GSK3B AS A TARGET FOR PRO-NEURONAL SURVIVAL IN CNS NEURONS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。创伤性中枢神经系统损伤后的细胞死亡导致创伤幸存者的永久性神经功能障碍。在脊髓损伤(SCI)中，轴突损伤可能导致失神经诱导的大脑皮层神经元凋亡。因此，为了成功修复脊髓损伤，可能需要预防脊髓损伤所致的皮质神经元死亡。糖原合成酶3b(GSK3b)是一种蛋白激酶，参与营养支持撤退诱导的神经细胞凋亡。这表明GSK3b是神经保护干预的潜在靶点。在健康神经元中，GSK3b的高基础活性受到Akt介导的丝氨酸9残基(PSer9)磷酸化抑制的限制。此外，最近发表的具有PI贡献的研究表明，基础或BDNF刺激的ERK1/2信号活性在丝氨酸9磷酸化缺失的情况下抑制GSK3b的促凋亡作用。这表明了一种控制神经元GSK3b的新的促生存机制。我们的初步数据进一步支持了这一点，即适度刺激谷氨酸NMDA受体以pSer9依赖和ERK1/2依赖的方式抑制GSK3b介导的细胞凋亡。此外，我们发现GSK3b在体外被ERK1/2直接磷酸化，其位置不同于Ser9。因此，除了Ser9的磷酸化外，GSK3b可能还受到其他机制的调控。此外，对GSK介导的细胞死亡的完全控制可能需要pSer9依赖和独立的途径。因此，我们假设神经营养素或谷氨酸激活的抗凋亡机制包括ERK1/2介导的对GSK3b的抑制。此外，我们认为这种抑制是通过与Ser9不同的残基上的直接磷酸化来实现的。我们计划通过以下具体目标来进一步解决这一可能性：(I)剖析ERK1/2抑制GSK3b的分子机制，(Ii)鉴定ERK1/2对GSK3b的pSer9非依赖性调控的功能后果，以及(Iii)测试ERK1/2介导的GSK3b活性调节的普遍性。我们建议使用原代培养的大鼠皮质神经元作为主要系统来解决这些问题。我们还想通过用大鼠幼崽进行活体实验来验证在培养中所做的关键观察。这项研究的目的是识别支持大脑皮层神经元存活的新的信号机制。因此，它可能为脊髓损伤和神经退行性疾病的神经保护治疗提供潜在的靶点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cell death that follows traumatic CNS injuries contributes to permanent neurological deficits in trauma survivors. In spinal cord injury (SCI), axonal damage may result in denervation-induced neuronal apoptosis in cerebrocortical neurons. Therefore, prevention of SCI-induced neuronal death in the cortex may be required for the successful repair of the spinal cord damage. Glycogen Synthase Kinase 3b (GSK3b) is a protein kinase that is involved in neuronal apoptosis induced by trophic support withdrawal. This implicates GSK3b as a potential target for the neuroprotective interventions. In healthy neurons, the high basal activity of GSK3b is limited by inhibition through Akt-mediated phosphorylation at the serine 9 residue (pSer9). In addition, recently published studies with PI's contribution revealed that basal or BDNF-stimulated ERK1/2 signaling activity inhibited the pro-apoptotic action of GSK3b in the absence of serine 9 phosphorylation. This suggests a novel pro-survival mechanism that controls neuronal GSK3b. This is further supported by our preliminary data indicating that moderate stimulation of glutamate NMDA receptors inhibited GSK3b-mediated apoptosis in pSer9-independent and ERK1/2-dependent manner. Furthermore, we found that GSK3b is directly phosphorylated by ERK1/2 in vitro at a site that is distinct from Ser9. Therefore, in addition to Ser9 phosphorylation, GSK3b may be regulated by other mechanisms. In addition, the full control over GSK-mediated cell death may require both pSer9-dependent and independent pathways. Consequently, we hypothesize that the anti-apoptotic mechanisms activated by neurotrophins or glutamate include ERK1/2-mediated inhibition of GSK3b. Furthermore, we propose that this inhibition is by the direct phosphorylation at a residue different than Ser9. We plan to further address this possibility through the studies organized into following specific aims: (i) dissection of the molecular mechanism employed by ERK1/2 to inhibit GSK3b, (ii) identification of functional consequences of pSer9-independent regulation of GSK3b by ERK1/2, and (iii) testing the generality of ERK1/2-mediated regulation of GSK3b activity. We propose to use primary cultures of rat cortical neurons as a main system to address these issues. We would also like to validate the key observation done in cultures by in vivo experimentation using rat pups. This research is aimed on identification of novel signaling mechanisms that support neuronal survival in the cerebral cortex. Therefore, it may provide potential targets for neuroprotective therapies in SCI and neurodegenerative disorders.