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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 theformation of tau-positivetangles in zebrafish.(a) Histological section to demonstrate the individual cell layers ofthe zebrafish retina. The photoreceptor layer (PR, comprising rod and conephotoreceptors) lies immediately adjacent to the retinal pigment epithelium(RPE) at the outermost surface of the eye. Thioflavin-S labelling of retinalsections was used to identify neurofibrillary tangles in the photoreceptorlayer (marked with yellow dotted lines). No labelling was observed in theretina of rho::GFP at 8 d.p.f., whereas distinct thioflavin-S-positivetangles (arrows) were observed in the photoreceptor layer ofrho::GFP-tau fish.Note, the RPE is highly autofluorescent due to the presence of silverpigment. High power regions are shown in the top right of each panel.(b) Unilateral electroporation of CALM into the retina ofrho::GFP-tauzebrafish resulted in a marked increase in thioflavin-S positive tangles inthe electroporated retina in the photoreceptor layer (PR) compared with thecontrol side. Top panel are lower magnification images to show the retinalcell layers. Thioflavin-S labelling is restricted to the PR layer. Note theRPE is highly autofluorescent due to the presence of silver pigment. Bottompanel are higher magnification images to show individual thioflavin-Stangles the largest of which are indicated by arrows.
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f10: CALM modulates theformation of tau-positivetangles in zebrafish.(a) Histological section to demonstrate the individual cell layers ofthe zebrafish retina. The photoreceptor layer (PR, comprising rod and conephotoreceptors) lies immediately adjacent to the retinal pigment epithelium(RPE) at the outermost surface of the eye. Thioflavin-S labelling of retinalsections was used to identify neurofibrillary tangles in the photoreceptorlayer (marked with yellow dotted lines). No labelling was observed in theretina of rho::GFP at 8 d.p.f., whereas distinct thioflavin-S-positivetangles (arrows) were observed in the photoreceptor layer ofrho::GFP-tau fish.Note, the RPE is highly autofluorescent due to the presence of silverpigment. High power regions are shown in the top right of each panel.(b) Unilateral electroporation of CALM into the retina ofrho::GFP-tauzebrafish resulted in a marked increase in thioflavin-S positive tangles inthe electroporated retina in the photoreceptor layer (PR) compared with thecontrol side. Top panel are lower magnification images to show the retinalcell layers. Thioflavin-S labelling is restricted to the PR layer. Note theRPE is highly autofluorescent due to the presence of silver pigment. Bottompanel are higher magnification images to show individual thioflavin-Stangles the largest of which are indicated by arrows.

