Emerging structures and dynamic mechanisms of γ-secretase for Alzheimer's disease.

γ-Secretase, called "the proteasome of the membrane," is a membrane-embedded protease complex that cleaves 150+ peptide substrates with central roles in biology and medicine, including amyloid precursor protein and the Notch family of cell-surface receptors. Mutations in γ-secretase and amyloid precursor protein lead to early-onset familial Alzheimer's disease. γ-Secretase has thus served as a critical drug target for treating familial Alzheimer's disease and the more common late-onset Alzheimer's disease as well. However, critical gaps remain in understanding the mechanisms of processive proteolysis of substrates, the effects of familial Alzheimer's disease mutations, and allosteric modulation of substrate cleavage by γ-secretase. In this review, we focus on recent studies of structural dynamic mechanisms of γ-secretase. Different mechanisms, including the "Fit-Stay-Trim," "Sliding-Unwinding," and "Tilting-Unwinding," have been proposed for substrate proteolysis of amyloid precursor protein by γ-secretase based on all-atom molecular dynamics simulations. While an incorrect registry of the Notch1 substrate was identified in the cryo-electron microscopy structure of Notch1-bound γ-secretase, molecular dynamics simulations on a resolved model of Notch1-bound γ-secretase that was reconstructed using the amyloid precursor protein-bound γ-secretase as a template successfully captured γ-secretase activation for proper cleavages of both wildtype and mutant Notch, being consistent with biochemical experimental findings. The approach could be potentially applied to decipher the processing mechanisms of various substrates by γ-secretase. In addition, controversy over the effects of familial Alzheimer's disease mutations, particularly the issue of whether they stabilize or destabilize γ-secretase-substrate complexes, is discussed. Finally, an outlook is provided for future studies of γ-secretase, including pathways of substrate binding and product release, effects of modulators on familial Alzheimer's disease mutations of the γ-secretase-substrate complexes. Comprehensive understanding of the functional mechanisms of γ-secretase will greatly facilitate the rational design of effective drug molecules for treating familial Alzheimer's disease and perhaps Alzheimer's disease in general.

Familial Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes.

Mutations that cause familial Alzheimer's disease (FAD) are found in amyloid precursor protein (APP) and presenilin, the catalytic component of γ-secretase, that together produce amyloid ß-peptide (Aß). Nevertheless, whether Aß is the primary disease driver remains controversial. We report here that FAD mutations disrupt initial proteolytic events in the multistep processing of APP substrate C99 by γ-secretase. Cryoelectron microscopy reveals that a substrate mimetic traps γ-secretase during the transition state, and this structure aligns with activated enzyme-substrate complex captured by molecular dynamics simulations. In silico simulations and in cellulo fluorescence microscopy support stabilization of enzyme-substrate complexes by FAD mutations. Neuronal expression of C99 and/or presenilin-1 in Caenorhabditis elegans leads to synaptic loss only with FAD-mutant transgenes. Designed mutations that stabilize the enzyme-substrate complex and block Aß production likewise led to synaptic loss. Collectively, these findings implicate the stalled process-not the products-of γ-secretase cleavage of substrates in FAD pathogenesis.

Molecular Dynamics Activation of γ-Secretase for Cleavage of the Notch1 Substrate.

γ-Secretase is an intramembrane aspartyl protease complex that cleaves the transmembrane domain of over 150 peptide substrates, including amyloid precursor protein (APP) and the Notch family of receptors, via two conserved aspartates D257 and D385 in the presenilin-1 (PS1) catalytic subunit. However, while the activation of γ-secretase for cleavage of APP has been widely studied, the cleavage of Notch by γ-secretase remains poorly explored. Here, we combined Gaussian accelerated molecular dynamics (GaMD) simulations and mass spectrometry (MS) analysis of proteolytic products to present the first dynamic models for cleavage of Notch by γ-secretase. MS showed that γ-secretase cleaved the WT Notch at Notch residue G34, while cleavage of the L36F mutant Notch occurred at Notch residue C33. Initially, we prepared our simulation systems starting from the cryoEM structure of Notch-bound γ-secretase (PDB: 6IDF) and failed to capture the proper cleavages of WT and L36F Notch by γ-secretase. We then discovered an incorrect registry of the Notch substrate in the PS1 active site through alignment of the experimental structure of Notch-bound (PDB: 6IDF) and APP-bound γ-secretase (PDB: 6IYC). Every residue of the APP substrate was systematically mutated to the corresponding Notch residue to prepare a resolved model of Notch-bound γ-secretase complexes. GaMD simulations of the resolved model successfully captured γ-secretase activation for proper cleavages of both WT and L36F mutant Notch. Our findings presented here provided mechanistic insights into the structural dynamics and enzyme-substrate interactions required for γ-secretase activation for cleavage of Notch and other substrates.

