Abstract WP268: Leukemia Inhibitory Factor Upregulates Superoxide Dismutase 3 in Neurons via Activation of Myeloid Zinc Finger-1

Abstract

Objective: To determine whether leukemia inhibitory factor (LIF) upregulates superoxide dismutase 3 (SOD3), a neuroprotective antioxidant enzyme, through the transcription factor myeloid zinc finger-1 (MZF-1). Hypothesis: MZF-1 facilitates LIF-mediated neuroprotection by increasing transcription of the SOD3 gene in neurons. Methods: After middle cerebral artery occlusion or sham surgery, male Sprague-Dawley rats were injected with PBS or LIF (125 μg/kg) (n=4 per group). Rats were euthanized 72 h after injury and western blotting was used to measure MZF-1 protein expression in brain tissue. For in vitro studies, rat neurons were transfected with scrambled or MZF-1 siRNA (n=3 per group). Neurons were treated with PBS or 200 ng/mL LIF prior to 24 h in vitro ischemia induced by oxygen glucose deprivation. Lactate dehydrogenase (LDH) release was measured to assess neuronal death. MZF-1 levels were quantified in cultured neurons with immunocytochemistry. SOD3 and MZF-1 mRNA levels were measured with real-time PCR. Results: LIF (0.938 OD units ± 0.170), but not PBS (0.562 OD units ± 0.223), significantly increased MZF-1 protein expression in ipsilateral tissue 72 h after stroke compared to sham surgery (0.411 OD units ± 0.039, p<0.05). LIF treatment prior to 24 h in vitro ischemia significantly increased the percentage of MZF-1-positive neurons (52.17 % ± 0.93) compared to PBS 44.84 % ± 1.11, p<0.01). PCR results confirmed the increase in MZF-1 mRNA (1.89 fold change ± 0.33) in LIF-treated neurons compared to PBS-treated neurons (1.00 fold change ± 0.23, p<0.05). Moreover, LIF increased SOD3 mRNA after 24 h ischemia (1.35 fold change ± 0.03) compared to PBS (1.00 fold change ± 0.13, p<0.05). LIF decreased LDH release (363,967 ± 68,557 neuronal units) compared to PBS (559,856 ± 60,555 neuronal units, p<0.05) among neurons transfected with scrambled siRNA. However, MZF-1 siRNA attenuated the neuroprotective effect of LIF (690,633 neuronal units ± 19,167; p<0.05). Conclusions: MZF-1 plays a fundamental role in a novel neuroprotective pathway by enhancing SOD3 expression and provides insight regarding protection by antioxidant enzymes during stroke.