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: 8668720
• 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/8668720
• 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; 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; neuroprotection; novel; overexpression; prevent; public health relevance; 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）： 在美国和退伍军人事务部社区中，缺血性脑损伤会导致心脏骤停和栓塞性中风患者死亡和长期残疾。受影响神经元中缺血引起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、谷氨酸分泌过多、离子型谷氨酸受体过度活跃以及 GABA 信号传导丧失被认为会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 C 家族的成员，该家族还包括细胞外 Ca2+ 感应受体 (CaSR)。 GABA-B-R 通常以由 GABA-B-R1 和 GABA-B-R2 亚基组成的异二聚体形式发挥作用，这是稳定的细胞表面表达和受体复合物信号传导所必需的，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（包括海马体）中共表达。与 GABA-B-R1/R2 异二聚体不同，CaSR 可以以同二聚体的形式发挥作用，发挥兴奋性信号反应——激活 Ca2+ 和非选择性阳离子通道、刺激磷脂酶 C、增加 [Ca2+]i 并增加神经元和其他细胞系统的细胞兴奋性。 GABA-B-R1 可以与 CaSR 形成异二聚体，并抑制转染的 HEK-293 细胞中 CaSR 蛋白的总表达和细胞表面表达及其信号传导反应。相反，敲除海马神经元中的 GABA-B-R1 基因会上调 CaSR 表达。我们进一步观察到缺血海马神经元中 CaSR 过度表达，显示 GABA-B-R1 表达减少，表明 CaSR 和 GABA-B-R1 表达之间存在抵消相互作用。 CaSR 表达和活性的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，导致缺血神经元过度活跃，从而减少 GABA 反应。为了支持后一个观点，我们发现在海马神经元中删除了 CaSR 基因的 HippCaSR-KO 小鼠中，缺血不再抑制 GABA-B-R1 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 介导的信号反应以及通过化学计量竞争与 GABA-B-R2 结合抑制 GABA-B-R1 表达而导致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必需的。我们的建议将 (1) 确定删除 CaSR 基因或通过特定拮抗剂（或钙阻滞剂）阻断 CaSR 活性是否可以防止缺血引起的神经元损伤，并减弱缺血对 GABA-B-R1/R2 的表达、运输和二聚化的抑制作用，以及（2）确定是否需要维持 GABA-B-R1 表达来对抗脑缺血的神经保护以及是否需要联合治疗 与钙阻药和 GABA-B-R1 激动剂一起使用可进一步增强针对缺血引起的脑损伤的神经保护作用。该研究的成功完成将建立一条产生缺血性神经元损伤的新途径，并将开发治疗缺血性脑损伤引起的疾病的新疗法。