Generation of Abeta38 and Abeta42 is independently and differentially affected by familial Alzheimer disease-associated presenilin mutations and gamma-secretase modulation

Alzheimer disease amyloid beta-peptide (Abeta) is generated via proteolytic processing of the beta-amyloid precursor protein by beta- and gamma-secretase. Gamma-secretase can be blocked by selective inhibitors but can also be modulated by a subset of non-steroidal anti-inflammatory drugs, including sulindac sulfide. These drugs selectively reduce the generation of the aggregation-prone 42-amino acid Abeta(42) and concomitantly increase the levels of the rather benign Abeta(38). Here we show that Abeta(42) and Abeta(38) generation occur independently from each other. The amount of Abeta(42) produced by cells expressing 10 different familial Alzheimer disease (FAD)-associated mutations in presenilin (PS) 1, the catalytic subunit of gamma-secretase, appeared to correlate with the respective age of onset in patients. However, Abeta(38) levels did not show a negative correlation with the age of onset. Modulation of gamma-secretase activity by sulindac sulfide reduced Abeta(42) in the case of wild type PS1 and two FAD-associated PS1 mutations (M146L and A285V). The remaining eight PS1 FAD mutants showed either no reduction of Abeta(42) or only rather subtle effects. Strikingly, even the mutations that showed no effect on Abeta(42) levels allowed a robust increase of Abeta(38) upon treatment with sulindac sulfide. Similar observations were made for fenofibrate, a compound known to increase Abeta(42) and to decrease Abeta(38). For mutants that predominantly produce Abeta(42), the ability of fenofibrate to further increase Abeta(42) levels became diminished, whereas Abeta(38) levels were altered to varying extents for all mutants analyzed. Thus, we conclude that Abeta(38) and Abeta(42) production do not depend on each other. Using an independent non-steroidal anti-inflammatory drug derivative, we obtained similar results for PS1 as well as for PS2. These in vitro results were confirmed by in vivo experiments in transgenic mice expressing the PS2 N141I FAD mutant. Our findings therefore have strong implications on the selection of transgenic mouse models used for screening of the Abeta(42)-lowering capacity of gamma-secretase modulators. Furthermore, human patients with certain PS mutations may not respond to gamma-secretase modulators.     

Somatostatin regulates brain amyloid beta peptide Abeta42 through modulation of proteolytic degradation

Expression of somatostatin in the brain declines during aging in various mammals including apes and humans. A prominent decrease in this neuropeptide also represents a pathological characteristic of Alzheimer disease. Using in vitro and in vivo paradigms, we show that somatostatin regulates the metabolism of amyloid beta peptide (Abeta), the primary pathogenic agent of Alzheimer disease, in the brain through modulating proteolytic degradation catalyzed by neprilysin. Among various effector candidates, only somatostatin upregulated neprilysin activity in primary cortical neurons. A genetic deficiency of somatostatin altered hippocampal neprilysin activity and localization, and increased the quantity of a hydrophobic 42-mer form of Abeta, Abeta(42), in a manner similar to presenilin gene mutations that cause familial Alzheimer disease. These results indicate that the aging-induced downregulation of somatostatin expression may be a trigger for Abeta accumulation leading to late-onset sporadic Alzheimer disease, and suggest that somatostatin receptors may be pharmacological-target candidates for prevention and treatment of Alzheimer disease.     

FAD mutants unable to increase neurotoxic Abeta 42 suggest that mutation effects on neurodegeneration may be independent of effects on Abeta

Strong support for a primary causative role of the Abeta peptides in the development of Alzheimer's disease (AD) neurodegeneration derives from reports that presenilin familial AD (FAD) mutants alter amyloid precursor protein processing, thus increasing production of neurotoxic Abeta 1-42 (Abeta 42). This effect of FAD mutants is also reflected in an increased ratio of peptides Abeta 42 over Abeta 1-40 (Abeta 40). In the present study, we show that several presenilin 1 FAD mutants failed to increase production of Abeta 42 or the Abeta 42/40 ratio. Our data suggest that the mechanism by which FAD mutations promote neurodegeneration and AD may be independent of their effects on Abeta production.     

