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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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Intralumenal vesicle formation and sequestration of the EGF receptor is intact in CHMP2B mutant cells. (A) FTD-3 and control fibroblast cell lines were stimulated with 100 ng/ml EGF with anti-EGFR-gold for 2–4 h. EM analysis of control cells showed that EGFR moved onto the internal vesicles of MVBs (a and b) within this time frame. EGFR was also observed on ILVs within normal MVBs and abnormal structures in two different FTD-3 patients (c–e), and the Q165X patient (f, g). (c) A relatively normal MVB with peripheral ILVs associated with gold particles; (d, e) Abnormal structures containing unusual membranes, but also ILVs associated with gold particles. (f) Enlarged structures containing membranes and ILVs associated with gold particles. (g) EGFR–gold is clearly identifiable on ILVs in MVBs. Anti-EGFR, 10 nm gold; examples of gold particles indicated by arrows; scale bar = 500 nm. (B) Wildtype-293 and Intron5-293 cells were not stimulated (0) or stimulated with EGF for 5 min and then chased for 5, 10, 30 or 60 min. Western blotting with antibodies against phosphorylated ERK1/2 and total ERK1/2 (C) was quantitated using LI-COR Odyssey software. Phosphorylated ERK1/2 was corrected against total ERK1/2 and a time course of ERK1/2 activation was plotted as a percentage of the respective phosphorylation intensities after 5 min of EGF stimulation and 5 min of chase. Wildtype-293 cells, filled symbols; Intron5-293 cells unfilled symbols; error bars represent the SEM of three experiments.
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DDQ100F7: Intralumenal vesicle formation and sequestration of the EGF receptor is intact in CHMP2B mutant cells. (A) FTD-3 and control fibroblast cell lines were stimulated with 100 ng/ml EGF with anti-EGFR-gold for 2–4 h. EM analysis of control cells showed that EGFR moved onto the internal vesicles of MVBs (a and b) within this time frame. EGFR was also observed on ILVs within normal MVBs and abnormal structures in two different FTD-3 patients (c–e), and the Q165X patient (f, g). (c) A relatively normal MVB with peripheral ILVs associated with gold particles; (d, e) Abnormal structures containing unusual membranes, but also ILVs associated with gold particles. (f) Enlarged structures containing membranes and ILVs associated with gold particles. (g) EGFR–gold is clearly identifiable on ILVs in MVBs. Anti-EGFR, 10 nm gold; examples of gold particles indicated by arrows; scale bar = 500 nm. (B) Wildtype-293 and Intron5-293 cells were not stimulated (0) or stimulated with EGF for 5 min and then chased for 5, 10, 30 or 60 min. Western blotting with antibodies against phosphorylated ERK1/2 and total ERK1/2 (C) was quantitated using LI-COR Odyssey software. Phosphorylated ERK1/2 was corrected against total ERK1/2 and a time course of ERK1/2 activation was plotted as a percentage of the respective phosphorylation intensities after 5 min of EGF stimulation and 5 min of chase. Wildtype-293 cells, filled symbols; Intron5-293 cells unfilled symbols; error bars represent the SEM of three experiments.

Mentions: EGF-induced phosphorylation of ERK1/2 was not affected by the expression of CHMP2BIntron5. Both Wildtype-293 and Intron5-293 cells showed a rapid peak in phosphorylation after 5 min, which rapidly returned to baseline levels (Fig. 7B and C). This suggests that EGF-induced signalling was silenced in both lines, due to the sequestration of EGFR in the ILVs. To determine whether EGFR was sequestered on ILVs in patient tissue, FTD-3 patient fibroblasts and age-matched control fibroblasts were incubated with 100 ng/ml EGF and a gold-conjugated anti-EGFR antibody. The EGFR–gold complexes are endocytosed in response to the EGF stimulation and can be used to monitor endosomal trafficking by electron microscopy (13). Sorting of EGFR onto internal vesicles of both normal and abnormal MVBs was observed in three patient fibroblasts lines: two FTD-3 patients and one CHMP2BQ165X patient (Fig. 7). This suggests that impaired degradation of EGFR is due to decreased fusion with lysosomes rather than impaired sorting onto ILVs.


