c-Abl phosphorylates α-synuclein and regulates its degradation: implication for α-synuclein clearance and contribution to the pathogenesis of Parkinson's disease

Increasing evidence suggests that the c-Abl protein tyrosine kinase could play a role in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. c-Abl has been shown to regulate the degradation of two proteins implicated in the pathogenesis of PD, parkin and α-synuclein (α-syn). The inhibition of parkin's neuroprotective functions is regulated by c-Abl-mediated phosphorylation of parkin. However, the molecular mechanisms by which c-Abl activity regulates α-syn toxicity and clearance remain unknown. Herein, using NMR spectroscopy, mass spectrometry, in vitro enzymatic assays and cell-based studies, we established that α-syn is a bona fide substrate for c-Abl. In vitro studies demonstrate that c-Abl directly interacts with α-syn and catalyzes its phosphorylation mainly at tyrosine 39 (pY39) and to a lesser extent at tyrosine 125 (pY125). Analysis of human brain tissues showed that pY39 α-syn is detected in the brains of healthy individuals and those with PD. However, only c-Abl protein levels were found to be upregulated in PD brains. Interestingly, nilotinib, a specific inhibitor of c-Abl kinase activity, induces α-syn protein degradation via the autophagy and proteasome pathways, whereas the overexpression of α-syn in the rat midbrains enhances c-Abl expression. Together, these data suggest that changes in c-Abl expression, activation and/or c-Abl-mediated phosphorylation of Y39 play a role in regulating α-syn clearance and contribute to the pathogenesis of P

FAS-dependent cell death in α-synuclein transgenic oligodendrocyte models of multiple system atrophy

Multiple system atrophy is a parkinsonian neurodegenerative disorder. It is cytopathologically characterized by accumulation of the protein p25α in cell bodies of oligodendrocytes followed by accumulation of aggregated α-synuclein in so-called glial cytoplasmic inclusions. p25α is a stimulator of α-synuclein aggregation, and coexpression of α-synuclein and p25α in the oligodendroglial OLN-t40-AS cell line causes α-synuclein aggregate-dependent toxicity. In this study, we investigated whether the FAS system is involved in α-synuclein aggregate dependent degeneration in oligodendrocytes and may play a role in multiple system atrophy. Using rat oligodendroglial OLN-t40-AS cells we demonstrate that the cytotoxicity caused by coexpressing α-synuclein and p25α relies on stimulation of the death domain receptor FAS and caspase-8 activation. Using primary oligodendrocytes derived from PLP-α-synuclein transgenic mice we demonstrate that they exist in a sensitized state expressing pro-apoptotic FAS receptor, which makes them sensitive to FAS ligand-mediated apoptosis. Immunoblot analysis shows an increase in FAS in brain extracts from multiple system atrophy cases. Immunohistochemical analysis demonstrated enhanced FAS expression in multiple system atrophy brains notably in oligodendrocytes harboring the earliest stages of glial cytoplasmic inclusion formation. Oligodendroglial FAS expression is an early hallmark of oligodendroglial pathology in multiple system atrophy that mechanistically may be coupled to α-synuclein dependent degeneration and thus represent a potential target for protective intervention

c-Abl phosphorylates α-synuclein and regulates its degradation: implication for α-synuclein clearance and contribution to the pathogenesis of Parkinson's disease

Increasing evidence suggests that the c-Abl protein tyrosine kinase could play a role in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. c-Abl has been shown to regulate the degradation of two proteins implicated in the pathogenesis of PD, parkin and α-synuclein (α-syn). The inhibition of parkin's neuroprotective functions is regulated by c-Abl-mediated phosphorylation of parkin. However, the molecular mechanisms by which c-Abl activity regulates α-syn toxicity and clearance remain unknown. Herein, using NMR spectroscopy, mass spectrometry, in vitro enzymatic assays and cell-based studies, we established that α-syn is a bona fide substrate for c-Abl. In vitro studies demonstrate that c-Abl directly interacts with α-syn and catalyzes its phosphorylation mainly at tyrosine 39 (pY39) and to a lesser extent at tyrosine 125 (pY125). Analysis of human brain tissues showed that pY39 α-syn is detected in the brains of healthy individuals and those with PD. However, only c-Abl protein levels were found to be upregulated in PD brains. Interestingly, nilotinib, a specific inhibitor of c-Abl kinase activity, induces α-syn protein degradation via the autophagy and proteasome pathways, whereas the overexpression of α-syn in the rat midbrains enhances c-Abl expression. Together, these data suggest that changes in c-Abl expression, activation and/or c-Abl-mediated phosphorylation of Y39 play a role in regulating α-syn clearance and contribute to the pathogenesis of PD

