Rili Ahmad

Formation of a stabilized cysteine sulfinic acid is critical for the mitochondrial function of the parkinsonism protein DJ-1.

The formation of cysteine-sulfinic acid has recently become appreciated as a modification that links protein function to cellular oxidative status. Human DJ-1, a protein associated with inherited parkinsonism, readily forms cysteine-sulfinic acid at a conserved cysteine residue (Cys106 in human DJ-1). Mutation of Cys106 causes the protein to lose its normal protective function in cell culture and model organisms. However, it is unknown whether the loss of DJ-1 protective function in these mutants is due to the absence of Cys106 oxidation or the absence of the cysteine residue itself. To address this question, we designed a series of substitutions at a proximal glutamic acid residue (Glu18) in human DJ-1 that alter the oxidative propensity of Cys106 through changes in hydrogen bonding. We show that two mutations, E18N and E18Q, allow Cys106 to be oxidized to Cys106-sulfinic acid under mild conditions. In contrast, the E18D mutation stabilizes a cysteine-sulfenic acid that is readily reduced to the thiol in solution and in vivo. We show that E18N and E18Q can both partially substitute for wild-type DJ-1 using mitochondrial fission and cell viability assays. In contrast, the oxidatively impaired E18D mutant behaves as an inactive C106A mutant and fails to protect cells. We therefore conclude that formation of Cys106-sulfinic acid is a key modification that regulates the protective function of DJ-1.

RNA binding activity of the recessive parkinsonism protein DJ-1 supports involvement in multiple cellular pathways

Parkinsons disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanisms involved in the degeneration of neurons remain unclear. DJ-1 mutations are one cause of recessive parkinsonism, but this gene is also reported to be involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of DJ-1 is unknown, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here, we show, using four independent methods, that DJ-1 associates with RNA targets in cells and the brain, including mitochondrial genes, genes involved in glutathione metabolism, and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that DJ-1 is sufficient for RNA binding at nanomolar concentrations. Further, we show that DJ-1 binds RNA but dissociates after oxidative stress. These data implicate a single mechanism for the pleiotropic effects of DJ-1 in different model systems, namely that the protein binds multiple RNA targets in an oxidation-dependent manner.

Mitochondrial Alterations in PINK1 Deficient Cells Are Influenced by Calcineurin-Dependent Dephosphorylation of Dynamin-Related Protein 1

PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.

Co‐ordinate transcriptional regulation of dopamine synthesis genes by α‐synuclein in human neuroblastoma cell lines

Abnormal accumulation of α‐synuclein in Lewy bodies is a neuropathological hallmark of both sporadic and familial Parkinsons disease (PD). Although mutations in α‐synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic cell death occurs remains unknown. We investigated transcriptional changes in neuroblastoma cell lines transfected with either normal or mutant (A30P or A53T) α‐synuclein using microarrays, with confirmation of selected genes by quantitative RT‐PCR. Gene products whose expression was found to be significantly altered included members of diverse functional groups such as stress response, transcription regulators, apoptosis‐inducing molecules, transcription factors and membrane‐bound proteins. We also found evidence of altered expression of dihydropteridine reductase, which indirectly regulates the synthesis of dopamine. Because of the importance of dopamine in PD, we investigated the expression of all the known genes in dopamine synthesis. We found co‐ordinated downregulation of mRNA for GTP cyclohydrolase, sepiapterin reductase (SR), tyrosine hydroxylase (TH) and aromatic acid decarboxylase by wild‐type but not mutant α‐synuclein. These were confirmed at the protein level for SR and TH. Reduced expression of the orphan nuclear receptor Nurr1 was also noted, suggesting that the co‐ordinate regulation of dopamine synthesis is regulated through this transcription factor.

Mutations in LRRK2/dardarin associated with Parkinson disease are more toxic than equivalent mutations in the homologous kinase LRRK1

Several mutations have been found in the leucine‐rich repeat kinase 2 gene (LRRK2), encoding the protein dardarin, which are associated with autosomal dominant Parkinson disease. We have previously shown that mutant LRRK2/dardarin is toxic to neurons and neuron‐like cell lines in culture and that some mutations are also associated with an inclusion‐body phenotype. There is a homologous kinase, LRRK1, which has a similar domain structure but is not known to carry mutations causing Parkinson disease. In the current study, we introduced mutations at equivalent residues in both LRRK2 and LRRK1 to determine their effects in cells. We show that mutations in dardarin are more prone to form inclusion bodies in transfected cells and are more toxic than equivalent mutations in LRRK1. This work suggests that dardarin/LRRK2 is inherently more damaging than LRRK1.

Tyrosinase exacerbates dopamine toxicity but is not genetically associated with Parkinson's disease

Tyrosinase is a key enzyme in the synthesis of melanin in skin and hair and has also been proposed to contribute to the formation of neuromelanin (NM). The presence of NM, which is biochemically similar to melanin in peripheral tissues, identifies groups of neurons susceptible in Parkinsons disease (PD). Whether tyrosinase is beneficial or detrimental to neurons is unclear; whilst the enzyme activity of tyrosinase generates dopamine‐quinones and other oxidizing compounds, NM may form a sink for such radical species. In the present study, we demonstrated that tyrosinase is expressed at low levels in the human brain. We found that mRNA, protein and enzyme activity are all present but at barely detectable levels. In cell culture systems, expression of tyrosinase increases neuronal susceptibility to oxidizing conditions, including dopamine itself. We related these in vitro observations to the human disease by assessing whether there was any genetic association between the gene encoding tyrosinase and idiopathic PD. We found neither genotypic or haplotypic association with three polymorphic markers of the gene. This argues against a strong genetic association between tyrosinase and PD, although the observed contribution to cellular toxicity suggests that a biochemical association is likely.

Post-transcriptional regulation of mRNA associated with DJ-1 in Sporadic Parkinson disease

Mutations in DJ-1 lead to a monogenic form of early onset recessive parkinsonism. DJ-1 can respond to oxidative stress, which has been proposed to be involved in the pathogenesis of sporadic Parkinson disease (PD). We have recently reported that DJ-1 interacts with mRNA in an oxidation-dependent manner. Here, we confirm interaction of DJ-1 and RNA in human brain using immunoprecipitation followed by quantitative real time PCR. We confirmed previous reports that DJ-1 is more oxidized in cortex from cases of sporadic PD compared to controls. In the same samples, protein and RNA expression was measured for four DJ-1 target genes GPx4, MAPK8IP1, ND2 and ND5. While no alterations in mRNA expression were observed, an increase in protein expression was observed in PD cases for GPx4 and MAPK8IP1. In the same patients, we saw decreased mRNA and protein levels of two mitochondrial targets, ND2 and ND5. These results suggest that these proteins undergo regulation at the post-transcriptional level that may involve translational regulation by DJ-1.