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      PSEN2 (presenilin 2) mutants linked to familial Alzheimer disease impair autophagy by altering Ca2+ homeostasis

      1 , 1 , 1 , 1 , 1 , 2

      Autophagy

      Informa UK Limited

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          Abstract

          PSEN2 (presenilin 2) is one of the 3 proteins that, when mutated, causes early onset familial Alzheimer disease (FAD) cases. In addition to its well-known role within the γ-secretase complex (the enzyme ultimately responsible for Aβ peptides formation), PSEN2 is endowed with some γ-secretase-independent functions in distinct cell signaling pathways, such as the modulation of intracellular Ca 2+ homeostasis. Here, by using different FAD-PSEN2 cell models, we demonstrate that mutated PSEN2 impairs autophagy by causing a block in the degradative flux at the level of the autophagosome-lysosome fusion step. The defect does not depend on an altered lysosomal functionality but rather on a decreased recruitment of the small GTPase RAB7 to autophagosomes, a key event for normal autophagy progression. Importantly, FAD-PSEN2 action on autophagy is unrelated to its γ-secretase activity but depends on its previously reported ability to partially deplete ER Ca 2+ content, thus reducing cytosolic Ca 2+ response upon IP3-linked cell stimulations. Our data sustain the pivotal role for Ca 2+ signaling in autophagy and reveal a novel mechanism by which FAD-linked presenilins alter the degradative process, reinforcing the view of a causative role for a dysfunctional quality control pathway in AD neurodegeneration. Abbreviations: Aβ: amyloid β; AD: Alzheimer disease; ACTB: actin beta; AMPK: AMP-activated protein kinase; APP: amyloid-beta precursor protein; BafA: bafilomycin A 1 ; BAPTA-AM: 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester; CFP: cyan fluorescent protein; EGTA-AM: ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid acetoxymethyl ester; ER: endoplasmic reticulum; EGFP-HDQ74: enhanced GFP-huntingtin exon 1 containing 74 polyglutamine repeats; FAD: familial Alzheimer disease; FCS: fetal calf serum; FRET: fluorescence/Förster resonance energy transfer; GFP: green fluorescent protein; IP3: inositol trisphosphate; KD: knockdown; LAMP1: lysosomal associated membrane protein 1; MAP1LC3-II/LC3-II: lipidated microtubule-associated protein 1 light chain 3; MCU: mitochondrial calcium uniporter; MICU1: mitochondrial calcium uptake 1; MEFs: mouse embryonic fibroblasts; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; SQSTM1/p62: sequestosome 1; PSEN1: presenilin 1; PSEN2: presenilin 2; RAB7: RAB7A: member RAS oncogene family; RFP: red fluorescent protein; ATP2A/SERCA: ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting; siRNA: small interference RNA; V-ATPase: vacuolar-type H + -ATPase; WT: wild type

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          Most cited references 51

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          Essential regulation of cell bioenergetics by constitutive InsP3 receptor Ca2+ transfer to mitochondria.

          Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role for constitutive InsP(3)R-mediated Ca(2+) release in maintaining cellular bioenergetics. Macroautophagy provides eukaryotes with an adaptive response to nutrient deprivation that prolongs survival. Constitutive InsP(3)R Ca(2+) signaling is required for macroautophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to diminished mitochondrial Ca(2+) uptake. Mitochondrial uptake of InsP(3)R-released Ca(2+) is fundamentally required to provide optimal bioenergetics by providing sufficient reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer results in enhanced phosphorylation of pyruvate dehydrogenase and activation of AMPK, which activates prosurvival macroautophagy. Thus, constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics. Copyright 2010 Elsevier Inc. All rights reserved.
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            Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2.

            Macroautophagy is an evolutionary conserved lysosomal pathway involved in the turnover of cellular macromolecules and organelles. In spite of its essential role in tissue homeostasis, the molecular mechanisms regulating mammalian macroautophagy are poorly understood. Here, we demonstrate that a rise in the free cytosolic calcium ([Ca(2+)](c)) is a potent inducer of macroautophagy. Various Ca(2+) mobilizing agents (vitamin D(3) compounds, ionomycin, ATP, and thapsigargin) inhibit the activity of mammalian target of rapamycin, a negative regulator of macroautophagy, and induce massive accumulation of autophagosomes in a Beclin 1- and Atg7-dependent manner. This process is mediated by Ca(2+)/calmodulin-dependent kinase kinase-beta and AMP-activated protein kinase and inhibited by ectopic Bcl-2 located in the endoplasmatic reticulum (ER), where it lowers the [Ca(2+)](ER) and attenuates agonist-induced Ca(2+) fluxes. Thus, an increase in the [Ca(2+)](c) serves as a potent inducer of macroautophagy and as a target for the antiautophagy action of ER-located Bcl-2.
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              Role for Rab7 in maturation of late autophagic vacuoles.

              The small GTP binding protein Rab7 has a role in the late endocytic pathway and lysosome biogenesis. The role of mammalian Rab7 in autophagy is, however, unknown. We have addressed this by inhibiting Rab7 function with RNA interference and overexpression of dominant negative Rab7. We show here that Rab7 was needed for the formation of preferably perinuclear, large aggregates, where the autophagosome marker LC3 colocalised with Rab7 and late endosomal and lysosomal markers. By electron microscopy we showed that these large aggregates corresponded to autophagic vacuoles surrounding late endosomal or lysosomal vesicles. Our experiments with quantitative electron microscopy showed that Rab7 was not needed for the initial maturation of early autophagosomes to late autophagic vacuoles, but that it participated in the final maturation of late autophagic vacuoles. Finally, we showed that the recruitment of Rab7 to autophagic vacuoles was retarded in cells deficient in the lysosomal membrane proteins Lamp1 and Lamp2, which we have recently shown to accumulate late autophagic vacuoles during starvation. In conclusion, our results showed a role for Rab7 in the final maturation of late autophagic vacuoles.
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                Author and article information

                Journal
                Autophagy
                Autophagy
                Informa UK Limited
                1554-8627
                1554-8635
                March 20 2019
                March 20 2019
                : 1-19
                Affiliations
                [1 ] Department of Biomedical Sciences, University of Padua, Padua, Italy
                [2 ] Neuroscience Institute – Italian National Research Council (CNR), Padua, Italy
                Article
                10.1080/15548627.2019.1596489
                6844518
                30892128
                9f5d4016-91ef-492c-b370-940d61725df8
                © 2019

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