The Parkinson's Disease-Linked Protein DJ-1 Associates with Cytoplasmic mRNP Granules During Stress and Neurodegeneration.

Mutations in the gene encoding DJ-1 are associated with autosomal recessive forms of Parkinson's disease (PD). DJ-1 plays a role in protection from oxidative stress, but how it functions as an "upstream" oxidative stress sensor and whether this relates to PD is still unclear. Intriguingly, DJ-1 may act as an RNA binding protein associating with specific mRNA transcripts in the human brain. Moreover, we previously reported that the yeast DJ-1 homolog Hsp31 localizes to stress granules (SGs) after glucose starvation, suggesting a role for DJ-1 in RNA dynamics. Here, we report that DJ-1 interacts with several SG components in mammalian cells and localizes to SGs, as well as P-bodies, upon induction of either osmotic or oxidative stress. By purifying the mRNA associated with DJ-1 in mammalian cells, we detected several transcripts and found that subpopulations of these localize to SGs after stress, suggesting that DJ-1 may target specific mRNAs to mRNP granules. Notably, we find that DJ-1 associates with SGs arising from N-methyl-D-aspartate (NMDA) excitotoxicity in primary neurons and parkinsonism-inducing toxins in dopaminergic cell cultures. Thus, our results indicate that DJ-1 is associated with cytoplasmic RNA granules arising during stress and neurodegeneration, providing a possible link between DJ-1 and RNA dynamics which may be relevant for PD pathogenesis

Molecular characterization of the Parkinson’s associated protein DJ-1

Mutations in DJ-1 (PARK7), a conserved protein of 189 amino acids, cause autosomal recessive cases of Parkinson’s disease (PD). DJ-1 appears to play a central role in protecting cells from oxidative stress, which likely has relevance for its role in PD pathogenesis. Biochemical and crystallographic approaches indicate that DJ-1 dimerizes, which is likely fundamental for its stability and normal function. A main focus of this thesis was identifying and characterizing DJ-1 dimerization modifiers through an unbiased screen of a kinase and phosphatase inhibitor library, taking advantage of bimolecular fluorescence complementation (BiFC) as a readout for DJ-1 dimerisation in living cells. To address this aim, we generated HEK 293T cell clones stably over-expressing DJ-1 BiFC constructs and optimised high throughput Cell^RScan^ R screening. This approach identified two kinase inhibitors (Bosutinib and KW2449) which decrease DJ-1 dimerisation in an oxidative stress-dependent manner. Furthermore, to indicate whether or not the observed effects of the kinase inhibitors on DJ-1 dimerization were due to a direct alteration of DJ-1 phosphorylation status or were indirect effects, we studied DJ-1 phosphorylation at all potential sites on its dimerization and stability by generating phosphomimic and phosphoblocking mutants. This work indicates that phosphorylation of key residues of DJ-1 likely dramatically reduces its stablilty. Additionally, by using the BiFC approach we found a direct interaction in living cells of DJ-1 with microtubule-associated protein tau, which is known to be hyper phosphorylated and aggregated in neurodegenerative disorders like Alzheimer’s disease and Parkinson’s disease. These analyses suggest that alterations in DJ-1 dimerization, stability and phosphorylation status in normal conditions and in response to oxidative stress may shed more light on DJ-1 function and its role associated with PD pathogenesis