Ischemia-induced Interaction Of NNOS With PSD95 In Neurons Negatively Controls The Fate Of Neural Stem Cells

Ischemic stroke is a kind of central nervous system disease, which is the third leading cause of death. There have been substantial advances in understanding molecular mechanisms of ischemia induced damage. It is recognized that glutamate induced excitatory toxicity is the major process underlying ischemic injury. Cerebral ischemia triggers glutamatergic storm. Receptors for the excitatory neurotransmitter glutamate such as the N-Methyl-D-aspartate receptor (NMDAR) are overactivated. NMDAR activation stimulates the interaction of neuronal nitric oxide synthase (nNOS) and postsynaptic density 95 (PSD95), and further activates nNOS. Massive nitric oxide (NO) is produced in this process, leading to neuronal damage. Our previous studies have demonstrated that disrupting nNOS-PSD95 interaction can ameliorate focal cerebral ischemic damage. Also, there are substantial evidence suggest that nNOS negatively regulate neurogenesis. According to these facts, we reasonably speculate that uncoupling of nNOS-PSD95 interaction after stroke may affect neurogenesis by reducing NO production from nNOS.Here, we investigated the following questions:(1) Whether reducing nNOS-PSD95 interaction after ischemia will affect endogenous neurogenesis. (2) Will nNOS-PSD95 uncoupling affect exogenous neural stem cells (NSCs) transplanted to the ischemic brain? (3) What is the role of nNOS-PSD95 in fate determination of NSCs in vitro studies.Chapter 1. Down-regulation of ischemia-induced nNOS-PSD95 association promotes neurogenesisTo determine whether disrupting the ischemia-induced nNOS-PSD95 association affects the fate of cells born before ischemia, we treated rats with BrdU to label proliferating cells at d 5 and d 4 before MCAO. ZL006 was administrated for 7 d beginning immediately after reperfusion. These rats were sacrificed at d14 after the last BrdU injection to estimate the number of BrdU+ and BrdU+/DCX+(a marker for immature neurons) cells. Our results showed that treatment with ZL006 significantly increased BrdU+ and BrdU+/DCX+ cells in the hippocampus and striatum. ZL006 also increased BrdU+ cells in the granular cell layer (GCL) of dentate gyrus (DG). In addition, we found that ZL006 significantly enhanced migration distance of newborn cells as well as increased neurite length of DCX+cells in the ischemic striatum and ischemic DG.In the normal brain, ZL006 treatment displayed a tendency to enhance neurogenesis in the DG, although without significant difference. Newborn cells were scarce in both vehicle- and ZL006-treated animals in the normal striatum, and there was no difference between groups.To further strengthen these findings, we generated a lentiviral vector (LV-nNOS-N1-133-GFP) that selectively expresses nNOS-N1-133, a region crucial for nNOS-PSD95 interaction. We delivered LV-nNOS-N1-133-GFP into the striatum of rats by microinjection. Also, we treated rats with BrdU to label proliferating cells. Rats were subjected to MCAO and sacrificed at d 14 after BrdU injection to estimate the number of BrdU+ and BrdU+/DCX+ cells. LV-nNOS-N1-133-GFP infection significantly increased the number of BrdU+ and BrdU+/DCX+ cells in the striatum but not in the hippocampus, suggesting a selective enhancement of neurogenesis in the LV-nNOS-N1-133-GFP-infected area. Moreover, this treatment significantly increased migration distance and neurite length of DCX+ cells in the ischemic striatum.Together, these data suggest that disrupting nNOS-PSD95 interaction after stroke benefits the survival and neuronal differentiation of cells which born before stroke, promotes migration of newborn cells into injured area and neurite growth of newborn neurons in the ischemic brain.To examine the effect of disassociating nNOS-PSD95 on ischemia-stimulated newborn cells, we treated rats with BrdU at d 6 and d 7 after MCAO to label proliferating cells. In this part of investigation, we showed that ZL006 significantly increased the number of BrdU+ cells in the ipsilateral hippocampal DG 4 weeks but not 2 h after BrdU labeling, and moreover, ZL006 increased survival rate of newborn cells in both contralateral