CaMKII, endocannabinoids, synaptic plasticity and motor function

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起关键作用。运动活动的正常纹状体驱动 需要精确平衡来自表达D1-和D2-多巴胺（DA）受体的两种类型的纹状体中型多刺神经元（MSN）的相反输出。在D1-和D2-MSN中谷氨酸和DA之间的复杂信号传导串扰涉及“按需”Ca 2+依赖性内源性大麻素（eCB）合成。因此，DA，谷氨酸和eCB合作，以平衡短期和长期控制的两个纹状体输出途径，通过从事不同的信号传导机制在两个MSN亚型。这些机制的破坏可以诱导运动缺陷（例如，帕金森氏病）或其他异常的基于纹状体的行为。Ca 2 +/钙调素依赖性蛋白激酶II（CaMKII）在海马、皮质和小脑中具有多种双向作用，控制兴奋性突触。虽然CaMKII在两种纹状体MSN亚型中表达，表明它调节纹状体MSN和运动活动的兴奋性输入，但纹状体CaMKII的确切功能知之甚少。我们对CaMKIIα中Thr 286自磷酸化位点被Ala取代的敲入突变小鼠（T286 A-KI小鼠）的分析揭示了CaMKII在长期和短期eCB依赖性控制D1和D2-MSNs兴奋性输入中的特定作用。我们还发现CaMK II α与二酰基甘油脂肪酶α（DGLα）结合并磷酸化，DGL α是钙依赖性合成脑中最丰富的eCB 2-花生四烯酸甘油（2-AG）的限速酶。此外，T286 A-KI小鼠的基线活动过度可以通过抑制2-AG分解来挽救。这些初步的研究结果有力地支持了一个新的假设，即CaMKII是突触后Ca 2+和2-AG信号的启动之间的关键环节，控制纹状体突触和纹状体为基础的行为。我们还创建了新型转基因eAC 3 I小鼠，其选择性地表达与纹状体MSN中的eGFP融合的短CaMKII抑制剂肽。三个具体的目标将利用T286 A-KI和eAC 3 I小鼠的独特功能来测试关于纹状体CaMKII自磷酸化和活性的作用的具体假设。1.检验Ca 2+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结合结构域。将在异源细胞和WT、T286 A-KI和eAC 3 I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MSNs中eCB依赖性突触调节的假设。通过比较WT、T286 A-KI和eAC 3 I小鼠纹状体切片中D1-和D2-MSN的兴奋性突触输入的性质，并通过使用CaMKII抑制剂肽，确定CaMKII的短期和长期作用。3.检验CaMKII调节eCB依赖性运动活动的假设。我们将在WT、T286 A-KI和eAC 3 I小鼠中评价基础条件下和2-AG代谢药理学调节后的运动活性和协调性。

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.