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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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Autophagy and CALMcontribute to tau degradationin vivo.(ai) Western blot analysis of tubulin, LC3-I and LC3-II in zebrafishlarvae where ATG7 wasdownregulated, as indicated. (ii) Western blot analysis of CALM-HA, actin and LC3-II inzebrafish larvae where CALM-HA was expressed, as indicated. The larvae weretreated with ammoniumchloride (NH4Cl), as indicated. (SE, shortexposure; LE, longer exposure). Quantification of LC3-II/actin ratio isshown. Data are representative of three independent experiments and shown asmean ±s.d. (*P<0.05; two-tailed t-test).(b) Modulation of autophagy alters Dendra-tau clearance dynamics. Thefluorescence intensity of each individual cell was quantified at eachtimepoint (n≥ 31 cells, ≥9 larvae per treatmentgroup) and mean cell intensity values for each drug treatment at eachtimepoint were calculated. Images were taken immediately afterphotoconversion and at 24, 30 and 48 h intervals thereafter.Rapamycin treatmentsignificantly increased the rate of Dendra-tau clearance. Ammonium chloride (NH4Cl) treatmentsignificantly decreases the rate of Dendra-tau clearance(**P<0.01, ***P<0.001, one-way analysis ofvariance (ANOVA)). Error bars are ±s.e.m. (c)Dendra-tau clearancein the presence of CALM:representative images of larvae with mosaic expression ofDendra-tau andfull-length CALM takenimmediately after photoconversion and at 6, 24, 30, 48 and 54 hafter conversion. The fluorescence intensity of individual cells wasquantified and mean fluorescent intensity of cells co-expressingDendra-tau and eitherfull-length CALM orΔ–ANTH CALM (CALM-ANTH-HA) constructs (n≥ 100cells, ≥9 larvae per treatment group) at each time point wascalculated. (i) Expression of full-length CALM significantly delayed theclearance of Dendra-tauat all time points compared with Δ–ANTH CALM (***P<0.001,one-way ANOVA). One graph, representative of three independent experiments,is presented. (CALM-ANTH-HA: 132 cells, 20 fishes; CALM-HA: 18 cells, 8 fishes).Another two experiments are shown in Supplementary Fig. S5C. Error bars are mean ±s.d. (ii)Treatment with ammoniumchloride alters the dynamics of Dendra-tau clearance. Expression offull-length CALMsignificantly delayed the clearance of Dendra-tau at all time points comparedwith Δ–ANTH CALM, as observed in i (P<0.001, one-wayANOVA). However, treatment of Δ–ANTH CALM injected larvae withammonium chlorideslows the Dendra-tauclearance by 70% to a level comparable to that observed in larvae injectedwith full-length CALM.Ammonium chloridetreatment of larvae injected with full-length CALM results in a modest (22%)decrease in Dendra-tauclearance, suggesting that CALM overexpression and ammonium chloride treatment have acumulative effect (n≥25 cells, n≥9larvae per treatment group). Error bars are mean ±s.d. Note that iand ii are distinct experiments.
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f8: Autophagy and CALMcontribute to tau degradationin vivo.(ai) Western blot analysis of tubulin, LC3-I and LC3-II in zebrafishlarvae where ATG7 wasdownregulated, as indicated. (ii) Western blot analysis of CALM-HA, actin and LC3-II inzebrafish larvae where CALM-HA was expressed, as indicated. The larvae weretreated with ammoniumchloride (NH4Cl), as indicated. (SE, shortexposure; LE, longer exposure). Quantification of LC3-II/actin ratio isshown. Data are representative of three independent experiments and shown asmean ±s.d. (*P<0.05; two-tailed t-test).(b) Modulation of autophagy alters Dendra-tau clearance dynamics. Thefluorescence intensity of each individual cell was quantified at eachtimepoint (n≥ 31 cells, ≥9 larvae per treatmentgroup) and mean cell intensity values for each drug treatment at eachtimepoint were calculated. Images were taken immediately afterphotoconversion and at 24, 30 and 48 h intervals thereafter.Rapamycin treatmentsignificantly increased the rate of Dendra-tau clearance. Ammonium chloride (NH4Cl) treatmentsignificantly decreases the rate of Dendra-tau clearance(**P<0.01, ***P<0.001, one-way analysis ofvariance (ANOVA)). Error bars are ±s.e.m. (c)Dendra-tau clearancein the presence of CALM:representative images of larvae with mosaic expression ofDendra-tau andfull-length CALM takenimmediately after photoconversion and at 6, 24, 30, 48 and 54 hafter conversion. The fluorescence intensity of individual cells wasquantified and mean fluorescent intensity of cells co-expressingDendra-tau and eitherfull-length CALM orΔ–ANTH CALM (CALM-ANTH-HA) constructs (n≥ 100cells, ≥9 larvae per treatment group) at each time point wascalculated. (i) Expression of full-length CALM significantly delayed theclearance of Dendra-tauat all time points compared with Δ–ANTH CALM (***P<0.001,one-way ANOVA). One graph, representative of three independent experiments,is presented. (CALM-ANTH-HA: 132 cells, 20 fishes; CALM-HA: 18 cells, 8 fishes).Another two experiments are shown in Supplementary Fig. S5C. Error bars are mean ±s.d. (ii)Treatment with ammoniumchloride alters the dynamics of Dendra-tau clearance. Expression offull-length CALMsignificantly delayed the clearance of Dendra-tau at all time points comparedwith Δ–ANTH CALM, as observed in i (P<0.001, one-wayANOVA). However, treatment of Δ–ANTH CALM injected larvae withammonium chlorideslows the Dendra-tauclearance by 70% to a level comparable to that observed in larvae injectedwith full-length CALM.Ammonium chloridetreatment of larvae injected with full-length CALM results in a modest (22%)decrease in Dendra-tauclearance, suggesting that CALM overexpression and ammonium chloride treatment have acumulative effect (n≥25 cells, n≥9larvae per treatment group). Error bars are mean ±s.d. Note that iand ii are distinct experiments.

