Specific Mutations in Aph1 Cause γ-Secretase Activation

Amyloid beta peptides (Aβs) are generated from amyloid precursor protein (APP) through multiple cleavage steps mediated by γ-secretase, including endoproteolysis and carboxypeptidase-like trimming. The generation of neurotoxic Aβ42/43 species is enhanced by familial Alzheimer’s disease (FAD) mutations within the catalytic subunit of γ-secretase, presenilin 1 (PS1). FAD mutations of PS1 cause partial loss-of-function and decrease the cleavage activity. Activating mutations, which have the opposite effect of FAD mutations, are important for studying Aβ production. Aph1 is a regulatory subunit of γ-secretase; it is presumed to function as a scaffold of the complex. In this study, we identified Aph1 mutations that are active in the absence of nicastrin (NCT) using a yeast γ-secretase assay. We analyzed these Aph1 mutations in the presence of NCT; we found that the L30F/T164A mutation is activating. When introduced in mouse embryonic fibroblasts, the mutation enhanced cleavage. The Aph1 mutants produced more short and long Aβs than did the wild-type Aph1, without an apparent modulatory function. The mutants did not change the amount of γ-secretase complex, suggesting that L30F/T164A enhances catalytic activity. Our results provide insights into the regulatory function of Aph1 in γ-secretase activity.

The evolved divergence of γ-secretase-susceptibility of homologous proteins Ngfrb and Nradd in zebrafish

Objective NGFR/p75NTR and NRADD/NRH proteins are closely related structurally and are encoded by genes that arose from a duplication event early in vertebrate evolution. The transmembrane domain (TMD) of NGFR is cleaved by γ-secretase but there is conflicting data around the susceptibility to γ-secretase cleavage of NRADD proteins. If NGFR and NRADD show differential susceptibility to γ-secretase, then they can be used to dissect the structural constraints determining substrate susceptibility. We sought to test this differential susceptibility. Results We developed labelled, lumenally-truncated forms of zebrafish Ngfrb and Nradd and a chimeric protein in which the TMD of Nradd was replaced with the TMD of Ngfrb. We expressed these in zebrafish embryos to test their susceptibility to γ-secretase cleavage by monitoring their stability using western immunoblotting. Inhibition of γ-secretase activity using DAPT increased the stability of only the Ngfrb construct. Our results support that only NGFR is cleaved by γ-secretase. Either NGFR evolved γ-secretase-susceptibility since its creation by gene duplication, or NRADD evolved to be refractory to γ-secretase. Protein structure outside of the TMD of NGFR is likely required for susceptibility to γ-secretase.

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

γ-Secretase, a membrane-embedded aspartyl protease complex with presenilin as the catalytic component, cleaves within the transmembrane domain (TMD) of its many substrates, which include the amyloid precursor protein (APP) of Alzheimer's disease (AD). APP TMD is processively cut by γ-secretase through endoproteolysis followed by tricarboxypeptidase "trimming", the latter function being deficient with mutations causing hereditary AD. Toward better understanding of substrate recognition and hydrolytic reactions catalyzed by this enzyme within its hydrophobic milieu, we recently developed a prototype substrate TMD mimetic as a chemical tool for structural analysis (Bhattarai S et al. J. Am. Chem. Soc., 2020, 142(7): 3351-3355). This TMD mimetic--composed of a helical peptide inhibitor (HPI) connected through a linker to a transition-state analog inhibitor (TSA)--simultaneously engages the substrate docking exosite and the active site and is pre-organized to trap the protease transition state of carboxypeptidase trimming. In this study, we developed variants of this prototype designed to allow visualization of transition states for endoproteolysis, TMD helix unwinding, and lateral gating of substrate. New HPI-TSA conjugates were synthesized using an earlier established divergent strategy, which involved the construction of a tripeptidomimetic building block followed by solid-phase peptide synthesis (SPPS). For each class of structural probe, SAR analysis led to discovery of highly potent prototypes with stoichiometric or low-nanomolar inhibition. These TMD mimetics exhibited non-competitive inhibition of γ-secretase activity and occupy both docking and active site as demonstrated by enzyme 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.

Amyloidogenic, neuroinflammatory and memory dysfunction effects of HIV-1 gp120

Human immunodeficiency virus 1 (HIV-1) infection can cause several HIV-associated neurocognitive disorders a variety of neurological impairments characterized by the loss of cortical and subcortical neurons and decreased cognitive and motor function. HIV-1 gp120, the major envelope glycoprotein on viral particles, acts as a binding protein for viral entry and is known to be an agent of neuronal cell death. To determine the mechanism of HIV-1 gp120-induced memory dysfunction, we performed mouse intracerebroventricular (i.c.v.) infusion with HIV-1 gp120 protein (300 ng per mouse) and investigated memory impairment and amyloidogenesis. Infusion of the HIV-1 gp120 protein induced memory dysfunction, which was evaluated using passive avoidance and water maze tests. Infusion of HIV-1 gp120 induced neuroinflammation, such as the release of iNOS and COX-2 and the activation of astrocytes and microglia and increased the mRNA and protein levels of IL-6, ICAM-1, M-CSF, TIM, and IL-2. In particular, we found that the infusion of HIV-1 gp120 induced the accumulation of amyloid plaques and signs of elevated amyloidogenesis, such as increased expression of amyloid precursor protein and BACE1 and increased β-secretase activity. Therefore, these studies suggest that HIV-1 gp120 may induce memory impairment through Aβ accumulation and neuroinflammation.

