CaMKII, endocannabinoids, synaptic plasticity and motor function

CaMKII、内源性大麻素、突触可塑性和运动功能

• 批准号: 9102281
• 负责人: ROGER J COLBRAN
• 金额: $ 38.25万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9102281
• 关键词: 2-arachidonylglycerol; Ataxia; Basal Ganglia; Behavior; Binding; Biological Assay; Brain; Brain region; Ca(2+)-Calmodulin Dependent Protein Kinase; Calcium/calmodulin-dependent protein kinase; Cells; Cerebellum; Complex; Corpus striatum structure; Depressed mood; Dopamine; Dopamine D2 Receptor; Electrophysiology (science); Endocannabinoids; Enzymes; Equilibrium; Excitatory Synapse; Exhibits; Follow-Up Studies; Functional disorder; Glutamates; Goals; Health; Hippocampus (Brain); Home environment; Huntington Disease; Hyperactive behavior; Knock-in; Knock-in Mouse; Learning; Link; Memory; Metabolism; Molecular; Monitor; Motor; Motor Activity; Motor Cortex; Movement; Movement Disorders; Mus; Mutant Strains Mice; Mutation; Neurons; Output; Parkinson Disease; Pathway interactions; Peptides; Phosphorylation; Phosphorylation Site; Physiological; Play; Property; Proteins; Regulation; Reporter; Role; Signal Transduction; Site; Slice; Synapses; Synaptic plasticity; Testing; Thalamic structure; Tracer; Transgenic Organisms; base; calmodulin-dependent protein kinase II; endocannabinoid signaling; gamma-Aminobutyric Acid; in vivo; inhibitor/antagonist; lipoprotein lipase; motor deficit; motor learning; nervous system disorder; new therapeutic target; novel; postsynaptic; selective expression; synaptic depression; synaptic function; tool

DESCRIPTION (provided by applicant): The striatum plays a key role in motor activity/coordination and goal-directed, habitual learning. Normal striatal drive of motor activity requires precisely balanced opposing outputs from two types of striatal medium spiny neurons (MSNs) that express D1- and D2-dopamine(DA) receptors. A complex signaling cross-talk between glutamate and DA in D1- and D2-MSNs involves "on demand" Ca2+-dependent endocannabinoid (eCB) synthesis. Thus, DA, glutamate and eCBs collaborate to balance short- and long-term control of the two striatal output pathways by engaging distinct signaling mechanisms in the two MSN subtypes. Disruption of these mechanisms can induce motor deficits (e.g., Parkinson's Disease) or other abnormal striatal-based behaviors. Ca2+/calmodulin-dependent protein kinase II (CaMKII) has diverse bidirectional roles controlling excitatory synapses in hippocampus, cortex and cerebellum. While CaMKII is expressed in both striatal MSN subtypes, suggesting that it regulates excitatory inputs to striatal MSNs and motor activity, the precise functions of striatal CaMKII are poorly understood. Our analyses of knockin mutant mice with the Thr286 autophosphorylation site in CaMKIIα replaced by Ala (T286A-KI mice) revealed specific roles for CaMKII in long- and short term eCB- dependent control of excitatory inputs to D1- and D2-MSNs. We also found that CaMKIIα associates with and phosphorylates diacylglycerol lipase α (DGLα), the rate-limiting enzyme for Ca2+-dependent synthesis of the most abundant brain eCB, 2-arachidonyl glycerol (2-AG). In addition, baseline hyperactivity of T286A-KI mice can be rescued by inhibiting 2-AG breakdown. These initial findings strongly support a novel hypothesis that CaMKII is a critical link between postsynaptic Ca2+ and the initiation of 2-AG signaling that controls striatal synapses and striatal based-behaviors. We also created novel transgenic eAC3I mice that selectively express a short CaMKII inhibitor peptide fused to eGFP in striatal MSNs. Three specific aims will exploit unique features of T286A-KI and eAC3I mice to test specific hypotheses about the roles of striatal CaMKII autophosphorylation and activity. 1. Test the hypothesis that Ca2+-dependent 2-AG synthesis is modulated by CaMKII. We will identify sites of phosphorylation and CaMKII-binding domains in DGLα. DGLα phosphorylation, DGLα activity and 2-AG synthesis will be investigated in heterologous cells and in striatal slices from WT, T286A-KI and eAC3I mice. 2. Test the hypothesis that CaMKII modulates eCB-dependent synaptic regulation in striatal MSNs. Short and long-term roles of CaMKII will be determined by comparing the properties of excitatory synaptic inputs to D1- and D2-MSNs in striatal slices from WT, T286A-KI and eAC3I mice, and by using CaMKII inhibitor peptides. 3. Test the hypothesis that CaMKII modulates eCB-dependent motor activity. We will evaluate motor activity and coordination under basal conditions and following pharmacological modulation of 2-AG metabolism in WT, T286A-KI and eAC3I mice.

