Biology subjects

Maesako, M.

Publications and source records attributed to Maesako, M..

5 recordsLinked to original sources

bioRxiv2024

Alzheimer-mutant γ-secretase complexes stall amyloid β-peptide production

Missense mutations in the amyloid precursor protein (APP) and presenilin-1 (PSEN1) cause early-onset familial Alzheimers disease (FAD) and alter proteolytic production of secreted 38-to-43-residue amyloid {beta}-peptides (A{beta}) by the PSEN1-containing {gamma}-secretase complex, ostensibly supporting the amyloid hypothesis of pathogenesis. However, proteolysis of APP substrate by {gamma}-secretase is processive, involving initial endoproteolysis to produce long A{beta} peptides of 48 or 49 residues followed by carboxypeptidase trimming in mostly tripeptide increments. We recently reported evidence that FAD mutations in APP and PSEN1 cause deficiencies in early steps in processive proteolysis of APP substrate C99 and that this results from stalled {gamma}-secretase enzyme-substrate and/or enzyme-intermediate complexes. These stalled complexes triggered synaptic degeneration in a C. elegans model of FAD independently of A{beta} production. Here we conducted full quantitative analysis of all proteolytic events on APP substrate by {gamma}-secretase with six additional PSEN1 FAD mutations and found that all six are deficient in multiple processing steps. However, only one of these (F386S) was deficient in certain trimming steps but not in endoproteolysis. Fluorescence lifetime imaging microscopy in intact cells revealed that all six PSEN1 FAD mutations lead to stalled {gamma}-secretase enzyme-substrate/intermediate complexes. The F386S mutation, however, does so only in A{beta}-rich regions of the cells, not in C99-rich regions, consistent with the deficiencies of this mutant enzyme only in trimming of A{beta} intermediates. These findings provide further evidence that FAD mutations lead to stalled and stabilized {gamma}-secretase enzyme-substrate and/or enzyme-intermediate complexes and are consistent with the stalled process rather than the products of {gamma}-secretase proteolysis as the pathogenic trigger.

biochemistry↗

bioRxiv2024

Recording gamma-secretase activity in living mouse brains

{gamma}-Secretase plays a pivotal role in the central nervous system. Our recent development of genetically encoded Forster resonance energy transfer (FRET)-based biosensors has enabled the spatiotemporal recording of {gamma}-secretase activity on a cell-by-cell basis in live neurons in culture. Nevertheless, how {gamma}-secretase activity is regulated in vivo remains unclear. Here we employ the near-infrared (NIR) C99 720-670 biosensor and NIR confocal microscopy to quantitatively record {gamma}-secretase activity in individual neurons in living mouse brains. Intriguingly, we uncovered that {gamma}-secretase activity may influence the activity of {gamma}-secretase in neighboring neurons, suggesting a potential "cell non-autonomous" regulation of {gamma}-secretase in mouse brains. Given that {gamma}-secretase plays critical roles in important biological events and various diseases, our new assay in vivo would become a new platform that enables dissecting the essential roles of {gamma}-secretase in normal health and diseases.

neuroscience↗

bioRxiv2023

Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes

Alzheimers disease is characterized pathologically by cerebral deposition of 42-residue amyloid {beta}-peptide (A{beta}42), proteolytically produced from amyloid precursor protein (APP) by {beta}- and {gamma}-secretases.1 Although mutations in APP and presenilin, the catalytic component of {gamma}-secretase, cause familial Alzheimers disease (FAD), a role for A{beta}42 as the primary disease driver has not been clearly established and remains controversial.2,3 Here we show through comprehensive analysis of the multi-step proteolysis of APP substrate C99 by {gamma}-secretase that FAD mutations are consistently deficient in early proteolytic events, not later events that produce secreted A{beta} peptides. Cryo-electron microscopy revealed that a substrate mimetic traps {gamma}-secretase at the transition state for intramembrane proteolysis, and this structure closely aligns with activated enzyme-substrate complex captured by molecular dynamics simulations. In silico simulations and fluorescence lifetime imaging microscopy in cultured cells support stabilization by FAD mutations of enzyme-substrate and/or enzyme-intermediate complexes. Neuronal expression of C99 and/or presenilin-1 in Caenorabditis elegans led to age-dependent synaptic loss only when one of the transgenes carried an FAD mutation. Designed mutations that stabilize the enzyme-substrate complex and block proteolysis likewise led to synaptic loss. Collectively, these findings implicate the stalled process--not the released products--of {gamma}-secretase cleavage of substrates in FAD pathogenesis.

neuroscience↗

bioRxiv2023

Alzheimer's disease linked Aβ42 exerts product feedback inhibition on γsecretase impairing downstream cell signaling

Amyloid {beta} (A{beta}) peptides accumulating in the brain are proposed to trigger Alzheimers disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for A{beta}42 toxicity that arises from its proven affinity for {gamma}-secretases. We hypothesized that the reported increases in A{beta}42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on {gamma}-secretases, and thereby impair downstream signaling events. We show that human A{beta}42 peptides, but neither murine A{beta}42 nor human A{beta}17-42 (p3), inhibit {gamma}-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75 and pan-cadherin. Moreover, A{beta}42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in A{beta}42 contribute to cellular toxicity via the {gamma}-secretase inhibition, and provide a novel conceptual framework to address A{beta} toxicity in the context of {gamma}-secretase-dependent homeostatic signaling.

biochemistry↗

bioRxiv2023

Identification of PS1/gamma-secretase and glutamate transporter GLT-1 interaction sites

The recently discovered interaction between Presenilin 1 (PS1), a catalytic subunit of {gamma}-secretase responsible for generating amyloid-{beta} (A{beta}) peptides, and GLT-1, a major glutamate transporter in the brain (EAAT2) provides a mechanistic link between these two key factors involved in Alzheimers disease (AD) pathology. Modulating this interaction can be crucial to understand the consequence of such crosstalk in AD context and beyond. However, the interaction sites between these two proteins are unknown. Herein, we utilized an alanine scanning approach coupled with FRET-based fluorescence lifetime imaging microscopy (FLIM) to identify the interaction sites between PS1 and GLT-1 in their native environment within intact cells. We found that GLT-1 residues at position 276 to 279 (TM5) and PS1 residues at position 249 to 252 (TM6) are crucial for GLT-1/PS1 interaction. These results have been cross validated using AlphaFold Multimer prediction. To further investigate whether this interaction of endogenously expressed GLT-1 and PS1 can be prevented in primary neurons, we designed PS1/GLT-1 cell-permeable peptides (CPPs) targeting the PS1 or GLT-1 binding site. We used HIV TAT domain to allow for cell penetration which was assayed in neurons. First, we assessed the toxicity and penetration of CPPs by confocal microscopy. Next, to ensure the efficiency of CPPs, we monitored the modulation of GLT-1/PS1 interaction in intact neurons by FLIM. We saw significantly less interaction between PS1 and GLT-1 with both CPPs. Our study establishes a new tool to study the functional aspect of GLT-1/PS1 interaction and its relevance in normal physiology and AD models.

biochemistry↗