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Analysis of targeted mutation in DJ-1 on cellular function in primary astrocytes.

ABSTRACT: DJ-1 mutation induces early-onset Parkinson's disease, and conversely over-expression of DJ-1 is associated with cancer in numerous tissues. A gene-trap screening library conducted in embryonic stem cells was utilized for generation of a DJ-1 mutant mouse. Real-time PCR and immunoblotting were utilized to confirm functional mutation of the DJ-1 gene. Normal DJ-1 protein expression in adult mouse tissue was characterized and demonstrates high expression in brain tissue with wide systemic distribution. Primary astrocytes isolated from DJ-1(-/-) mice reveal a decreased nuclear localization of DJ-1 protein in response to rotenone or LPS, with a concomitant increase in mitochondrial localization of DJ-1 found only in the rotenone exposure. Resting mitochondrial membrane potential was significantly lower in DJ-1(-/-) astrocytes, as compared to controls. Our DJ-1 knockout mouse provides an exciting tool for exploring the molecular and physiological roles of DJ-1 to further explicate its functions in neurodegeneration.

SUBMITTER: Ashley AK

PROVIDER: S-EPMC4632527 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Analysis of targeted mutation in DJ-1 on cellular function in primary astrocytes.

Ashley Amanda K AK Hanneman William H WH Katoh Takeshi T Moreno Julie A JA Pollack Ashley A Tjalkens Ronald B RB Legare Marie E ME

Toxicology letters 20081121 3

DJ-1 mutation induces early-onset Parkinson's disease, and conversely over-expression of DJ-1 is associated with cancer in numerous tissues. A gene-trap screening library conducted in embryonic stem cells was utilized for generation of a DJ-1 mutant mouse. Real-time PCR and immunoblotting were utilized to confirm functional mutation of the DJ-1 gene. Normal DJ-1 protein expression in adult mouse tissue was characterized and demonstrates high expression in brain tissue with wide systemic distribu ...[more]

PMID: 19063952

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Project description:BackgroundComplex interactions involving genetic susceptibility and environmental factors are thought to underlie the pathogenesis of Parkinson's disease (PD). Although the role of inflammatory processes in modulating risk for development of PD has yet to be fully understood, prospective studies suggest that chronic use of NSAIDs reduce the incidence of PD. Loss-of-function mutations in the DJ-1 gene cause a rare form of familial PD with an autosomal recessive pattern of inheritance; however, DJ-1-/- mice do not display nigrostriatal pathway degeneration, suggesting that additional factors such as inflammation may be needed to induce neurodegeneration on the background of DJ-1 gene mutations. Neuroinflammation causes oxidative stress and, based on evidence that DJ-1 plays a protective role against oxidative stress, we investigated whether DJ-1-/- mice display increased vulnerability to inflammation-induced nigral degeneration.MethodsWe exposed adult wild-type and DJ-1-/- mice to repeated intranasal administration of soluble TNF (inTNF) or repeated intraperitoneal injections of low-dose lipopolysaccharide (LPS) or saline vehicle. We measured locomotor performance using a variety of behavior tasks, striatal dopamine (DA) content by HPLC, DA neuron (TH+ cells) and total neuron (NeuN+ cells) number in the substantia nigra pars compacta and ventral tegmental area by unbiased stereology, number of Iba1-positive microglia, and mRNA levels of inflammatory and oxidative stress genes by quantitative PCR in the midbrain, cortex and isolated peritoneal macrophages of DJ-1-/- and wild-type mice.ResultsWe found that chronic LPS injections induced similar neuroinflammatory responses in the midbrains of DJ-1-/- mice and wild-type mice and neither group developed locomotor deficits or nigral degeneration. inTNF administration did not appear to induce neuroinflammatory responses in LPS-treated wild-type or DJ-1-/- mice. The lack of vulnerability to inflammation-induced nigral degeneration was not due to enhanced anti-oxidant gene responses in the midbrains of DJ-1-/- mice which, in fact, displayed a blunted response relative to that of wild-type mice. Peripheral macrophages from wild-type and DJ-1-/- mice displayed similar basal and LPS-induced inflammatory and oxidative stress markers in vitro.ConclusionsOur studies indicate that DJ-1-/- mice do not display increased vulnerability to inflammation-related nigral degeneration in contrast to what has been reported for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine. We conclude that either DJ-1 does not have a critical role in protecting DA neurons against inflammation-induced oxidative stress and/or there is compensatory gene expression in the midbrain of DJ-1-/- mice that renders them resistant to the cytotoxic effects triggered by chronic peripheral inflammation.

Project description: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.

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Analysis of targeted mutation in DJ-1 on cellular function in primary astrocytes.

Publications

Analysis of targeted mutation in DJ-1 on cellular function in primary astrocytes.

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