Search

A series of analogs based on a prototype aryl aminothiazole γ-secretase modulator (GSM) were synthesized and tested for their effects on the profile of 37-to-42-residue amyloid β-peptides (Aβ), generated through processive proteolysis of precursor protein substrate by γ-secretase. Certain substitutions on the terminal aryl D ring resulted in an altered profile of Aβ production compared to that seen with the parent molecule. Small structural changes led to concentration-dependent increases in Aβ37 and Aβ38 production without parallel decreases in their precursors Aβ40 and Aβ42, respectively. The new compounds therefore apparently also stimulate carboxypeptidase trimming of Aβ peptides ≥ 43 residues, providing novel chemical tools for mechanistic studies of processive proteolysis by γ-secretase., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Dominant missense mutations in the amyloid-β protein precursor (AβPP) cause early-onset familial Alzheimer's disease (FAD) and are associated with changes in the production or properties of the amyloid-β peptide (Aβ), particularly of the 42-residue variant (Aβ42) that deposits in the Alzheimer's disease (AD) brain. Recent findings, however, show that FAD mutations in AβPP also lead to increased production of longer Aβ variants of 45-49 residues in length., Objective: We aimed to test neurotoxicity of Aβ42 vis-á-vis longer variants, focusing specifically on mitochondrial function, as dysfunctional mitochondria are implicated in the pathogenesis of AD., Methods: We generated SH-SY5Y human neuroblastoma cells stably expressing AβPP mutations that lead to increased production of long Aβ peptides with or without Aβ42. These AβPP-expressing cells were tested for oxygen consumption rates (OCR) under different conditions designed to interrogate mitochondrial function. These cell lines were also examined for expression of genes important for mitochondrial or neuronal structure and function., Results: The mutant AβPP-expressing cells showed decreased basal OCRs as well as decreased OCRs associated with mitochondrial ATP production, even more so in the absence of Aβ42 production. Moreover, mutant AβPP-expressing cells producing longer forms of Aβ displayed altered expression of certain mitochondrial- and neuronal-associated genes, whether or not Aβ42 was produced., Conclusion: These findings suggest that mutant AβPP can cause mitochondrial dysfunction that is associated with long Aβ but not with Aβ42.

Production of amyloid β-protein (Aβ) is carried out by the membrane-embedded γ-secretase complex. Mutations in the transmembrane domain of amyloid β-protein precursor (APP) associated with early-onset familial Alzheimer's disease (FAD) can alter the ratio of aggregation-prone 42-residue Aβ (Aβ42) to 40-residue Aβ (Aβ40). However, APP substrate is proteolyzed processively by γ-secretase along two pathways: Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42→Aβ38. Effects of FAD mutations on each proteolytic step are unknown, largely due to difficulties in detecting and quantifying longer Aβ peptides. To address this, we carried out systematic and quantitative analyses of all tri- and tetrapeptide coproducts from proteolysis of wild-type and 14 FAD-mutant APP substrates by purified γ-secretase. These small peptides, including FAD-mutant forms, were detected by tandem mass spectrometry and quantified by establishing concentration curves for each of 32 standards. APP intracellular domain (AICD) coproducts were quantified by immunoblot, and the ratio of AICD products corresponding to Aβ48 and Aβ49 was determined by mass spectrometry. Levels of individual Aβ peptides were determined by subtracting levels of peptide coproducts associated with degradation from those associated with production. This method was validated for Aβ40 and Aβ42 by specific ELISAs and production of equimolar levels of Aβ and AICD. Not all mutant substrates led to increased Aβ42/40. However, all 14 disease-causing mutations led to inefficient processing of longer forms of Aβ ≥ 45 residues. In addition, the effects of certain mutations provided insight into the mechanism of processive proteolysis: intermediate Aβ peptides apparently remain bound for subsequent trimming and are not released and reassociated., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