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)

Intralumenal vesicle formation and sequestration of the EGF receptor is intact in CHMP2B mutant cells. (A) FTD-3 and control fibroblast cell lines were stimulated with 100 ng/ml EGF with anti-EGFR-gold for 2–4 h. EM analysis of control cells showed that EGFR moved onto the internal vesicles of MVBs (a and b) within this time frame. EGFR was also observed on ILVs within normal MVBs and abnormal structures in two different FTD-3 patients (c–e), and the Q165X patient (f, g). (c) A relatively normal MVB with peripheral ILVs associated with gold particles; (d, e) Abnormal structures containing unusual membranes, but also ILVs associated with gold particles. (f) Enlarged structures containing membranes and ILVs associated with gold particles. (g) EGFR–gold is clearly identifiable on ILVs in MVBs. Anti-EGFR, 10 nm gold; examples of gold particles indicated by arrows; scale bar = 500 nm. (B) Wildtype-293 and Intron5-293 cells were not stimulated (0) or stimulated with EGF for 5 min and then chased for 5, 10, 30 or 60 min. Western blotting with antibodies against phosphorylated ERK1/2 and total ERK1/2 (C) was quantitated using LI-COR Odyssey software. Phosphorylated ERK1/2 was corrected against total ERK1/2 and a time course of ERK1/2 activation was plotted as a percentage of the respective phosphorylation intensities after 5 min of EGF stimulation and 5 min of chase. Wildtype-293 cells, filled symbols; Intron5-293 cells unfilled symbols; error bars represent the SEM of three experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2865375&req=5

DDQ100F7: Intralumenal vesicle formation and sequestration of the EGF receptor is intact in CHMP2B mutant cells. (A) FTD-3 and control fibroblast cell lines were stimulated with 100 ng/ml EGF with anti-EGFR-gold for 2–4 h. EM analysis of control cells showed that EGFR moved onto the internal vesicles of MVBs (a and b) within this time frame. EGFR was also observed on ILVs within normal MVBs and abnormal structures in two different FTD-3 patients (c–e), and the Q165X patient (f, g). (c) A relatively normal MVB with peripheral ILVs associated with gold particles; (d, e) Abnormal structures containing unusual membranes, but also ILVs associated with gold particles. (f) Enlarged structures containing membranes and ILVs associated with gold particles. (g) EGFR–gold is clearly identifiable on ILVs in MVBs. Anti-EGFR, 10 nm gold; examples of gold particles indicated by arrows; scale bar = 500 nm. (B) Wildtype-293 and Intron5-293 cells were not stimulated (0) or stimulated with EGF for 5 min and then chased for 5, 10, 30 or 60 min. Western blotting with antibodies against phosphorylated ERK1/2 and total ERK1/2 (C) was quantitated using LI-COR Odyssey software. Phosphorylated ERK1/2 was corrected against total ERK1/2 and a time course of ERK1/2 activation was plotted as a percentage of the respective phosphorylation intensities after 5 min of EGF stimulation and 5 min of chase. Wildtype-293 cells, filled symbols; Intron5-293 cells unfilled symbols; error bars represent the SEM of three experiments.
Mentions: EGF-induced phosphorylation of ERK1/2 was not affected by the expression of CHMP2BIntron5. Both Wildtype-293 and Intron5-293 cells showed a rapid peak in phosphorylation after 5 min, which rapidly returned to baseline levels (Fig. 7B and C). This suggests that EGF-induced signalling was silenced in both lines, due to the sequestration of EGFR in the ILVs. To determine whether EGFR was sequestered on ILVs in patient tissue, FTD-3 patient fibroblasts and age-matched control fibroblasts were incubated with 100 ng/ml EGF and a gold-conjugated anti-EGFR antibody. The EGFR–gold complexes are endocytosed in response to the EGF stimulation and can be used to monitor endosomal trafficking by electron microscopy (13). Sorting of EGFR onto internal vesicles of both normal and abnormal MVBs was observed in three patient fibroblasts lines: two FTD-3 patients and one CHMP2BQ165X patient (Fig. 7). This suggests that impaired degradation of EGFR is due to decreased fusion with lysosomes rather than impaired sorting onto ILVs.

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