P/Q-Type Channels and Cerebellar Synaptic Transmission

描述（由申请方提供）：在获得性Ca 2+通道病如副肿瘤性小脑变性（PCD）和Lambert-Eaton肌无力综合征（LEMS）中，神经元功能障碍可能是由质膜上抗PQCC的自身抗体结合引发的。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的免疫介导过程。虽然尚不清楚通道功能障碍如何导致共济失调表型，但发病机制可能与突触生理学改变有关，因为PQCC是突触传递和可塑性中重要的突触前膜和突触后膜的关键参与者。由于不可能研究人类小脑神经传递，因此该资助申请提出使用抗PQCC抗体来敲低通道功能来创建PCD的细胞模型。采用电生理记录和FM 1 - 43实验等成熟技术，研究抗PQCC抗体抑制PQCC的机制，并评价其对平行纤维-浦肯野细胞突触活性依赖性Ca 2+内流、神经递质释放和突触生理学的影响。特别是，平行纤维的重复刺激将用于观察在该PCD模型中是否存在谷氨酸释放的LEMS型促进。除抗体敲低模型外，还将使用PQCC功能障碍的替代模型，即α 1A-/-和ω-Agatoxin-IVA处理的小脑。α 1A-/-和ω-Agatoxin-IVA模型模拟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，并且可用于在真实的时间内剖析通道功能丧失的后果。小脑突触前和突触后生理学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中的共济失调表型可能是神经递质释放和突触后神经元对重合信号检测的翻译中的多种错误的顶点。

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.