γ-Secretase is responsible for the proteolysis of amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides, which are centrally implicated in the pathogenesis of Alzheimer's disease (AD). The biochemical mechanism of how processing by γ-secretase is regulated, especially as regards the interaction between enzyme and substrate, remains largely unknown. Here, mutagenesis reveals that the hydrophilic loop-1 (HL-1) of presenilin-1 (PS1) is critical for both γ-secretase step-wise cleavages (processivity) and its allosteric modulation by heterocyclic γ-modulatory compounds. Systematic mutagenesis of HL-1, including all of its familial AD mutations and additional engineered variants, and quantification of the resultant Aβ products show that HL-1 is necessary for proper sequential γ-secretase processivity. We identify Y106, L113, and Y115 in HL-1 as key targets for heterocyclic γ-secretase modulators (GSMs) to stimulate processing of pathogenic Aβ peptides. Further, we confirm that the GxxxG domain in the APP transmembrane region functions as a critical substrate motif for γ-secretase processivity: a G29A substitution in APP-C99 mimics the beneficial effects of GSMs. Together, these findings provide a molecular basis for the structural regulation of γ-processivity by enzyme and substrate, facilitating the rational design of new GSMs that lower AD-initiating amyloidogenic Aβ peptides., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Intramembrane proteolysis of transmembrane substrates by the presenilin-γ-secretase complex is preceded and regulated by shedding of the substrate's ectodomain by α- or β-secretase. We asked whether β- and γ-secretases interact to mediate efficient sequential processing of APP, generating the amyloid β (Aβ) peptides that initiate Alzheimer's disease. We describe a hitherto unrecognized multiprotease complex containing active β- and γ-secretases. BACE1 coimmunoprecipitated and cofractionated with γ-secretase in cultured cells and in mouse and human brain. An endogenous high molecular weight (HMW) complex (∼5 MD) containing β- and γ-secretases and holo-APP was catalytically active in vitro and generated a full array of Aβ peptides, with physiological Aβ42/40 ratios. The isolated complex responded properly to γ-secretase modulators. Alzheimer's-causing mutations in presenilin altered the Aβ42/40 peptide ratio generated by the HMW β/γ-secretase complex indistinguishably from that observed in whole cells. Thus, Aβ is generated from holo-APP by a BACE1-γ-secretase complex that provides sequential, efficient RIP processing of full-length substrates to final products., (© 2019 Liu et al.)

Early-onset familial Alzheimer's disease (FAD) is pathologically and clinically similar to the more common late-onset sporadic form of the disease. The study of rare genetic mutations that cause FAD should provide insight into the pathogenesis of sporadic Alzheimer's disease. FAD mutations have only been found in the substrate (amyloid precursor protein, APP) and protease (γ-secretase) that produces the amyloid-β peptide (Aβ). The secreted, aggregation-prone 42-residue Aβ peptide (Aβ42) has long been considered the pathogenic entity in Alzheimer's disease. However, recent understanding of the complexity of the processing of APP by γ-secretase and the effects of FAD mutations on this processing suggest other forms of Aβ as potentially pathogenic., (© 2019 Elsevier Inc. All rights reserved.)

