tg2576 mice.

BACKGROUND: A hallmark of Alzheimer's disease is the presence of senile plaques in human brain primarily containing the amyloid peptides Aß42 and Aß40. Many drug discovery efforts have focused on decreasing the production of Aß42 through γ-secretase inhibition. However, identification of γ-secretase inhibitors has also uncovered mechanism-based side effects. One approach to circumvent these side effects has been modulation of γ-secretase to shift Aß production to favor shorter, less amyloidogenic peptides than Aß42, without affecting the overall cleavage efficiency of the enzyme. This approach, frequently called γ-secretase modulation, appears more promising and has lead to the development of new therapeutic candidates for disease modification in Alzheimer's disease. RESULTS: Here we describe EVP-0015962, a novel small molecule γ-secretase modulator. EVP-0015962 decreased Aß42 in H4 cells (IC50 = 67 nM) and increased the shorter Aß38 by 1.7 fold at the IC50 for lowering of Aß42. AßTotal, as well as other carboxyl-terminal fragments of amyloid precursor protein, were not changed. EVP-0015962 did not cause the accumulation of other γ-secretase substrates, such as the Notch and ephrin A4 receptors, whereas a γ-secretase inhibitor reduced processing of both. A single oral dose of EVP-0015962 (30 mg/kg) decreased Aß42 and did not alter AßTotal peptide levels in a dose-dependent manner in Tg2576 mouse brain at an age when overt Aß deposition was not present. In Tg2576 mice, chronic treatment with EVP-0015962 (20 or 60 mg/kg/day in a food formulation) reduced Aß aggregates, amyloid plaques, inflammatory markers, and cognitive deficits. CONCLUSIONS: EVP-0015962 is orally bioavailable, detected in brain, and a potent, selective γ-secretase modulator in vitro and in vivo. Chronic treatment with EVP-0015962 was well tolerated in mice and lowered the production of Aß42, attenuated memory deficits, and reduced Aß plaque formation and inflammation in Tg2576 transgenic animals. In summary, these data suggest that γ-secretase modulation with EVP-0015962 represents a viable therapeutic alternative for disease modification in Alzheimer's disease.