David W. Miller

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.

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.

Glial cell inclusions and the pathogenesis of neurodegenerative diseases.

In this review, we discuss examples that show how glial-cell pathology is increasingly recognized in several neurodegenerative diseases. We also discuss the more provocative idea that some of the disorders that are currently considered to be neurodegenerative diseases might, in fact, be due to primary abnormalities in glia. Although the mechanism of glial pathology (i.e. modulating glutamate excitotoxicity) might be better established for amyotrophic lateral sclerosis (ALS), a role for neuronal-glial interactions in the pathogenesis of most neurodegenerative diseases is plausible. This burgeoning area of neuroscience will receive much attention in the future and it is expected that further understanding of basic neuronal-glial interactions will have a significant impact on the understanding of the fundamental nature of human neurodegenerative disorders.

α-Synuclein overexpression increases dopamine toxicity in BE(2)-M17 cells

BackgroundOxidative stress has been proposed to be involved in the pathogenesis of Parkinsons disease (PD). A plausible source of oxidative stress in nigral dopaminergic neurons is the redox reactions that specifically involve dopamine and produce various toxic molecules, i.e., free radicals and quinone species. α-Synuclein, a protein found in Lewy bodies characteristic of PD, is also thought to be involved in the pathogenesis of PD and point mutations and multiplications in the gene coding for α-synuclein have been found in familial forms of PD.ResultsWe used dopaminergic human neuroblastoma BE(2)-M17 cell lines stably transfected with WT or A30P mutant α-synuclein to characterize the effect of α-synuclein on dopamine toxicity. Cellular toxicity was analyzed by lactate dehydrogenase assay and by fluorescence-activated cell sorter analysis. Increased expression of either wild-type or mutant α-synuclein enhances the cellular toxicity induced by the accumulation of intracellular dopamine or DOPA.ConclusionsOur results suggest that an interplay between dopamine and α-synuclein can cause cell death in a neuron-like background. The data presented here are compatible with several models of cytotoxicity, including the formation of α-synuclein oligomers and impairment of the lysosomal degradation.

Caspase-1 causes truncation and aggregation of the Parkinson’s disease-associated protein α-synuclein

Significance The aggregation of α-synuclein (aSyn) is a pathological hallmark of Parkinson’s disease. Here we show that the enzymatic component of the innate inflammation system, known as caspase-1, hydrolyzes aSyn, rendering it aggregation-prone. The aggregation of α-synuclein (aSyn) leading to the formation of Lewy bodies is the defining pathological hallmark of Parkinson’s disease (PD). Rare familial PD-associated mutations in aSyn render it aggregation-prone; however, PD patients carrying wild type (WT) aSyn also have aggregated aSyn in Lewy bodies. The mechanisms by which WT aSyn aggregates are unclear. Here, we report that inflammation can play a role in causing the aggregation of WT aSyn. We show that activation of the inflammasome with known stimuli results in the aggregation of aSyn in a neuronal cell model of PD. The insoluble aggregates are enriched with truncated aSyn as found in Lewy bodies of the PD brain. Inhibition of the inflammasome enzyme caspase-1 by chemical inhibition or genetic knockdown with shRNA abated aSyn truncation. In vitro characterization confirmed that caspase-1 directly cleaves aSyn, generating a highly aggregation-prone species. The truncation-induced aggregation of aSyn is toxic to neuronal culture, and inhibition of caspase-1 by shRNA or a specific chemical inhibitor improved the survival of a neuronal PD cell model. This study provides a molecular link for the role of inflammation in aSyn aggregation, and perhaps in the pathogenesis of sporadic PD as well.

Unaltered α-synuclein blood levels in juvenile Parkinsonism with a parkin exon 4 deletion

We recently reported here that SNCA triplication results in a doubling in the amount of α-synuclein protein in blood from cases with hereditary Lewy body disease. This observation shows that α-synuclein levels in blood accurately reflect gene dosage, which we assume drives pathogenesis in these individuals. A previous report has suggested that parkin can affect α-synuclein metabolism in human brain. Here we have tested whether there is also an increase of α-synuclein in autosomal recessive juvenile Parkinsonism (ARJP). We find there is not and discuss this result in terms of the putative relationships between α-synuclein and parkin.

Parkin and -Synuclein: Opponent Actions in The Pathogenesis of Parkinson’S Disease

Dominant mutations in the gene for •-synuclein, a small presynaptic protein, can cause Parkinson’s disease. Although there is still substantial debate about the precise mechanisms, •-synuclein is toxic to vulnerable neurons, probably as a result of its tendency to aggregate. Opposing this is another gene product that, when mutated, causes a recessive form of parkinsonism, parkin. Parkin has been recently shown to protect cells against •-synuclein toxicity. However, the precise details of the mechanism are unclear. This review will discuss the concept that there are multiple neuronal functions that are targeted by mutant •-synuclein, and in many cases, there is evidence that parkin can protect cells against damage to the same systems. The authors will also discuss ways in which to test some of these ideas, by using newly identified genes such as DJ-1 that cause similar phenotypes.

Identification of the epitope of a monoclonal antibody to DJ-1.

Mutations in DJ-1 can cause early onset parkinsonism. Various antibodies have been generated to detect this protein, one of which is a commonly used monoclonal antibody (clone 3E8). Since results of in situ examinations of DJ-1 expression with this antibody have differed from analyses with species-specific antibodies (e.g. rat), it would be useful to know the epitope for this antibody. Using GFP-tagged deletion constructs of human DJ-1, we have localized the epitope region for this antibody to within residues 56-78 of human DJ-1. Mapping this region to the published three-dimensional structure of DJ-1 indicates that this is a solvent-accessible surface epitope. Immunonegativity of E64D mutant DJ-1 with the monoclonal antibody suggests that glutamate 64 of human DJ-1 contributes to the epitope recognized by this antibody. Moreover, the loss of immunoreactivity due to such a small substitution demonstrates the remarkable sensitivity of the monoclonal antibody 3E8 to DJ-1.