Regulation of PINK1 by NR2B-containing NMDA receptors in ischemic neuronal injury

Dysfunction of PTEN-induced kinase-1 (PINK1) is implicated in neurodegeneration. We report here that oxygen-glucose deprivation (OGD), an in vitro insult mimicking ischemic neuron injury, resulted in a significant reduction of PINK1 protein expression in cultured cortical neurons. The decrease of PINK1 expression was blocked by the antagonists of NMDA receptors. We revealed that the overactivation of NR2B-containing NMDA receptors (NR2BRs) was responsible for the OGD-induced PINK1 reduction. The overactivated NR2BRs also inhibited the phosphorylation, but not the protein expression, of the cell survival-promoting kinase Akt after OGD insult, indicating that OGD-induced reduction of PINK1 protein is specific in the injury paradigm. We further showed that enhancing the protein expression of PINK1 antagonized OGD-induced reduction of Akt phosphorylation, suggesting that Akt may be a downstream target of PINK1 in ischemic neuron injury. Importantly, we provided evidence that both NR2BR antagonist and PINK1 over-expression protected against OGD-induced neuronal death. These results suggest that the overactivation of NR2BRs may contribute to ischemic neuron death through suppressing PINK1-dependent survival signaling. Thus, selectively antagonizing NR2BR signal pathway-induced neurotoxicity may be a potential neuroprotection strategy.     

Combination therapy with bone marrow stromal cells and FK506 enhanced amelioration of ischemic brain damage in rats

AimsWe previously reported that cysteinyl leukotriene receptor 2 (CysLT₂) mediates ischemic astrocyte injury, and leukotriene D₄-activated CysLT₂ receptor up-regulates the water channel aquaporin 4 (AQP4). Here we investigated the mechanism underlying CysLT₂ receptor-mediated ischemic astrocyte injury induced by 4-h oxygen-glucose deprivation and 24-h recovery (OGD/R).Main methodsPrimary cultures of rat astrocytes were treated by OGD/R to construct the cell injury model. AQP4 expression was inhibited by small interfering RNA (siRNA). The expressions of AQP4 and CysLTs receptors, and the MAPK signaling pathway were determined.Key findingsOGD/R induced astrocyte injury, and increased expression of the CysLT₂ (but not CysLT₁) receptor and AQP4. OGD/R-induced cell injury and AQP4 up-regulation were inhibited by a CysLT₂ receptor antagonist (Bay cysLT2) and a non-selective CysLT receptor antagonist (Bay u9773), but not by a CysLT₁ receptor antagonist (montelukast). Knockdown of AQP4 by siRNA attenuated OGD/R injury. Furthermore, OGD/R increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the cell injury and AQP4 up-regulation.SignificanceThe CysLT₂ receptor mediates AQP4 up-regulation in astrocytes, and up-regulated AQP4 leads to OGD/R-induced injury, which results from activation of the ERK1/2 and p38 MAPK pathways.     

Prevention of cell death by the zinc ion chelating agent TPEN in cultured PC12 cells exposed to Oxygen–Glucose Deprivation (OGD)

To elucidate the role of Zn²⁺-associated glutamate signaling pathway and voltage-dependent outward potassium ion currents in neuronal death induced by hypoxia–ischemia, PC12 cells were exposed to Oxygen–Glucose Deprivation (OGD) solution mimicking the hypoxic–ischemic condition in neuron, and the effect of N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a specific Zn²⁺ chelating agent on OGD-induced neuronal death was assessed in the present study. The cell survival rate, apoptosis status, potassium channel currents, intracellular free glutamate concentration and GluR2 expression in PC12 cells exposed to OGD in the absence or presence of TPEN for different time were investigated. The results showed that OGD exposure increased apoptosis, reduced the cell viability (P < 0.01 at 3 h, 6 h and 24 h, respectively compared to control), changed the voltage-dependent outward potassium ion current (increase at 1 h, but decrease at 3 h) and decreased the concentration of intracellular glutamate (P < 0.05 at 3 h and 6 h, P < 0.01 at 24 h respectively compared to control) and GluR2 expression (P < 0.05 at 3 h, 6 h and 24 h, respectively compared to control) in PC12 cells. TPEN partially reversed the influence resulted from OGD. These results suggest that OGD-induced cell apoptosis and/or death is mediated by the alteration in glutamate signaling pathway and the voltage-dependent outward potassium ion currents, while TPEN effectively prevent cell apoptosis and/or death under hypoxic–ischemic condition.     

