Modulation of dendritic CaMKII by dopamine

描述(由申请人提供)： 帕金森病(PD)的多巴胺(DA)替代疗法通常在疾病进展后期由于耐药性和运动障碍的发展而无效。我们推测这是由于树突状细胞蛋白表达和/或功能的改变引起纹状体中棘神经元形态的改变。初步研究利用单侧黑质注射6-羟基佐巴胺(6-OHDA)所产生的帕金森病大鼠模型。注射6-OHDA 9个月后，损毁侧纹状体CaMKIIa总水平较对侧纹状体降低30%，其他几种蛋白无明显变化。CaMKII对海马区突触可塑性至关重要：NMDA受体激活诱导Thr286自动磷酸化，产生一种钙非依赖性形式的CaMKII，它移位到突触后的密度，磷酸化AMPA受体(GluRl)，是长时程增强所必需的。虽然纹状体CaMKII的功能仍然不清楚，但初步数据表明，在6-OHDA损伤的纹状体中，CaMKII的Thr286自磷酸化增加。总而言之，这些令人兴奋的发现表明，慢性多巴胺 PD缺乏或由6-OHDA诱导，扰乱了关键的CaMKII信号通路的正常生理调节，这将通过三个AIMS进行测试。 1.研究CaMKII在6-羟色胺损毁的纹状体中的表达。CaMKII在6-OHDA损伤的纹状体中的表达/定位将与对侧大脑半球的对照纹状体和车辆注射的动物的纹状体进行比较。将研究i-DOPA或谷氨酸受体阻滞剂对改变的可能改善。 2.确定DA在正常纹状体CaMKII急性调节中的作用(S)。分离的纹状体脑片将被用来研究D1和D2DA受体在调节基础和谷氨酸刺激的CaMKII自磷酸化以及该信号通路上游(DARPP32)和下游(GluRl，%)组分的磷酸化中的作用。 3.明确慢性DA耗竭对CaMKII信号转导的影响(S)。损毁纹状体6-OHDA或长期给予D1/D2 DA受体拮抗剂对基础CaMKII自磷酸化和DARPP32、GluR1和NR2B的磷酸化的影响将被确定。急性DA信号的病理缺陷将通过比较6-OHDA损伤和对照纹状体急性分离脑片中CaMKII的调节来确定。所有数据将与本PPG项目1和3中确定的脊柱形态和突触传递的可塑性相关。DA信号的慢性干扰预计会对CaMKII信号产生重大影响，这可能会导致晚期PD的并发症。这些见解可能有助于开发改进的帕金森病治疗策略。

Description (provided by applicant): Dopamine (DA) replacement therapy of Parkinson's Disease (PD) often becomes ineffective late in disease progression due to development of drug resistance and dyskinesias. We hypothesize that this is due to altered morphology of striatal medium spiny neurons arising from changes in expression andlor function of dendritic proteins. Preliminary Studies exploit a rat model of PD generated by unilateral injection of 6-hydroxyzopamine (6-OHDA) into substantia nigra. Total CaMKIIa levels in lesioned striaturn were decreased by 30% relative to contralateral control striaturn 9 months after 6-OHDA-injection; several other proteins were unchanged. CaMKII is critical for hippocampal synaTtic plasticity: NMDA receptor activation induces Thr 286 autophosphorylation, generating a Ca2+- independent form of CaMKII that translocates to postsynaptic densities, phsphorylates AMPA receptors (GluRl) and is required for long term potentiation. Although striatal CaMKII functions remain obscure, preliminary data indicate that Thr 286 autophosphorylation of CaMKII is increased in 6-OHDA lesioned striatum. Together, these exciting findings suggest the specific hypothesis that chronic dopamine deficiency in PD or induced by 6 OHDA perturbs normal physiological regulation of the key CaMKII signaling pathway, which will be tested using three Aims. 1. Characterize CaMKII expression in 6-OHDA-lesioned striaturn. CaMKII expression/localization in 6-OHDA lesioned striaturn will be compared to control striatum from the contralateral hemisphere and from vehicle-injected animals. Possible amelioration of changes by I-DOPA, or glutamate-receptor blockers will be investigated. 2. Determine role(s) of DA in acute regulation of CaMKII in normal striaturn. Isolated striatal slices will be used to investigate roles of D1 and D2 DA receptors in regulating basal and glutamate-stimulated CaMKII autophosphorylation as well as phosphorylation of upstream (DARPP32) and downstream (GluRl, %) components of this signaling pathway. 3. Define effect(s) of chronic DA depletion on CaMKII signaling. Effects of striatal 6-OHDA lesion or chronic administration of D1/D2 DA receptor antagonists on basal CaMKII autophosphorylation and phosphorylation of DARPP32, GluRl and NR2B will be determined. Pathological defects in acute DA signaling will be determined by comparing CaMKII regulation in acutely isolated slices from 6-OHDA lesioned and control striaturn. All data will be correlated with plasticity of spine morphology and synaptic transmission determined in Projects 1 and 3 of this PPG. Chronic disruption of DA signaling is predicted to substantially impact CaMKIi signaling, which may contribute to complications of late-phase PD. These insights may help develop improved PD treatment strategies.