Lysosomal-associated membrane protein 2 isoforms are differentially affected in early Parkinson\u27s disease

Lysosomes are the primary catabolic compartment for the degradation of intracellular proteins through autophagy. The presence of abnormal intracellular -synuclein-positive aggregates in Parkinson\u27s disease (PD) indicates that the degradative capacity of lysosomes is impaired in PD. Specific dysfunction of chaperone-mediated autophagy (CMA) in PD is suggested by reductions in the CMA membrane receptor, lysosomal-associated membrane protein (LAMP) 2A, although whether LAMP2A is the only LAMP2 isoform affected by PD is unknown. Messenger RNA (mRNA) and protein expression of all three LAMP2 isoforms was assessed in brain extracts from regions with and without PD-related increases in -synuclein in autopsy samples from subjects in the early pathological stage of PD (n=9), compared to age- and postmortem delay-matched controls (n=10). In the early stages of PD, mRNA expression of all LAMP2 isoforms was not different from controls, with LAMP2B and LAMP2C protein levels also unchanged in PD. The selective loss of LAMP2A protein directly correlated with the increased levels of -synuclein and decreased levels of the CMA chaperone heat shock cognate protein 70 in the same PD samples, as well as with the accumulation of cytosolic CMA substrate proteins. Our data show that LAMP2 protein isoforms are differentially affected in the early stages of PD, with LAMP2A selectively reduced in association with increased -synuclein, and suggests that dysregulation of CMA-mediated protein degradation occurs before substantial -synuclein aggregation in PD. (c) 2015 International Parkinson and Movement Disorder Society

Lysosomal-associated membrane protein 2 isoforms are differentially affected in early Parkinson\u27s disease

Lysosomes are the primary catabolic compartment for the degradation of intracellular proteins through autophagy. The presence of abnormal intracellular -synuclein-positive aggregates in Parkinson\u27s disease (PD) indicates that the degradative capacity of lysosomes is impaired in PD. Specific dysfunction of chaperone-mediated autophagy (CMA) in PD is suggested by reductions in the CMA membrane receptor, lysosomal-associated membrane protein (LAMP) 2A, although whether LAMP2A is the only LAMP2 isoform affected by PD is unknown. Messenger RNA (mRNA) and protein expression of all three LAMP2 isoforms was assessed in brain extracts from regions with and without PD-related increases in -synuclein in autopsy samples from subjects in the early pathological stage of PD (n=9), compared to age- and postmortem delay-matched controls (n=10). In the early stages of PD, mRNA expression of all LAMP2 isoforms was not different from controls, with LAMP2B and LAMP2C protein levels also unchanged in PD. The selective loss of LAMP2A protein directly correlated with the increased levels of -synuclein and decreased levels of the CMA chaperone heat shock cognate protein 70 in the same PD samples, as well as with the accumulation of cytosolic CMA substrate proteins. Our data show that LAMP2 protein isoforms are differentially affected in the early stages of PD, with LAMP2A selectively reduced in association with increased -synuclein, and suggests that dysregulation of CMA-mediated protein degradation occurs before substantial -synuclein aggregation in PD. (c) 2015 International Parkinson and Movement Disorder Society

Reduced glucocerebrosidase is associated with increased α-synuclein in sporadic Parkinson\u27s disease

