Alexander Mongin - US grants

potassium; phosphorylation; glia; biological signal transduction; intracellular transport; cell morphology; protein tyrosine kinase; electrophysiology; enzyme inhibitors; calmodulin dependent protein kinase; tissue /cell culture; voltage /patch clamp;

[unreadable] DESCRIPTION (provided by applicant): Stroke is a pathological reduction in blood flow which causes irreversible damage of brain tissue, long-term disruptions of brain functions, and frequently patient death. The major focus of ischemia research is on the mechanisms of cell death and the search for pharmacological approaches to salvage vulnerable neural cells. Substantially less attention is paid to long-term impairment of neural cell function that is not associated with cell death. Existing literature suggest, nonetheless, that even relatively mild ischemia causes long-lasting suppression of synaptic communication. For unknown reasons, only presynaptic release is affected, while postsynaptic responses remain preserved. In the current study we hypothesize that suppression of synaptic transmission occurs due to nitrosation of cysteine residues in N-ethylmaleimide sensitive fusion protein (NSF) by nitric oxide (NO) and NO-related reactive nitrogen species. The NSF protein is a trimeric ATPase which is crucial for continuous synaptic vesicle docking/fusion. Modification of a critical cysteine residues in the NSF by N-ethylmaleimide causes irreversible inhibition of vesicle docking and fusion. Similarly, NOdependent modification of NSF thiols may cause long-term inhibition of vesicular neurotransmitter release. To test our hypothesis we propose the following specific aims. (1) We will test for the increased modification of thiols in the ischemic tissue up to 24 hours after ischemia and will further demonstrate thiol modification in the NSF protein immunoprecipitated from the ischemic brain. (2) Using synaptosomal preparation we will test whether nitric oxide and related reactive nitrogen species modify NSF and via this mechanism inhibit vesicular neurotransmitter release. We will further attempt to reverse NSF nitrosation and defects in neurotransmitter release by applying thiol reducing agents. This project will clarify the molecular mechanisms of less understood long-term disruption of synaptic transmission in ischemia and may suggest additional approaches for patient treatment and rehabilitation after stroke and transient ischemic attacks. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Stroke is the third leading cause of death and the leading cause for long-term disability in the U.S. and other industrialized nations. Each year 750,000 Americans suffer, and more than 150,000 die as a result of new and recurrent strokes. In spite of extensive research efforts, to date only one drug, the thrombolytic agent tPA, has been approved by the FDA for stroke treatment. A number of alternative neuroprotective approaches have been identified in animal ischemia models, but failed in clinical trials. Therefore there is continuous need for development and improvement of novel stroke therapies. In this study we propose to test the novel HYPOTHESIS that oxidative stress potently augments and propagates ischemic brain damage by enhancing glial glutamate release via activation of redox-sensitive glutamate transport pathways. Studies from this and other laboratories has identified three redox-sensitive glutamate release pathways: volume-regulated anion channels (VRAC), the cystine/glutamate antiporter (xCT), and the connexin hemichannels (Cxs). In the proposed work we will use pure and mixed primary cultures of rat astrocytes and microglial cells to find the optimal approaches for reducing pathological glutamate release from glia. This will be followed by testing novel pharmacological treatments in vivo, in a rat model of transient focal ischemia. The following SPECIFIC AIMS are proposed: (AIM 1) to elucidate relative contribution of VRAC, xCT, and Cxs to glutamate release in response to oxidative stress in primary glial cultures, using pharmacological tools and an siRNA approach;(AIM 2) to identify the relationship between oxidative stress and pathological glutamate release in vivo in a rat model of transient focal ischemia, using a microdialysis approach and the intra-dialysate drug delivery;(AIM 3) to test the neuroprotective potential of the pharmacological treatments validated in AIMS 1 and 2, by measuring brain infarction volumes in the control ischemia group and after treatment with neuroprotectants. The major objective of this study is to establish a new fundamental relationship between tissue production of reactive oxygen species and pathological glutamate release from glial cells, and to identify novel pharmacological targets in stroke and other neurological disorders (such as multiple sclerosis, Alzheimer's disease, and traumatic brain injury), which involve oxidative stress. PUBLIC HEALTH RELEVANCE: Stroke is a devastating brain disorder that affects 750,000 Americans every year, killing more than 150,000 people and leaving many others permanently debilitated. Presently, only one pharmacological agent, tissue plasminogen activator (tPA), has been approved for acute stroke treatment. Because of several clinical limitations, tPA is used in only 5 to 8% of stroke patients. The present project will study previously unrecognized mechanisms of brain damage and seek to develop new pharmacological agents for stroke treatment.