Mechanism of Cellular Formation and In Vivo Seeding Effects of Hexameric β-amyloid Assemblies

The β-amyloid peptide (Aβ) is the main constituent of senile plaques, a typical hallmark of Alzheimer’s disease (AD). Monomeric Aβ is generated through sequential processing of the amyloid precursor protein (APP), with a final step involving γ-secretase activity. In AD, Aβ monomers assemble in oligomers and ultimately fibrils depositing in plaques. Importantly, Aβ toxicity appears related to its soluble oligomeric intermediates. In particular, recombinant Aβ studies described Aβ hexamers as critical oligomeric nuclei. We recently identified hexameric Aβ assemblies in a cellular model, and revealed their ability to enhance recombinant Aβ aggregation in vitro. Here, we assessed the contribution of similar hexameric-like Aβ assemblies to the development of amyloid pathology. We report their early presence in both transgenic mice brains exhibiting human Aβ pathology and cerebrospinal fluid of AD patients, suggesting hexameric Aβ as a putative novel AD biomarker. Using isolated cell-derived hexameric Aβ, we report the potential of these assemblies to seed other human Aβ species, resulting in neuronal toxicity in vitro and amyloid deposition aggravation in vivo. In order to identify key contributors to their formation in a cellular context, we investigated the role of presenilin-1 (PS1) and presenilin-2 (PS2) in the formation of hexameric-like Aβ assemblies. As catalytic subunits of the γ-secretase complex, PS1 and PS2 can differentially participate in Aβ generation. Using CRISPR-Cas9-modified neuronal-like cell lines knockdown for each of the two presenilins, we present experimental evidence suggesting a direct link between the PS2-dependent pathway and the release of hexameric-like Aβ assemblies in extracellular vesicles.

A precise regulation of presenilin expression is required for the sea urchin early development

Presenilins or PSENs homologues are widely expressed across eukaryotes. Two PSEN are expressed in humans where they play a crucial role in Alzheimer's disease (AD). Each PSEN can be part of the γ-secretase complex that has multiple substrates such as Notch or the amyloid precursor protein (AβPP) which gives the Aβ peptides composing the senile plaques during AD. PSENs also interact with various proteins independently of their γ-secretase activity. They can then be involved in numerous cellular functions, which makes their role in a given cell and/or organism complex to decipher. We settled the sea urchin embryo as a new model to study the role of PSEN. PSEN is present in unduplicated form and highly similar to that of humans. Our results suggest that its expression must be precisely tuned to control the course of the first mitotic cycles and the associated Cai transients, gastrulation execution and, probably in association with ciliated cells, the establishment of the pluteus. We suggest that it would be relevant to study the role of PSEN within the GRN deciphered in the sea urchin.

An evaluation of the self-assembly enhancing properties of cell-derived hexameric amyloid-β

A key hallmark of Alzheimer’s disease is the extracellular deposition of amyloid plaques composed primarily of the amyloidogenic amyloid-β (Aβ) peptide. The Aβ peptide is a product of sequential cleavage of the Amyloid Precursor Protein, the first step of which gives rise to a C-terminal Fragment (C99). Cleavage of C99 by γ-secretase activity releases Aβ of several lengths and the Aβ42 isoform in particular has been identified as being neurotoxic. The misfolding of Aβ leads to subsequent amyloid fibril formation by nucleated polymerisation. This requires an initial and critical nucleus for self-assembly. Here, we identify and characterise the composition and self-assembly properties of cell-derived hexameric Aβ42 and show its assembly enhancing properties which are dependent on the Aβ monomer availability. Identification of nucleating assemblies that contribute to self-assembly in this way may serve as therapeutic targets to prevent the formation of toxic oligomers.

Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells

Presenilin (PS)/γ-secretase is an aspartyl protease that processes a wide range of transmembrane proteins such as the amyloid precursor protein (APP) and Notch1, playing essential roles in normal biological events and diseases. However, whether there is a substrate preference for PS/γ-secretase processing in cells is not fully understood. Structural studies of PS/γ-secretase enfolding a fragment of APP or Notch1 showed that the two substrates engage the protease in broadly similar ways, suggesting the limited substrate specificity of PS/γ-secretase. In the present study, we developed a new multiplexed imaging platform that, for the first time, allowed us to quantitatively monitor how PS/γ-secretase processes two different substrates (e.g., APP vs. Notch1) in the same cell. In this assay, we utilized the recently reported, spectrally compatible visible and near-infrared (NIR)-range Förster resonance energy transfer (FRET) biosensors that permit quantitative recording of PS/γ-secretase activity in live cells. Here, we show that, overall, PS/γ-secretase similarly cleaves Notch1 N100, wild-type APP C99, and familial Alzheimer’s disease (FAD)-linked APP C99 mutants in Chinese hamster ovary (CHO) cells, which further supports the limited PS/γ-secretase substrate specificity. On the other hand, a cell-by-cell basis analysis demonstrates a certain degree of variability in substrate recognition and processing by PS/γ-secretase among different cells. Our new multiplexed FRET assay could be a useful tool to better understand how PS/γ-secretase processes its multiple substrates in normal and disease conditions in live, intact cells.