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PICALM modulates autophagy activity and tau accumulation.

Moreau K, Fleming A, Imarisio S, Lopez Ramirez A, Mercer JL, Jimenez-Sanchez M, Bento CF, Puri C, Zavodszky E, Siddiqi F, Lavau CP, Betton M, O'Kane CJ, Wechsler DS, Rubinsztein DC - Nat Commun (2014)

Bottom Line: Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models.CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation.This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, University of Cambridge, Cambridge Institute for Medical Research, Addenbrooke's Hospital, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.

ABSTRACT
Genome-wide association studies have identified several loci associated with Alzheimer's disease (AD), including proteins involved in endocytic trafficking such as PICALM/CALM (phosphatidylinositol binding clathrin assembly protein). It is unclear how these loci may contribute to AD pathology. Here we show that CALM modulates autophagy and alters clearance of tau, a protein which is a known autophagy substrate and which is causatively linked to AD, both in vitro and in vivo. Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models. CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation. This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover.

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CALM modulates autophagysubstrate clearance in vitro.(a) p62 vesicleformation in CALMknockdown HeLa cells. Confocal pictures are shown. #CALM-downregulated cells wherep62 vesiclesaccumulate. Data are representative of three independent experiments andshown as mean ±s.e.m. (n≥500 cells;*P<0.01; two-tailed t-test). Scale bars,5 μm. (b) Western blot analysis of p62, actin and GAPDH in HeLa cellsand HEK cells (basal conditions, BC, or without serum for 1 h)where CALM was knockeddown using shRNA or siRNA, as indicated. Data are mean ±s.d(n=3 experiments for HeLa cells and HEK cells;*P<0.05; two-tailed t-test). (c) Percentage ofQ74-expressing cells with aggregates in CALM knockdown HeLa cells. Data depict onerepresentative experiments performed in triplicate, out of three independentexperiments and shown as mean ±s.d. (*P<0.05;two-tailed t-test). (d) Tau-positive tangle formation in CALM knockdown cells. HeLa cellstransiently expressing DsRed-tau 4R were treated with Baf A1 for 4 h asindicated. Cells were fixed and analysed by confocal microscopy afterimmnunostaining for phosphorylated tau using PHF1 antibody (green). Data represent thenumber of cells with phosphorylated tau-positive tangles as mean ±s.e.m.(n=3 experiments; *P<0.01; two-tailed t-test).Scale bars, 5 μm.
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f2: CALM modulates autophagysubstrate clearance in vitro.(a) p62 vesicleformation in CALMknockdown HeLa cells. Confocal pictures are shown. #CALM-downregulated cells wherep62 vesiclesaccumulate. Data are representative of three independent experiments andshown as mean ±s.e.m. (n≥500 cells;*P<0.01; two-tailed t-test). Scale bars,5 μm. (b) Western blot analysis of p62, actin and GAPDH in HeLa cellsand HEK cells (basal conditions, BC, or without serum for 1 h)where CALM was knockeddown using shRNA or siRNA, as indicated. Data are mean ±s.d(n=3 experiments for HeLa cells and HEK cells;*P<0.05; two-tailed t-test). (c) Percentage ofQ74-expressing cells with aggregates in CALM knockdown HeLa cells. Data depict onerepresentative experiments performed in triplicate, out of three independentexperiments and shown as mean ±s.d. (*P<0.05;two-tailed t-test). (d) Tau-positive tangle formation in CALM knockdown cells. HeLa cellstransiently expressing DsRed-tau 4R were treated with Baf A1 for 4 h asindicated. Cells were fixed and analysed by confocal microscopy afterimmnunostaining for phosphorylated tau using PHF1 antibody (green). Data represent thenumber of cells with phosphorylated tau-positive tangles as mean ±s.e.m.(n=3 experiments; *P<0.01; two-tailed t-test).Scale bars, 5 μm.

Mentions: The inhibition of autophagosome formation and degradation when CALM was downregulated was associatedwith the accumulation of autophagic substrates, as measured by the number ofendogenous p62 dots per cell,p62 levels by westernblotting or the number of mutant huntingtin (Q74) aggregates per cell (huntingtin mutant with 74 glutamine repeats) (Fig.2ac). Both of these proteins aggregate when autophagy is impaired andthe number of mutant huntingtin aggregates correlates linearly with proteinabundance under normal conditions34. We also observed anaccumulation of AD associated tau, another autophagy substrate, and its phosphorylatedform (using PHF1 antibody) by immunofluorescence in structures resemblingtau tangles (a hallmarkof AD) in cells expressing DsRed-tau 4R when autophagy was inhibited by Baf A1 or when CALM was knocked down (Fig. 2d).


