Lysosomal alkalization and dysfunction in human fibroblasts with the Alzheimer’s disease-linked presenilin 1 A246E mutation can be reversed with cAMP

Abstract

Mutation in presenilin 1 (PS1) is one of the leading causes of familial Alzheimer’s disease (fAD). PS1 mutation exacerbates the autophagic and lysosomal pathology in AD patients, leading to accumulation of partially degraded material in bloated lysosomes and autophagosomes – a pathology that bears some resemblance to other diseases characterized by elevated lysosomal pH, like age-related macular degeneration. In this study, we examined the effect of the PS1-fAD mutation A246E on lysosomal pH and lysosomal function, and asked whether restoration of lysosomal pH could reverse some of these changes. Lysosomal pH was elevated by 0.2-0.3 pH units in human fibroblasts with the PS1-fAD mutation. The lysosomal alkalization in PS1-fAD fibroblasts was supported by a reduction in the pH-dependent cleavage of cathepsin D and by a reduction in binding of BODIPY FL-pepstatin A to the cathepsin D active site. PS1-fAD cells had increased LC3B-II/-I ratios and p62 levels, consistent with impaired lysosomal degradation and analogous to changes induced by lysosomal alkalinization with chloroquine. PS1-fAD fibroblasts had increased expression of ATP6V1B2, ATG5, BECN1 TFEB mRNA, and of ATP6V1B2, ATG5 and beclin at the protein level, consistent with chronic impairment of autophagic and lysosomal functions in the mutant cells. Critically, cAMP treatment reacidified lysosomal pH in mutant PS1-fAD; cAMP also increased the availability of active cathepsin D and lowered the LC3B-II/-I ratio. These results confirm a small elevation in the lysosomal pH of human PS1-fAD fibroblasts, demonstrate that this lysosomal alkalization is associated with chronic changes in autophagy and degradation, and suggest that treatment to reacidify the lysosomes with cAMP can reverse these changes.

Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

“…Moreover, stable transfection of PS1 into PS1/2 DKO cells reverses these features and restores V0a1 subunit glycosylation, lysosomal acidification and autophagic clearance (Lee et al, 2010). Importantly, additional studies have also demonstrated impaired vATPase V0a1 subunit maturation and endolysosomal acidification in multiple models, including PS1 ablated cells or mutant PS1 models (Avrahami et al, 2013; Coffey et al, 2014; Dobrowolski et al, 2012; Torres et al, 2012; Wolfe et al, 2013), underscoring that the acidification defect induced by PS1 loss of function is generalisable across various cell types. In one study, PS1 and vATPase V0a1 subunit were reported to have no role in lysosomal pH regulation (Coen et al, 2012) and differences between methods and cell culture conditions might explain some discrepancies between this study and our studies.…”

Autophagy and neurodegeneration

“…Rescue of lysosomal defects can restore autophagic activity. For example cAMP treatment decreased lysosomal pH in patient fibroblasts (16). Further, deletion of cystatin B (an inhibitor of lysosomal cysteine proteases) in an AD mouse model enhanced defective lysosomal turnover, promoted Aβ clearance, and improved mouse cognitive performance (17).…”

IL13 activates autophagy to regulate secretion in airway epithelial cells

“…Sequence-specific primers for MUC5AC and ATG5 were used as previously published. 11,63 Gene expression was normalized to OAZ1. 64 …”