The Role of CaSR and GABA-B-R in Neuronal Responses to Ischemic Brain Injury

描述(由申请人提供)： 在美国和我们的退伍军人事务部，缺血性脑损伤会导致心脏骤停和栓塞性中风患者的死亡和长期残疾。缺血诱导的神经元过度活动是神经退行性变发展的关键步骤，因为它会导致细胞死亡、神经元回路不可逆转的丢失和重组，最终导致神经元缺陷。缺血后即刻，细胞膜去极化、谷氨酸分泌过多、离子型谷氨酸受体过度活动和GABA信号丧失被认为是导致神经元过度活动的原因。GABA-B-R(R1和R2)是G蛋白偶联受体(GPCR)超家族中C家族的成员，该超家族还包括细胞外钙敏感受体(CaSR)。GABA-B-Rs通常以由GABA-B-R1和GABA-B-R2亚基组成的异源二聚体的形式发挥作用，这是稳定的细胞表面表达和受体复合体信号传递所必需的，从而产生抑制性神经元输入和防止神经元过度活动。GABA-B-Rs和CaSR在大脑的许多区域共表达，包括海马区。与GABA-B-R1/R2异源二聚体不同，CaSR可以同源二聚体的形式发挥作用，发挥兴奋性信号反应的作用--激活钙离子通道和非选择性阳离子通道，刺激磷脂酶C，增加[钙]i，增加神经元和其他细胞系统中细胞的兴奋性。在HEK-293细胞中，GABA-B-R1可与CaSR异二聚化，抑制CaSR蛋白的表达和细胞表面的信号转导。相反，敲除海马神经元中的GABA-B-R1基因会上调CaSR的表达。我们进一步观察到CaSR在缺血海马神经元中的过度表达，表现为GABA-B-R1表达降低，表明CaSR和GABA-B-R1表达之间存在相互抵消的作用。CaSR的表达和活性增加可能通过其自身的兴奋作用和/或其干扰GABA-B-R1/R2异源二聚体的形成而导致缺血神经元的过度活动，从而降低GABA的反应。为了支持后一种观点，我们发现在海马神经元CaSR基因缺失的HippCaSR-KO小鼠中，缺血不再抑制GABA-B-R1的表达或导致细胞死亡。我们假设，缺血诱导的CaSR过度表达通过刺激CaSR介导的信号反应和通过化学计量竞争与GABA-B-R2结合来抑制GABA-B-R1的表达而导致神经元过度活动和细胞死亡，而阻断CaSR的表达或活性与增强GABA-B-R信号是抗缺血损伤的最佳神经保护所必需的。我们的建议将(1) 确定删除CaSR基因或用特定拮抗剂(或解钙剂)阻断CaSR活性是否可保护缺血所致的神经元损伤，并钝化缺血对GABA-B-R1/R2表达、转运和二聚化的抑制作用；(2)确定持续的GABA-B-R1表达是否为抗脑缺血神经保护所必需，以及解钙剂和GABA-B-R1激动剂联合治疗是否进一步加强了抗脑缺血损伤的神经保护。这项研究的成功完成将建立一条产生缺血诱导神经元损伤的新途径，并将开发一种治疗缺血性脑损伤疾病的新方法。

DESCRIPTION (provided by applicant): Ischemic brain injury causes death and long-term disability in patients who suffer cardiac arrest and embolism stroke in the United States and in our VA community. Ischemia-induced neuronal hyperactivity in the affected neurons is a key step in the development of neurodegeneration as it leads to cell death, irreversible loss and reorganization of neuronal circuits, and eventually neuronal deficiency. Immediately after Ischemia, cell membrane depolarization, excess glutamate secretion, overactivity of ionotropic glutamate receptor, and loss of GABA signaling are thought to cause neuronal hyperactivity. GABA-B-Rs (R1 and R2) are members of the family C of the G-protein coupled receptor (GPCR) superfamily, which also includes the extracellular Ca2+- sensing receptor (CaSR). GABA-B-Rs generally function in the form of heterodimer comprised of GABA-B-R1 and GABA-B-R2 subunit that is required for stable cell-surface expression and signaling of the receptor complex to produce inhibitory neuronal input and prevent neuronal overactivity. The GABA-B-Rs and the CaSR are co-expressed in many regions of the brain, including hippocampus. Unlike the GABA-B-R1/R2 heterodimer, the CaSR can function in the form of homodimer to exert excitatory signaling responses -- activation of Ca2+ and non-selective cation channels, stimulation of phospholipase C, increases in [Ca2+]i, and increasing cell excitability in neurons and other cell systems. GABA-B-R1 can heterodimerize with CaSR and suppress the total and cell-surface expression of CaSR protein and its signaling responses in transfected HEK- 293 cells. Contrarily, knocking out GABA-B-R1 gene in hippocampal neurons up-regulates CaSR expression. We further observed CaSR overexpression in ischemic hippocampal neurons, which showed reduced GABA- B-R1 expression, suggesting a counteracting interaction between the CaSR and GABA-B-R1 expression. The increased expression and activity of CaSR could contribute to the hyperactivity of the ischemic neurons via its own excitatory actions and/or its ability to interfere with the formation of GABA-B-R1/R2 heterodimers, therefore reducing GABA responses. In supporting the latter notion, we found that in the HippCaSR-KO mice, which have their CaSR genes deleted in hippocampal neurons, ischemia no longer inhibited the GABA-B-R1 expression or caused cell death. We hypothesize that ischemia-induced CaSR overexpression causes neuronal hyperactivity and cell death by stimulating CaSR-mediated signaling responses and by inhibiting GABA-B-R1 expression via stoichiometric competition for binding to GABA-B-R2, and that blocking the expression or activity of CaSR together with enhancement of GABA-B-R signaling are required for optimal neuroprotection against ischemic injuries. Our proposal will (1) determine whether deleting CaSR gene or blocking CaSR activity by specific antagonists (or calcilytics) protects against the ischemia-induced neuronal injury and blunts the inhibitory effect of ischemia on the expression, trafficking, and dimerization of GABA-B-R1/R2 and (2) determine whether sustaining GABA-B-R1 expression is required for neuroprotection against cerebral ischemia and whether a combined therapy with calcilytics and GABA-B-R1 agonists further enhances neuroprotection against ischemia-induced brain injury. The successful completion of the study will establish a novel pathway that produces ischemia-induced neuronal injury and will develop a new therapy for treating diseases due to ischemic brain injury.