A neurodegeneration checkpoint mediated by REST protects against the onset of Alzheimer's disease.

Many aging individuals accumulate the pathology of Alzheimer's disease (AD) without evidence of cognitive decline. Here we describe an integrated neurodegeneration checkpoint response to early pathological changes that restricts further disease progression and preserves cognitive function. Checkpoint activation is mediated by the REST transcriptional repressor, which is induced in cognitively-intact aging humans and AD mouse models at the onset of amyloid ß-protein (Aß) deposition and tau accumulation. REST induction is mediated by the unfolded protein response together with ß-catenin signaling. A consequence of this response is the targeting of REST to genes involved in key pathogenic pathways, resulting in downregulation of gamma secretase, tau kinases, and pro-apoptotic proteins. Deletion of REST in the 3xTg and J20 AD mouse models accelerates Aß deposition and the accumulation of misfolded and phosphorylated tau, leading to neurodegeneration and cognitive decline. Conversely, viral-mediated overexpression of REST in the hippocampus suppresses Aß and tau pathology. Thus, REST mediates a neurodegeneration checkpoint response with multiple molecular targets that may protect against the onset of AD.

Effects of presenilin-1 familial Alzheimer's disease mutations on γ-secretase activation for cleavage of amyloid precursor protein.

Presenilin-1 (PS1) is the catalytic subunit of γ-secretase which cleaves within the transmembrane domain of over 150 peptide substrates. Dominant missense mutations in PS1 cause early-onset familial Alzheimer's disease (FAD); however, the exact pathogenic mechanism remains unknown. Here we combined Gaussian accelerated molecular dynamics (GaMD) simulations and biochemical experiments to determine the effects of six representative PS1 FAD mutations (P117L, I143T, L166P, G384A, L435F, and L286V) on the enzyme-substrate interactions between γ-secretase and amyloid precursor protein (APP). Biochemical experiments showed that all six PS1 FAD mutations rendered γ-secretase less active for the endoproteolytic (ε) cleavage of APP. Distinct low-energy conformational states were identified from the free energy profiles of wildtype and PS1 FAD-mutant γ-secretase. The P117L and L286V FAD mutants could still sample the "Active" state for substrate cleavage, but with noticeably reduced conformational space compared with the wildtype. The other mutants hardly visited the "Active" state. The PS1 FAD mutants were found to reduce γ-secretase proteolytic activity by hindering APP residue L49 from proper orientation in the active site and/or disrupting the distance between the catalytic aspartates. Therefore, our findings provide mechanistic insights into how PS1 FAD mutations affect structural dynamics and enzyme-substrate interactions of γ-secretase and APP.

Identification of the Aß37/42 peptide ratio in CSF as an improved Aß biomarker for Alzheimer's disease.

INTRODUCTION: Identifying CSF-based biomarkers for the ß-amyloidosis that initiates Alzheimer's disease (AD) could provide inexpensive and dynamic tests to distinguish AD from normal aging and predict future cognitive decline. METHODS: We developed immunoassays specifically detecting all C-terminal variants of secreted amyloid ß-protein and identified a novel biomarker, the Aß 37/42 ratio, that outperforms the canonical Aß42/40 ratio as a means to evaluate the γ-secretase activity and brain Aß accumulation. RESULTS: We show that Aß 37/42 can distinguish physiological and pathological status in (1) presenilin-1 mutant vs wild-type cultured cells, (2) AD vs control brain tissue, and (3) AD versus cognitively normal (CN) subjects in CSF, where 37/42 (AUC 0.9622) outperformed 42/40 (AUC 0.8651) in distinguishing CN from AD. DISCUSSION: We conclude that the Aß 37/42 ratio sensitively detects presenilin/γ-secretase dysfunction and better distinguishes CN from AD than Aß42/40 in CSF. Measuring this novel ratio alongside promising phospho-tau analytes may provide highly discriminatory fluid biomarkers for AD.