氨磷汀对氧糖剥夺引起的PC12细胞缺血再灌注损伤的保护作用研究

目的：研究氨磷汀对体外培养的神经元样细胞的缺血再灌注损伤的保护作用,为其最终用于临床脑缺血的治疗打下基础。方法：体外培养的PC12细胞氧糖剥夺4h后复氧复糖,给予不同浓度的氨磷汀处理,20h后镜下观察细胞形态学变化,用MTT和LDH检测细胞活力和损伤情况,免疫荧光染色观察凋亡细胞,流式细胞仪计数凋亡细胞的比例。结果：高浓度氨磷汀对正常PC12细胞活力有抑制作用（P〈0.05）,而低浓度则无。氨磷汀可以提高缺血再灌注损伤PC12细胞活力（P〈0.05）,减少LDH释放（P〈0.05）,保护细胞正常形态,抑制细胞凋亡（P〈0.05）。结论：氨磷汀对氧糖剥夺引起的神经元样细胞的缺血再灌注损伤具有保护作用。     

Role of Bcl-2 family of proteins in mediating apoptotic death of PC12 cells exposed to oxygen and glucose deprivation

Apoptotic cell death has been observed in many in vivo and in vitro models of ischemia. However, the molecular pathways involved in ischemia-induced apoptosis remain unclear. We have examined the role of Bcl-2 family of proteins in mediating apoptosis of PC12 cells exposed to the conditions of oxygen and glucose deprivation (OGD) or OGD followed by restoration of oxygen and glucose (OGD-restoration, OGD-R). OGD decreased mitochondrial membrane potential and induced necrosis of PC12 cells, which were both prevented by the overexpression of Bcl-2 proteins. OGD-R caused apoptotic cell death, induced cytochrome C release from mitochondria and caspase-3 activation, decreased mitochondrial membrane potential, and increased levels of pro-apoptotic Bax translocated to the mitochondrial membrane, all of which were reversed by overexpression of Bcl-2. These results demonstrate that the cell death induced by OGD and OGD-R in PC12 cells is potentially mediated through the regulation of mitochondrial membrane potential by the Bcl-2 family of proteins. It also reveals the importance of developing therapeutic strategies for maintaining the mitochondrial membrane potential as a possible way of reducing necrotic and apoptotic cell death that occurs following an ischemic insult.     

The effects of NONRATT021972 lncRNA siRNA on PC12 neuronal injury mediated by P2X<Subscript>7</Subscript> receptor after exposure to oxygen-glucose deprivation

Adenosine triphosphate (ATP) participates in signal transmission by acting on P2X receptors, and the P2X₇ receptor is involved in the pathophysiological changes of ischemic injury. The PC12 cell line is a popular model system to study sympathetic neuronal function. Long noncoding RNAs (lncRNAs) are highly expressed in the nervous system and serve as regulatory RNAs. In this study, the effects of NONRATT021972 lncRNA siRNA on P2X₇-mediated PC12 neuronal injury after exposure to oxygen-glucose deprivation (OGD) were investigated. Our results showed that the viability of PC12 cells cultured with OGD or the P2X₇ agonist BzATP was significantly decreased. Treatment with NONRATT021972 siRNA reversed the decreased viability of PC12 cells under OGD conditions. The upregulated P2X₇ mRNA and protein levels in PC12 cells under OGD conditions or BzATP treatment were significantly decreased when pretreated with NONRATT021972 siRNA. Moreover, NONRATT021972 siRNA treatment effectively suppressed the increase in [Ca²⁺]_i induced by OGD or P2X₇ agonists (ATP or BzATP) in PC12 cells. Therefore, treatment with NONRATT021972 siRNA may decrease sympathetic neuronal injury induced by ischemia.     

