Gabaergic Rescue of Associative Motor Learning in Spinocerebellar Ataxia Type 6 Mice

脊髓小脑型共济失调6型(SCA6)是一种进行性退行性神经系统疾病，起病晚，几乎为单纯小脑性共济失调，由P/Q型钙通道基因第47外显子扩增的CAG重复序列引起。P/Q型钙通道α亚基(A1ACT)的羧基末端(CT)包含CAG重复序列，并从CACNA1a内的IRES(基因内核糖体进入位点)翻译而来，导致更稳定的病态CT(A1ACTSCA6)肽片段，该片段特异性地积累在胞浆中，并在SCA6患者的核浦肯野细胞(PC)蛋白聚集体中少量积累。这些患病的CT片段可能作为显性负性突变体，干扰P/Q型钙通道与其底物(结合伙伴)之间的正常相互作用，最终导致SCA6疾病状态。以往的研究表明，皮质小脑萎缩患者和SCA1小鼠的GABA能药物治疗(如加巴喷丁、普瑞巴林或巴氯芬)可改善其共济失调症状。总之，这些研究表明GABABR在共济失调中存在功能障碍。在这项研究中，我们想要确定A1ACTSCA6损伤的特定蛋白靶点和第二信使通路，以便我们可以开发GPCR特异性的光遗传学工具来挽救小脑皮质的关联性运动学习。我们初步的眨眼条件作用(EBC)数据显示，在SCA6小鼠中，GABABR光遗传工具的光激活可以部分恢复EBC反应，这表明a1ACTSCA6干扰了GABR或其下游效应器的GI/O途径。为了证明a1ACTSCA6正在扰乱正常的GABR功能，我们将测量在a1ACTSCA6存在的情况下Gi/o诱导的GIRK活性，并与GABABR刺激后的对照a1ACT进行比较。其次，我们希望通过免疫沉淀(IP)分析从表达a1ACTSCA6的小鼠的小脑裂解物中显示a1ACTSCA6与GABABR之间的直接相互作用。然后，我们想通过在光激活的GABABR存在的情况下通过测量PF-PC突触上LTD的诱导来研究光激活的GABABR是否可以恢复表达PC的1ACTSCA6受损的LTD。最后，我们将缓解SCA6小鼠的类似SCA6的症状，将我们的光激活GABR与运动学习联系在一起。这些研究将可能为SCA6患者提供一种有益的治疗工具。

Spinocerebellar ataxia type 6 (SCA6) is a progressive, degenerative neurological disorder characterized by its late onset and almost pure cerebellar ataxia, which is caused by an expanded CAG repeat in exon 47 of the P/Q type calcium channel gene. The carboxy terminus (CT) of the alpha subunit of the P/Q type calcium channel (a1ACT) contains the CAG repeats and is translated from an IRES (intragenic internal ribosome entry site) within the CACNA1A, leading to a more stable diseased CT (a1ACTSCA6) peptide fragment which specifically accumulates in cytosolic and to a lesser extent in nuclear Purkinje cells (PC) protein aggregates from SCA6 patients. These diseased CT fragments may potentially be acting as a dominant negative mutant to interfere with the normal interactions between P/Q type calcium channels and its substrates (binding partners) which eventually leads to the SCA6 disease state. Past work showed that GABAergic drug treatment (ie gabapentin, pregabalin or baclofen) of cortical cerebellar atrophy patients and SCA1 mice improved their ataxic symptoms. Together these studies implicate a misfunction of GABABR in ataxia. In this study we would like to identify the specific protein targets and second messenger pathways which a1ACTSCA6 is impairing, so we can develop GPCR specific optogenetic tools to rescue associative motor learning from the cerebellar cortex. Our preliminary eye blink conditioning (EBC) data where we demonstrate a partial recovery of EBC responses with light activation of GABABR optogenetic tool in SCA6 mice suggests that a1ACTSCA6 interferes with the Gi/o-pathway of the GABABR or its downstream effectors. To demonstrate that a1ACTSCA6 is disrupting normal GABABR function, we will measure Gi/o-induced GIRK activity in the presence of a1ACTSCA6 compared to control a1ACT following GABABR stimulation. Secondly, we would like to show a direct interaction between the a1ACTSCA6 and GABABR by immunoprecipitation (IP) assays from cerebellar lysates of a1ACTSCA6 expressing mice. Then we would like to investigate whether the light-activated GABABR can recover impaired LTD in a1ACTSCA6 expressing PCs by measuring LTD induction at the PF-PC synapses in the presence of the light-activated GABABR. Lastly, we would relieve SCA6-like symptoms for associative motor learning with our light-activated GABABR in SCA6 mice. These studies will potentially provide a beneficial therapeutic tool for SCA6 patients.