Abstract
Loss-of-function mutations in PINK1 are causally linked to recessively inherited Parkinson’s disease (PD), with marked loss of dopaminergic neurons in the substantia nigra that are required for normal movement. PINK1 is a nuclear-encoded mitochondrial-targeted kinase that phosphorylates a conserved serine at amino acid 65 (pS65) in ubiquitin as well as Parkin, another gene with loss-of-function mutations linked to recessive parkinsonism. The steady-state levels of PINK1 protein are very low, even in cells that express PINK1, because PINK1 is normally targeted for degradation after mitochondrial import by a process that is dependent upon mitochondrial membrane potential. Dissipation of the mitochondrial membrane potential with ionophores, such as CCCP and valinomycin, causes the accumulation of PINK1 on the outer mitochondrial membrane, a marked increase of pS65-ubiquitin and the recruitment of Parkin, which targets dysfunctional mitochondria for degradation by autophagy. While the high penetrance of PINK1 mutations establish its critical function for maintaining neurons, the activity of PINK1 in primary neurons has been difficult to detect. Mounting evidence implicates non-neuronal cells, including astrocytes and microglia, in the pathogenesis of both idiopathic and inherited PD. Herein we used both western analysis and immunofluorescence of pS65-ubiquitin to directly compare the activity of PINK1 in primary neurons, astrocytes, microglia, and oligodendrocyte progenitor cells cultured from the brains of wild-type (WT) and PINK1 knockout (KO) rat pups. Our findings that PINK1-dependent ubiquitin phosphorylation is predominantly in astrocytes supports increased priority for research on the function of PINK1 in astrocytes and the contribution of astrocyte dysfunction to PD pathogenesis.
Highlights
Loss-of-function mutations in the gene encoding PTEN-induced kinase 1 (PINK1) are causally linked to a recessively inherited form of Parkinson’s disease (PD) clinically similar to idiopathic PD with earlier onset.[1]
Unlike idiopathic PD brains, which by definition have Lewy body pathology in addition to nigral cell loss, some—but not all—autopsy reports of PINK1 and Parkin-linked PD brains show severe nigral cell loss without apparent Lewy body pathology.[20,21,22]. This suggests that PINK1 and Parkin are required for the long-term survival of dopaminergic neurons irrespective of Lewy body pathology
The most prominent neuropathological feature of PD is the striking loss of dopaminergic neurons in the substantia nigra, neurodegeneration occurs in many other brain regions and cell types.[34]
Summary
Loss-of-function mutations in the gene encoding PTEN-induced kinase 1 (PINK1) are causally linked to a recessively inherited form of Parkinson’s disease (PD) clinically similar to idiopathic PD with earlier onset.[1]. Western analysis of cell lysates using an antibody specific for pS65-Ub showed a high molecular weight smear typical of poly-ubiquitinated proteins only in cells from WT rats treated with valinomycin to induce PINK1 activity (Fig. 1a). Valinomycin treatment induced PINK1 activity predominantly in astrocytes This was consistently observed in western blots from three additional independent experiments (Supplementary Fig. 1). To further assess the relative enrichment of each culture and to localize the pS65-Ub immunoreactivity at the cellular and subcellular level, we analyzed the cells plated on coverslips from each culture using confocal microscopy and immunofluorescence with antibodies specific for pS65-Ub as a measure of PINK1 activity, TIM23 as a marker for mitochondria, microtubule‐ associated protein 2A (MAP2A) as a marker for neurons, glial fibrillary acidic protein (GFAP) as a marker for astrocytes, OX-42 as a marker for microglia, and 4D4 as a marker for OPCs (Fig. 2).
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