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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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Delayed degradation of EGF in CHMP2B mutant cells. (A) SK-N-SH cells were transiently transfected with CHMP2BWildtype (a–f), CHMP2BIntron5 (g–l) or CHMP2BΔ10 (m–r), and used 24 h later for an EGF-488 trafficking assay. Cells were fed 100 ng/ml EGF-488 (green) and fixed at either 30 (left panel) or 90 min (right panel). Fixed cells were immunostained with an anti-myc antibody to visualize myc-CHMP2B transfected cells (red) and the nuclei counterstained with Hoecsht (blue). At 30 min, all cells show punctate localization of EGF-488. In CHMP2BWildtype-expressing cells at 90 mins (d–f), the EGF-488 signal is degraded. However, in cells overexpressing CHMP2BIntron5 (j–l) or CHMP2BΔ10 (p–r), the majority of EGF-488 remains undegraded. Insets show magnification of EGF-488 colocalizing with CHMP2BIntron5-positive compartments. Scale bar = 20 µm. (B) LSM 510 Meta software was used to quantify this effect. ∼95% EGF-488 is degraded in CHMP2BWildtype expressing cells, compared with only 25–35% in cells expressing CHMP2BIntron5 or CHMP2BΔ10 (t-tests, both P < 0.0001).
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DDQ100F6: Delayed degradation of EGF in CHMP2B mutant cells. (A) SK-N-SH cells were transiently transfected with CHMP2BWildtype (a–f), CHMP2BIntron5 (g–l) or CHMP2BΔ10 (m–r), and used 24 h later for an EGF-488 trafficking assay. Cells were fed 100 ng/ml EGF-488 (green) and fixed at either 30 (left panel) or 90 min (right panel). Fixed cells were immunostained with an anti-myc antibody to visualize myc-CHMP2B transfected cells (red) and the nuclei counterstained with Hoecsht (blue). At 30 min, all cells show punctate localization of EGF-488. In CHMP2BWildtype-expressing cells at 90 mins (d–f), the EGF-488 signal is degraded. However, in cells overexpressing CHMP2BIntron5 (j–l) or CHMP2BΔ10 (p–r), the majority of EGF-488 remains undegraded. Insets show magnification of EGF-488 colocalizing with CHMP2BIntron5-positive compartments. Scale bar = 20 µm. (B) LSM 510 Meta software was used to quantify this effect. ∼95% EGF-488 is degraded in CHMP2BWildtype expressing cells, compared with only 25–35% in cells expressing CHMP2BIntron5 or CHMP2BΔ10 (t-tests, both P < 0.0001).

Mentions: Impaired fusion was confirmed by delayed degradation of EGF and its receptor epidermal growth factor receptor (EGFR). Tetracycline-inducible HEK-293 cell lines expressing HA-tagged CHMP2BWildtype (Wildtype-293) or CHMP2BIntron5 (Intron5-293) showed downregulation of EGFR in response to stimulation with EGF, a process requiring delivery of EGFR from the MVB to the lysosome. However, this process was delayed in Intron5-293 cells when compared with Wildtype-293 cells (Fig. 5C and D), also suggesting that fusion is impaired. Impairment of EGF-488 degradation was also shown in transfected SK-N-SH cells. Levels of cellular EGF, as measured by fluorescence intensity, were recorded after 30 and 90 min chase (Fig. 6B). In CHMP2BWildtype expressing cells, ∼90% of the EGF signal present at 30 min was degraded at 90 min. In contrast, in cells expressing CHMP2BIntron5 or CHMP2BΔ10, much less (25–30%) degradation of EGF was observed. Interestingly, there was partial colocalization of EGF with CHMP2BIntron5-positive compartments at 90 min (Fig. 6A, inset), suggesting that the failure of degradation may be due to EGF being trapped in these aberrant compartments.


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)

Delayed degradation of EGF in CHMP2B mutant cells. (A) SK-N-SH cells were transiently transfected with CHMP2BWildtype (a–f), CHMP2BIntron5 (g–l) or CHMP2BΔ10 (m–r), and used 24 h later for an EGF-488 trafficking assay. Cells were fed 100 ng/ml EGF-488 (green) and fixed at either 30 (left panel) or 90 min (right panel). Fixed cells were immunostained with an anti-myc antibody to visualize myc-CHMP2B transfected cells (red) and the nuclei counterstained with Hoecsht (blue). At 30 min, all cells show punctate localization of EGF-488. In CHMP2BWildtype-expressing cells at 90 mins (d–f), the EGF-488 signal is degraded. However, in cells overexpressing CHMP2BIntron5 (j–l) or CHMP2BΔ10 (p–r), the majority of EGF-488 remains undegraded. Insets show magnification of EGF-488 colocalizing with CHMP2BIntron5-positive compartments. Scale bar = 20 µm. (B) LSM 510 Meta software was used to quantify this effect. ∼95% EGF-488 is degraded in CHMP2BWildtype expressing cells, compared with only 25–35% in cells expressing CHMP2BIntron5 or CHMP2BΔ10 (t-tests, both P < 0.0001).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DDQ100F6: Delayed degradation of EGF in CHMP2B mutant cells. (A) SK-N-SH cells were transiently transfected with CHMP2BWildtype (a–f), CHMP2BIntron5 (g–l) or CHMP2BΔ10 (m–r), and used 24 h later for an EGF-488 trafficking assay. Cells were fed 100 ng/ml EGF-488 (green) and fixed at either 30 (left panel) or 90 min (right panel). Fixed cells were immunostained with an anti-myc antibody to visualize myc-CHMP2B transfected cells (red) and the nuclei counterstained with Hoecsht (blue). At 30 min, all cells show punctate localization of EGF-488. In CHMP2BWildtype-expressing cells at 90 mins (d–f), the EGF-488 signal is degraded. However, in cells overexpressing CHMP2BIntron5 (j–l) or CHMP2BΔ10 (p–r), the majority of EGF-488 remains undegraded. Insets show magnification of EGF-488 colocalizing with CHMP2BIntron5-positive compartments. Scale bar = 20 µm. (B) LSM 510 Meta software was used to quantify this effect. ∼95% EGF-488 is degraded in CHMP2BWildtype expressing cells, compared with only 25–35% in cells expressing CHMP2BIntron5 or CHMP2BΔ10 (t-tests, both P < 0.0001).
Mentions: Impaired fusion was confirmed by delayed degradation of EGF and its receptor epidermal growth factor receptor (EGFR). Tetracycline-inducible HEK-293 cell lines expressing HA-tagged CHMP2BWildtype (Wildtype-293) or CHMP2BIntron5 (Intron5-293) showed downregulation of EGFR in response to stimulation with EGF, a process requiring delivery of EGFR from the MVB to the lysosome. However, this process was delayed in Intron5-293 cells when compared with Wildtype-293 cells (Fig. 5C and D), also suggesting that fusion is impaired. Impairment of EGF-488 degradation was also shown in transfected SK-N-SH cells. Levels of cellular EGF, as measured by fluorescence intensity, were recorded after 30 and 90 min chase (Fig. 6B). In CHMP2BWildtype expressing cells, ∼90% of the EGF signal present at 30 min was degraded at 90 min. In contrast, in cells expressing CHMP2BIntron5 or CHMP2BΔ10, much less (25–30%) degradation of EGF was observed. Interestingly, there was partial colocalization of EGF with CHMP2BIntron5-positive compartments at 90 min (Fig. 6A, inset), suggesting that the failure of degradation may be due to EGF being trapped in these aberrant compartments.

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