In-Depth Characterization of Endo-Lysosomal Aß in Intact Neurons.

Amyloid-beta (Aß) peptides are produced within neurons. Some peptides are released into the brain parenchyma, while others are retained inside the neurons. However, the detection of intracellular Aß remains a challenge since antibodies against Aß capture Aß and its precursor proteins (i.e., APP and C99). To overcome this drawback, we recently developed 1) the C99 720-670 biosensor for recording γ-secretase activity and 2) a unique multiplexed immunostaining platform that enables the selective detection of intracellular Aß with subcellular resolution. Using these new assays, we showed that C99 is predominantly processed by γ-secretase in late endosomes and lysosomes, and intracellular Aß is enriched in the same subcellular loci in intact neurons. However, the detailed properties of Aß in the acidic compartments remain unclear. Here, we report using fluorescent lifetime imaging microscopy (FLIM) that intracellular Aß includes both long Aß intermediates bound to γ-secretase and short peptides dissociated from the protease complex. Surprisingly, our results also suggest that the dissociated Aß is bound to the glycoproteins on the inner membrane of lysosomes. Furthermore, we show striking cell-to-cell heterogeneity in intracellular Aß levels in primary neurons and APP transgenic mouse brains. These findings provide a basis for the further investigation of the role(s) of intracellular Aß and its relevance to Alzheimer's disease (AD).

Mechanism of Tripeptide Trimming of Amyloid ß-Peptide 49 by γ-Secretase.

The membrane-embedded γ-secretase complex processively cleaves within the transmembrane domain of amyloid precursor protein (APP) to produce 37-to-43-residue amyloid ß-peptides (Aß) of Alzheimer's disease (AD). Despite its importance in pathogenesis, the mechanism of processive proteolysis by γ-secretase remains poorly understood. Here, mass spectrometry and Western blotting were used to quantify the efficiency of tripeptide trimming of wild-type (WT) and familial AD (FAD) mutant Aß49. In comparison to WT Aß49, the efficiency of tripeptide trimming was similar for the I45F, A42T, and V46F Aß49 FAD mutants but substantially diminished for the I45T and T48P mutants. In parallel with biochemical experiments, all-atom simulations using a novel peptide Gaussian accelerated molecular dynamics (Pep-GaMD) method were applied to investigate the tripeptide trimming of Aß49 by γ-secretase. The starting structure was the active γ-secretase bound to Aß49 and APP intracellular domain (AICD), as generated from our previous study that captured the activation of γ-secretase for the initial endoproteolytic cleavage of APP (Bhattarai, A., ACS Cent. Sci. 2020, 6, 969-983). Pep-GaMD simulations captured remarkable structural rearrangements of both the enzyme and substrate, in which hydrogen-bonded catalytic aspartates and water became poised for tripeptide trimming of Aß49 to Aß46. These structural changes required a positively charged N-terminus of endoproteolytic coproduct AICD, which could dissociate during conformational rearrangements of the protease and Aß49. The simulation findings were highly consistent with biochemical experimental data. Taken together, our complementary biochemical experiments and Pep-GaMD simulations have enabled elucidation of the mechanism of tripeptide trimming of Aß49 by γ-secretase.

Structure and mechanism of the γ-secretase intramembrane protease complex.

γ-Secretase is a membrane protein complex that proteolyzes within the transmembrane domain of >100 substrates, including those derived from the amyloid precursor protein and the Notch family of cell surface receptors. The nine-transmembrane presenilin is the catalytic component of this aspartyl protease complex that carries out hydrolysis in the lipid bilayer. Advances in cryoelectron microscopy have led to the elucidation of the structure of the γ-secretase complex at atomic resolution. Recently, structures of the enzyme have been determined with bound APP- or Notch-derived substrates, providing insight into the nature of substrate recognition and processing. Molecular dynamics simulations of substrate-bound enzymes suggest dynamic mechanisms of intramembrane proteolysis. Structures of the enzyme bound to small-molecule inhibitors and modulators have also been solved, setting the stage for rational structure-based drug discovery targeting γ-secretase.

Verteporfin is a substrate-selective γ-secretase inhibitor that binds the amyloid precursor protein transmembrane domain.

