P/Q-Type Channels and Cerebellar Synaptic Transmission

描述(申请人提供)：获得性钙通道病，如副肿瘤性小脑变性(PCD)和Lambert-Eaton肌无力综合征(LEMS)，神经元功能障碍可能是由质膜上抗PQCC的自身抗体结合引起的。PCD的发病机制可能包括通道生理改变、通道蛋白下调或抗体结合后的免疫介导过程。尽管目前尚不清楚通道功能障碍如何导致共济失调表型，但其发病机制可能与突触生理改变有关，因为PQCC是突触前膜和突触后膜的关键角色，在突触传递和可塑性中起重要作用。由于不可能研究人类小脑的神经传递，这项拨款申请建议使用抗PQCC抗体来抑制通道功能，建立PCD的细胞模型。包括电生理记录和FM1-43实验在内的成熟技术将被用来确定抗PQCC抗体抑制的机制，并评估其对平行纤维-浦肯野细胞突触的活性依赖性钙进入、神经递质释放和突触生理的影响。特别是，重复刺激平行纤维将被用来观察在这种PCD模型中是否存在LEMS类型的谷氨酸释放促进作用。除了抗体敲除模型外，还将使用PQCC功能障碍的替代模型，即Alpha1A-/-和omega-Agatoxin-IVA治疗的小脑。Alpha1a-/-和omega-Agatoxin-IVA模型例证了通道功能的最终丧失，而抗体敲除方法最接近于获得性人类通道病，可用于实时剖析通道功能丧失的后果。小脑突触前后的生理改变可能会对突触可塑性、小脑回路和运动学习产生复杂的影响，因为它们依赖于来自不同路径的一致信号。PCD的共济失调表型可能是神经递质释放和突触后神经元对一致信号检测的翻译错误导致的。

DESCRIPTION (provided by applicant): In acquired Ca 2+ channelopathies such as paraneoplastic cerebellar degeneration (PCD) and Lambert-Eaton myasthenic syndrome (LEMS), neuronal dysfunction is presumably initiated by the binding of autoantibodies against PQCC at the plasma membrane. Pathogenesis of PCD may include alteration of channel physiology, down-regulation of channel proteins, or immune-mediated processes following antibody binding. Although it is not clear how dysfunction of a channel leads to the ataxic phenotype, the pathogenesis is likely related to altered synaptic physiology, since PQCC is a key player in the pre- and post-synaptic membranes important in synaptic transmission and plasticity. Because it is impossible to study human cerebellar neurotransmission, this grant application proposes the creation of a cellular model of PCD using anti-PQCC antibodies to knockdown channel function. Well-established techniques including electrophysiological recordings and FM1- 43 experiments will be used to determine the mechanisms of anti-PQCC antibody inhibition and to evaluate its effects on activity-dependent Ca 2+ entry, neurotransmitter release, and synaptic physiology at the parallel fiber-Purkinje cell synapse. In particular, repetitive stimulation of the parallel fibers will be used to see if a LEMS-type facilitation of glutamate release may exist in this model of PCD. In addition to the antibody knockdown model, alternative models of PQCC dysfunction, the alpha1A -/- and omega-Agatoxin-IVA-treated cerebella, will be used. The alpha1A -/- and omega-Agatoxin-IVA models exemplify the ultimate loss of channel function, while the antibody knockdown approach most closely resembles an acquired human channelopathy and can be used to dissect the consequences of loss of channel function in real time. Altered pre- and post-synaptic physiology in the cerebellum will likely have complex impacts on synaptic plasticity, cerebellar circuitry, and motor learning because of their dependence on coincident signals from different pathways. The ataxic phenotype in PCD may be the culmination of a multiplicity of errors in neurotransmitter release and in the translation of coincident signal detection by the post-synaptic neurons.