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Parkinson's disease-linked mutations in VPS35 induce dopaminergic neurodegeneration.

Tsika E, Glauser L, Moser R, Fiser A, Daniel G, Sheerin UM, Lees A, Troncoso JC, Lewis PA, Bandopadhyay R, Schneider BL, Moore DJ - Hum. Mol. Genet. (2014)

Bottom Line: The common D620N missense mutation in VPS35 does not compromise its protein stability or localization to endosomal and lysosomal vesicles, or the vesicular sorting of the retromer cargo, sortilin, SorLA and cation-independent mannose 6-phosphate receptor, in rodent primary neurons or patient-derived human fibroblasts.In yeast we show that PD-linked VPS35 mutations are functional and can normally complement VPS35 phenotypes suggesting that they do not result in a loss-of-function.Collectively, these studies establish that dominant VPS35 mutations lead to neurodegeneration in PD consistent with a gain-of-function mechanism, and support a key role for VPS35 in the development of PD.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neurodegenerative Research.

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Normal steady-state levels and vesicular localization of VPS35D620N in cortical neurons. (A) Western blot analysis of soluble extracts from primary cortical neurons infected with lentiviral vectors expressing V5-tagged human VPS35 (WT or D620N) or control virus with anti-V5 or β-tubulin antibodies. Densitometric analysis of human VPS35 normalized to β-tubulin levels indicates the equivalent expression of WT and D620N variants (mean ± SEM, n = 4 experiments). n.s., non-significant by unpaired, two-tailed Student's t-test. (B) Representative confocal microscopic images of primary cortical neurons co-labeled for WT or D620N human VPS35 (V5) and RFP-Rab5, GFP-Rab7, RFP-LAMP1 or trans-Golgi protein Giantin, and DAPI. Inset indicates enlarged boxed area in merged images. Cytofluorograms and correlation coefficients (Rcoloc, mean ± SEM, n ≥ 5 neurons) indicate the degree of co-localization of fluorescence signals for V5 and each marker. Scale bars: 10 μm. (C) Graph showing co-localization coefficients (mean ± SEM, n ≥ 5 neurons/group) of WT or D620N VPS35 with each vesicular marker in cortical neurons. n.s., non-significant by unpaired, two-tailed Student's t-test as indicated.
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DDU178F2: Normal steady-state levels and vesicular localization of VPS35D620N in cortical neurons. (A) Western blot analysis of soluble extracts from primary cortical neurons infected with lentiviral vectors expressing V5-tagged human VPS35 (WT or D620N) or control virus with anti-V5 or β-tubulin antibodies. Densitometric analysis of human VPS35 normalized to β-tubulin levels indicates the equivalent expression of WT and D620N variants (mean ± SEM, n = 4 experiments). n.s., non-significant by unpaired, two-tailed Student's t-test. (B) Representative confocal microscopic images of primary cortical neurons co-labeled for WT or D620N human VPS35 (V5) and RFP-Rab5, GFP-Rab7, RFP-LAMP1 or trans-Golgi protein Giantin, and DAPI. Inset indicates enlarged boxed area in merged images. Cytofluorograms and correlation coefficients (Rcoloc, mean ± SEM, n ≥ 5 neurons) indicate the degree of co-localization of fluorescence signals for V5 and each marker. Scale bars: 10 μm. (C) Graph showing co-localization coefficients (mean ± SEM, n ≥ 5 neurons/group) of WT or D620N VPS35 with each vesicular marker in cortical neurons. n.s., non-significant by unpaired, two-tailed Student's t-test as indicated.

Mentions: To explore the putative pathogenic effects of dominant familial PD mutations in VPS35, we generated lentiviral vectors expressing V5-tagged human VPS35 harboring the common D620N mutation or wild-type (WT) protein. The D620N mutation does not influence the steady-state levels of human VPS35 protein exogenously expressed in rat primary cortical neurons (Fig. 2A). Furthermore, the D620N mutation fails to significantly alter the vesicular localization of human VPS35 (Fig. 2B–C). WT and D620N variants of VPS35 display a similar degree of co-localization with multiple vesicular or membranous compartments, including early (Rab5), late (Rab7) and recycling (Rab9) endosomes, lysosomes (LAMP1) and the trans-Golgi network (Giantin/GOLGB1 and Golgin/GOLGA4) in primary cortical neurons (Fig. 2B–C). We do not observe any clear differences in the subcellular distribution of VPS35-positive endosomal and lysosomal vesicles between the WT and D620N variants (Fig. 2B). In general, the D620N mutation does not compromise the protein stability or vesicular localization of VPS35.Figure 2.