Mentions: Although the Drosophila tools enable study of the effects of CALM knockdown in relation totau, we used zebrafish toexamine the effects of CALMoverexpression. We used this approach in favour of performing knockdown withmorpholino oligonucleotides, as the two PICALM-like genes in zebrafish would probably necessitatesilencing both genes, and the ‘gold standard’ validationfor these assays (performing rescue experiments by co-injection of messengerRNA) would be practically impossible due to the effects resulting fromoverexpression. CALMoverexpression decreased LC3-II levels in the zebrafish with or withoutammonium chloride(NH4Cl), whichmimics Baf A1 treatment(Fig. 8a), similar to what we observed in HeLa cells(Supplementary Fig. 1c).


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)

Autophagy and CALMcontribute to tau degradationin vivo.(ai) Western blot analysis of tubulin, LC3-I and LC3-II in zebrafishlarvae where ATG7 wasdownregulated, as indicated. (ii) Western blot analysis of CALM-HA, actin and LC3-II inzebrafish larvae where CALM-HA was expressed, as indicated. The larvae weretreated with ammoniumchloride (NH4Cl), as indicated. (SE, shortexposure; LE, longer exposure). Quantification of LC3-II/actin ratio isshown. Data are representative of three independent experiments and shown asmean ±s.d. (*P<0.05; two-tailed t-test).(b) Modulation of autophagy alters Dendra-tau clearance dynamics. Thefluorescence intensity of each individual cell was quantified at eachtimepoint (n≥ 31 cells, ≥9 larvae per treatmentgroup) and mean cell intensity values for each drug treatment at eachtimepoint were calculated. Images were taken immediately afterphotoconversion and at 24, 30 and 48 h intervals thereafter.Rapamycin treatmentsignificantly increased the rate of Dendra-tau clearance. Ammonium chloride (NH4Cl) treatmentsignificantly decreases the rate of Dendra-tau clearance(**P<0.01, ***P<0.001, one-way analysis ofvariance (ANOVA)). Error bars are ±s.e.m. (c)Dendra-tau clearancein the presence of CALM:representative images of larvae with mosaic expression ofDendra-tau andfull-length CALM takenimmediately after photoconversion and at 6, 24, 30, 48 and 54 hafter conversion. The fluorescence intensity of individual cells wasquantified and mean fluorescent intensity of cells co-expressingDendra-tau and eitherfull-length CALM orΔ–ANTH CALM (CALM-ANTH-HA) constructs (n≥ 100cells, ≥9 larvae per treatment group) at each time point wascalculated. (i) Expression of full-length CALM significantly delayed theclearance of Dendra-tauat all time points compared with Δ–ANTH CALM (***P<0.001,one-way ANOVA). One graph, representative of three independent experiments,is presented. (CALM-ANTH-HA: 132 cells, 20 fishes; CALM-HA: 18 cells, 8 fishes).Another two experiments are shown in Supplementary Fig. S5C. Error bars are mean ±s.d. (ii)Treatment with ammoniumchloride alters the dynamics of Dendra-tau clearance. Expression offull-length CALMsignificantly delayed the clearance of Dendra-tau at all time points comparedwith Δ–ANTH CALM, as observed in i (P<0.001, one-wayANOVA). However, treatment of Δ–ANTH CALM injected larvae withammonium chlorideslows the Dendra-tauclearance by 70% to a level comparable to that observed in larvae injectedwith full-length CALM.Ammonium chloridetreatment of larvae injected with full-length CALM results in a modest (22%)decrease in Dendra-tauclearance, suggesting that CALM overexpression and ammonium chloride treatment have acumulative effect (n≥25 cells, n≥9larvae per treatment group). Error bars are mean ±s.d. Note that iand ii are distinct experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199285&req=5