Amyloid plaques, a neuropathological hallmark of Alzheimer's disease, are largely composed of amyloid β (Aβ) peptide, derived from cleavage of amyloid precursor protein (APP) by β- and γ-secretases. The endosome is increasingly recognized as an important crossroad for APP and these secretases, with major implications for APP processing and amyloidogenesis. Among various post-translational modifications affecting APP accumulation, ubiquitination of cytodomain lysines may represent a key signal controlling APP endosomal sorting. Here, we show that substitution of APP C-terminal lysines with arginine disrupts APP ubiquitination and that an increase in the number of substituted lysines tends to increase APP metabolism. An APP mutant lacking all C-terminal lysines underwent the most pronounced increase in processing, leading to accumulation of both secreted and intracellular Aβ40. Artificial APP ubiquitination with rapalog-mediated proximity inducers reduced Aβ40 generation. A lack of APP C-terminal lysines caused APP redistribution from endosomal intraluminal vesicles (ILVs) to the endosomal limiting membrane, with a subsequent decrease in APP C-terminal fragment (CTF) content in secreted exosomes, but had minimal effects on APP lysosomal degradation. Both the increases in secreted and intracellular Aβ40 were abolished by depletion of presenilin 2 (PSEN2), recently shown to be enriched on the endosomal limiting membrane compared with PSEN1. Our findings demonstrate that ubiquitin can act as a signal at five cytodomain-located lysines for endosomal sorting of APP. They further suggest that disruption of APP endosomal sorting reduces its sequestration in ILVs and results in PSEN2-mediated processing of a larger pool of APP-CTF on the endosomal membrane., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

γ-secretase is responsible for the proteolysis of amyloid precursor protein (APP) into short, aggregation-prone amyloid-beta (Aβ) peptides, which are centrally implicated in the pathogenesis of Alzheimer's disease (AD). Despite considerable interest in developing γ-secretase targeting therapeutics for the treatment of AD, the precise mechanism by which γ-secretase produces Aβ has remained elusive. Herein, we demonstrate that γ-secretase catalysis is driven by the stabilization of an enzyme-substrate scission complex via three distinct amino-acid-binding pockets in the enzyme's active site, providing the mechanism by which γ-secretase preferentially cleaves APP in three amino acid increments. Substrate occupancy of these three pockets occurs after initial substrate binding but precedes catalysis, suggesting a conformational change in substrate may be required for cleavage. We uncover and exploit substrate cleavage preferences dictated by these three pockets to investigate the mechanism by which familial Alzheimer's disease mutations within APP increase the production of pathogenic Aβ species., Competing Interests: The authors declare that no competing interests exist.

Two proteolytic enzymes, β- and γ-secretases, work together to produce the amyloid β-peptide of Alzheimer's disease. New evidence suggests that these proteases directly interact and compounds that disrupt this interaction reduce amyloid β-peptide levels without directly blocking either enzyme's solo 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-β (Aβ) production. In cells, CRF treatment increases Aβ 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 β-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 Aβ. These data collectively link CRF to increased Aβ 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 Aβ and in some cases preferentially increase Aβ42 via complex effects on γ-secretase., (© 2015 The Authors.)

The presenilin-containing γ-secretase complex produces the amyloid β-peptide (Aβ) through intramembrane proteolysis, and >100 presenilin mutations are associated with familial early-onset Alzheimer disease (AD). The question of whether these mutations result in AD through a gain or a loss of function remains highly controversial. Mutations in presenilins increase ratios of 42- to 40-residue Aβ critical to pathogenesis, but other Aβs of 38-49 residues are also formed by γ-secretase. Evidence in cells suggests the protease first cleaves substrate within the transmembrane domain at the ϵ site to form 48- or 49-residue Aβ. Subsequent cleavage almost every three residues from the C terminus is thought to occur along two pathways toward shorter secreted forms of Aβ: Aβ49 → Aβ46 → Aβ43 → Aβ40 and Aβ48 → Aβ45 → Aβ42 → Aβ38. Here we show that the addition of synthetic long Aβ peptides (Aβ45-49) directly into purified preparations of γ-secretase leads to the formation of Aβ40 and Aβ42 whether the protease complex is detergent-solubilized or reconstituted into lipid vesicles, and the ratios of products Aβ42 to Aβ40 follow a pattern consistent with the dual-pathway hypothesis. Kinetic analysis of five different AD-causing mutations in presenilin-1 revealed that all result in drastic reduction of normal carboxypeptidase function. Altered trimming of long Aβ peptides to Aβ40 and Aβ42 by mutant proteases occurs at multiple levels, independent of the effects on initial endoproteolysis at the ϵ site, all conspiring to increase the critical Aβ42/Aβ40 ratio implicated in AD pathogenesis. Taken together, these results suggest that specific reduction of carboxypeptidase function of γ-secretase leads to the gain of toxic Aβ42/Aβ40., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The amyloid-beta peptide (Abeta), implicated in the pathogenesis of Alzheimer's disease (AD), is produced through sequential proteolysis of the Abeta precursor protein (APP) by beta- and gamma-secretases. Thus, blocking either of these two proteases, directly or indirectly, is potentially worthwhile toward developing AD therapeutics. beta-Secretase is a membrane-tethered pepsin-like aspartyl protease suitable for structure-based design, whereas gamma-secretase is an unusual, heterotetrameric membrane-embedded aspartyl protease. While gamma-secretase inhibitors entered clinical trials first due to their superior pharmacological properties (for example, brain penetration) over beta-secretase inhibitors, it has since become clear that gamma-secretase inhibitors can cause mechanism-based toxicities owing to interference with the proteolysis of another gamma-secretase substrate, the Notch receptor. Strategies for targeting Abeta production at the gamma-secretase level without blocking Notch signalling will be discussed. Other strategies utilizing cell-based screening have led to the identification of novel Abeta lowering agents that likewise leave Notch proteolysis intact. The mechanism by which these agents lower Abeta is unknown, but these compounds may ultimately reveal new targets for AD therapeutics.