PICALM modulates autophagy activity and tau accumulation.

Moreau K, Fleming A, Imarisio S, Lopez Ramirez A, Mercer JL, Jimenez-Sanchez M, Bento CF, Puri C, Zavodszky E, Siddiqi F, Lavau CP, Betton M, O'Kane CJ, Wechsler DS, Rubinsztein DC - Nat Commun (2014)

CALM modulates autophagysubstrate clearance in vitro.(a) p62 vesicleformation in CALMknockdown HeLa cells. Confocal pictures are shown. #CALM-downregulated cells wherep62 vesiclesaccumulate. Data are representative of three independent experiments andshown as mean ±s.e.m. (n≥500 cells;*P<0.01; two-tailed t-test). Scale bars,5 μm. (b) Western blot analysis of p62, actin and GAPDH in HeLa cellsand HEK cells (basal conditions, BC, or without serum for 1 h)where CALM was knockeddown using shRNA or siRNA, as indicated. Data are mean ±s.d(n=3 experiments for HeLa cells and HEK cells;*P<0.05; two-tailed t-test). (c) Percentage ofQ74-expressing cells with aggregates in CALM knockdown HeLa cells. Data depict onerepresentative experiments performed in triplicate, out of three independentexperiments and shown as mean ±s.d. (*P<0.05;two-tailed t-test). (d) Tau-positive tangle formation in CALM knockdown cells. HeLa cellstransiently expressing DsRed-tau 4R were treated with Baf A1 for 4 h asindicated. Cells were fixed and analysed by confocal microscopy afterimmnunostaining for phosphorylated tau using PHF1 antibody (green). Data represent thenumber of cells with phosphorylated tau-positive tangles as mean ±s.e.m.(n=3 experiments; *P<0.01; two-tailed t-test).Scale bars, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199285&req=5

f2: CALM modulates autophagysubstrate clearance in vitro.(a) p62 vesicleformation in CALMknockdown HeLa cells. Confocal pictures are shown. #CALM-downregulated cells wherep62 vesiclesaccumulate. Data are representative of three independent experiments andshown as mean ±s.e.m. (n≥500 cells;*P<0.01; two-tailed t-test). Scale bars,5 μm. (b) Western blot analysis of p62, actin and GAPDH in HeLa cellsand HEK cells (basal conditions, BC, or without serum for 1 h)where CALM was knockeddown using shRNA or siRNA, as indicated. Data are mean ±s.d(n=3 experiments for HeLa cells and HEK cells;*P<0.05; two-tailed t-test). (c) Percentage ofQ74-expressing cells with aggregates in CALM knockdown HeLa cells. Data depict onerepresentative experiments performed in triplicate, out of three independentexperiments and shown as mean ±s.d. (*P<0.05;two-tailed t-test). (d) Tau-positive tangle formation in CALM knockdown cells. HeLa cellstransiently expressing DsRed-tau 4R were treated with Baf A1 for 4 h asindicated. Cells were fixed and analysed by confocal microscopy afterimmnunostaining for phosphorylated tau using PHF1 antibody (green). Data represent thenumber of cells with phosphorylated tau-positive tangles as mean ±s.e.m.(n=3 experiments; *P<0.01; two-tailed t-test).Scale bars, 5 μm.
Mentions: The inhibition of autophagosome formation and degradation when CALM was downregulated was associatedwith the accumulation of autophagic substrates, as measured by the number ofendogenous p62 dots per cell,p62 levels by westernblotting or the number of mutant huntingtin (Q74) aggregates per cell (huntingtin mutant with 74 glutamine repeats) (Fig.2ac). Both of these proteins aggregate when autophagy is impaired andthe number of mutant huntingtin aggregates correlates linearly with proteinabundance under normal conditions34. We also observed anaccumulation of AD associated tau, another autophagy substrate, and its phosphorylatedform (using PHF1 antibody) by immunofluorescence in structures resemblingtau tangles (a hallmarkof AD) in cells expressing DsRed-tau 4R when autophagy was inhibited by Baf A1 or when CALM was knocked down (Fig. 2d).

Bottom Line: Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models.CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation.This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, University of Cambridge, Cambridge Institute for Medical Research, Addenbrooke's Hospital, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.

ABSTRACT
Genome-wide association studies have identified several loci associated with Alzheimer's disease (AD), including proteins involved in endocytic trafficking such as PICALM/CALM (phosphatidylinositol binding clathrin assembly protein). It is unclear how these loci may contribute to AD pathology. Here we show that CALM modulates autophagy and alters clearance of tau, a protein which is a known autophagy substrate and which is causatively linked to AD, both in vitro and in vivo. Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models. CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation. This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover.

Show MeSH
Related in: MedlinePlus