APH1, PEN2, and Nicastrin increase Abeta levels and gamma-secretase activity

A major component of the amyloid plaque core in Alzheimer's disease (AD) is the 40-42-residue amyloid beta peptide (Abeta). Mutations linked to AD such as those in presenilins 1 (PS1) and 2 (PS2) invariably increase the longer Abeta42 species that forms neurotoxic oligomers. It is believed that PS1/2 constitute the catalytic subunit of the gamma-secretase responsible for the final step in Abeta biogenesis. Recent genetic studies have identified a number of additional genes encoding APH1a, APH1b, PEN2, and Nicastrin proteins, which are part of the gamma-secretase complex with PS1. Further, knockout studies using RNAi showed that these components are essential for gamma-secretase activity. However, the nature of gamma-secretase and how the aforementioned proteins regulate its activity are still incompletely understood. Here we present evidence that unlike PS1, overexpression of these proteins can increase the levels of Abeta, suggesting that these proteins are limiting for gamma-secretase activity. In addition, our studies also suggest that the presenilin partners regulate the relative levels of Abeta40 and Abeta42.     

{gamma}-Secretase Substrate Concentration Modulates the Abeta42/Abeta40 Ratio: IMPLICATIONS FOR ALZHEIMER DISEASE

Mutation of the amyloid precursor protein (APP), presenilin-1, or presenilin-2 results in the development of early onset autosomal dominant forms of Alzheimer disease (AD). These mutations lead to an increased Abeta42/Abeta40 ratio that correlates with the onset of disease. However, it remains unknown how these mutations affect gamma-secretase, a protease that generates the termini of Abeta40 and Abeta42. Here we have determined the reaction mechanism of gamma-secretase with wild type and three mutated APP substrates. Our findings indicate that despite the overall outcome of an increased Abeta42/Abeta40 ratio, these mutations each display rather distinct reactivity to gamma-secretase. Intriguingly, we found that the ratio of Abeta42/Abeta40 is variable with substrate concentration; increased substrate concentrations result in higher ratios of Abeta42/Abeta40. Moreover, we demonstrated that reduction of gamma-secretase substrate concentration by BACE1 inhibition in cells decreased the Abeta42/Abeta40 ratio. This study indicates that biological factors affecting targets such as BACE1 and APP, which ultimately cause an increased concentration of gamma-secretase substrate, can augment the Abeta42/Abeta40 ratio and may play a causative role in sporadic AD. Therefore, strategies lowering the Abeta42/Abeta40 ratio through partial reduction of gamma-secretase substrate production may introduce a practical therapeutic modality for treatment of AD.     

Nonsteroidal anti-inflammatory drugs lower Abeta42 and change presenilin 1 conformation

Recent reports suggest that some commonly used nonsteroidal anti-inflammatory drugs (NSAIDs) unexpectedly shift the cleavage products of amyloid precursor protein (APP) to shorter, less fibrillogenic forms, although the underlying mechanism remains unknown. We now demonstrate, using a fluorescence resonance energy transfer method, that Abeta(42)-lowering NSAIDs specifically affect the proximity between APP and presenilin 1 and alter presenilin 1 conformation both in vitro and in vivo, suggesting a novel allosteric mechanism of action.     

