Flexible and Accurate Substrate Processing with Distinct Presenilin/γ-Secretases in Human Cortical Neurons

Abstract

Mutations in the presenilin genes (PS1, PS2) have been linked to the majority of familial Alzheimer's disease (AD). Although great efforts have been made to investigate pathogenic PS mutations, which ultimately cause an increase in the toxic form of β-amyloid (Aβ), the intrinsic physiological functions of PS in human neurons remain to be determined. In this study, to investigate the physiological roles of PS in human neurons, we generated PS1 conditional knockout induced pluripotent stem cells (iPSCs), in which PS1 can be selectively abrogated under Cre transduction with or without additional PS2 knockout. We showed that iPSC-derived neural progenitor cells do not confer a maintenance ability in the absence of both PS1 and PS2, showing the essential role of PS in Notch signaling. We then generated PS-null human cortical neurons, where PS1 was intact until full neuronal differentiation occurred. Aβ40 production was reduced exclusively in human PS1/PS2-null neurons along with a concomitant accumulation of APP-CTFs, whereas Aβ42 was decreased in neurons devoid of PS2. Unlike previous studies in mice, in which APP cleavage is largely attributable to PS1, γ-secretase activity seemed to be comparable between PS1 and PS2. In contrast, cleavage of another substrate, N-cadherin, was impaired only in neurons devoid of PS1. Moreover, PS2/γ-secretase exists largely in late endosomes/lysosomes, as measured by specific antibody against the γ-secretase complex, in which Aβ42 species are supposedly produced. Using this novel stem cell-based platform, we assessed important physiological PS1/PS2 functions in mature human neurons, the dysfunction of which could underlie AD pathogenesis. Significance Statement Presenilins are crucial catalytic subunits of γ-secretase, an intramembranous protease complex, whose mutations underlie AD pathogenesis via the dysregulation of Aβ generation. The γ-secretase complex exhibits heterogeneity via the assembly of PS1 or PS2, but the correlation of γ-secretase heterogeneity with substrate processing remains to be established in human neurons. Here, using a novel iPSC-derived cellular model carrying PS1 and/or PS2 conditional knockout alleles, we uncovered the unique processing of three substrates, Notch, APP and N-cadherin, by PS1 or PS2 in human neural cell contexts. Furthermore, the intrinsic subcellular localization of γ-secretase depends on PS1 or PS2, leading to putative differences in the processing of substrates. This novel 3 platform will help ensure the correlation of γ-secretase/substrates in human neurons.

Different transmembrane domains determine the specificity and efficiency of the cleavage activity of the γ-secretase subunit presenilin

“…Although low signal intensities of Aβ42 prevented us to calculate reliable Aβ42/Aβ40 ratios from IP-MS measurements, qualitatively, Aβ42 levels relative to Aβ40 appeared to be similar for PS1 and PS2. This is in line with studies reporting a similar Aβ42/Aβ40 ratio for both PS paralogs ( 22 , 23 ) but inconsistent with other studies describing a higher Aβ42/Aβ40 ratio for PS2-containing γ-secretase ( 52 , 53 ). For both paralogs, ε49 appears to be the major cleavage site, followed by ε48, which agrees with previous reports ( 28 , 54 , 55 ).…”

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

“…c-Secretase primarily processes APP C99 in acidic compartments of neurons While FRET/FLIM and super-resolution microscopy revealed closer proximity between APP and PSEN1 near and/or at the cell surface (Berezovska et al, 2003;Escamilla-Ayala et al, 2020), earlier biochemical studies suggested that Ab is generated rather in the endo-lysosomal compartments (Haass et al, 1992;Koo and Squazzo, 1994;Schrader-Fischer and Paganetti, 1996;Koo et al, 1996;Pasternak et al, 2003). Recent studies shed light on the restricted localization of PSEN2 in late endosomes and lysosomes (Meckler and Checler, 2016;Sannerud et al, 2016;Watanabe et al, 2021), and the role of PSEN2/g -secretase in the generation of intracellular Ab (Sannerud et al, 2016). To directly visualize where g -secretase processes C99 and thus generates Ab peptides, we employed high-resolution confocal microscopy and our recently developed C99 720-670 biosensor (Houser et al, 2020).…”

“…On the other hand, g -secretase has a broad pH range and retains considerable activity at lower pHs. Furthermore, previous studies illustrated that, while PSEN1/g -secretase is broadly expressed within the entire cells, PSEN2/g -secretase is predominantly localized in late endosomes and lysosomes (Meckler and Checler, 2016;Sannerud et al, 2016;Watanabe et al, 2021). Therefore, we predict the prominent processing of C99 720-670 biosensor by g -secretase and the accumulation of Ab in these compartments could be because the actual expression of functional g -secretase complexes (i.e., PSEN1 "plus" PSEN2/ g -secretase) is higher in the acidic subcellular loci than other areas.…”

“…Electron microscopy and biochemical analysis demonstrated the enrichment of Nicastrin expression, a subunit of g -secretase, and increased g -secretase activity in lysosomes (Pasternak et al, 2003). Recently, it was also illustrated that while PSEN1 is broadly distributed in the cell, PSEN2 is predominantly localized in late endosomes and lysosomes (Meckler and Checler, 2016;Sannerud et al, 2016;Watanabe et al, 2021), where it is responsible for the generation of an intracellular pool of Ab (Sannerud et al, 2016). Importantly, FAD-causing PSEN2 mutations promote the production of intracellular Ab (Ab 42 in particular) and several PSEN1 FAD mutants phenocopy FAD PSEN2 with respect to of subcellular localization and intracellular Ab 42 generation (Sannerud et al, 2016).…”

Flying under the radar: CDH2 (N-cadherin), an important hub molecule in neurodevelopmental and neurodegenerative diseases

“…PSEN1 is the proteolytic subunit of the γ-secretase complex which cleaves transmembrane proteins by initiating a wide range of indispensable biochemical processes and pathways. However, this complex also cleaves the transmembrane domain of the APP protein which produces the toxic and accumulative form of the amyloid-β-peptide (Aβ1-42) forming the characteristic plaque depositions in Alzheimer's disease (Takasugi et al, 2003;Watanabe et al, 2021). Besides its plasma membrane localization, PSEN1 is also present in the endoplasmic reticulum, where it is responsible for posttranslational endoproteolytic processing by cleaving the N-or C-terminal fragments (NTF or CTF, respectively) of various proteins (Wolfe, 2019).…”