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?-Secretase modulators reduce endogenous amyloid ?42 levels in human neural progenitor cells without altering neuronal differentiation.

ABSTRACT: Soluble ?-secretase modulators (SGSMs) selectively decrease toxic amyloid ? (A?) peptides (A?42). However, their effect on the physiologic functions of ?-secretase has not been tested in human model systems. ?-Secretase regulates fate determination of neural progenitor cells. Thus, we studied the impact of SGSMs on the neuronal differentiation of ReNcell VM (ReN) human neural progenitor cells (hNPCs). Quantitative PCR analysis showed that treatment of neurosphere-like ReN cell aggregate cultures with ?-secretase inhibitors (GSIs), but not SGSMs, induced a 2- to 4-fold increase in the expression of the neuronal markers Tuj1 and doublecortin. GSI treatment also induced neuronal marker protein expression, as shown by Western blot analysis. In the same conditions, SGSM treatment selectively reduced endogenous A?42 levels by ?80%. Mechanistically, we found that Notch target gene expressions were selectively inhibited by a GSI, not by SGSM treatment. We can assert, for the first time, that SGSMs do not affect the neuronal differentiation of hNPCs while selectively decreasing endogenous A?42 levels in the same conditions. Our results suggest that our hNPC differentiation system can serve as a useful model to test the impact of GSIs and SGSMs on both endogenous A? levels and ?-secretase physiologic functions including endogenous Notch signaling.

SUBMITTER: D'Avanzo C

PROVIDER: S-EPMC4511200 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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γ-Secretase modulators reduce endogenous amyloid β42 levels in human neural progenitor cells without altering neuronal differentiation.

D'Avanzo Carla C Sliwinski Christopher C Wagner Steven L SL Tanzi Rudolph E RE Kim Doo Yeon DY Kovacs Dora M DM

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20150422 8

Soluble γ-secretase modulators (SGSMs) selectively decrease toxic amyloid β (Aβ) peptides (Aβ42). However, their effect on the physiologic functions of γ-secretase has not been tested in human model systems. γ-Secretase regulates fate determination of neural progenitor cells. Thus, we studied the impact of SGSMs on the neuronal differentiation of ReNcell VM (ReN) human neural progenitor cells (hNPCs). Quantitative PCR analysis showed that treatment of neurosphere-like ReN cell aggregate cultures ...[more]

PMID: 25903103

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Project description:BACKGROUND:γ-Secretase is a multiprotein protease that cleaves amyloid protein precursor (APP) and other type I transmembrane proteins. It has two catalytic subunits, presenilins 1 and 2 (PS1 and 2). In our previous report, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates and slightly different potencies of PS1 versus PS2 inhibition for select γ-secretase inhibitors (GSIs) on various substrates. In this study, we investigated whether γ-secretase modulators (GSMs) and inverse γ-secretase modulators (iGSMs) modulate γ-secretase processivity using multiple different substrates. We next used HEK 293T cell lines in which PSEN1 or PSEN2 was selectively knocked out to investigate processivity and response to GSMs and iGSMs. METHODS:For cell-free γ-secretase cleavage assay, recombinant substrates were incubated with CHAPSO-solubilized CHO or HEK 293T cell membrane with GSMs or iGSMs in suitable buffer. For cell-based assay, cDNA encoding substrates were transfected into HEK 293T cells. Cells were then treated with GSMs or iGSMs, and conditioned media were collected. Aβ and Aβ-like peptide production from cell-free and cell-based assay were measured by ELISA and mass spectrometry. RESULT:These studies demonstrated that GSMs are highly selective for effects on APP, whereas iGSMs have a more promiscuous effect on many substrates. Surprisingly, iGSMs actually appear to act as like GSIs on select substrates. The data with PSEN1 or PSEN2 knocked out HEK 293T reveal that PS1 has higher processivity and response to GSMs than PS2, but PS2 has higher response to iGSM. CONCLUSION:Collectively, these data indicate that GSMs are likely to have limited target-based toxicity. In addition, they show that iGSMs may act as substrate-selective GSIs providing a potential new route to identify leads for substrate-selective inhibitors of certain γ-secretase-mediated signaling events. With growing concerns that long-term β-secretase inhibitor is limited by target-based toxicities, such data supports continued development of GSMs as AD prophylactics.

Dataset Information

?-Secretase modulators reduce endogenous amyloid ?42 levels in human neural progenitor cells without altering neuronal differentiation.

Publications

γ-Secretase modulators reduce endogenous amyloid β42 levels in human neural progenitor cells without altering neuronal differentiation.

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