This work reports substrate-selective inhibition of a protease with broad substrate specificity based on direct binding of a small-molecule inhibitor to the substrate. The target for these studies was γ-secretase protease, which cleaves dozens of different single-span membrane protein substrates, including both the C99 domain of the human amyloid precursor protein and the Notch receptor. Substrate-specific inhibition of C99 cleavage is desirable to reduce production of the amyloid-ß polypeptide without inhibiting Notch cleavage, a major source of toxicity associated with broad specificity γ-secretase inhibitors. In order to identify a C99-selective inhibitors of the human γ-secretase, we conducted an NMR-based screen of FDA-approved drugs against C99 in model membranes. From this screen, we identified the small-molecule verteporfin with these properties. We observed that verteporfin formed a direct 1:1 complex with C99, with a KD of 15-47 µM (depending on the membrane mimetic used), and that it did not bind the transmembrane domain of the Notch-1 receptor. Biochemical assays showed that direct binding of verteporfin to C99 inhibits γ-secretase cleavage of C99 with IC50 values in the range of 15-164 µM, while Notch-1 cleavage was inhibited only at higher concentrations, and likely via a mechanism that does not involve binding to Notch-1. This work documents a robust NMR-based approach to discovery of small-molecule binders to single-span membrane proteins and confirmed that it is possible to inhibit γ-secretase in a substrate-specific manner.

Presenilin/γ-Secretase Activity Is Located in Acidic Compartments of Live Neurons.

Presenilin (PSEN)/γ-secretase is a protease complex responsible for the proteolytic processing of numerous substrates. These substrates include the amyloid precursor protein (APP), the cleavage of which by γ-secretase results in the production of ß-amyloid (Aß) peptides. However, exactly where within the neuron γ-secretase processes APP C99 to generate Aß and APP intracellular domain (AICD) is still not fully understood. Here, we employ novel Förster resonance energy transfer (FRET)-based multiplexed imaging assays to directly "visualize" the subcellular compartment(s) in which γ-secretase primarily cleaves C99 in mouse cortex primary neurons (from both male and female embryos). Our results demonstrate that γ-secretase processes C99 mainly in LysoTracker-positive low-pH compartments. Using a new immunostaining protocol which distinguishes Aß from C99, we also show that intracellular Aß is significantly accumulated in the same subcellular loci. Furthermore, we found functional correlation between the endo-lysosomal pH and cellular γ-secretase activity. Taken together, our findings are consistent with Aß being produced from C99 by γ-secretase within acidic compartments such as lysosomes and late endosomes in living neurons.SIGNIFICANCE STATEMENT Alzheimer's disease (AD) genetics and histopathology highlight the importance of amyloid precursor protein (APP) processing by γ-secretase in pathogenesis. For the first time, this study has enabled us to directly "visualize" that γ-secretase processes C99 mainly in acidic compartments such as late endosomes and lysosomes in live neurons. Furthermore, we uncovered that intracellular ß-amyloid (Aß) is significantly accumulated in the same subcellular loci. Emerging evidence proposes the great importance of the endo-lysosomal pathway in mechanisms of misfolded proteins propagation (e.g., Tau, α-Syn). Therefore, the predominant processing of C99 and enrichment of Aß in late endosomes and lysosomes may be critical events in the molecular cascade leading to AD.

Discovery of aryl aminothiazole γ-secretase modulators with novel effects on amyloid ß-peptide production.

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 ß-peptides (Aß), 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 Aß production compared to that seen with the parent molecule. Small structural changes led to concentration-dependent increases in Aß37 and Aß38 production without parallel decreases in their precursors Aß40 and Aß42, respectively. The new compounds therefore apparently also stimulate carboxypeptidase trimming of Aß peptides ≥ 43 residues, providing novel chemical tools for mechanistic studies of processive proteolysis by γ-secretase.

Design of Transmembrane Mimetic Structural Probes to Trap Different Stages of γ-Secretase-Substrate Interaction.

The transmembrane domain (TMD) of the amyloid precursor protein of Alzheimer's disease is cut processively by γ-secretase through endoproteolysis and tricarboxypeptidase "trimming". We recently developed a prototype substrate TMD mimetic for structural analysis-composed of a helical peptide inhibitor linked to a transition-state analogue-that simultaneously engages a substrate exosite and the active site and is pre-organized to trap the carboxypeptidase transition state. Here, we developed variants of this prototype designed to allow visualization of transition states for endoproteolysis, TMD helix unwinding, and lateral gating of the substrate, identifying potent inhibitors for each class. These TMD mimetics exhibited non-competitive inhibition and occupy both the exosite and the active site, as demonstrated by inhibitor cross-competition experiments and photoaffinity probe binding assays. The new probes should be important structural tools for trapping different stages of substrate recognition and processing via ongoing cryo-electron microscopy with γ-secretase, ultimately aiding rational drug design.