描述（由申请人提供）：纹状体在运动活动/协调和目标导向的习惯性学习中发挥着关键作用。运动活动的正常纹状体驱动 需要来自表达 D1 和 D2 多巴胺 (DA) 受体的两种纹状体中型多巴胺神经元 (MSN) 的精确平衡的相反输出。 D1-和 D2-MSN 中谷氨酸和 DA 之间复杂的信号串扰涉及“按需”Ca2+ 依赖性内源性大麻素 (eCB) 合成。因此，DA、谷氨酸和 eCB 通过参与两种 MSN 亚型中不同的信号传导机制，协作平衡两种纹状体输出途径的短期和长期控制。这些机制的破坏可能会导致运动缺陷（例如帕金森病）或其他基于纹状体的异常行为。 Ca2+/钙调蛋白依赖性蛋白激酶 II (CaMKII) 具有多种双向作用，控制海马、皮质和小脑的兴奋性突触。虽然 CaMKII 在两种纹状体 MSN 亚型中都有表达，表明它调节纹状体 MSN 的兴奋性输入和运动活动，但纹状体 CaMKII 的精确功能尚不清楚。我们对 CaMKIIα 中 Thr286 自磷酸化位点被 Ala 取代的敲入突变小鼠（T286A-KI 小鼠）的分析揭示了 CaMKII 在 D1- 和 D2-MSN 兴奋性输入的长期和短期 eCB 依赖性控制中的特定作用。我们还发现 CaMKIIα 与二酰基甘油脂肪酶 α (DGLα) 结合并磷酸化，DGLα 是最丰富的脑 eCB、2-花生四烯酰甘油 (2-AG) 的 Ca2+ 依赖性合成的限速酶。此外，T286A-KI 小鼠的基线过度活跃可以通过抑制 2-AG 分解来挽救。这些初步发现有力地支持了一个新的假设，即 CaMKII 是突触后 Ca2+ 和控制纹状体突触和纹状体行为的 2-AG 信号传导启动之间的关键联系。我们还创建了新型转基因 eAC3I 小鼠，其在纹状体 MSN 中选择性表达与 eGFP 融合的短 CaMKII 抑制肽。三个具体目标将利用 T286A-KI 和 eAC3I 小鼠的独特特征来测试有关纹状体 CaMKII 自磷酸化和活性的作用的具体假设。 1. 检验 Ca2+ 依赖性 2-AG 合成受 CaMKII 调节的假设。我们将鉴定 DGLα 中的磷酸化位点和 CaMKII 结合域。将在异源细胞和 WT、T286A-KI 和 eAC3I 小鼠的纹状体切片中研究 DGLα 磷酸化、DGLα 活性和 2-AG 合成。 2. 检验 CaMKII 调节纹状体 MSN 中 eCB 依赖性突触调节的假设。 CaMKII 的短期和长期作用将通过比较 WT、T286A-KI 和 eAC3I 小鼠纹状体切片中 D1-和 D2-MSN 的兴奋性突触输入的特性并使用 CaMKII 抑制剂肽来确定。 3. 检验 CaMKII 调节 eCB 依赖性运动活动的假设。我们将评估 WT、T286A-KI 和 eAC3I 小鼠在基础条件下以及 2-AG 代谢的药理调节后的运动活动和协调性。