Beneficial Effect of Astragalosides on Stroke Condition Using PC12 Cells under Oxygen Glucose Deprivation and Reperfusion

Astragalosides (AST) are reported to be neuroprotective in focal cerebral ischemic models in vivo. In this study, the direct effect of AST against oxygen and glucose deprivation (OGD) including neuronal injury and the underlying mechanisms in vitro were investigated. 5 h OGD followed by 24 h of reperfusion [adding back oxygen and glucose (OGD-R)] was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. AST (1, 100, and 200 µg/mL) were added to the culture after 5 h of the OGD ischemic insult and was present during the reoxygenation phases. A key finding was that OGD-R decreased cell viability, increased lactate dehydrogenase, increased reactive oxygen species, apoptosis, autophagy, functional impairment of mitochondria, and endoplasmic reticulum stress in PC12 cells, all of which AST treatment significantly reduced. In addition, AST attenuated OGD-R-induced cell loss through P38 MAPK activation a neuroprotective effect blunted by SB203580, a specific inhibitor of P38 MAPK. Our data suggest that both apoptosis and autophagy are important characteristics of OGD-R-induced PC12 death and that treating PC12 cells with AST blocked OGD-R-induced apoptosis and autophagy by suppressing intracellular oxidative stress, functional impairment of mitochondria, and endoplasmic reticulum stress. Our data provide identification of AST that can concomitantly inhibit multiple cells death pathways following OGD injuries in neural cells.     

Mesenchymal stem cells protect neurons against hypoxic-ischemic injury via inhibiting parthanatos,necroptosis, and apoptosis,but not autophagy

Cellular therapy with mesenchymal stem cells (MSCs) protects cortical neurons against hypoxic-ischemic injury of stroke. Although sorts of efforts have been made to confirm the neuroprotective effect of MSCs on neurons against hypoxic-ischemic injury, the mechanism is until now far away from clear. Here in this study, oxygen-glucose deprivation (OGD)-injured neuron model was applied to mimic the neuronal hypoxic-ischemic injury in vitro. Co-culturing with MSCs in a transwell co-culture system, the OGD injured neurons were rescued by 75.0 %. Further data demonstrated that co-culturing with MSCs protected the cortical neurons from the OGD-induced parthanatos by alleviating apoptosis-inducing factor (AIF) nuclear translocation; attenuated the neuronal necroptosis by down-regulating the expression of the two essential kinases in necroptosis, receptor interacting protein kinase1 (RIP1) and 3 (RIP3); rescued the neurons from apoptosis by deactivating caspase-3; whilst performed no significant influence on OGD-induced neuronal autophagy, according to its failed regulation on Beclin1. In conclusion, MSCs potentially protect the cortical neurons from OGD-injury in vitro, through rescuing neurons from the cell death of parthanatos, necroptosis, and apoptosis, but not autophagy, which could provide some evidence to the mechanism explanation on stem cell treatment for ischemic stroke.     

Tyrosine Kinase Activity Is Essential for Interleukin-1β-Stimulated Production of Interleukin-6 in U373 Human Astrocytoma Cells

Abstract: Previous studies have shown that PC12 cells depend on growth factors for their survival. When deprived of growth factors, the cells undergo a dying process termed "apoptosis" (programed cell death). We show here that muscarinic agonists inhibited the apoptotic death of growth factor-deprived PC12M1 cells (PC12 cells stably expressing cloned m1 muscarinic acetylcholine receptors). This protective effect of the muscarinic agonists was observed in both proliferating and neuronal PC12M1 cells, was blocked by the muscarinic antagonist atropine, and was not observed in PC12 cells lacking m1 receptors. Muscarinic receptors therefore mediate inhibition of apoptosis in these cells. In addition to its effect on survival, the muscarinic agonist oxotremorine induced inhibition of DNA synthesis as well as growth arrest of exponentially growing PC12M1 cells at the S and G₂/M phases of the cell cycle. Muscarinic receptors in these cells may therefore mediate inhibition of cell cycle progression.