Protease Omi cleaving Hax-1 protein contributes to OGD/R-induced mitochondrial damage in neuroblastoma N2a cells and cerebral injury in MCAO mice

Abstract

Aim: In the penumbra after focal cerebral ischemia, an increase of protease Omi is linked to a decrease of Hs1-associated protein X-1 (Hax-1), a protein belonging to the Bcl-2 family. In this study we investigated the mechanisms underlying the regulation of Hax-1 by protease Omi in cerebral ischemia/reperfusion (I/R) injury. Methods: Mouse neuroblastoma N2a cells were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R); cell viability was assessed with MTT assay. Mice underwent 2-h middle cerebral artery occlusion (MCAO) and reperfusion, and the infarct volume was determined with TTC staining. The expression of Omi and Hax-1 was detected using immunoblot and immunofluorescence assays. The mitochondrial membrane potential was measured using TMRM staining. Results: In the brains of MCAO mice, the protein level of Omi was significantly increased, while the protein level of Hax-1 was decreased. Similar changes were observed in OGD/R-treated N2a cells, but the mRNA level of Hax-1 was not changed. Furthermore, in OGD/R-treated N2a cells, knockdown of Omi significantly increased Hax-1 protein level. Immunofluorescence assay showed that Omi and Hax-1 were co-localized in mitochondria of N2a cells. OGD/R caused marked mitochondrial damage and apoptosis in N2a cells, while inhibition of Omi protease activity with UCF-101 (10 μmol/L) or overexpression of Hax-1 could restore the mitochondrial membrane potential and attenuate cell apoptosis. Moreover, pretreatment of MCAO mice with UCF-101 (7.15 mg/kg, ip) could restore Hax-1 expression, inhibit caspase activation, and significantly reduce the infarct volume. Conclusion: Protease Omi impairs mitochondrial function by cleaving Hax-1, which induces apoptosis in OGD/R-treated N2a cells and causes I/R injury in MCAO mice.

Humanin analogue, S14G-humanin, has neuroprotective effects against oxygen glucose deprivation/reoxygenation by reactivating Jak2/Stat3 signaling through the PI3K/AKT pathway

“…Brain nerve cells are characterized by high sensitivity to deficiency of oxygen and glucose in cerebral ischemia (5). Recovering the blood supply, termed reperfusion, is a key step to rescue nerve cells from ischemia-induced death, however, it also raises serious problems linked to oxidative stress, inflammation disorder and ion overloading, thus the onset of reperfusion usually produces more severe damage to nerve cells (2). Apoptosis of nerve cells is a common event in stroke, according to a previous report (2).…”

“…Recovering the blood supply, termed reperfusion, is a key step to rescue nerve cells from ischemia-induced death, however, it also raises serious problems linked to oxidative stress, inflammation disorder and ion overloading, thus the onset of reperfusion usually produces more severe damage to nerve cells (2). Apoptosis of nerve cells is a common event in stroke, according to a previous report (2). Apoptotic alterations of neurons, including the condensation of nuclei and the activation of caspases, are observed in the ischemic core in the early stage of focal cerebral ischemia (19,20).…”

“…Multiple studies have evidenced that oxidative stress is closely associated with stroke damage (2,18). Reasons behind the occurrence of oxidative stress in stroke are basically elucidated.…”

“…Of these, 5 million succumb and a further 5 million are permanently disabled (1). However, no effective approach has been established to prevent or treat this brain damage (2). Stroke may be classified into ischemic stroke and hemorrhagic stroke.…”

“…Cerebral ischemia in strokes blocks delivery of oxygen and nutrients, and the brain has a considerably low tolerance to lack of these substances as it does not store oxygen and glycogen, but has a high oxygen consumption rate and rapid metabolism (4,5). Although rapid restoration of blood supply normalizes the demands for physiological processes, this reperfusion gives rise to other serious problems, such as oxidative stress, inflammatory responses, mitochondrial dysfunction and ion imbalances, which paradoxically antagonize the beneficial effect, resulting in more severe pathological state directly related to the intensity of stroke injury (2). Therefore, suppression of a series of disastrous effects induced by ischemia/reperfusion is essential for the treatment of strokes.…”