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Disruption of endocytic trafficking in frontotemporal dementia with CHMP2B mutations.

Urwin H, Authier A, Nielsen JE, Metcalf D, Powell C, Froud K, Malcolm DS, Holm I, Johannsen P, Brown J, Fisher EM, van der Zee J, Bruyland M, FReJA ConsortiumVan Broeckhoven C, Collinge J, Brandner S, Futter C, Isaacs AM - Hum. Mol. Genet. (2010)

Bottom Line: Mutations in CHMP2B cause frontotemporal dementia (FTD) in a large Danish pedigree, which is termed FTD linked to chromosome 3 (FTD-3), and also in an unrelated familial FTD patient.CHMP2B is a component of the ESCRT-III complex, which is required for function of the multivesicular body (MVB), an endosomal structure that fuses with the lysosome to degrade endocytosed proteins.We report a novel endosomal pathology in CHMP2B mutation-positive patient brains and also identify and characterize abnormal endosomes in patient fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: MRC Prion Unit, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK.

ABSTRACT
Mutations in CHMP2B cause frontotemporal dementia (FTD) in a large Danish pedigree, which is termed FTD linked to chromosome 3 (FTD-3), and also in an unrelated familial FTD patient. CHMP2B is a component of the ESCRT-III complex, which is required for function of the multivesicular body (MVB), an endosomal structure that fuses with the lysosome to degrade endocytosed proteins. We report a novel endosomal pathology in CHMP2B mutation-positive patient brains and also identify and characterize abnormal endosomes in patient fibroblasts. Functional studies demonstrate a specific disruption of endosome-lysosome fusion but not protein sorting by the MVB. We provide evidence for a mechanism for impaired endosome-lysosome fusion whereby mutant CHMP2B constitutively binds to MVBs and prevents recruitment of proteins necessary for fusion to occur, such as Rab7. The fusion of endosomes with lysosomes is required for neuronal function and the data presented therefore suggest a pathogenic mechanism for FTD caused by CHMP2B mutations.

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Impaired recruitment of Rab7 onto endosomes in mutant CHMP2B expressing cells. (A) SK-N-SH cells were co-transfected with EGFP-Rab7 (green), HA-CHMP2BWildtype (a, b), HA-CHMP2BIntron5 (c, d) or CHMP2BΔ10 (e, f) and then immunostained for CD63 to visualise endosomes (red) and HA. All CD63-positive endosomes within each of 40–50 cells per condition were scored for colocalization of EGFP-Rab7 by an experimenter blind to conditions. (B) There was a significant reduction in the percentage of endosomes that recruited EGFP-Rab7 in cells expressing CHMP2BIntron5 (P < 0.0001) or CHMP2BΔ10 (P < 0.001). Recruitment of endogenous Rab7 was similarly impaired (P < 0.0001 for both CHMP2BIntron5 and CHMP2BΔ10). Scale bar = 10 µm.
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DDQ100F8: Impaired recruitment of Rab7 onto endosomes in mutant CHMP2B expressing cells. (A) SK-N-SH cells were co-transfected with EGFP-Rab7 (green), HA-CHMP2BWildtype (a, b), HA-CHMP2BIntron5 (c, d) or CHMP2BΔ10 (e, f) and then immunostained for CD63 to visualise endosomes (red) and HA. All CD63-positive endosomes within each of 40–50 cells per condition were scored for colocalization of EGFP-Rab7 by an experimenter blind to conditions. (B) There was a significant reduction in the percentage of endosomes that recruited EGFP-Rab7 in cells expressing CHMP2BIntron5 (P < 0.0001) or CHMP2BΔ10 (P < 0.001). Recruitment of endogenous Rab7 was similarly impaired (P < 0.0001 for both CHMP2BIntron5 and CHMP2BΔ10). Scale bar = 10 µm.

Mentions: We hypothesised that the impaired fusion of endosomes with lysosomes may be due to a failure of CHMP2BIntron5-positive endosomes to recruit proteins necessary for heterotypic fusion. Endosome–lysosome fusion requires recruitment of Rab7, a recognized marker of late, fusion-competent endosomes (14). SK-N-SH cells were co-transfected with EGFP-Rab7 and CHMP2BWildtype, CHMP2BIntron5 or CHMP2BΔ10 and then immunostained for CD63 to visualize late endosomes (Fig. 8A). There was an approximate one-third reduction in the recruitment of EGFP-Rab7 onto endosomes in CHMP2BIntron5 cells (t-test, P < 0.0001) and an approximate one-quarter reduction in CHMP2BΔ10 cells (test, P < 0.001) when compared with cells expressing CHMP2BWildtype. This assay was repeated using an anti-Rab7 antibody to show endogenous Rab7 rather than using overexpression (Supplementary Material, Fig. S2). Both CHMP2BIntron5 and CHMP2BΔ10 cells showed at least a one-third reduction in recruitment of endogenous Rab7 onto endosomes compared with CHMP2BWildtype cells (t-tests, both P < 0.0001) (Fig. 8B). There were no significant differences when comparing between experiments, i.e. CHMP2BIntron5 or CHMP2BΔ10 cells showed a similar reduction in both EGFP-Rab7 and endogenous Rab7 recruitment (Fig. 8B).


