Cyclin-G-associated kinase modifies alpha-synuclein expression levels and toxicity in Parkinson's disease: results from the GenePD Study

Although family history is a well-established risk factor for Parkinson's disease (PD), fewer than 5% of PD cases can be attributed to known genetic mutations. The etiology for the remainder of PD cases is unclear; however, neuronal accumulation of the protein α-synuclein is common to nearly all patients, implicating pathways that influence α-synuclein in PD pathogenesis. We report a genome-wide significant association (P = 3.97 × 10−8) between a polymorphism, rs1564282, in the cyclin-G-associated kinase (GAK) gene and increased PD risk, with a meta-analysis odds ratio of 1.48. This association result is based on the meta-analysis of three publicly available PD case–control genome-wide association study and genotyping from a new, independent Italian cohort. Microarray expression analysis of post-mortem frontal cortex from PD and control brains demonstrates a significant association between rs1564282 and higher α-synuclein expression, a known cause of early onset PD. Functional knockdown of GAK in cell culture causes a significant increase in toxicity when α-synuclein is over-expressed. Furthermore, knockdown of GAK in rat primary neurons expressing the A53T mutation of α-synuclein, a well-established model for PD, decreases cell viability. These observations provide evidence that GAK is associated with PD risk and suggest that GAK and α-synuclein interact in a pathway involved in PD pathogenesis. The GAK protein, a serine/threonine kinase, belongs to a family of proteins commonly targeted for drug development. This, combined with GAK's observed relationship to the levels of α-synuclein expression and toxicity, suggests that the protein is an attractive therapeutic target for the treatment of PD.Robert P. & Judith N. Goldberg FoundationWilliam N. & Bernice E. Bumpus FoundationHoward Hughes Medical Institute (Collaborative Innovation Award)National Science Foundation (U.S.) (R01-NS036711

Two C-terminal Sequence Variations Determine Differential Neurotoxicity Between Human and Mouse α-synuclein

BACKGROUND: α-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinson\u27s disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here, we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models, as well as their biochemical properties relevant to aSyn pathobiology. METHODS: Primary midbrain cultures transduced with aSyn-encoding adenoviruses were analyzed immunocytochemically to determine relative dopaminergic neuron viability. Brain sections prepared from rats injected intranigrally with aSyn-encoding adeno-associated viruses were analyzed immunohistochemically to determine nigral dopaminergic neuron viability and striatal dopaminergic terminal density. Recombinant aSyn variants were characterized in terms of fibrillization rates by measuring thioflavin T fluorescence, fibril morphologies via electron microscopy and atomic force microscopy, and protein-lipid interactions by monitoring membrane-induced aSyn aggregation and aSyn-mediated vesicle disruption. Statistical tests consisted of ANOVA followed by Tukey\u27s multiple comparisons post hoc test and the Kruskal-Wallis test followed by a Dunn\u27s multiple comparisons test or a two-tailed Mann-Whitney test. RESULTS: Mouse aSyn was less neurotoxic than human aSyn A53T in cell culture and in rat midbrain, and data obtained for the chimeric variants indicated that the human-to-mouse substitutions D121G and N122S were at least partially responsible for this decrease in neurotoxicity. Human aSyn A53T and a chimeric variant with the human residues D and N at positions 121 and 122 (respectively) showed a greater propensity to undergo membrane-induced aggregation and to elicit vesicle disruption. Differences in neurotoxicity among the human, mouse, and chimeric aSyn variants correlated weakly with differences in fibrillization rate or fibril morphology. CONCLUSIONS: Mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders

Compounds from an Unbiased Chemical Screen Reverse Both Er-to-Golgi Trafficking Defects and Mitochondrial Dysfunction in Parkinson's Disease Models

α-Synuclein (α-syn) is a small lipid-binding protein involved in vesicle trafficking whose function is poorly characterized. It is of great interest to human biology and medicine because α-syn dysfunction is associated with several neurodegenerative disorders, including Parkinson’s disease (PD). We previously created a yeast model of α-syn pathobiology, which established vesicle trafficking as a process that is particularly sensitive to α-syn expression. We also uncovered a core group of proteins with diverse activities related to α-syn toxicity that is conserved from yeast to mammalian neurons. Here, we report that a yeast strain expressing a somewhat higher level of α-syn also exhibits strong defects in mitochondrial function. Unlike our previous strain, genetic suppression of endoplasmic reticulum (ER)-to-Golgi trafficking alone does not suppress α-syn toxicity in this strain. In an effort to identify individual compounds that could simultaneously rescue these apparently disparate pathological effects of α-syn, we screened a library of 115,000 compounds. We identified a class of small molecules that reduced α-syn toxicity at micromolar concentrations in this higher toxicity strain. These compounds reduced the formation of α-syn foci, re-established ER-to-Golgi trafficking and ameliorated α-syn-mediated damage to mitochondria. They also corrected the toxicity of α-syn in nematode neurons and in primary rat neuronal midbrain cultures. Remarkably, the compounds also protected neurons against rotenone-induced toxicity, which has been used to model the mitochondrial defects associated with PD in humans. That single compounds are capable of rescuing the diverse toxicities of α-syn in yeast and neurons suggests that they are acting on deeply rooted biological processes that connect these toxicities and have been conserved for a billion years of eukaryotic evolution. Thus, it seems possible to develop novel therapeutic strategies to simultaneously target the multiple pathological features of PD.MGH/MIT Morris Udall Center of Excellence in Parkinson Disease Research (NS038372)Michael J. Fox Foundation for Parkinson's ResearchHoward Hughes Medical InstituteUnited States. National Institutes of Health (NS049221)American Parkinson Disease Association, Inc

