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

CaSR 和 GABA-B-R 在缺血性脑损伤神经元反应中的作用

• 批准号: 8803349
• 负责人: Wenhan Chang
• 金额: --
• 依托单位: VETERANS AFFAIRS MED CTR SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8803349
• 关键词: Adverse effects; Affect; Agonist; Aminobutyric Acids; Animal Model; Animals; Baclofen; Binding; Brain; Brain Injuries; Brain Ischemia; Brain region; Calcium-Sensing Receptors; Cations; Cause of Death; Cell Death; Cell membrane; Cell physiology; Cell surface; Cells; Cerebral Ischemia; Cicatrix; Communities; Complementary DNA; Complex; Cytoplasm; Development; Dimerization; Disease; Dose; Down-Regulation; Effectiveness; Embolism; Family member; G-Protein-Coupled Receptors; GABA Receptor; GABA-B Receptor; Germ Cells; Gliosis; Glutamate Receptor; Glutamates; Health; Heart Arrest; Hippocampus (Brain); Hyperactive behavior; Injury; Ion Channel; Ischemia; Ischemic Brain Injury; Knock-out; Lead; Learning; Mediating; Mus; Nerve Degeneration; Neuronal Injury; Neurons; Pathway interactions; Patients; Phospholipase C; Play; Population; RRM1 gene; Reactive Oxygen Species; Receptor Gene; Receptor Signaling; Regimen; Role; Signal Transduction; Stroke; Synaptic Transmission; System; Testing; Tissues; United States; cell injury; design; disability; extracellular; gamma-Aminobutyric Acid; improved; in vivo; injured; mouse model; neuronal circuitry; neuroprotection; novel; overexpression; prevent; receptor; receptor expression; receptor-mediated signaling; response; response to injury; subunit 1 GABA type B receptor; therapy development; trafficking; voltage

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.

描述（由申请人提供）： 在美国和我们的VA社区，缺血性脑损伤导致心脏骤停和栓塞性中风患者死亡和长期残疾。受影响神经元中的缺血诱导的神经元过度活跃是神经变性发展的关键步骤，因为它导致细胞死亡、神经元回路的不可逆损失和重组，并最终导致神经元缺陷。缺血后，细胞膜去极化，过量的谷氨酸分泌，离子型谷氨酸受体的过度活性，和GABA信号转导的损失被认为是导致神经元过度活跃。GABA-B-R（R1和R2）是G蛋白偶联受体（GPCR）超家族的C家族成员，其还包括细胞外Ca 2+敏感受体（CaSR）。GABA-B-R通常以由GABA-B-R1和GABA-B-R2亚基组成的异二聚体的形式起作用，所述异二聚体是稳定的细胞表面表达和受体复合物的信号传导所需的，以产生抑制性神经元输入并防止神经元过度活动。GABA-B-R和CaSR在大脑的许多区域中共表达，包括海马。与GABA-B-R1/R2异二聚体不同，CaSR可以同二聚体的形式发挥作用，以产生兴奋性信号传导反应-激活Ca 2+和非选择性阳离子通道，刺激磷脂酶C，增加[Ca 2 +] i，并增加神经元和其他细胞系统中的细胞兴奋性。GABA-B-R1可与CaSR异源二聚体化，并抑制转染HEK-293细胞中CaSR蛋白的总表达和细胞表面表达及其信号转导。同样，敲除海马神经元GABA-B-R1基因可上调CaSR表达。我们进一步观察到CaSR在缺血海马神经元中的过表达，其显示GABA-B-R1表达减少，表明CaSR和GABA-B-R1表达之间的相互抵消作用。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异二聚体形成的能力，从而降低GABA反应，从而导致缺血神经元的过度活动。为了支持后一种观点，我们发现在海马神经元中CaSR基因缺失的HippCaSR-KO小鼠中，缺血不再抑制GABA-B-R1表达或引起细胞死亡。我们假设缺血诱导的CaSR过表达通过刺激CaSR介导的信号传导反应和通过化学计量竞争结合GABA-B-R2抑制GABA-B-R1表达而引起神经元过度活跃和细胞死亡，并且阻断CaSR的表达或活性以及增强GABA-B-R信号传导是针对缺血性损伤的最佳神经保护所需的。我们的建议将（1） 确定是否删除CaSR基因或用特异性拮抗剂阻断CaSR活性（或calcilytics）保护免于缺血诱导的神经元损伤，并减弱缺血对表达，运输，和GABA-B-R1/R2的二聚化，以及（2）确定持续的GABA-B-R1表达是否是针对脑缺血的神经保护所必需的，以及用calcilytics和GABA-B-R1/R2的联合治疗是否是有效的。R1激动剂进一步增强对缺血诱导的脑损伤的神经保护。该研究的成功完成将建立一个新的途径，产生缺血诱导的神经元损伤，并将开发一种新的治疗缺血性脑损伤疾病的疗法。