P/Q-Type Channels and Cerebellar Synaptic Transmission

描述（由申请人提供）：在获得性ca2 +通道病变中，如副肿瘤小脑变性（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针对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学的改变、通道蛋白的下调或抗体结合后免疫介导的过程。尽管尚不清楚通道功能障碍如何导致失联表型，但其发病机制可能与突触生理学改变有关，因为PQCC是突触前和突触后膜的关键参与者，对突触传递和可塑性至关重要。由于研究人类小脑神经传递是不可能的，这项拨款申请建议使用抗pqcc抗体来敲除通道功能，创建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用于确定抗pqcc抗体抑制的机制，并评估其对活性依赖性ca2 +进入、神经递质释放和平行纤维-浦肯野细胞突触突触生理学的影响。特别是，平行纤维的重复刺激将被用来观察在这种PCD模型中是否存在lems型谷氨酸释放的促进作用。除了抗体敲除模型，PQCC功能障碍的替代模型，阿尔法1a -/-和omega- agatoxin - iva治疗的小脑，将被使用。alpha1A -/-和omega-Agatoxin-IVA模型举例说明了通道功能的最终丧失，而抗体敲低方法最接近于获得性人类通道病，可用于实时分析通道功能丧失的后果。小脑突触前和突触后生理的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通路的一致信号。PCD的共济失调表型可能是神经递质释放和突触后神经元同步信号检测翻译中多种错误的结果。

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.