CaMKII, endocannabinoids, synaptic plasticity and motor function

描述(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着关键作用。运动活动的正常纹状体驱动 需要两种纹状体中棘神经元(MSN)精确平衡的相反输出，这些神经元表达D1-和D2-多巴胺(DA)受体。在D_1和D_2-MSN中，谷氨酸和DA之间复杂的信号串扰涉及“按需”依赖的内源性大麻素(ECB)的合成。因此，DA、谷氨酸和ECB通过在两个MSN亚型中使用不同的信号机制来协作平衡对两个纹状体输出通路的短期和长期控制。这些机制的破坏会导致运动障碍(如帕金森氏病)或其他基于纹状体的异常行为。Ca2/钙调蛋白依赖的蛋白激酶II(CaMKII)在海马区、大脑皮层和小脑中具有不同的双向调控兴奋性突触的作用。虽然CaMKII在两种纹状体MSN亚型中都有表达，这表明它调节纹状体MSN的兴奋性输入和运动活动，但纹状体CaMKII的确切功能尚不清楚。我们对CaMKIIα中Thr286自动磷酸化位点被ALA取代的敲门突变小鼠(T286A-Ki小鼠)的分析揭示了CaMKII在依赖于欧洲央行的长期和短期控制兴奋输入到D1-和D2-MSN中的特定作用。我们还发现，CaMKIIα与二酰基甘油脂肪酶α(Dglα)结合并使其磷酸化，Dgl Dgl是合成脑内最丰富的ECB-2-花生四烯基甘油(2-AG)的限速酶。此外，T286A-Ki小鼠的基线多动可通过抑制2-AG的降解来挽救。这些初步发现有力地支持了一种新的假设，即CaMKII是突触后钙和2-AG信号启动之间的关键联系，2-AG信号控制纹状体突触和基于纹状体的行为。我们还创造了新的转基因eAC3I小鼠，在纹状体MSN中选择性地表达与EGFP融合的CaMKII抑制短肽。三个特定的目标将利用T286A-Ki和eAC3I小鼠的独特特征来测试关于纹状体CaMKII自动磷酸化和活性的特定假设。1.验证钙依赖的2-AG合成受CaMKII调控的假说。我们将确定Dglα中的磷酸化和CaMKII结合域的位置。将在异种细胞和WT、T286A-KI和eAC3I小鼠的纹状体切片中研究Dglα的磷酸化、Dglα的活性和2-AG的合成。2.验证CaMKII调节纹状体MSN中ECB依赖的突触调节的假设。CaMKII的短期和长期作用将通过比较WT、T286A-KI和eAC3I小鼠纹状体切片中兴奋性突触输入到D1和D2-MSN的特性以及使用CaMKII抑制肽来确定。3.验证CaMKII调节ECB依赖的运动活动的假设。我们将评估WT、T286A-KI和eAC3I小鼠在基础条件下的运动活性和协调性，以及药物对2-AG代谢的调节。

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.