DJ-1 knock-down impairs astrocyte mitochondrial function.

Mitochondrial dysfunction has long been implicated in the pathogenesis of Parkinson's disease (PD). PD brain tissues show evidence for mitochondrial respiratory chain Complex I deficiency. Pharmacological inhibitors of Complex I, such as rotenone, cause experimental parkinsonism. The cytoprotective protein DJ-1, whose deletion is sufficient to cause genetic PD, is also known to have mitochondria-stabilizing properties. We have previously shown that DJ-1 is over-expressed in PD astrocytes, and that DJ-1 deficiency impairs the capacity of astrocytes to protect co-cultured neurons against rotenone. Since DJ-1 modulated, astrocyte-mediated neuroprotection against rotenone may depend upon proper astrocytic mitochondrial functioning, we hypothesized that DJ-1 deficiency would impair astrocyte mitochondrial motility, fission/fusion dynamics, membrane potential maintenance, and respiration, both at baseline and as an enhancement of rotenone-induced mitochondrial dysfunction. In astrocyte-enriched cultures, we observed that DJ-1 knock-down reduced mitochondrial motility primarily in the cellular processes of both untreated and rotenone treated cells. In these same cultures, DJ-1 knock-down did not appreciably affect mitochondrial fission, fusion, or respiration, but did enhance rotenone-induced reductions in the mitochondrial membrane potential. In neuron-astrocyte co-cultures, astrocytic DJ-1 knock-down reduced astrocyte process mitochondrial motility in untreated cells, but this effect was not maintained in the presence of rotenone. In the same co-cultures, astrocytic DJ-1 knock-down significantly reduced mitochondrial fusion in the astrocyte cell bodies, but not the processes, under the same conditions of rotenone treatment in which DJ-1 deficiency is known to impair astrocyte-mediated neuroprotection. Our studies therefore demonstrated the following new findings: (i) DJ-1 deficiency can impair astrocyte mitochondrial physiology at multiple levels, (ii) astrocyte mitochondrial dynamics vary with sub-cellular region, and (iii) the physical presence of neurons can affect astrocyte mitochondrial behavior.

Mitochondrial factors with dual roles in death and survival.

At least in mammals, we have some understanding of how caspases facilitate mitochondria-mediated cell death, but the biochemical mechanisms by which other factors promote or inhibit programmed cell death are not understood. Moreover, most of these factors are only studied after treating cells with a death stimulus. A growing body of new evidence suggests that cell death regulators also have 'day jobs' in healthy cells. Even caspases, mitochondrial fission proteins and pro-death Bcl-2 family proteins appear to have normal cellular functions that promote cell survival. Here, we review some of the supporting evidence and stretch beyond the evidence to seek an understanding of the remaining questions.

Quantitative biochemical and ultrastructural comparison of mitochondrial permeability transition in isolated brain and liver mitochondria: evidence for reduced sensitivity of brain mitochondria.

Opening of the mitochondrial permeability transition pore has increasingly been implicated in excitotoxic, ischemic, and apoptotic cell death, as well as in several neurodegenerative disease processes. However, much of the work directly characterizing properties of the transition pore has been performed in isolated liver mitochondria. Because of suggestions of tissue-specific differences in pore properties, we directly compared isolated brain mitochondria with liver mitochondria and used three quantitative biochemical and ultrastructural measurements of permeability transition. We provide evidence that brain mitochondria do not readily undergo permeability transition upon exposure to conditions that rapidly induce the opening of the transition pore in liver mitochondria. Exposure of liver mitochondria to transition-inducing agents led to a large, cyclosporin A-inhibitable decrease in spectrophotometric absorbance, a loss of mitochondrial glutathione, and morphologic evidence of matrix swelling and disruption, as expected. However, we found that similarly treated brain mitochondria showed very little absorbance change and no loss of glutathione. The absence of response in brain was not simply due to structural limitations, since large-amplitude swelling and release of glutathione occurred when membrane pores unrelated to the transition pore were formed. Additionally, electron microscopy revealed that the majority of brain mitochondria appeared morphologically unchanged following treatment to induce permeability transition. These findings show that isolated brain mitochondria are more resistant to induction of permeability transition than mitochondria from liver, which may have important implications for the study of the mechanisms involved in neuronal cell death.

Modification of dopamine transporter function: effect of reactive oxygen species and dopamine.

Dopamine can oxidize to form reactive oxygen species and quinones, and we have previously shown that dopamine quinones bind covalently to cysteinyl residues on striatal proteins. The dopamine transporter is one of the proteins at risk for this modification, because it has a high affinity for dopamine and contains several cysteinyl residues. Therefore, we tested whether dopamine transport in rat striatal synaptosomes could be affected by generators of reactive oxygen species, including dopamine. Uptake of [3H]dopamine (250 nM) was inhibited by ascorbate (0.85 mM; -44%), and this inhibition was prevented by the iron chelator diethylenetriaminepentaacetic acid (1 mM), suggesting that ascorbate was acting as a prooxidant in the presence of iron. Preincubation with xanthine (500 microM) and xanthine oxidase (50 mU/ml) also reduced [3H]dopamine uptake (-76%). Preincubation with dopamine (100 microM) caused a 60% inhibition of subsequent [3H]dopamine uptake. This dopamine-induced inhibition was attenuated by diethylenetriaminepentaacetic acid (1 mM), which can prevent iron-catalyzed oxidation of dopamine during the preincubation, but was unaffected by the monoamine oxidase inhibitor pargyline (10 microM). None of these incubations caused a loss of membrane integrity as indicated by lactate dehydrogenase release. These findings suggest that reactive oxygen species and possibly dopamine quinones can modify dopamine transport function.