Role of alpha-synuclein primary structure and oxidative stress in Parkinson\u27s disease pathology

Parkinson\u27s disease (PD) is a neurodegenerative disorder which affects approximately 1% of the population 65 years of age and younger. Two classic hallmarks of PD include loss of dopaminergic neurons from the substantia nigra and the presence in surviving neurons of Lewy Bodies. Lewy bodies are cellular inclusions which contain fibrillar forms of the presynaptic protein, alpha-synuclein (aSyn). aSyn mutants involved in familial PD (e.g. A53T, A30P, E46K) have a greater tendency to self-associate than the wild-type (WT) protein, suggesting that aSyn aggregation contributes to PD pathogenesis. Accordingly, understanding more about how the primary structure of aSyn relates to aggregation and neurotoxicity can provide greater insight into the pathology of PD. Whereas patients carrying the A53T mutation typically suffer from PD, aged mice that also carry this mutation do not show PD phenotypes. Comparison of the human and mouse aSyn sequences reveals seven mismatches including A53T. Moreover, 5 of the 6 additional mismatches are located in the C-terminal region (residues 100, 103, 107, 121, and 122). We chose to focus on the role of residues 121 and 122 because evidence suggests that the C-terminal region encompassing these two residues modulates aSyn self-assembly’ (or ‘aggregation’). Primary midbrain cultures were transduced with adenoviruses encoding Chimera 1 (human A53T with mouse residues at 121 and 122), Chimera 2 (mouse aSyn with human residues at 121 and 122), mouse aSyn, and A53T aSyn. Cultures were analyzed immunocytochemically to assess relative dopaminergic cell viability. Our data showed that mouse and Chimera 1 had no effect on dopaminergic cell viability compared to that in the untransduced control, whereas A53T and Chimera 2 were significantly toxic compared to mouse and Chimera 1. Taken together, these results identified two key C-terminal residues, D121 and N122, as being involved in aSyn-mediated toxicity. To further elucidate the role of the C-terminal region, we investigated the impact of oxidative stress and C-terminal post-translational modifications on aSyn aggregation and toxicity. Previous studies in the lab revealed that aSyn undergoes post-translational modifications, including phosphorylation of Ser 129 and nitration or phosphorylation of Tyr 125, 133, and 136, in neuronal cells exposed to rotenone, an inhibitor of mitochondrial complex I that causes oxidative stress. We found that the appearance of these modifications correlated with the formation of membrane-bound and cytosolic aSyn aggregates in rotenone-treated PC12 cells. To determine the effects of tyrosine modifications on aSyn aggregation, we constructed variants in which the three tyrosine residues were replaced with phenylalanine (‘3YF’, to eliminate post-translational modifications) or aspartate (‘3YD’, to mimic the introduction of a negative charge by tyrosine phosphorylation), and we examined the impact of these substitutions on aSyn aggregation. PC12 cells expressing each variant were cultured in the absence or presence of rotenone and separated into membrane and cytosolic fractions, and levels of SDS-resistant aSyn oligomers in each fraction were determined via Western blotting. Results suggested that 3YF and 3YD have reduced and increased propensities (respectively) to form membrane-bound aSyn aggregates in rotenone-treated cells. Additional experiments revealed that 3YF and 3YD exhibit reduced and enhanced dopaminergic cell killing (respectively) in primary midbrain cultures, These results imply that (i) phosphorylation and/or nitration of the three C-terminal tyrosine residues leads to an increase in aSyn self-assembly and neurotoxicity, and (ii) a build-up of membrane-bound aggregates correlates with aSyn-mediated dopaminergic cell death. Given the importance of oxidative stress in PD pathogenesis as outlined above, we hypothesized that natural products with strong antioxidant properties (e.g. phenolic compounds) may protect dopaminergic neurons from PD related-insults. To address this hypothesis, we examined the effects of extracts enriched in anthocyanins (ANC) (e.g. a blueberry (BB) extract) proanthocyanidins (PAC), phenolic acids (PA), or stilbenes on rotenone neurotoxicity in primary midbrain cultures. We found that in general dopaminergic cell death elicited by rotenone was suppressed to a greater extent by extracts enriched in ANC than those enriched in PAC, PA, or stilbenes. In other studies, we showed that a BB alleviated aSyn neurotoxicity in the primary cell culture model and inhibited aSyn fibril formation in a test-tube model. Collectively, our data indicate that the C-terminal region of aSyn, specifically residues 121 and 122, play an important role in aSyn neurotoxicity. Additionally, our results suggest that oxidative stress stimulates the formation of toxic aSyn aggregates by promoting harmful post-translational modifications in the C-terminal region, and dopaminergic neurons may be protected from PD-related insults by phenolic-rich extracts. Our findings provide insight into PD pathogenesis and suggest that antioxidant therapies may help slow disease progression