Parkinson's disease-linked mutations in VPS35 induce dopaminergic neurodegeneration.

Tsika E, Glauser L, Moser R, Fiser A, Daniel G, Sheerin UM, Lees A, Troncoso JC, Lewis PA, Bandopadhyay R, Schneider BL, Moore DJ - Hum. Mol. Genet. (2014)

Normal steady-state levels and vesicular localization of VPS35D620N in cortical neurons. (A) Western blot analysis of soluble extracts from primary cortical neurons infected with lentiviral vectors expressing V5-tagged human VPS35 (WT or D620N) or control virus with anti-V5 or β-tubulin antibodies. Densitometric analysis of human VPS35 normalized to β-tubulin levels indicates the equivalent expression of WT and D620N variants (mean ± SEM, n = 4 experiments). n.s., non-significant by unpaired, two-tailed Student's t-test. (B) Representative confocal microscopic images of primary cortical neurons co-labeled for WT or D620N human VPS35 (V5) and RFP-Rab5, GFP-Rab7, RFP-LAMP1 or trans-Golgi protein Giantin, and DAPI. Inset indicates enlarged boxed area in merged images. Cytofluorograms and correlation coefficients (Rcoloc, mean ± SEM, n ≥ 5 neurons) indicate the degree of co-localization of fluorescence signals for V5 and each marker. Scale bars: 10 μm. (C) Graph showing co-localization coefficients (mean ± SEM, n ≥ 5 neurons/group) of WT or D620N VPS35 with each vesicular marker in cortical neurons. n.s., non-significant by unpaired, two-tailed Student's t-test as indicated.
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DDU178F2: Normal steady-state levels and vesicular localization of VPS35D620N in cortical neurons. (A) Western blot analysis of soluble extracts from primary cortical neurons infected with lentiviral vectors expressing V5-tagged human VPS35 (WT or D620N) or control virus with anti-V5 or β-tubulin antibodies. Densitometric analysis of human VPS35 normalized to β-tubulin levels indicates the equivalent expression of WT and D620N variants (mean ± SEM, n = 4 experiments). n.s., non-significant by unpaired, two-tailed Student's t-test. (B) Representative confocal microscopic images of primary cortical neurons co-labeled for WT or D620N human VPS35 (V5) and RFP-Rab5, GFP-Rab7, RFP-LAMP1 or trans-Golgi protein Giantin, and DAPI. Inset indicates enlarged boxed area in merged images. Cytofluorograms and correlation coefficients (Rcoloc, mean ± SEM, n ≥ 5 neurons) indicate the degree of co-localization of fluorescence signals for V5 and each marker. Scale bars: 10 μm. (C) Graph showing co-localization coefficients (mean ± SEM, n ≥ 5 neurons/group) of WT or D620N VPS35 with each vesicular marker in cortical neurons. n.s., non-significant by unpaired, two-tailed Student's t-test as indicated.
Mentions: To explore the putative pathogenic effects of dominant familial PD mutations in VPS35, we generated lentiviral vectors expressing V5-tagged human VPS35 harboring the common D620N mutation or wild-type (WT) protein. The D620N mutation does not influence the steady-state levels of human VPS35 protein exogenously expressed in rat primary cortical neurons (Fig. 2A). Furthermore, the D620N mutation fails to significantly alter the vesicular localization of human VPS35 (Fig. 2B–C). WT and D620N variants of VPS35 display a similar degree of co-localization with multiple vesicular or membranous compartments, including early (Rab5), late (Rab7) and recycling (Rab9) endosomes, lysosomes (LAMP1) and the trans-Golgi network (Giantin/GOLGB1 and Golgin/GOLGA4) in primary cortical neurons (Fig. 2B–C). We do not observe any clear differences in the subcellular distribution of VPS35-positive endosomal and lysosomal vesicles between the WT and D620N variants (Fig. 2B). In general, the D620N mutation does not compromise the protein stability or vesicular localization of VPS35.Figure 2.

Bottom Line: The common D620N missense mutation in VPS35 does not compromise its protein stability or localization to endosomal and lysosomal vesicles, or the vesicular sorting of the retromer cargo, sortilin, SorLA and cation-independent mannose 6-phosphate receptor, in rodent primary neurons or patient-derived human fibroblasts.In yeast we show that PD-linked VPS35 mutations are functional and can normally complement VPS35 phenotypes suggesting that they do not result in a loss-of-function.Collectively, these studies establish that dominant VPS35 mutations lead to neurodegeneration in PD consistent with a gain-of-function mechanism, and support a key role for VPS35 in the development of PD.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neurodegenerative Research.

Show MeSH
Related in: MedlinePlus