and ipsilateral hippocampus, suggesting that disrupting nNOS-PSD95 coupling also benefits the survival of the cells which born after stroke.Chapter 2. Ischemia-Induced nNOS-PSD95 association affects the fate of transplanted NSCsTo determine whether nNOS-PSD95 interaction affects the fate of transplanted NSCs in ischemic brain, we transplanted GFP+ NSCs into the ipsilateral hemisphere of ischemic rats. Rats were treated with ZL006 after ischemia. Four weeks after transplantation, we estimate the number of GFP+ and GFP+/NeuN+ cells in the brain. Treatment with ZL006 significantly increased GFP+ cells in the striatum, suggesting an increased migration. More importantly, although no GFP+/NeuN+ cell was existed in vehicle-treated rats, several GFP+/NeuN+ cells were found in the injured striatum in ZL006-treated animals, and some of them had multiple or long projection and expressed GAD67 (marker of GABAergic neurons), indicating that disrupting nNOS-PSD95 interaction can improve the fate of transplanted NSCs in ischemic brain.Chapter 3. Interaction of nNOS-PSD95 in neurons negatively controls the fate of NSCsTo determine whether nNOS-PSD95 interaction affects the fate of NSCs through intercellular communication between neurons and NSCs, we established a co-culture of NSCs with GFP+ neurons. We demonstrated that interaction of nNOS with PSD95 in neurons is critical for hypoxia-induced NO production. Thus, it is reasonable that NO, as a downstream molecule of nNOS-PSD95 interaction, mediates intercellular communication between neurons and NSCs. The co-cultures were suffered oxygen-glucose deprivation (OGD), a cell model to mimic ischemia in vitro. ZL006 or Tat-nNOS-N1-133 (a fusion peptide of nNOS-N1-133 and Tat protein) was introduced at the beginning of OGD. It was displayed that OGD exposure caused a significant decrease in β-Ⅲ-tubulin+ newborn cells, but not in GFAP+ or GalC+ newborn cells, suggesting decreased neuronal differentiation. Treatment with ZL006 or Tat-nNOS-N1-133 reversed the OGD-induced reduction in neuronal differentiation. Moreover, the number of newborn neurons with multi-neurites were decreased in OGD-treated co-cultures, and ZL006 or Tat-nNOS-N1-133 ameliorated the neurite growth. We also measured neurite length of the newborn neurons in each groups, and results revealed that ZL006 or Tat-nNOS-N1-133 improved the OGD-induced neurite growth impairment. These data suggest that nNOS-PSD95 interaction in neurons contributes to the OGD-induced changes in NSCs fate.To heighten our findings, we co-cultured NSCs with neurons from nNOS gene knockout mice. The co-cultures were subjected to OGD and treated with ZL006. We found that OGD with or without ZL006 did not change the number of β-Ⅲ-tubulin+newborn cells and the number of newborn neurons with multi-neurites, suggesting that nNOS-PSD95 interaction in neurons is crucial for the effects of OGD and ZL006 on NSCs fate.It is possible that blockers of nNOS-PSD95 may change NSCs fate by directly targeting on NSCs in the co-cultures. To exclude this possibility, we subjected only cultured NSCs to OGD and treated them with ZL006 or Tat-nNOS-N1-133. OGD significantly increased neuronal differentiation, but reduced neurite length of newborn neurons. Neither ZL006 nor Tat-nNOS-N1-133 affected the OGD-induced alterations. Thus, NSCs are not the direct target of nNOS-PSD95 blockers.Next, we investigated the roles of nNOS-PSD95 uncoupling on original neurons (GFP+) in the OGD-treated co-cultures. OGD exposure not only caused neurons’ death but also decreased process length and dendrite spine density of original neurons significantly. Treatment with ZL006 ameliorated the destructive effects of OGD on process and dendrite spine formation. Similarly, LV-nNOS-N1-133-GFP abolished ischemia-induced spine density decrease in vivo. Thus, neurons are direct target of nNOS-PSD95 blockers.Conclusion:(1) Reducing ischemia-induced nNOS-PSD95 interaction promotes endogenous neurogenesis. (2) Disrupting nNOS-PSD95 interaction can improve the fate of transplanted NSCs in ischemic brain. (3) Uncoupling of nNOS-PSD95 interaction in neurons benefits both neuronal differentiation of NSCs and dendritic growth of newborn neurons.