Role of the hydrogen sulfide-releasing donor ADT-OH in the regulation of mammal neural precursor cells.

Neural precursor cells (NPCs) generate new neurons to supplement neuronal loss as well as to repair damaged neural circuits. Therefore, NPCs have potential applications in a variety of neurological diseases, such as spinal cord injury, traumatic brain injury, and glaucoma. Specifically, improving NPCs proliferation and manipulating their differentiated cell types can be a beneficial therapy for a variety of these diseases. ADT-OH is a slow-releasing organic H2 S donor that produces a slow and continuous release of H2 S to maintain normal brain functions. In this study, we aimed to explore the effect of ADT-OH on NPCs. Our results demonstrated that ADT-OH promotes self-renewal and antiapoptosis ability of cultured NPCs. Additionally, it facilitates more NPCs to differentiate into neurons and oligodendrocytes, while inhibiting their differentiation into astrocytes. Furthermore, it enhances axonal growth. Moreover, we discovered that the mRNA and protein expression of ß-catenin, TCF7L2, c-Myc, Ngn1, and Ngn2, which are key genes that regulate NPCs self-renewal and differentiation, were increased in the presence of ADT-OH. Altogether, these results indicate that ADT-OH may be a promising drug to regulate the neurogenesis of NPCs, and needs to be studied in the future for clinical application potential.

TRAF2 protects against cerebral ischemia-induced brain injury by suppressing necroptosis.

Necroptosis contributes to ischemia-induced brain injury. Tumor necrosis factor (TNF) receptor associated factor 2 (TRAF2) has been reported to suppress necroptotic cell death under several pathological conditions. In this study, we investigated the role of TRAF2 in experimental stroke using a mouse middle cerebral artery occlusion (MCAO) model and in vitro cellular models. TRAF2 expression in the ischemic brain was assessed with western blot and real-time RT-PCR. Gene knockdown of TRAF2 by lentivirus was utilized to investigate the role of TRAF2 in stroke outcomes. The expression of TRAF2 was significantly induced in the ischemic brain at 24 h after reperfusion, and neurons and microglia were two of the cellular sources of TRAF2 induction. Striatal knockdown of TRAF2 increased infarction size, cell death, microglial activation and the expression of pro-inflammatory markers at 24 h after reperfusion. TRAF2 expression and necroptosis were induced in mouse primary microglia treated with conditioned medium collected from neurons subject to oxygen and glucose deprivation (OGD) and in TNFα-treated mouse hippocampal neuronal HT-22 cells in the presence of the pan-caspase inhibitor Z-VAD. In addition, TRAF2 knockdown exacerbated microglial cell death and neuronal cell death under these conditions. Moreover, pre-treatment with a specific necroptosis inhibitor necrostatin-1 (nec-1) suppressed the cell death exacerbated by TRAF2 knockdown in the brain following MCAO, indicating that TRAF2 impacted ischemic brain damage through necroptosis mechanism. Taken together, our results demonstrate that TRAF2 is a novel regulator of cerebral ischemic injury.

Sphingosine kinase 1 mediates neuroinflammation following cerebral ischemia.

Sphingosine kinases (Sphks) are the rate-limiting kinases in the generation of sphingosine-1-phosphate, which is a well-established intracellular pro-survival lipid mediator. Sphk2 has been reported to be protective following experimental stroke. We investigated the role of Sphk1 in cerebral ischemia using a mouse middle cerebral artery occlusion (MCAO) model and an in vitro glucose-oxygen deprivation (OGD) model. Sphk expression and activity were assessed in the ischemic brain with quantitative PCR (qPCR), Western blot, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Pharmacological and gene knockdown approaches were utilized to investigate the effects of Sphk1 on stroke outcomes. The expression of Sphk1 but not that of Sphk2 was rapidly induced in the cortical penumbra over 96h after MCAO, and the microglia were one of the major cellular sources of Sphk1 induction. Consistently, Sphk activity was enhanced in the cortical penumbra. In contrast to the protective role of Sphk2, pharmacological inhibition and cortical knockdown of Sphk1 reduced infarction at 24 and 96h after reperfusion. Additionally, the Sphk1 inhibitor improved the neurological deficits at 96h after reperfusion. Mechanistically, Sphk1 inhibition and knockdown significantly attenuated MCAO-induced expression of inflammatory mediators in the cortical penumbra. Moreover, using a conditioned medium transfer approach, we demonstrated that OGD-treated neurons induced the expression of Sphk1 and pro-inflammatory mediators in primary microglia, and the microglial induction of pro-inflammatory mediators by ischemic neurons was blunted by Sphk1 inhibition. Taken together, our results indicate that Sphk1 plays an essential role in mediating post-stroke neuroinflammation.

Improvement of functional recovery by chronic metformin treatment is associated with enhanced alternative activation of microglia/macrophages and increased angiogenesis and neurogenesis following experimental stroke.

Acute AMPK activation exacerbates ischemic brain damage experimentally. Paradoxically, the clinical use of an AMPK activator metformin reduces the incidence of stroke. We investigated whether post-stroke chronic metformin treatment promotes functional recovery and tissue repair via an M2-polarization mechanism following experimental stroke. Mice were randomly divided to receive metformin or vehicle daily beginning at 24h after middle cerebral artery occlusion (MCAO). Neurological deficits were monitored for 30days following MCAO. To characterize the polarization of the microglia and infiltrating macrophages, the expression of the M1 and M2 signature genes was analyzed with qPCR, ELISA and immunohistochemistry. Post-MCAO angiogenesis and neurogenesis were examined immunohistochemically. An in vitro angiogenesis model was employed to examine whether metformin promoted angiogenesis in a M2 polarization-dependent manner. Post-stroke chronic metformin treatment had no impact on acute infarction but enhanced cerebral AMPK activation, promoted functional recovery and skewed the microglia/macrophages toward an M2 phenotype following MCAO. Metformin also significantly increased angiogenesis and neurogenesis in the ischemic brain. Consistently, metformin-induced M2 polarization of BV2 microglial cells depended on AMPK activation in vitro. Furthermore, treatment of brain endothelial cells with conditioned media collected from metformin-polarized BV2 cells promoted angiogenesis in vitro. In conclusion, post-stroke chronic metformin treatment improved functional recovery following MCAO via AMPK-dependent M2 polarization. Modulation of microglia/macrophage polarization represents a novel therapeutic strategy for stroke.