Formation of tau inclusions in knock-in mice with familial Alzheimer disease (FAD) mutation of presenilin 1 (PS1)

Mutations in the presenilin 1 (PS1) gene are responsible for the early onset of familial Alzheimer disease (FAD). Accumulating evidence shows that PS1 is involved in gamma-secretase activity and that FAD-associated mutations of PS1 commonly accelerate Abeta(1-42) production, which causes Alzheimer disease (AD). Recent studies suggest, however, that PS1 is involved not only in Abeta production but also in other processes that lead to neurodegeneration. To better understand the causes of neurodegeneration linked to the PS1 mutation, we analyzed the development of tau pathology, another key feature of AD, in PS1 knock-in mice. Hippocampal samples taken from FAD mutant (I213T) PS1 knock-in mice contained hyperphosphorylated tau that reacted with various phosphodependent tau antibodies and with Alz50, which recognizes the conformational change of PHF tau. Some neurons exhibited Congo red birefringence and Thioflavin T reactivity, both of which are histological criteria for neurofibrillary tangles (NFTs). Biochemical analysis of the samples revealed SDS-insoluble tau, which under electron microscopy examination, resembled tau fibrils. These results indicate that our mutant PS1 knock-in mice exhibited NFT-like tau pathology in the absence of Abeta deposition, suggesting that PS1 mutations contribute to the onset of AD not only by enhancing Abeta(1-42) production but by also accelerating the formation and accumulation of filamentous tau.     

Independent generation of Abeta42 and Abeta38 peptide species by gamma-secretase

Proteolytic processing of the amyloid precursor protein by beta- and gamma-secretase generates the amyloid-beta (Abeta) peptides, which are principal drug targets in Alzheimer disease therapeutics. gamma-Secretase has imprecise cleavage specificity and generates the most abundant Abeta40 and Abeta42 species together with longer and shorter peptides such as Abeta38. Several mechanisms could explain the production of multiple Abeta peptides by gamma-secretase, including sequential processing of longer into shorter Abeta peptides. A novel class of gamma-secretase modulators (GSMs) that includes some non-steroidal anti-inflammatory drugs has been shown to selectively lower Abeta42 levels without a change in Abeta40 levels. A signature of GSMs is the concomitant increase in shorter Abeta peptides, such as Abeta38, leading to the suggestion that generation of Abeta42 and Abeta38 peptide species by gamma-secretase is coordinately regulated. However, no evidence for or against such a precursor-product relationship has been provided. We have previously shown that stable overexpression of aggressive presenilin-1 (PS1) mutations associated with early-onset familial Alzheimer disease attenuated the cellular response to GSMs, resulting in greatly diminished Abeta42 reductions as compared with wild type PS1. We have now used this model system to investigate whether Abeta38 production would be similarly affected indicating coupled generation of Abeta42 and Abeta38 peptides. Surprisingly, treatment with the GSM sulindac sulfide increased Abeta38 production to similar levels in four different PS1 mutant cell lines as compared with wild type PS1 cells. This was confirmed with the structurally divergent GSMs ibuprofen and indomethacin. Mass spectrometry analysis and high resolution urea gel electrophoresis further demonstrated that sulindac sulfide did not induce detectable compensatory changes in levels of other Abeta peptide species. These data provide evidence that Abeta42 and Abeta38 species can be independently generated by gamma-secretase and argue against a precursor-product relationship between these peptides.     

Wild-type presenilin 1 protects against Alzheimer disease mutation-induced amyloid pathology

Mutations in presenilin 1 (PS1) lead to dominant inheritance of early onset familial Alzheimer disease (FAD). These mutations are known to alter the gamma-secretase cleavage of the amyloid precursor protein, resulting in increased ratio of Abeta42/Abeta40 and accelerated amyloid plaque pathology in transgenic mouse models. To investigate the factors that drive the Abeta42/Abeta40 ratio and amyloid pathogenesis and to investigate the possible interactions between wild-type and FAD mutant PS1, which are co-expressed in transgenic animals, we expressed the PS1 M146V knock-in allele either on wild-type PS1 (PS1M146V/+) or PS1 null (PS1M146V/-) background and crossed these alleles with the Tg2576 APP transgenic mice. Introduction of the PS1 M146V mutation on Tg2576 background resulted in earlier onset of plaque pathology. Surprisingly, removing the wild-type PS1 in the presence of the PS1 M146V mutation (PS1M146V/-) greatly exacerbated the amyloid burden; and this was attributed to a reduction of gamma-secretase activity rather than an increase in Abeta42. Our findings establish a protective role of the wild-type PS1 against the FAD mutation-induced amyloid pathology through a partial loss-of-function mechanism.     

