Abstract 3599: Cyclosporine A Treatment Abrogates Ischemia-Induced Neuronal Cell Death By Preserving Mitochondrial Function By Upregulating The Parkinson's Disease Protein DJ-1

Abstract

Background: Stroke is characterized by neural tissue death after deprivation of oxygen, glucose, and other nutrients due to a reduction in blood flow to the brain. Disease manifestations of stroke involve primarily an infarcted core, and subsequently the formation of an ischemic penumbra which over a sub-acute period remains as salvageable neural tissue, thereby amenable to therapeutic intervention. Secondary cell death processes, including oxidative stress-induced mitochondrial dysfunction, can endanger the penumbra thereby limiting neuroprotection. Exploring the direct effects of drugs, such as Cyclosporine A (CsA), acting on neuronal mitochondria may reveal novel mitochondria-based cell death signaling therapeutic targets for stroke. Objective: Under hypoxic ischemia condition, the mitochondrial membrane potential (Δψ m ), respiratory-related enzymes, and mitochondrial DNA deteriorate resulting in the aberrant accumulation of free radicals and reactive oxygen species. CsA, traditionally considered a robust immunosuppressant drug, has been shown as a potent neuroprotectant against neuronal cell death. However, the molecular mechanism by which CsA interacts with mitochondrial membrane-associated proteins remains not fully understood. Using the in vitro stroke model of oxygen glucose deprivation (OGD), we examined here this interaction between CsA and the Parkinson’s disease protein DJ-1, which has been recently implicated in the regulation of mitochondrial function. Methods: Primary rat astrocytes/neurons (6/4) were exposed to the OGD and processed for immunocytochemistry and ELISA to reveal the role of DJ-1 in CsA modulation of mitochondrial function. In addition, ATP content, hexokinase activity (accumulation of intracellular glucose 6-phosphate), and mitochondrial DNA (mtDNA) stability were measured by a bioluminescence assay kit. Results: Administration of CsA before stroke onset (24 hrs pre-OGD), but not after stroke (2 hrs post-OGD), afforded significant neuroprotective effects compared to OGD-treated cells without CsA, characterized by the following cellular and molecular events: (1) CsA prevented the mitochondria-dependent cell death signaling pathway involved in cytochrome c induced apoptosis; (2) CsA protected cellular ATP decline (two-fold higher) without altering the hexokinase activity and the Δψ m ; (3) blocked mtDNA degradation, and, interestingly; (4) enhanced secretion of DJ-1into neighboring cells. This neuroprotection was achieved with the effective dose range of 500 nM - 10 μ M of CsA. Conclusion: These novel observations indicate that CsA- and DJ-1-neuroprotection amplify the maintenance of mitochondrial function, and that mitochondria-based treatments targeting the early phase of disease progression may prove beneficial in stroke.