Mutant SOD1 alters the motor neuronal transcriptome: implications for familial ALS.

Familial amyotrophic lateral sclerosis (FALS) is caused, in 20% of cases, by mutations in the Cu/Zn superoxide dismutase gene (SOD1). Although motor neuron injury occurs through a toxic gain of function, the precise mechanism(s) remains unclear. Using an established NSC34 cellular model for SOD1-associated FALS, we investigated the effects of mutant SOD1 specifically in cells modelling the vulnerable cell population, the motor neurons, without contamination from non-neuronal cells present in CNS. Using gene expression profiling, 268 transcripts were differentially expressed in the presence of mutant human G93A SOD1. Of these, 197 were decreased, demonstrating that the presence of mutant SOD1 leads to a marked degree of transcriptional repression. Amongst these were a group of antioxidant response element (ARE) genes encoding phase II detoxifying enzymes and antioxidant response proteins (so-called 'programmed cell life' genes), the expression of which is regulated by the transcription factor NRF2. We provide evidence that dysregulation of Nrf2 and the ARE, coupled with reduced pentose phosphate pathway activity and decreased generation of NADPH, represent significant and hitherto unrecognized components of the toxic gain of function of mutant SOD1. Other genes of interest significantly altered in the presence of mutant SOD1 include several previously implicated in neurodegeneration, as well as genes involved in protein degradation, the immune response, cell death/survival and the heat shock response. Preliminary studies on isolated motor neurons from SOD1-associated motor neuron disease cases suggest key genes are also differently expressed in the human disease.

The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization.

Loss-of-function DJ-1 mutations can cause early-onset Parkinson's disease. The function of DJ-1 is unknown, but an acidic isoform accumulates after oxidative stress, leading to the suggestion that DJ-1 is protective under these conditions. We addressed whether this represents a posttranslational modification at cysteine residues by systematically mutating cysteine residues in human DJ-1. WT or C53A DJ-1 was readily oxidized in cultured cells, generating a pI 5.8 isoform, but an artificial C106A mutant was not. We observed a cysteine-sulfinic acid at C106 in crystalline DJ-1 but no modification of C53 or C46. Oxidation of DJ-1 was promoted by the crystallization procedure. In addition, oxidation-induced mitochondrial relocalization of DJ-1 and protection against cell death were abrogated in C106A but not C53A or C46A. We suggest that DJ-1 protects against neuronal death, and that this is signaled by acidification of the key cysteine residue, C106.

Parkin and alpha-synuclein: opponent actions in the pathogenesis of Parkinson's disease.

Dominant mutations in the gene for alpha-synuclein, a small presynaptic protein, can cause Parkinson's disease. Although there is still substantial debate about the precise mechanisms, alpha-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 alpha-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 alpha-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.

L166P mutant DJ-1, causative for recessive Parkinson's disease, is degraded through the ubiquitin-proteasome system.

Mutations in a gene on chromosome 1, DJ-1, have been reported recently to be associated with recessive, earlyonset Parkinson's disease. While one mutation is a large deletion that is predicted to produce an effective knockout of the gene, the second is a point mutation, L166P, whose precise effects on protein function are unclear. In the present study, we show that L166P destabilizes DJ-1 protein and promotes its degradation through the ubiquitin-proteasome system. A double mutant (K130R, L166P) was more stable than L166P, suggesting that this lysine residue contributes to stability of the protein. Subcellular localization was broadly similar for both wild type and L166P forms of the protein, indicating that the effect of the mutation is predominantly on protein stability. These observations are reminiscent of other recessive gene mutations that produce an effective loss of function. The L166P mutation has the simple effect of promoting DJ-1 degradation, thereby reducing net DJ-1 protein within the cell.

Microarray analysis reveals induction of heat shock proteins mRNAs by the torsion dystonia protein, TorsinA.

An in-frame deletion (Delta E302/303) in the TorsinA gene has been demonstrated to be responsible for primary torsion dystonia, showing dominant inheritance with reduced penetrance. The Delta E302/303 torsinA mutation forms intracellular ER derived inclusions in a variety of cultured cells, which may suggest that the mutations might evoke ER stress. We used microarray analysis of human derived cell lines expressing the Delta E302/303 torsinA mutation in order to reveal alterations in gene expression in the hope of identifying genetic modifying loci or novel markers for disease pathogenesis. We identified transcriptional changes in multiple members of the heat shock protein family of genes, confirmed by reverse transcription-polymerase chain reaction, which could be indicative of ER stress. However, both wild type and mutant torsinA were affected to a similar extent, suggesting that this is not related to either disease state or the formation of ER-derived inclusions.

Co-ordinate transcriptional regulation of dopamine synthesis genes by alpha-synuclein in human neuroblastoma cell lines.

Abnormal accumulation of alpha-synuclein in Lewy bodies is a neuropathological hallmark of both sporadic and familial Parkinson's disease (PD). Although mutations in alpha-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) alpha-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 alpha-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.

Parkin protects against the toxicity associated with mutant alpha-synuclein: proteasome dysfunction selectively affects catecholaminergic neurons.

One hypothesis for the etiology of Parkinson's disease (PD) is that subsets of neurons are vulnerable to a failure in proteasome-mediated protein turnover. Here we show that overexpression of mutant alpha-synuclein increases sensitivity to proteasome inhibitors by decreasing proteasome function. Overexpression of parkin decreases sensitivity to proteasome inhibitors in a manner dependent on parkin's ubiquitin-protein E3 ligase activity, and antisense knockdown of parkin increases sensitivity to proteasome inhibitors. Mutant alpha-synuclein also causes selective toxicity to catecholaminergic neurons in primary midbrain cultures, an effect that can be mimicked by the application of proteasome inhibitors. Parkin is capable of rescuing the toxic effects of mutant alpha-synuclein or proteasome inhibition in these cells. Therefore, parkin and alpha-synuclein are linked by common effects on a pathway associated with selective cell death in catecholaminergic neurons.

An in vitro model of Parkinson's disease: linking mitochondrial impairment to altered alpha-synuclein metabolism and oxidative damage.

Chronic systemic complex I inhibition caused by rotenone exposure induces features of Parkinson's disease (PD) in rats, including selective nigrostriatal dopaminergic degeneration and formation of ubiquitin- and alpha-synuclein-positive inclusions (Betarbet et al., 2000). To determine underlying mechanisms of rotenone-induced cell death, we developed a chronic in vitro model based on treating human neuroblastoma cells with 5 nm rotenone for 1-4 weeks. For up to 4 weeks, cells grown in the presence of rotenone had normal morphology and growth kinetics, but at this time point, approximately 5% of cells began to undergo apoptosis. Short-term rotenone treatment (1 week) elevated soluble alpha-synuclein protein levels without changing message levels, suggesting that alpha-synuclein degradation was retarded. Chronic rotenone exposure (4 weeks) increased levels of SDS-insoluble alpha-synuclein and ubiquitin. After a latency of >2 weeks, rotenone-treated cells showed evidence of oxidative stress, including loss of glutathione and increased oxidative DNA and protein damage. Chronic rotenone treatment (4 weeks) caused a slight elevation in basal apoptosis and markedly sensitized cells to further oxidative challenge. In response to H2O2, there was cytochrome c release from mitochondria, caspase-3 activation, and apoptosis, all of which occurred earlier and to a much greater extent in rotenone-treated cells; caspase inhibition provided substantial protection. These studies indicate that chronic low-grade complex I inhibition caused by rotenone exposure induces accumulation and aggregation of alpha-synuclein and ubiquitin, progressive oxidative damage, and caspase-dependent death, mechanisms that may be central to PD pathogenesis.