Mentions: To examine the consequences of CALM overexpression on tau toxicity, we developed a zebrafish model expressingenhanced GFP (EGFP)-tagged human tau under the control of the rhodopsin promoter to driveexpression in the rod photoreceptors (rho::GFP-tau). We observed normal development ofthe rod photoreceptors expressing EGFP-tau from 3 days post fertilization (d.p.f.) to 6 d.p.f.,then degeneration from 7 d.p.f. onwards, initially within the central retinalregion and then at the margins (Supplementary Fig. 7a,b). Western blotting and immunohistochemistryfor rhodopsin, the major component of the rod outer segment (Supplementary Fig. 7c), confirmed that theloss of EGFP-tauphotoreceptors was truly degeneration of the rods rather than transgenedownregulation. Accordingly, in subsequent experiments, we used the presence ofEGFP-tau-positivephotoreceptors as a proxy for assessing neurodegeneration. Before degeneration,we observed phosphorylation of the transgenic human tau protein at specific serine and threonine residues (Supplementary Fig. 7d); such phosphorylationis a hallmark of taupathology in human and can be used as an indicator of disease progression4142. The degeneration was confirmed by increased numbers ofapoptotic cells in the retina of rho::GFP-tau compared with rho::GFP fish (a control transgenic lineexpressing GFP under the rhodopsin promoter) at 7 d.p.f. (Supplementary Fig. 7e). Rod photoreceptordegeneration was prevented by treatment with rapamycin (Fig. 9a) and exacerbated bythe autophagy blockers wortmannin and NH4Cl (Fig. 9a),suggesting that autophagy manipulation can alter disease progression in thismodel, as described in other systems10. We developed a novelelectroporation technique to deliver exogenous DNA to the photoreceptor layer oflarval zebrafish (Supplementary Fig.7f) and used this approach to overexpress CALM in the photoreceptors ofrho::GFP-tau fish orrho::GFP fish to investigate the effects of CALM expression on tau-induced pathology. CALM overexpression in the photoreceptor layer acceleratedrod photoreceptor degeneration and increased the number of apoptoticphotoreceptors in rho::GFP-tau but did not cause any signs of pathology in the rho::GFPfish (Fig. 9b and Supplementary Figs 7e and 8a,b). To investigate whetherneurofibrillary tangles were evident, we blocked cell death by treatment withthe caspase inhibitor Z-VAD-FMK, to prolong photoreceptor survival.Thioflavin-S-positive tangles were observed in the photoreceptor layer ofrho::GFP-tau fish, butnot rho::GFP fish (Fig. 10). The accelerated degenerationfollowing CALM overexpressionwas associated with increased tau phosphorylation and accumulation ofthioflavin-S-positive tangles (Fig. 10b and Supplementary Fig. 8c). To validatethat this degeneration was caused by autophagic impairment, we treatedelectroporated fish with autophagy-inducing (rapamycin) or -inhibiting (NH4Cl) drugs that modulatedtau toxicity (Fig. 9a). NH4Cl exacerbated the photoreceptor degenerationin the control eye (as observed previously, Fig. 9a) suchthat the control eye and the electroporated eye showed equal levels ofdegeneration (Fig. 9c). Rapamycin rescued photoreceptordegeneration in the control eye but was unable to rescue the degeneration in theelectroporated eye (Fig. 9c). Although rapamycin can induce autophagy andrescue degeneration in the control eye, our data suggest that CALM disrupts autophagosome biogenesisand therefore rapamycintreatment is unable to rescue degeneration in the CALM-electroporated eye, as autophagyupregulation via target of rapamycin inhibition cannot overcome the deficit inautophagosome formation caused by CALM. These data suggest that the effects of CALM on tau toxicity in this model areautophagy dependent.


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 theformation of tau-positivetangles in zebrafish.(a) Histological section to demonstrate the individual cell layers ofthe zebrafish retina. The photoreceptor layer (PR, comprising rod and conephotoreceptors) lies immediately adjacent to the retinal pigment epithelium(RPE) at the outermost surface of the eye. Thioflavin-S labelling of retinalsections was used to identify neurofibrillary tangles in the photoreceptorlayer (marked with yellow dotted lines). No labelling was observed in theretina of rho::GFP at 8 d.p.f., whereas distinct thioflavin-S-positivetangles (arrows) were observed in the photoreceptor layer ofrho::GFP-tau fish.Note, the RPE is highly autofluorescent due to the presence of silverpigment. High power regions are shown in the top right of each panel.(b) Unilateral electroporation of CALM into the retina ofrho::GFP-tauzebrafish resulted in a marked increase in thioflavin-S positive tangles inthe electroporated retina in the photoreceptor layer (PR) compared with thecontrol side. Top panel are lower magnification images to show the retinalcell layers. Thioflavin-S labelling is restricted to the PR layer. Note theRPE is highly autofluorescent due to the presence of silver pigment. Bottompanel are higher magnification images to show individual thioflavin-Stangles the largest of which are indicated by arrows.
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Related In: Results  -  Collection