f8: Autophagy and CALMcontribute to tau degradationin vivo.(ai) Western blot analysis of tubulin, LC3-I and LC3-II in zebrafishlarvae where ATG7 wasdownregulated, as indicated. (ii) Western blot analysis of CALM-HA, actin and LC3-II inzebrafish larvae where CALM-HA was expressed, as indicated. The larvae weretreated with ammoniumchloride (NH4Cl), as indicated. (SE, shortexposure; LE, longer exposure). Quantification of LC3-II/actin ratio isshown. Data are representative of three independent experiments and shown asmean ±s.d. (*P<0.05; two-tailed t-test).(b) Modulation of autophagy alters Dendra-tau clearance dynamics. Thefluorescence intensity of each individual cell was quantified at eachtimepoint (n≥ 31 cells, ≥9 larvae per treatmentgroup) and mean cell intensity values for each drug treatment at eachtimepoint were calculated. Images were taken immediately afterphotoconversion and at 24, 30 and 48 h intervals thereafter.Rapamycin treatmentsignificantly increased the rate of Dendra-tau clearance. Ammonium chloride (NH4Cl) treatmentsignificantly decreases the rate of Dendra-tau clearance(**P<0.01, ***P<0.001, one-way analysis ofvariance (ANOVA)). Error bars are ±s.e.m. (c)Dendra-tau clearancein the presence of CALM:representative images of larvae with mosaic expression ofDendra-tau andfull-length CALM takenimmediately after photoconversion and at 6, 24, 30, 48 and 54 hafter conversion. The fluorescence intensity of individual cells wasquantified and mean fluorescent intensity of cells co-expressingDendra-tau and eitherfull-length CALM orΔ–ANTH CALM (CALM-ANTH-HA) constructs (n≥ 100cells, ≥9 larvae per treatment group) at each time point wascalculated. (i) Expression of full-length CALM significantly delayed theclearance of Dendra-tauat all time points compared with Δ–ANTH CALM (***P<0.001,one-way ANOVA). One graph, representative of three independent experiments,is presented. (CALM-ANTH-HA: 132 cells, 20 fishes; CALM-HA: 18 cells, 8 fishes).Another two experiments are shown in Supplementary Fig. S5C. Error bars are mean ±s.d. (ii)Treatment with ammoniumchloride alters the dynamics of Dendra-tau clearance. Expression offull-length CALMsignificantly delayed the clearance of Dendra-tau at all time points comparedwith Δ–ANTH CALM, as observed in i (P<0.001, one-wayANOVA). However, treatment of Δ–ANTH CALM injected larvae withammonium chlorideslows the Dendra-tauclearance by 70% to a level comparable to that observed in larvae injectedwith full-length CALM.Ammonium chloridetreatment of larvae injected with full-length CALM results in a modest (22%)decrease in Dendra-tauclearance, suggesting that CALM overexpression and ammonium chloride treatment have acumulative effect (n≥25 cells, n≥9larvae per treatment group). Error bars are mean ±s.d. Note that iand ii are distinct experiments.
Mentions: Although the Drosophila tools enable study of the effects of CALM knockdown in relation totau, we used zebrafish toexamine the effects of CALMoverexpression. We used this approach in favour of performing knockdown withmorpholino oligonucleotides, as the two PICALM-like genes in zebrafish would probably necessitatesilencing both genes, and the ‘gold standard’ validationfor these assays (performing rescue experiments by co-injection of messengerRNA) would be practically impossible due to the effects resulting fromoverexpression. CALMoverexpression decreased LC3-II levels in the zebrafish with or withoutammonium chloride(NH4Cl), whichmimics Baf A1 treatment(Fig. 8a), similar to what we observed in HeLa cells(Supplementary Fig. 1c).

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