Loss-of-function presenilin mutations in Alzheimer disease. Talking Point on the role of presenilin mutations in Alzheimer disease

Presenilin mutations are the main cause of familial Alzheimer disease. From a genetic point of view, these mutations seem to result in a gain of toxic function; however, biochemically, they result in a partial loss of function in the gamma-secretase complex, which affects several downstream signalling pathways. Consequently, the current genetic terminology is misleading. In fact, the available data indicate that several clinical presenilin mutations also lead to a decrease in amyloid precursor protein-derived amyloid beta-peptide generation, further implying that presenilin mutations are indeed loss-of-function mutations. The loss of function of presenilin causes incomplete digestion of the amyloid beta-peptide and might contribute to an increased vulnerability of the brain, thereby explaining the early onset of the inherited form of Alzheimer disease. In this review, I evaluate the implications of this model for the amyloid-cascade hypothesis and for the efficacy of presenilin/gamma-secretase as a drug target.     

Diverse compounds mimic Alzheimer disease-causing mutations by augmenting Abeta42 production

Increased Abeta42 production has been linked to the development of Alzheimer disease. We now identify a number of compounds that raise Abeta42. Among the more potent Abeta42-raising agents identified are fenofibrate, an antilipidemic agent, and celecoxib, a COX-2-selective NSAID. Many COX-2-selective NSAIDs tested raised Abeta42, including multiple COX-2-selective derivatives of two Abeta42-lowering NSAIDs. Compounds devoid of COX activity and the endogenous isoprenoids FPP and GGPP also raised Abeta42. These compounds seem to target the gamma-secretase complex, increasing gamma-secretase-catalyzed production of Abeta42 in vitro. Short-term in vivo studies show that two Abeta42-raising compounds increase Abeta42 levels in the brains of mice. The elevations in Abeta42 by these compounds are comparable to the increases in Abeta42 induced by Alzheimer disease-causing mutations in the genes encoding amyloid beta protein precursor and presenilins, raising the possibility that exogenous compounds or naturally occurring isoprenoids might increase Abeta42 production in humans.     

Intracellular site of gamma-secretase cleavage for Abeta42 generation in neuro 2a cells harbouring a presenilin 1 mutation.

Previously, we reported that mutations in presenilin 1 (PS1) increased the intracellular levels of amyloid beta-protein (Abeta)42. However, it is still not known at which cellular site or how PS1 mutations exert their effect of enhancing Abeta42-gamma-secretase cleavage. In this study, to clarify the molecular mechanisms underlying this enhancement of Abeta42-gamma-secretase cleavage, we focused on determining the intracellular site of the cleavage. To address this issue, we used APP-C100 encoding the C-terminal beta-amyloid precursor protein (APP) fragment truncated at the N terminus of Abeta (C100); C100 requires only gamma-secretase cleavage to yield Abeta. Mutated PS1 (M146L)-induced Neuro 2a cells showed enhanced Abeta1-42 generation from transiently expressed C100 as well as from full-length APP, whereas the generation of Abeta1-40 was not increased. The intracellular generation of Abeta1-42 from transiently expressed C100 in both mutated PS1-induced and wild-type Neuro 2a cells was inhibited by brefeldin A. Moreover, the generation of Abeta1-42 and Abeta1-40 from a C100 mutant containing a di-lysine endoplasmic reticulum retention signal was greatly decreased, indicating that the major intracellular site of gamma-secretase cleavage is not the endoplasmic reticulum. The intracellular generation of Abeta1-42/40 from C100 was not influenced by monensin treatment, and the level of Abeta1-42/40 generated from C100 carrying a sorting signal for the trans-Golgi network was higher than that generated from wild-type C100. These results using PS1-mutation-harbouring and wild-type Neuro 2a cells suggest that Abeta42/40-gamma-secretase cleavages occur in the Golgi compartment and the trans-Golgi network, and that the PS1 mutation does not alter the intracelluar site of Abeta42-gamma-secretase cleavage in the normal APP proteolytic processing pathway.     