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

f10: CALM modulates theformation of tau-positivetangles in zebrafish.(a) Histological section to demonstrate the individual cell layers ofthe zebrafish retina. The photoreceptor layer (PR, comprising rod and conephotoreceptors) lies immediately adjacent to the retinal pigment epithelium(RPE) at the outermost surface of the eye. Thioflavin-S labelling of retinalsections was used to identify neurofibrillary tangles in the photoreceptorlayer (marked with yellow dotted lines). No labelling was observed in theretina of rho::GFP at 8 d.p.f., whereas distinct thioflavin-S-positivetangles (arrows) were observed in the photoreceptor layer ofrho::GFP-tau fish.Note, the RPE is highly autofluorescent due to the presence of silverpigment. High power regions are shown in the top right of each panel.(b) Unilateral electroporation of CALM into the retina ofrho::GFP-tauzebrafish resulted in a marked increase in thioflavin-S positive tangles inthe electroporated retina in the photoreceptor layer (PR) compared with thecontrol side. Top panel are lower magnification images to show the retinalcell layers. Thioflavin-S labelling is restricted to the PR layer. Note theRPE is highly autofluorescent due to the presence of silver pigment. Bottompanel are higher magnification images to show individual thioflavin-Stangles the largest of which are indicated by arrows.
Mentions: To examine the consequences of CALM overexpression on tau toxicity, we developed a zebrafish model expressingenhanced GFP (EGFP)-tagged human tau under the control of the rhodopsin promoter to driveexpression in the rod photoreceptors (rho::GFP-tau). We observed normal development ofthe rod photoreceptors expressing EGFP-tau from 3 days post fertilization (d.p.f.) to 6 d.p.f.,then degeneration from 7 d.p.f. onwards, initially within the central retinalregion and then at the margins (Supplementary Fig. 7a,b). Western blotting and immunohistochemistryfor rhodopsin, the major component of the rod outer segment (Supplementary Fig. 7c), confirmed that theloss of EGFP-tauphotoreceptors was truly degeneration of the rods rather than transgenedownregulation. Accordingly, in subsequent experiments, we used the presence ofEGFP-tau-positivephotoreceptors as a proxy for assessing neurodegeneration. Before degeneration,we observed phosphorylation of the transgenic human tau protein at specific serine and threonine residues (Supplementary Fig. 7d); such phosphorylationis a hallmark of taupathology in human and can be used as an indicator of disease progression4142. The degeneration was confirmed by increased numbers ofapoptotic cells in the retina of rho::GFP-tau compared with rho::GFP fish (a control transgenic lineexpressing GFP under the rhodopsin promoter) at 7 d.p.f. (Supplementary Fig. 7e). Rod photoreceptordegeneration was prevented by treatment with rapamycin (Fig. 9a) and exacerbated bythe autophagy blockers wortmannin and NH4Cl (Fig. 9a),suggesting that autophagy manipulation can alter disease progression in thismodel, as described in other systems10. We developed a novelelectroporation technique to deliver exogenous DNA to the photoreceptor layer oflarval zebrafish (Supplementary Fig.7f) and used this approach to overexpress CALM in the photoreceptors ofrho::GFP-tau fish orrho::GFP fish to investigate the effects of CALM expression on tau-induced pathology. CALM overexpression in the photoreceptor layer acceleratedrod photoreceptor degeneration and increased the number of apoptoticphotoreceptors in rho::GFP-tau but did not cause any signs of pathology in the rho::GFPfish (Fig. 9b and Supplementary Figs 7e and 8a,b). To investigate whetherneurofibrillary tangles were evident, we blocked cell death by treatment withthe caspase inhibitor Z-VAD-FMK, to prolong photoreceptor survival.Thioflavin-S-positive tangles were observed in the photoreceptor layer ofrho::GFP-tau fish, butnot rho::GFP fish (Fig. 10). The accelerated degenerationfollowing CALM overexpressionwas associated with increased tau phosphorylation and accumulation ofthioflavin-S-positive tangles (Fig. 10b and Supplementary Fig. 8c). To validatethat this degeneration was caused by autophagic impairment, we treatedelectroporated fish with autophagy-inducing (rapamycin) or -inhibiting (NH4Cl) drugs that modulatedtau toxicity (Fig. 9a). NH4Cl exacerbated the photoreceptor degenerationin the control eye (as observed previously, Fig. 9a) suchthat the control eye and the electroporated eye showed equal levels ofdegeneration (Fig. 9c). Rapamycin rescued photoreceptordegeneration in the control eye but was unable to rescue the degeneration in theelectroporated eye (Fig. 9c). Although rapamycin can induce autophagy andrescue degeneration in the control eye, our data suggest that CALM disrupts autophagosome biogenesisand therefore rapamycintreatment is unable to rescue degeneration in the CALM-electroporated eye, as autophagyupregulation via target of rapamycin inhibition cannot overcome the deficit inautophagosome formation caused by CALM. These data suggest that the effects of CALM on tau toxicity in this model areautophagy dependent.

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