| Abstract No.: | A-B1062 |
| Country: | Canada |
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| Title: | PEROXYNITRITE EFFECTS ON SYNAPSIN PROVIDE AN ENDOGENOUS NEUROPROTECTIVE EFFECT FOLLOWING STROKE |
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| Authors/Affiliations: | 1 Joan Forder*; 4 Lidong Liu; 1 Ibrahim Khambati; 3 Hong Sun; 4 Yu-Tian Wang; 2 Michael Tymianski;
1 University Health Network, Toronto, ON, Canada; 2 University Health Network and University of Toronto, ON, Canada; 3 University of Toronto, ON, Canada; 4 Vancouver Coastal Health Research Institute and University of British Columbia, BC, Canada
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| Content: | Both physiological synaptic activity, such as long term potentiation (LTP) and stroke involve glutamate release, leading to postsynaptic changes mediated, in part, by nitric oxide (NO) and its reaction products. Yet, only cells in strokes die. One toxic reaction product of NO is the oxidant peroxynitrite (PN), which nitrates tyrosineresidues in a large number of proteins.
OBJECTIVES: The objective of this study was to determine the differences in PN effects during stroke and LTP. We hypothesized that synapsin inactivation may reflect an endogenous neuroprotective mechanism aimed at limiting excitotoxic damage in stroke.
MATERIALS AND METHODS: To probe differences between stroke and nonlethal synaptic activity we used immunoprecipitation and mass spectrometry to profile protein nitration in brain slices subjected to LTP and in rats subjected to stroke. Once a target protein was selected, the specific effects of this protein were explored both in vitro and in vivo models of stroke. To separate the putative protective effects of PN from its damaging effects in-vivo, we administered low and high PN concentrations directly to rat strokes. Whereas high PN concentrations increased infarct size, the low concentrations resulted in significantly decreased stroke damage. In-vivo, protein nitration following stroke preceded brain infarct development, and paralleled a decrease in synapsin phosphorylation, indicating a loss of synapsin activity. To further investigate the involvement of synapsin in neuroprotection, three separate synapsin siRNA constructs were utilized to knockdown synapsin in vitro. To separate the putative protective effects of PN from its damaging effects in-vivo, we administered low: and high PN concentrations directly to rat strokes. To further investigate the involvement of synapsin in neuroprotection, three separate synapsin siRNA constructs were utilized to knockdown synapsin in vitro.
RESULTS: Of 26 nitrated proteins identified, 23 were common to both stroke and LTP whereas 3 proteins were nitrated exclusively in stroke. Of these, the effect of synapsin nitration in stroke has become the focus on this study. Consistent with this, cultured cortical neurons tolerated low PN concentrations (0.5-1 mM) without cell death and this treatment prior to oxygen-glucose deprivation (OGD) reduced neuronal glutamate release, and enhanced neuronal survival. , cultured cortical neurons tolerated low PN concentrations (0.5-1 mM) without cell death and this treatment prior to oxygen-glucose deprivation (OGD) reduced neuronal glutamate release, and enhanced neuronal survival. Whereas high PN concentrations increased infarct size, the low concentrations resulted in significantly decreased stroke damage. In-vivo, protein nitration following stroke preceded brain infarct development, and paralleled a decrease in synapsin phosphorylation, indicating a loss of synapsin activity. When these cells were subjected to OGD, the neuroprotective results were again seen.
CONCLUSIONS: Our results reveal the positive and negative differences between synaptic and excitotoxic mechanisms in vivo, and reveal for the first time that PN, previously considered a toxic stroke mediator, provokes an endogenous neuroprotective response that limits the damaging effects of a stroke. We provide convincing evidence for nitration of synapsin by PN may be involved in this neuroprotective response.
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