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Background: Sirtuin3 (Sirt3), NAD+-dependent deacetylase, regulates several key proteins in both mitochondria and nucleus. The majority of early studies have shown neuroprotective effects of Sirt3 activation in various models of CNS injury. Recently, it was found that genetic loss of Sirt3 is beneficial in ischemia/reperfusion (I/R) injury. Therefore, this study was designed to explore the effects of both genetic and pharmacological inhibition of Sirt3 in a middle cerebral artery occlusion (MCAo) model of stroke.Methods: Focal cerebral ischemia was induced by transient right MCAo for 60min followed by 0, 6, 24 and 72 hrs of reperfusion. A 72hrs post ischemic injury time point was chosen to measure infarction in various treatment groups. The Sirt3 inhibitor AGK7 (sc- 204281 Santa cruz,) that selectively inhibits Sirt3 over Sirt1 and Sirt2, was given as single intraperitoneal injection (0.15, 0.5 and 1.5mg/kg) 3hrs after stroke onset. Activity or expression of Sirt3, was examined with Sirt3 activity kit or western blot using anti-Sirt3, anti-acetylated lysine antibodies.Results: Sirt3 knockout mice showed a significant reduction in hemispheric infarct volume compared to WT littermate controls (37.72±5.21 vs 52.8±6.76, p<0.05). In the pharmacological study, a dose of 0.5 mg/kg i.p of inhibitor showed significant reduction in Sirt3 activity and but had no neuroprotective effect. Furthermore, a much higher dose (1.5mg/kg i.p) did not show any additional inhibitory effect on sirt3 activity but unexpectedly increased infarct volume (51.8±6.87 vs. 62.56± 7.89, P<0.05) and hemorrhagic transformation. Expression studies showed no overall change in Sirt3 expression in whole cell lysates at 0, 6, 24 or 72 hrs after reperfusion; however the expression pattern and activity varied with time in different subcellular compartments.Summary and Conclusions: Changes in subcellular translocation and activity of Sirt3 after I/R stress suggests it important role in target protein deacetylation in stroke pathophysiology. Surprisingly, genetic deletion but not pharmacological inhibition led to neuroprotection, indicating the need to carefully examine target protein by both genetic and pharmacological approaches.