Heterozygous mutations in GBA1, the gene encoding lysosomal glucocerebrosidase, are the most frequent known genetic risk factor for Parkinson’s disease. Reduced glucocerebrosidase and α-synuclein accumulation are directly related in cell models of Parkinson’s disease. We investigated relationships between Parkinson’s disease-specific glucocerebrosidase deficits, glucocerebrosidase-related pathways, and α-synuclein levels in brain tissue from subjects with sporadic Parkinson’s disease without GBA1 mutations. Brain regions with and without a Parkinson’s disease-related increase in α-synuclein levels were assessed in autopsy samples from subjects with sporadic Parkinson’s disease (n = 19) and age- and post-mortem delay-matched controls (n = 10). Levels of glucocerebrosidase, α-synuclein and related lysosomal and autophagic proteins were assessed by western blotting. Glucocerebrosidase enzyme activity was measured using a fluorimetric assay, and glucocerebrosidase and α-synuclein messenger RNA expression determined by quantitative polymerase chain reaction. Related sphingolipids were analysed by mass spectrometry. Multivariate statistical analyses were performed to identify differences between disease groups and regions, with non-parametric correlations used to identify relationships between variables. Glucocerebrosidase protein levels and enzyme activity were selectively reduced in the early stages of Parkinson’s disease in regions with increased α-synuclein levels although limited inclusion formation, whereas GBA1 messenger RNA expression was non-selectively reduced in Parkinson’s disease. The selective loss of lysosomal glucocerebrosidase was directly related to reduced lysosomal chaperone-mediated autophagy, increased α-synuclein and decreased ceramide. Glucocerebrosidase deficits in sporadic Parkinson’s disease are related to the abnormal accumulation of α-synuclein and are associated with substantial alterations in lysosomal chaperone-mediated autophagy pathways and lipid metabolism. Our data suggest that the early selective Parkinson’s disease changes are likely a result of the redistribution of cellular membrane proteins leading to a chronic reduction in lysosome function in brain regions vulnerable to Parkinson’s disease pathology

Ndfip1 is expressed in dopaminergic neurons containing α-synuclein deposits.

<p>(A) Bright field image of dopaminergic neurons in the substantia nigra of a PD brain. (B–D) Fluorescent labelling of α-synuclein and Ndfip1 from the bright field image shows deposits of α-synuclein which co-label with Ndfip1 in a dopaminergic neuron (arrow). Asterisk marks a dopaminergic neuron with neither α-synuclein nor Ndfip1 positive labelling. (E) Fluorescent labelling of Ndfip1 in a control brain. Scale bar: 25 µm.</p

Loss of Ndfip1 results in misregulation of DMT1 and increased susceptibility to iron induced death.

<p>(A) Staining of mouse dopaminergic neurons in culture showed that genetic deletion of Ndfip1 (Ndfip1^−/−) resulted in increased DMT1 staining compared to wild type (Ndfip1^+/+) dopaminergic neurons. (B) Increasing concentrations of iron resulted in a significant increase in dopaminergic neuronal cell death in Ndfip1 knockout neurons compared to wild type controls. Values are means ±SEM, ** p<0.005, *** p<0.001 un-paired Student t-test. Scale bar: 10 µm.</p

Ndfip1 is upregulated in the substantia nigra of PD brains.

<p>(A) Western blotting for Ndfip1 in the substantia nigra (SN) in both control and PD brains. (B) Quantification of Ndfip1 levels showed a significant increase of Ndfip1 in the substantia nigra of PD brains. (C) Iron content was analysed using ICP-MS on brain tissue from the substantia nigra of control and PD brains, a significant increase in iron levels in PD brains was observed. (D) Western blotting of Ndfip1 levels in the cortex of both control and PD brains. (E) Quantification of Ndfip1 levels showed no significant increase in Ndfip1 in the cortex in PD. (F) ICP-MS analysis for iron levels in the cortex showed no increase in iron concentrations between control and PD brains. (B, C, E, F) Values are means ±SEM, * p<0.05 un-paired Student t-test.</p

Ndfip1 and DMT1 are expressed in dopaminergic neurons of the substantia nigra.

<p>(A) Quantification of neuromelanin levels from the substantia nigra of control and PD brains showed a significant decrease in PD, indicating a loss of dopaminergic neurons. (B) Ndfip1 is expressed (arrows) in some but not all dopaminergic neurons of the substantia nigra (brown pigment represents neuromelanin). (C) High power image of Ndfip1 staining of dopaminergic neurons showed a predominant cytoplasmic distribution. Asterisks in B and C show dopaminergic neurons with no Ndfip1 staining. (D) Quantification of Ndfip1 positive dopaminergic neurons of the substantia nigra showed no significant difference between control and PD brains. (E) DMT1 is expressed in the cytoplasm (arrows) of dopaminergic neurons of the substantia nigra. Scale bars: 100 µm (B, E), 25 µm (C). (A, D) Values are the mean ±SD, * p<0.05 Mann Whitney U tests.</p