Mutations in the Amyloid-ß Protein Precursor Reduce Mitochondrial Function and Alter Gene Expression Independent of 42-Residue Amyloid-ß Peptide.

BACKGROUND: Dominant missense mutations in the amyloid-ß protein precursor (AßPP) cause early-onset familial Alzheimer's disease (FAD) and are associated with changes in the production or properties of the amyloid-ß peptide (Aß), particularly of the 42-residue variant (Aß42) that deposits in the Alzheimer's disease (AD) brain. Recent findings, however, show that FAD mutations in AßPP also lead to increased production of longer Aß variants of 45-49 residues in length. OBJECTIVE: We aimed to test neurotoxicity of Aß42 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 AßPP mutations that lead to increased production of long Aß peptides with or without Aß42. These AßPP-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 AßPP-expressing cells showed decreased basal OCRs as well as decreased OCRs associated with mitochondrial ATP production, even more so in the absence of Aß42 production. Moreover, mutant AßPP-expressing cells producing longer forms of Aß displayed altered expression of certain mitochondrial- and neuronal-associated genes, whether or not Aß42 was produced. CONCLUSION: These findings suggest that mutant AßPP can cause mitochondrial dysfunction that is associated with long Aß but not with Aß42.

Hydrophilic loop 1 of Presenilin-1 and the APP GxxxG transmembrane motif regulate γ-secretase function in generating Alzheimer-causing Aß peptides.

γ-Secretase is responsible for the proteolysis of amyloid precursor protein (APP) into amyloid-beta (Aß) 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 Aß 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 Aß 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 Aß peptides.

Familial Alzheimer's disease mutations in amyloid protein precursor alter proteolysis by γ-secretase to increase amyloid ß-peptides of ≥45 residues.

Production of amyloid ß-protein (Aß) is carried out by the membrane-embedded γ-secretase complex. Mutations in the transmembrane domain of amyloid ß-protein precursor (APP) associated with early-onset familial Alzheimer's disease (FAD) can alter the ratio of aggregation-prone 42-residue Aß (Aß42) to 40-residue Aß (Aß40). However, APP substrate is proteolyzed processively by γ-secretase along two pathways: Aß49→Aß46→Aß43→Aß40 and Aß48→Aß45→Aß42→Aß38. Effects of FAD mutations on each proteolytic step are unknown, largely due to difficulties in detecting and quantifying longer Aß 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 Aß48 and Aß49 was determined by mass spectrometry. Levels of individual Aß peptides were determined by subtracting levels of peptide coproducts associated with degradation from those associated with production. This method was validated for Aß40 and Aß42 by specific ELISAs and production of equimolar levels of Aß and AICD. Not all mutant substrates led to increased Aß42/40. However, all 14 disease-causing mutations led to inefficient processing of longer forms of Aß ≥ 45 residues. In addition, the effects of certain mutations provided insight into the mechanism of processive proteolysis: intermediate Aß peptides apparently remain bound for subsequent trimming and are not released and reassociated.

Probing Mechanisms and Therapeutic Potential of γ-Secretase in Alzheimer's Disease.

The membrane-embedded γ-secretase complex carries out hydrolysis within the lipid bilayer in proteolyzing nearly 150 different membrane protein substrates. Among these substrates, the amyloid precursor protein (APP) has been the most studied, as generation of aggregation-prone amyloid ß-protein (Aß) is a defining feature of Alzheimer's disease (AD). Mutations in APP and in presenilin, the catalytic component of γ-secretase, cause familial AD, strong evidence for a pathogenic role of Aß. Substrate-based chemical probes-synthetic peptides and peptidomimetics-have been critical to unraveling the complexity of γ-secretase, and small drug-like inhibitors and modulators of γ-secretase activity have been essential for exploring the potential of the protease as a therapeutic target for Alzheimer's disease. Such chemical probes and therapeutic prototypes will be reviewed here, with concluding commentary on the future directions in the study of this biologically important protease complex and the translation of basic findings into therapeutics.