Abstract 3144: Neuroglobin Inhibits Oxygen-Glucose Deprivation-induced Mitochondrial Permeability Transition Pore Opening in Primary Cultured Mouse Cortical Neurons

Background: Accumulating evidences from our laboratory and others have demonstrated that neuroglobin (Ngb) is protective against hypoxic/ischemic brain injury, thus targeting Ngb may be a novel approach for endogenous neuroprotection, but the molecular mechanisms remain undefined. Mitochondrial permeability transition pore (mPTP) opening is a key mitochondrial response to hypoxia/ischemia, followed by release of mitochondrial factors such as Cyt c and activation of Caspase-dependent apoptosis pathways. In this study, we test our hypothesis that Ngb inhibits OGD-induced mPTP opening in primary neurons. Method: Primary mouse cortical neurons were transduced with AAV-Ngb to achieve Ngb over-expression and Ngb-shRNA to achieve Ngb knockdown. At day 9 of culture, primary neurons were subjected to 4 hr OGD followed by reoxygenation. Neurotoxicity was measured after 20 hr reoxygenation by LDH release assay. mPTP opening was measured at 4 hr after reoxygenation by testing mitochondria swelling, NAD release and Cyt c release. NAD release was also measured using isolated mitochondria treated with OGD after incubation with recombinant Ngb protein. Results: (1) Four hour OGD followed by 20 hr reoxygenation caused 40±4.5% neuron death; Ngb overexpression significantly reduced the death rate to 18±5.2% (n=5, p<0.05). (2) Ngb-overexpression significantly reduced OGD-induced mitochondria swelling, and rescued OGD-induced mitochondrial NAD release (mitochondrial NAD content 4±0.56 nmol/mg protein at normoxia, 2.1±0.35 nmol/mg prot. after OGD, 3.4±0.46 nmol/mg prot. in Ngb-overexpressing group after OGD) (n=4, p<0.05). (3) Ngb-overexpression significantly reduced OGD-induced Cyt c release from mitochondria to 0.43±0.08 fold (n=3, p<0.05). (4) Incubation with recombinant Ngb significantly reduced OGD-induced mitochondria NAD release in isolated mitochondria (mitochondrial NAD content 3.5±0.46 nmol/mg protein at normoxia, 1.8±0.32 nmol/mg prot. after OGD, 3.0±0.45 nmol/mg prot. in Ngb-overexpressing group after OGD) (n=4, p<0.05). (5) Ngb knockdown significantly increased OGD-induced neuron death from 40±4.5% to 56.4± 7.2%, and worsened OGD-induced mitochondria NAD release, but these changes were rescued by CsA, an mPTP inhibitor (n=3, p<0.05). (6) Co-IP detected protein interaction between Ngb and VDAC, an mPTP component, and the interaction was increased after OGD.