Modulation of CaMKII and endocannabinoid signaling by Calcium Channels

描述（由申请人提供）：正常的习惯学习、运动控制和决策需要纹状体gaba -能中棘神经元将兴奋性输入与其他基底神经节结构的调节输入相结合。在这些过程中起关键作用的一个主要机制是内源性大麻素（eCB）依赖的可塑性。纹状体的主要eCB， 2-花生四烯酰基甘油（2-AG），在突触后由二酰基甘油脂肪酶-a （DGL）合成，响应突触活性，但反向抑制突触前谷氨酸释放并诱导突触抑制。纹状体l型电压门控钙通道（ltcc）介导钙内流，并被认为驱动2-AG的合成，从而促进ecb依赖性突触抑制。纹状体钙水平的另一个调节因子是t型电压门控钙通道（TTCCs），它可以对钙的进入做出重大贡献，也与eCB的可塑性有关。然而，很少有研究直接测量电压门控钙通道活性调节后2-AG水平的变化，也没有钙信号通路耦合ltcc和ttcc对纹状体eCB可塑性的很好表征。我们的实验室最近取得了令人兴奋的发现，Ca2+/钙调素依赖性蛋白激酶iia （CaMKII）抑制DGL活性和2-AG的产生，以限制ecb依赖性突触抑制（Nat Neurosci, 2013; 16(4):456- 63）。此外，我们实验室和其他人的工作表明，CaMKII与ltcc和ttcc的信号相关联并传播。我的初步研究发现，在基础条件下，高纹状体CaMKII活性和定位到突触后密度需要持续的细胞外钙进入，部分由ttcc介导。这个为期一年的项目的目标是测试ltcc和ttcc不同地激活CaMKII来调节ecb依赖性突触信号的总体假设，有两个具体目的：1。验证纹状体CaMKII与ttcc和ltcc有差异偶联的假设。急性分离纹状体切片将与这些通道的激动剂和拮抗剂孵育。提取物和亚细胞组分将通过磷酸化位点特异性抗体进行western blotting分析，通过Thr286的自磷酸化和已建立的突触底物（GluR1中的Ser831； NR2B中的Ser1303）的磷酸化，以及Thr286自磷酸化的免疫组织化学分析来监测CaMKII的激活。2. 验证ltcc和ttcc差异调节纹状体DGL和2-AG水平的假设。我将使用基于液相色谱/质谱的方法，在与Aim 1中定义的条件相似的条件下，检测纹状体切片提取物中的DGL活性和2-AG水平。综上所述，这些研究将首次直接探讨ltcc和ttcc对纹状体CaMKII Thr286自磷酸化的调节如何调节突触信号传导和2-AG合成。这将为未来研究纹状体为基础的神经和精神疾病中这些通路的潜在破坏提供坚实的基础。

DESCRIPTION (provided by applicant): Normal habit learning, motor control, and decision making requires striatal GABA-ergic medium spiny neurons to integrate excitatory inputs with modulatory inputs from other basal ganglia structures. One major mechanism playing a key role in these processes is endocannabinoid (eCB) dependent plasticity. The major striatal eCB, 2-arachidonylglycerol (2-AG), is synthesized postsynaptically by diacylglycerol lipase-a (DGL) in response to synaptic activity, but acts retrogradely to inhibit presynaptic glutamate release and induce synaptic depression. Striatal L-Type voltage gated calcium channels (LTCCs) mediate calcium influx and are thought to drive synthesis of 2-AG to promote eCB-dependent synaptic depression. Another regulator of striatal calcium levels are T-type voltage gated calcium channels (TTCCs), which can make substantial contributions to calcium entry and have also been linked to eCB plasticity. However, few studies have directly measured changes in 2-AG levels following modulation of voltage gated calcium channel activity, nor have the calcium signaling pathways coupling LTCCs and TTCCs to striatal eCB plasticity been well characterized. Our lab has recently made the exciting discovery that Ca2+/calmodulin-dependent protein kinase-IIa (CaMKII) restrains DGL activity and 2-AG production to limit eCB-dependent synaptic depression (Nat Neurosci, 2013; 16(4):456- 63). Furthermore, work from our lab and others shows that CaMKII associates with and propagates signaling from LTCCs and TTCCs. My preliminary studies found that high striatal CaMKII activity and localization to the postsynaptic density under basal conditions requires ongoing entry of extracellular calcium entry, partially mediated by TTCCs. The goal of the proposed 1 year project is to test the overarching hypothesis that LTCCs and TTCCs differentially activate CaMKII to regulate eCB-dependent synaptic signaling with two specific aims: 1. Test the hypothesis that striatal CaMKII is differentially coupled to TTCCs and LTCCs. Acutely isolated striatal slices will be incubated with agonists and antagonists of these channels. Extracts and subcellular fractions will be analyzed by western blotting using phospho-site specific antibodies to monitor the CaMKII activation by autophosphorylation at Thr286 and by phosphorylation of established synaptic substrates (Ser831 in GluR1; Ser1303 in NR2B), and by immunohistochemical analyses of Thr286 autophosphorylation. 2. Test the hypothesis that LTCCs and TTCCs differentially modulate striatal DGL and 2-AG levels. I will assay DGL activity and 2-AG levels in extracts of striatal slices incubated under conditions similar to those defined in Aim 1 using a liquid chromatography/mass spectrometry based method. In combination, these studies will be the first to directly investigate how the modulation of striatal CaMKII Thr286 autophosphorylation by LTCCs and TTCCs regulates synaptic signaling and 2-AG synthesis. This will provide a firm foundation for future studies of potential disruptions of these pathways in striatal-based neurological and psychiatric diseases.