Production of the amyloid beta-peptide (Abeta) via sequential proteolytic cleavage of the amyloid precursor protein by beta- and gamma-secretases is strongly implicated in the pathogenesis of Alzheimer disease. The beta-secretase that executes the first cleavage event is a transmembrane aspartyl protease known as beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1). BACE1 pre-mRNA is alternatively spliced through the use of alternative splice sites in exons 3 and 4, although the significance of these splicing events is unclear. Here, we quantitatively measured relative levels of BACE1 transcripts and identified a novel splice variant of BACE1. We found a subtle but significant difference in BACE1 splicing between brain and pancreas, indicating the cellular environment can affect BACE1 alternative splicing. Furthermore, we have shown that BACE1 proteins translated from alternatively spliced transcripts have dramatically reduced beta-secretase activity and promotion of BACE1 alternative splicing reduces Abeta production. These findings illustrate the importance of BACE1 alternative splicing in affecting the level of Abeta produced in cells and suggest that targeting regulation of BACE1 alternative splicing is a potential therapeutic strategy for lowering beta-secretase activity.

More than 100 missense mutations in presenilin 1 and 2 are associated with early-onset dominant Alzheimer disease. These proteins span the membrane several times and are ostensibly the catalytic component of the gamma-secretase complex, which is responsible for producing the amyloid beta-peptide (Abeta) that deposits in the Alzheimer brain. A common outcome of Alzheimer-associated presenilin mutations is an increase in the ratio of the more aggregation-prone 42-residue form of Abeta to the 40-residue variant, which is often referred to as a presenilin 'gain of function'. An apparent paradox is that most of these mutant presenilins have reduced proteolytic efficiency, which forms part of the counter argument that presenilin 'loss of function' can cause the neuronal dysfunction and death that lead to the disease. In this review, a unifying hypothesis is presented that puts forward a biochemical mechanism by which slower less-efficient forms of the protease can result in a greater proportion of 42-residue Abeta.

