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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。创伤性CNS损伤后的细胞死亡导致创伤幸存者的永久性神经功能缺损。在脊髓损伤（SCI）中，轴突损伤可导致失神经诱导的皮层神经元凋亡。因此，预防SCI诱导的皮层神经元死亡可能是成功修复脊髓损伤所必需的。糖原合成酶激酶3b（Glycogen Synthase Kinase 3b，GSK 3b）是一种参与营养支持戒断诱导的神经元凋亡的蛋白激酶。这暗示GSK 3b是神经保护干预的潜在靶点。在健康神经元中，GSK 3b的高基础活性受到通过Akt介导的丝氨酸9残基磷酸化（pSer 9）的抑制的限制。此外，最近发表的研究与PI的贡献表明，基础或BDNF刺激的ERK 1/2信号转导活性抑制促凋亡作用的GSK 3b的丝氨酸9磷酸化的情况下。这表明一种新的促生存机制，控制神经元GSK 3b。我们的初步数据进一步支持了这一点，表明谷氨酸NMDA受体的适度刺激以pSer 9非依赖性和ERK 1/2依赖性的方式抑制GSK 3b介导的凋亡。此外，我们发现GSK 3b在体外被ERK 1/2直接磷酸化，其位点不同于Ser 9。因此，除Ser 9磷酸化外，GSK 3b可能受其他机制调节。此外，对GSK介导的细胞死亡的完全控制可能需要pSer 9依赖性途径和独立途径。因此，我们推测神经营养因子或谷氨酸激活的抗凋亡机制包括ERK 1/2介导的GSK 3b抑制。此外，我们提出，这种抑制作用是通过在不同于Ser 9的残基上直接磷酸化。我们计划通过以下具体目的的研究进一步解决这种可能性：（i）解剖ERK 1/2抑制GSK 3b的分子机制，（ii）确定ERK 1/2对GSK 3b的pSer 9非依赖性调节的功能后果，（iii）测试ERK 1/2介导的GSK 3b活性调节的一般性。我们建议使用原代培养的大鼠皮层神经元作为一个主要的系统来解决这些问题。我们还希望通过使用幼鼠的体内实验来验证在培养物中进行的关键观察。这项研究的目的是识别支持大脑皮层神经元存活的新信号机制。因此，它可能为SCI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.