Antisense-induced reduction of presenilin 1 expression selectively increases the production of amyloid beta42 in transfected cells

Autosomal dominant mutations in the presenilin 1 (PS1) gene are associated with familial, early-onset Alzheimer's disease. Although the pathogenic mechanism of these mutations is unclear, their common feature is that they lead to an increased concentration of amyloid beta-peptide (Abeta) 42 in the plasma of early-onset patients, in the conditioned media of transfected cells, and in the brains of transgenic mice that overexpress mutant PS1. To address the mechanism(s) by which the pathogenic PS1 mutations increase Abeta42, we constructed human cell lines expressing a doxycyclin (dox)-inducible antisense PS1 RNA and measured its effects on the levels of PS1, amyloid precursor protein (APP), and Abeta. In time course experiments, we observed a statistically significant (p = 0.0038) more than twofold elevation in secreted Abeta42 as early as 12 days after addition of dox. This correlated with an 80% decrease in the 46-kDa PS1 holoprotein and a 30% decrease in the 26-kDa N-terminal fragment (NTF). Furthermore, there was a significant fivefold (p = 0.002) increase in Abeta42 after 14-day dox treatment; this correlated with a >90% decrease in PS1 holoprotein and 60% decrease in NTF. At no time point did we observe significant changes in Abeta40, APP holoprotein, presenilin 2, or tubulin. Ten days after the removal of dox, we observed a return to constitutive levels for Abeta42, PS1 holoprotein, and NTF. These results suggest that in human cell lines, the reduction of normal PS1 activity results in the increased production of Abeta42. Furthermore, our results are consistent with a loss of function or dominant negative mechanism for the pathogenic PS1 mutations.     

The presenilin 1 C92S mutation increases abeta 42 production

Although wild-type human presenilin 1 (PS1) rescues the C. elegans egg-laying (egl) phenotype that is caused by a loss of function mutation in the C. elegans presenilin homologue sel12, most familial Alzheimer's disease (FAD)-linked PS1 mutants only partially rescue this phenotype. To investigate the effects of the loss of function sel12 mutation on Abeta production in mammalian cells, we analyzed Abeta production in transfected H4 neuroglioma cells expressing the PS1 homologue of the sel12 C60S mutant, PS1 C92S. This analysis revealed that PS1 C92S increased Abeta42 levels in a similar fashion to other pathogenic Alzheimer's disease (AD) PS1 mutations. Significantly, the PS1 C92S mutation has recently been identified as the pathogenic mutation in an Italian family with FAD. Thus, placing a mutation that results in loss of function in C. elegans into a context whereby its effect on mammalian cells can be evaluated suggests that all FAD-linked PS1 mutants result in increased Abeta42 production through a partial loss of function mechanism.     

Abeta42 overproduction associated with structural changes in the catalytic pore of gamma-secretase: common effects of Pen-2 N-terminal elongation and fenofibrate

gamma-Secretase is an atypical aspartyl protease that cleaves amyloid beta-precursor protein to generate Abeta peptides that are causative for Alzheimer disease. gamma-Secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1, and Pen-2. Pen-2 directly binds to transmembrane domain 4 of PS and confers proteolytic activity on gamma-secretase, although the mechanism of activation and its role in catalysis remain unknown. Here we show that an addition of amino acid residues to the N terminus of Pen-2 specifically increases the generation of Abeta42, the longer and more aggregable species of Abeta. The effect of the N-terminal elongation of Pen-2 on Abeta42 generation was independent of the amino acid sequences, the expression system and the presenilin species. In vitro gamma-secretase assay revealed that Pen-2 directly affects the Abeta42-generating activity of gamma-secretase. The elongation of Pen-2 N terminus caused a reduction in the water accessibility of the luminal side of the catalytic pore of PS1 in a similar manner to that caused by an Abeta42-raising gamma-secretase modulator, fenofibrate, as determined by substituted cysteine accessibility method. These data suggest a unique mechanism of Abeta42 overproduction associated with structural changes in the catalytic pore of presenilins caused commonly by the N-terminal elongation of Pen-2 and fenofibrate.     

