Modulation of dendritic CaMKII by dopamine

描述（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变得无效。我们推测这是由于树突状蛋白表达和/或功能的改变引起纹状体中型棘状神经元形态的改变。初步研究利用单侧注射6-羟基唑帕明（6-OHDA）到黑质产生的PD大鼠模型。6-OHDA注射后9个月，相对于对侧对照纹状体，受损纹状体中的总CaMKIIa水平降低了30%;其他几种蛋白质没有变化。CaMKII对海马突触可塑性至关重要：NMDA受体激活诱导Thr 286自磷酸化，产生Ca 2+非依赖性形式的CaMKII，其易位至突触后密度，磷酸化AMPA受体（GluRl），并且是长时程增强所需的。虽然纹状体CaMKII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Thr 286自磷酸化增加。总之，这些令人兴奋的发现提出了一个特定的假设，即慢性多巴胺 PD中的或由6 OHDA诱导的缺陷扰乱了关键CaMKII信号传导途径的正常生理调节，这将使用三个目的进行测试。 1.表征6-OHDA损伤的纹状体中的CaMKII表达。将6-OHDA损伤的纹状体中的CaMKII表达/定位与来自对侧半球和来自媒介物注射的动物的对照纹状体进行比较。将研究I-DOPA或谷氨酸受体阻滞剂可能改善的变化。 2.确定DA在正常纹状体中对CaMKII的急性调节中的作用。分离的纹状体切片将用于研究D1和D2 DA受体在调节基础和谷氨酸刺激的CaMKII自磷酸化以及该信号传导途径的上游（DARPP 32）和下游（GluRl，%）组分的磷酸化中的作用。 3.定义慢性DA耗竭对CaMKII信号传导的影响。将确定纹状体6-OHDA损伤或D1/D2 DA受体拮抗剂的长期施用对基础CaMKII自磷酸化和DARPP 32、GluRl和NR 2B的磷酸化的影响。通过比较来自6-OHDA损伤和对照纹状体的急性分离的切片中的CaMK II调节来确定急性DA信号传导中的病理缺陷。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信号传导的慢性中断被预测会显著影响CaMKIi信号传导，这可能导致晚期PD的并发症。这些见解可能有助于开发改进的PD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.