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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 regulates autophagyand tau degradation inDrosophila.(a) Western blotting showing downregulation of lap (i) or increase inAtg8a-II (ii)expression level in adult fly heads on lap downregulation usingthe UAS-RNAi lines lapGD12732 orlapKK105767, or theheterozygous allele lap1. Quantification oflap/actin andAtg8a-II/actin isshown. Genotypes: Control w; elav-GAL4/+; for the RNAi lines:w;elav-GAL4/lapKK105767 andw;elav-GAL4/+;lapGD12732/+; forthe lap1 allele:w;elav-GAL4/+;lap1/+. (b)Western blotting showing the accumulation of tau in Drosophila adult flyheads on lapdownregulation using the UAS-RNAi lines lapGD12732 or lapKK105767, or theheterozygous allele lap1. Quantification oftau/actin is shown.Genotypes: Control w;elav-GAL4/+;for tau-WT:w;elav-GAL4/+;UAS-tau-WT/+; for RNAi lines: w; elav-GAL4/ lapKK105767;UAS-tau-WT/+and w; elav-GAL4/+;lapGD12732/tau-WT; for thelap1 allele:w;elav-GAL4/+;lap1/+ orw;elav-GAL4/+;lap1/UAS-tau-WT.
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f7: CALM regulates autophagyand tau degradation inDrosophila.(a) Western blotting showing downregulation of lap (i) or increase inAtg8a-II (ii)expression level in adult fly heads on lap downregulation usingthe UAS-RNAi lines lapGD12732 orlapKK105767, or theheterozygous allele lap1. Quantification oflap/actin andAtg8a-II/actin isshown. Genotypes: Control w; elav-GAL4/+; for the RNAi lines:w;elav-GAL4/lapKK105767 andw;elav-GAL4/+;lapGD12732/+; forthe lap1 allele:w;elav-GAL4/+;lap1/+. (b)Western blotting showing the accumulation of tau in Drosophila adult flyheads on lapdownregulation using the UAS-RNAi lines lapGD12732 or lapKK105767, or theheterozygous allele lap1. Quantification oftau/actin is shown.Genotypes: Control w;elav-GAL4/+;for tau-WT:w;elav-GAL4/+;UAS-tau-WT/+; for RNAi lines: w; elav-GAL4/ lapKK105767;UAS-tau-WT/+and w; elav-GAL4/+;lapGD12732/tau-WT; for thelap1 allele:w;elav-GAL4/+;lap1/+ orw;elav-GAL4/+;lap1/UAS-tau-WT.

Mentions: GWAS studies have identified that CALM is associated with AD risk2021.However, it is unclear how CALM impacts the development of AD. We hypothesized that itcould be via its regulation of autophagy activities, which would impacttau clearance. Invivo, we observed that downregulation of the DrosophilaCALM homologue,lap, using twoindependent RNA interference (RNAi) lines (lapGD12732 or lapKK105767, whose sequences donot overlap)37 or the heterozygous loss-of-functionlap1 allele38(Fig. 7ai), increases the level of Atg8a-II (Fig.7aii), the closest Drosophila homologue of mammalian LC3-II(ref. 39), like mammalian CALM knockdown (Fig.1). To study whether lap influences tau levels in vivo, we generated Drosophilaoverexpressing wild-type human tau (4R isoform). This transgenic line has no phenotype withregard to lethality, eye degeneration, locomotor activity, wing development andpseudopupil degeneration when we drive the transgene in diverse tissues(summarized in Supplementary Table1 and Supplementary Fig.5). This model expresses tau at much lower levels (<15%) compared with a modelwith overt phenotypes (Supplementary Fig.5a)40. When we reduced Drosophila lap levels (using either RNAior heterozygous allele lap1) in flies expressing humantau in neurons, weobserved an increased human tau/actin ratio (Fig. 7b).


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 regulates autophagyand tau degradation inDrosophila.(a) Western blotting showing downregulation of lap (i) or increase inAtg8a-II (ii)expression level in adult fly heads on lap downregulation usingthe UAS-RNAi lines lapGD12732 orlapKK105767, or theheterozygous allele lap1. Quantification oflap/actin andAtg8a-II/actin isshown. Genotypes: Control w; elav-GAL4/+; for the RNAi lines:w;elav-GAL4/lapKK105767 andw;elav-GAL4/+;lapGD12732/+; forthe lap1 allele:w;elav-GAL4/+;lap1/+. (b)Western blotting showing the accumulation of tau in Drosophila adult flyheads on lapdownregulation using the UAS-RNAi lines lapGD12732 or lapKK105767, or theheterozygous allele lap1. Quantification oftau/actin is shown.Genotypes: Control w;elav-GAL4/+;for tau-WT:w;elav-GAL4/+;UAS-tau-WT/+; for RNAi lines: w; elav-GAL4/ lapKK105767;UAS-tau-WT/+and w; elav-GAL4/+;lapGD12732/tau-WT; for thelap1 allele:w;elav-GAL4/+;lap1/+ orw;elav-GAL4/+;lap1/UAS-tau-WT.
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f7: CALM regulates autophagyand tau degradation inDrosophila.(a) Western blotting showing downregulation of lap (i) or increase inAtg8a-II (ii)expression level in adult fly heads on lap downregulation usingthe UAS-RNAi lines lapGD12732 orlapKK105767, or theheterozygous allele lap1. Quantification oflap/actin andAtg8a-II/actin isshown. Genotypes: Control w; elav-GAL4/+; for the RNAi lines:w;elav-GAL4/lapKK105767 andw;elav-GAL4/+;lapGD12732/+; forthe lap1 allele:w;elav-GAL4/+;lap1/+. (b)Western blotting showing the accumulation of tau in Drosophila adult flyheads on lapdownregulation using the UAS-RNAi lines lapGD12732 or lapKK105767, or theheterozygous allele lap1. Quantification oftau/actin is shown.Genotypes: Control w;elav-GAL4/+;for tau-WT:w;elav-GAL4/+;UAS-tau-WT/+; for RNAi lines: w; elav-GAL4/ lapKK105767;UAS-tau-WT/+and w; elav-GAL4/+;lapGD12732/tau-WT; for thelap1 allele:w;elav-GAL4/+;lap1/+ orw;elav-GAL4/+;lap1/UAS-tau-WT.
Mentions: GWAS studies have identified that CALM is associated with AD risk2021.However, it is unclear how CALM impacts the development of AD. We hypothesized that itcould be via its regulation of autophagy activities, which would impacttau clearance. Invivo, we observed that downregulation of the DrosophilaCALM homologue,lap, using twoindependent RNA interference (RNAi) lines (lapGD12732 or lapKK105767, whose sequences donot overlap)37 or the heterozygous loss-of-functionlap1 allele38(Fig. 7ai), increases the level of Atg8a-II (Fig.7aii), the closest Drosophila homologue of mammalian LC3-II(ref. 39), like mammalian CALM knockdown (Fig.1). To study whether lap influences tau levels in vivo, we generated Drosophilaoverexpressing wild-type human tau (4R isoform). This transgenic line has no phenotype withregard to lethality, eye degeneration, locomotor activity, wing development andpseudopupil degeneration when we drive the transgene in diverse tissues(summarized in Supplementary Table1 and Supplementary Fig.5). This model expresses tau at much lower levels (<15%) compared with a modelwith overt phenotypes (Supplementary Fig.5a)40. When we reduced Drosophila lap levels (using either RNAior heterozygous allele lap1) in flies expressing humantau in neurons, weobserved an increased human tau/actin ratio (Fig. 7b).

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