Parkinson's disease-linked mutations in VPS35 induce dopaminergic neurodegeneration.
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.
Affiliation: Laboratory of Molecular Neurodegenerative Research.
- Dopaminergic Neurons/metabolism/pathology*
- Nerve Degeneration/genetics*/pathology*
- Parkinson Disease/genetics*/pathology
- Vesicular Transport Proteins/genetics*
- Aged, 80 and over
- Cell Death
- Cells, Cultured
- HEK293 Cells
- Middle Aged
- Protein Stability
- Protein Transport
- Rats, Sprague-Dawley
- Saccharomyces cerevisiae/metabolism
- Stress, Physiological
- Transport Vesicles/metabolism
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DDU178F1: Cellular distribution and levels of endogenous VPS35 in normal and pathological mammalian brain. (A) Subcellular fractionation of endogenous VPS35 in mouse cerebral cortex. VPS35 is enriched in the microsomal (P3), synaptosomal (LP1) and synaptic vesicle (LP2) membrane fractions. Dynamin 1, TIM23, α-synuclein and synaptophysin serve as markers for microsomes, mitochondria, synaptic vesicle cytosolic and synaptosomal/synaptic vesicle membranes, respectively. Molecular mass is indicated in kDa. (B) Immunolabeling of endogenous VPS35 in the rat brain. VPS35 is detected in (i) pyramidal neurons of cortical layer III, (ii) pyramidal neurons of the hippocampus (CA1 region), (iii) ventral midbrain, (iv) brainstem (superior olivary complex), (v) Purkinje neurons in the cerebellum (granule cell layer, gcl; molecular layer, ml), (vi) deep cerebellar nuclei, and (vii) a sagittal section of rat brain (cerebral cortex, Ctx; hippocampal formation, Hip; cerebellum, Crb; deep cerebellar nuclei, DCN; caudate putamen, CPu; substantia nigra, SN. (C) Confocal microscopy analysis of rat primary midbrain cultures immunolabeled with VPS35 and the dopaminergic marker, tyrosine hydroxylase (TH). Nuclei are labeled with DAPI. VPS35 localizes to punctate intracellular vesicular structures within the soma and neuritic processes of TH-positive dopaminergic neurons. Scale bar: 10 μm. (D) Co-localization of endogenous VPS35 with TH-positive dopaminergic neurons in the substantia nigra pars compacta of adult rats. Scale bar: 10 μm. (E) Immunolabeling of endogenous VPS35 in the human cingulate cortex of control (1) and PD/DLB (3) subjects. Scale bar: 200 μm. High-magnification images of pyramidal neurons from cortical layer III are shown corresponding to the boxed area from control (2) and PD/DLB (4) brains. Scale bar: 50 μm. (F–G) Western blot analysis of soluble extracts from F human caudate putamen of control and idiopathic PD/DLB subjects, and G human frontal cortex of control, idiopathic PD (PD) or G2019S LRRK2-linked PD subjects, with antibodies to VPS35, and actin or β-tubulin as protein loading controls. Densitometric analysis of VPS35 normalized to actin or β-tubulin levels for individual subjects are shown expressed as a percent of the mean of control subjects. Horizontal bars represent mean ± SEM (n = 4–5 subjects/group) for each subject group. Subjects without VPS35 expression in F were excluded from the analysis (control = 4 from 5; PD = 5 from 7). ns, non-significant by one-way analysis of variance (ANOVA) with Dunnett's post hoc test. (H) Confocal microscopic analysis of VPS35 co-localization with Lewy bodies labeled with phospho-Ser129-α-synuclein in cortical layer III neurons from a PD/DLB subject. Correlation coefficients (Rcoloc) and cytofluorograms indicate a lack of co-localization of VPS35 with Lewy bodies. Scale bars: 20 μm (top panels) or 5 μm (bottom panels).
To begin to understand how familial VPS35 mutations precipitate neurodegeneration in PD, we investigated the normal distribution of endogenous VPS35 in the mammalian brain. Subcellular fractionation of mouse cerebral cortex reveals an enrichment of VPS35 in microsomal vesicles (P3) and at lower levels in crude synaptosomes (LP1) and synaptic vesicle membranes (LP2) (Fig. 1A). Within the rat brain, VPS35 is broadly distributed to multiple neuronal populations including those within the cerebral cortex, hippocampal formation, ventral midbrain, brainstem and cerebellum (Fig. 1B). VPS35 is not particularly enriched within neurons of the nigrostriatal dopaminergic pathway, which selectively degenerate in PD (Fig. 1B). However, confocal microscopic analyses reveal localization of VPS35 to intracellular punctate structures within dopaminergic neurons from rat primary midbrain cultures (Fig. 1C) or the intact rat substantia nigra (Fig. 1D), consistent with the localization of VPS35 to multiple vesicular compartments. Collectively, VPS35 is selectively localized to neuronal vesicular compartments throughout the rodent brain, including substantia nigra dopaminergic neurons that selectively degenerate in PD.Figure 1.