Disruption of endocytic trafficking in frontotemporal dementia with CHMP2B mutations.

Urwin H, Authier A, Nielsen JE, Metcalf D, Powell C, Froud K, Malcolm DS, Holm I, Johannsen P, Brown J, Fisher EM, van der Zee J, Bruyland M, FReJA ConsortiumVan Broeckhoven C, Collinge J, Brandner S, Futter C, Isaacs AM - Hum. Mol. Genet. (2010)

Impaired recruitment of Rab7 onto endosomes in mutant CHMP2B expressing cells. (A) SK-N-SH cells were co-transfected with EGFP-Rab7 (green), HA-CHMP2BWildtype (a, b), HA-CHMP2BIntron5 (c, d) or CHMP2BΔ10 (e, f) and then immunostained for CD63 to visualise endosomes (red) and HA. All CD63-positive endosomes within each of 40–50 cells per condition were scored for colocalization of EGFP-Rab7 by an experimenter blind to conditions. (B) There was a significant reduction in the percentage of endosomes that recruited EGFP-Rab7 in cells expressing CHMP2BIntron5 (P < 0.0001) or CHMP2BΔ10 (P < 0.001). Recruitment of endogenous Rab7 was similarly impaired (P < 0.0001 for both CHMP2BIntron5 and CHMP2BΔ10). Scale bar = 10 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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DDQ100F8: Impaired recruitment of Rab7 onto endosomes in mutant CHMP2B expressing cells. (A) SK-N-SH cells were co-transfected with EGFP-Rab7 (green), HA-CHMP2BWildtype (a, b), HA-CHMP2BIntron5 (c, d) or CHMP2BΔ10 (e, f) and then immunostained for CD63 to visualise endosomes (red) and HA. All CD63-positive endosomes within each of 40–50 cells per condition were scored for colocalization of EGFP-Rab7 by an experimenter blind to conditions. (B) There was a significant reduction in the percentage of endosomes that recruited EGFP-Rab7 in cells expressing CHMP2BIntron5 (P < 0.0001) or CHMP2BΔ10 (P < 0.001). Recruitment of endogenous Rab7 was similarly impaired (P < 0.0001 for both CHMP2BIntron5 and CHMP2BΔ10). Scale bar = 10 µm.
Mentions: We hypothesised that the impaired fusion of endosomes with lysosomes may be due to a failure of CHMP2BIntron5-positive endosomes to recruit proteins necessary for heterotypic fusion. Endosome–lysosome fusion requires recruitment of Rab7, a recognized marker of late, fusion-competent endosomes (14). SK-N-SH cells were co-transfected with EGFP-Rab7 and CHMP2BWildtype, CHMP2BIntron5 or CHMP2BΔ10 and then immunostained for CD63 to visualize late endosomes (Fig. 8A). There was an approximate one-third reduction in the recruitment of EGFP-Rab7 onto endosomes in CHMP2BIntron5 cells (t-test, P < 0.0001) and an approximate one-quarter reduction in CHMP2BΔ10 cells (test, P < 0.001) when compared with cells expressing CHMP2BWildtype. This assay was repeated using an anti-Rab7 antibody to show endogenous Rab7 rather than using overexpression (Supplementary Material, Fig. S2). Both CHMP2BIntron5 and CHMP2BΔ10 cells showed at least a one-third reduction in recruitment of endogenous Rab7 onto endosomes compared with CHMP2BWildtype cells (t-tests, both P < 0.0001) (Fig. 8B). There were no significant differences when comparing between experiments, i.e. CHMP2BIntron5 or CHMP2BΔ10 cells showed a similar reduction in both EGFP-Rab7 and endogenous Rab7 recruitment (Fig. 8B).

Bottom Line: Mutations in CHMP2B cause frontotemporal dementia (FTD) in a large Danish pedigree, which is termed FTD linked to chromosome 3 (FTD-3), and also in an unrelated familial FTD patient.CHMP2B is a component of the ESCRT-III complex, which is required for function of the multivesicular body (MVB), an endosomal structure that fuses with the lysosome to degrade endocytosed proteins.We report a novel endosomal pathology in CHMP2B mutation-positive patient brains and also identify and characterize abnormal endosomes in patient fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: MRC Prion Unit, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK.

ABSTRACT
Mutations in CHMP2B cause frontotemporal dementia (FTD) in a large Danish pedigree, which is termed FTD linked to chromosome 3 (FTD-3), and also in an unrelated familial FTD patient. CHMP2B is a component of the ESCRT-III complex, which is required for function of the multivesicular body (MVB), an endosomal structure that fuses with the lysosome to degrade endocytosed proteins. We report a novel endosomal pathology in CHMP2B mutation-positive patient brains and also identify and characterize abnormal endosomes in patient fibroblasts. Functional studies demonstrate a specific disruption of endosome-lysosome fusion but not protein sorting by the MVB. We provide evidence for a mechanism for impaired endosome-lysosome fusion whereby mutant CHMP2B constitutively binds to MVBs and prevents recruitment of proteins necessary for fusion to occur, such as Rab7. The fusion of endosomes with lysosomes is required for neuronal function and the data presented therefore suggest a pathogenic mechanism for FTD caused by CHMP2B mutations.

Show MeSH
Related in: MedlinePlus