Regulation of cholesterol and sphingomyelin metabolism by amyloid-beta and presenilin

Amyloid beta peptide (Abeta) has a key role in the pathological process of Alzheimer's disease (AD), but the physiological function of Abeta and of the amyloid precursor protein (APP) is unknown. Recently, it was shown that APP processing is sensitive to cholesterol and other lipids. Hydroxymethylglutaryl-CoA reductase (HMGR) and sphingomyelinases (SMases) are the main enzymes that regulate cholesterol biosynthesis and sphingomyelin (SM) levels, respectively. We show that control of cholesterol and SM metabolism involves APP processing. Abeta42 directly activates neutral SMase and downregulates SM levels, whereas Abeta40 reduces cholesterol de novo synthesis by inhibition of HMGR activity. This process strictly depends on gamma-secretase activity. In line with altered Abeta40/42 generation, pathological presenilin mutations result in increased cholesterol and decreased SM levels. Our results demonstrate a biological function for APP processing and also a functional basis for the link that has been observed between lipids and Alzheimer's disease (AD).     

Amino-terminally truncated Abeta peptide species are the main component of cotton wool plaques

Cotton wool plaques (CWPs) are round lesions that lack a central amyloid core. CWPs have been observed in individuals affected by early-onset familial Alzheimer disease (FAD) associated with mutations in the presenilin 1 (PSEN1) gene. Here we present the characterization of the amyloid-beta (Abeta) peptides deposited in the brain of an individual affected by FAD carrying the novel missense (V261I) mutation in the PSEN1 gene. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to determine the Abeta peptide species present in the cerebral and cerebellar cortices, in leptomeningeal vessels, and in CWPs isolated by laser microdissection (LMD). Our results indicate that amino-terminally truncated Abeta peptide species ending at residues 42 and 43 are the main Abeta peptides deposited in brain parenchyma and LMD-CWPs in association with the PSEN1 V261I mutation. Full-length Abeta1-42 and Abeta1-43 peptide species were underrepresented. CWPs were not found to be associated with vessels and did not contain Abeta1-40 peptides, the main component of the vascular deposits. Although Abeta deposits were present mostly in the form of CWPs in the cerebral cortex and as diffuse deposits in the cerebellar cortex, a similar array of amino-terminally truncated Abeta peptide species was seen in both cases. The biochemical data support the concept that parenchymal and vascular amyloid deposits are associated with a different array of Abeta peptide species. The generation and parenchymal deposition of highly insoluble amino-terminally truncated Abeta peptides may play an important role in the pathogenesis of AD and must be taken into consideration in developing new diagnostic and therapeutic strategies.     

Attenuated Abeta42 responses to low potency gamma-secretase modulators can be overcome for many pathogenic presenilin mutants by second-generation compounds

Sequential processing of the β-amyloid precursor protein by β- and γ-secretase generates the amyloid β-peptide (Aβ), which is widely believed to play a causative role in Alzheimer disease. Selective lowering of the pathogenic 42-amino acid variant of Aβ by γ-secretase modulators (GSMs) is a promising therapeutic strategy. Here we report that mutations in presenilin (PS), the catalytic subunit of γ-secretase, display differential responses to non-steroidal anti-inflammatory drug (NSAID)-type GSMs and more potent second-generation compounds. Although many pathogenic PS mutations resisted lowering of Aβ(42) generation by the NSAID sulindac sulfide, the potent NSAID-like second-generation compound GSM-1 was capable of lowering Aβ(42) for many but not all mutants. We further found that mutations at homologous positions in PS1 and PS2 can elicit differential Aβ(42) responses to GSM-1, suggesting that a positive GSM-1 response depends on the spatial environment in γ-secretase. The aggressive pathogenic PS1 L166P mutation was one of the few pathogenic mutations that resisted GSM-1, and Leu-166 was identified as a critical residue with respect to the Aβ(42)-lowering response of GSM-1. Finally, we found that GSM-1-responsive and -resistant PS mutants behave very similarly toward other potent second-generation compounds of different structural classes than GSM-1. Taken together, our data show that a positive Aβ(42) response for PS mutants depends both on the particular mutation and the GSM used and that attenuated Aβ(42) responses to low potency GSMs can be overcome for many PS mutants by second generation GSMs.     