Although extensive data support a central pathogenic role for amyloid beta protein (Abeta) in Alzheimer's disease, the amyloid hypothesis remains controversial, in part because a specific neurotoxic species of Abeta and the nature of its effects on synaptic function have not been defined in vivo. Here we report that natural oligomers of human Abeta are formed soon after generation of the peptide within specific intracellular vesicles and are subsequently secreted from the cell. Cerebral microinjection of cell medium containing these oligomers and abundant Abeta monomers but no amyloid fibrils markedly inhibited hippocampal long-term potentiation (LTP) in rats in vivo. Immunodepletion from the medium of all Abeta species completely abrogated this effect. Pretreatment of the medium with insulin-degrading enzyme, which degrades Abeta monomers but not oligomers, did not prevent the inhibition of LTP. Therefore, Abeta oligomers, in the absence of monomers and amyloid fibrils, disrupted synaptic plasticity in vivo at concentrations found in human brain and cerebrospinal fluid. Finally, treatment of cells with gamma-secretase inhibitors prevented oligomer formation at doses that allowed appreciable monomer production, and such medium no longer disrupted LTP, indicating that synaptotoxic Abeta oligomers can be targeted therapeutically.

Mutations in the presenilins cause Alzheimer's disease (AD) and alter gamma-secretase activity to increase the production of the 42-residue amyloid-beta peptide (Abeta) found disproportionally in the cerebral plaques that characterize the disease. The serpentine presenilins are required for transmembrane cleavage of both the amyloid-beta precursor protein (APP) and the Notch receptor by y-secretase, and presenilins are biochemically associated with the protease. Inhibitors of gamma-secretase have provided critical clues to the function of presenilins. Pharmacological profiling suggested that gamma-secretase is an aspartyl protease, leading to the identification of two conserved aspartates important to presenilin's role in proteolysis. Conversion of transition-state analogue inhibitors of gamma-secretase to affinity reagents resulted in specific tagging of the heterodimeric form of presenilins, strongly suggesting that the active site of gamma-secretase lies at the interface of the presenilin heterodimer. Heterodimeric presenilin appears to be the catalytic portion of a multi-protein gamma-secretase complex.

An unusual intramembranous cleavage of the beta-amyloid precursor protein (APP) by gamma-secretase is the final step in the generation of amyloid beta-peptide (Abeta). Two conserved aspartates in transmembrane (TM) domains 6 and 7 of presenilin (PS) 1 are required for Abeta production by gamma-secretase. Here we report that the APP C-terminal fragments, C83 and C99, which are the direct substrates of gamma-secretase, can be coimmunoprecipitated with both PS1 and PS2. PS/C83 complexes were detected in cells expressing endogenous levels of PS. The complexes accumulate when gamma-secretase is inactivated either pharmacologically or by mutating the PS aspartates. PS1/C83 and PS1/C99 complexes were detected in Golgi-rich and trans-Golgi network-rich vesicle fractions. In contrast, complexes of PS1 with APP holoprotein, which is not the immediate substrate of gamma-secretase, occurred earlier in endoplasmic reticulum-rich vesicles. The major portion of intracellular Abeta at steady state was found in the same Golgi/trans-Golgi network-rich vesicles, and Abeta levels in these fractions were markedly reduced when either PS1 TM aspartate was mutated to alanine. Furthermore, de novo generation of Abeta in a cell-free microsomal reaction occurred specifically in these same vesicle fractions and was markedly inhibited by mutating either TM aspartate. Thus, PSs are complexed with the gamma-secretase substrates C83 and C99 in the subcellular locations where Abeta is generated, indicating that PSs are directly involved in the pathogenically critical intramembranous proteolysis of APP.

The discovery that a deficiency of presenilin 1 (PS1) decreases the production of amyloid beta-protein (Abeta) identified the presenilins as important mediators of the gamma-secretase cleavage of beta-amyloid precursor protein (APP). Recently, we found that two conserved transmembrane (TM) aspartates in PS1 are critical for Abeta production, providing evidence that PS1 either functions as a required diaspartyl cofactor for gamma-secretase or is itself gamma-secretase. Presenilin 2 (PS2) shares substantial sequence and possibly functional homology with PS1. Here, we show that the two TM aspartates in PS2 are also critical for gamma-secretase activity, providing further evidence that PS2 is functionally homologous to PS1. Cells stably co-expressing TM Asp --> Ala mutations in both PS1 and PS2 show further accumulation of the APP-derived gamma-secretase substrates, C83 and C99. The production of Abeta is reduced to undetectable levels in the conditioned media of these cells. Furthermore, endoproteolysis of the exogenous Asp mutant PS2 is absent, and endogenous PS1 C-terminal fragments are diminished to undetectable levels. Therefore, the co-expression of PS1 and PS2 TM Asp --> Ala mutants suppresses the formation of any detectable PS1 or PS2 heterodimeric fragments and essentially abolishes the production of Abeta. These results explain the residual Abeta production seen in PS1-deficient cells and demonstrate the absolute requirement of functional presenilins for Abeta generation. We conclude that presenilins, and their TM aspartates in particular, are attractive targets for lowering Abeta therapeutically to prevent Alzheimer's disease.

The amyloid-beta protein (A beta), strongly implicated in the etiology of Alzheimer's disease (AD), is formed from the amyloid-beta precursor protein (APP) through sequential proteolysis by beta- and gamma-secretases. Cleavage by gamma-secretase takes place within the middle of the single transmembrane region of APP and results primarily in 40- and 42-amino acid A beta C-terminal variants, A beta 40 and A beta 42. The latter form of A beta is highly fibrillogenic, is invariably elevated in autosomal-dominant forms of AD, and is the major A beta component found presymptomatically in cerebral deposits. Thus, blocking production of A beta in general and A beta 42 in particular is considered an important therapeutic goal. We have developed transition-state analogue inhibitors of gamma-secretase as molecular probes for characterizing the active site of this enzyme, as pharmacological tools for understanding its role in biology, and as affinity labels toward its definitive identification. Specifically, we found that: (1) difluoro ketone and difluoro alcohol peptidomimetics are effective inhibitors of gamma-secretase activity in APP-transfected cells, strongly suggesting an aspartyl protease mechanism; (2) gamma-secretases that form A beta 40 and A beta 42 are pharmacologically distinct but are nevertheless closely similar; (3) large hydrophobic P1 substituents increase the inhibitory potency of these peptidomimetics, suggesting a large complementary S1 pocket for gamma-secretases; (4) A beta 42 production is increased several fold over control by these gamma-secretase inhibitors after replacement with inhibitor-free media; (5) a bromoacetamide derivative of one of these analogues continues to inhibit total A beta and A beta 42 production hours after replacement with compound-free media and should help identify the target(s) of these protease transition-state mimics.

Accumulation of the amyloid-beta protein (Abeta) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimer's disease. The final step in the generation of Abeta from the beta-amyloid precursor protein is an apparently intramembranous proteolysis by the elusive gamma-secretase(s). The most common cause of familial Alzheimer's disease is mutation of the genes encoding presenilins 1 and 2, which alters gamma-secretase activity to increase the production of the highly amyloidogenic Abeta42 isoform. Moreover, deletion of presenilin-1 in mice greatly reduces gamma-secretase activity, indicating that presenilin-1 mediates most of this proteolytic event. Here we report that mutation of either of two conserved transmembrane (TM) aspartate residues in presenilin-1, Asp 257 (in TM6) and Asp 385 (in TM7), substantially reduces Abeta production and increases the amounts of the carboxy-terminal fragments of beta-amyloid precursor protein that are the substrates of gamma-secretase. We observed these effects in three different cell lines as well as in cell-free microsomes. Either of the Asp --> Ala mutations also prevented the normal endoproteolysis of presenilin-1 in the TM6 --> TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimer's disease and which does not require this cleavage, the Asp 385 --> Ala mutation still inhibited gamma-secretase activity. Our results indicate that the two transmembrane aspartate residues are critical for both presenilin-1 endoproteolysis and gamma-secretase activity, and suggest that presenilin 1 is either a unique diaspartyl cofactor for gamma-secretase or is itself gamma-secretase, an autoactivated intramembranous aspartyl protease.