Functional implications of the presenilin dimerization: reconstitution of gamma-secretase activity by assembly of a catalytic site at the dimer interface of two catalytically inactive presenilins

Presenilins are the catalytic components of gamma-secretase, an intramembrane-cleaving protease whose substrates include beta-amyloid precursor protein (betaAPP) and the Notch receptors. These type I transmembrane proteins undergo two distinct presenilin-dependent cleavages within the transmembrane region, which result in the production of Abeta and APP intracellular domain (from betaAPP) and the Notch intracellular domain signaling peptide. Most cases of familial Alzheimer's disease are caused by presenilin mutations, which are scattered throughout the coding sequence. Although the underlying molecular mechanism is not yet known, the familial Alzheimer's disease mutations produce a shift in the ratio of the long and short forms of the Abeta peptide generated by the gamma-secretase. We and others have previously shown that presenilin homodimerizes and suggested that a presenilin dimer is at the catalytic core of gamma-secretase. Here, we demonstrate that presenilin transmembrane domains contribute to the formation of the dimer. In-frame substitution of the hydrophilic loop 1, located between transmembranes I and II, which modulates the interactions within the N-terminal fragment/N-terminal fragment dimer, abolishes both presenilinase and gamma-secretase activities. In addition, by reconstituting gamma-secretase activity from two catalytically inactive presenilin aspartic mutants, we provide evidence of an active diaspartyl group assembled at the interface between two presenilin monomers. Under our conditions, this catalytic group mediates the generation of APP intracellular domain and Abeta but not Notch intracellular domain, therefore suggesting that specific diaspartyl groups within the presenilin catalytic core of gamma-secretase mediate the cleavage of different substrates.     

Identification of gamma-secretase inhibitor potency determinants on presenilin

Production of amyloid beta peptides (Abeta), followed by their deposition in the brain as amyloid plaques, contributes to the hallmark pathology of Alzheimer disease. The enzymes responsible for production of Abeta, BACE1 and gamma-secretase, are therapeutic targets for treatment of Alzheimer disease. Two presenilin (PS) homologues, referred to as PS1 and PS2, comprise the catalytic core of gamma-secretase. In comparing presenilin selectivity of several classes of gamma-secretase inhibitors, we observed that sulfonamides in general tend to be more selective for inhibition of PS1-comprising gamma-secretase, as exemplified by ELN318463 and BMS299897. We employed a combination of chimeric constructs and point mutants to identify structural determinants for PS1-selective inhibition by ELN318463. Our studies identified amino acid residues Leu(172), Thr(281), and Leu(282) in PS1 as necessary for PS1-selective inhibition by ELN318463. These residues also contributed in part to the PS1-selective inhibition by BMS299897. Alanine scanning mutagenesis of areas flanking Leu(172), Thr(281), and Leu(282) identified additional amino acids that affect inhibitor potency of not only these sulfonamides but also nonsulfonamide inhibitors, without affecting Abeta production and presenilin endoproteolysis. Interestingly, many of these same residues have been identified previously to be important for gamma-secretase function. These findings implicate TM3 and a second region near the carboxyl terminus of PS1 aminoterminal fragment in mediating the activity of gamma-secretase inhibitors. Our observations demonstrate that PS-selective inhibitors of gamma-secretase are feasible, and such inhibitors may allow differential inhibition of Abeta peptide production and Notch signaling.