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

脊髓小脑性共济失调6型（SCA 6）是一种进行性、退行性神经系统疾病，其特征是迟发性和几乎纯小脑性共济失调，由P/Q型钙通道基因外显子47的CAG重复序列扩增引起。P/Q型钙通道α亚基（α 1 ACT）的羧基末端（CT）含有CAG重复序列，并从CACNA 1A内的IRES（基因内内部核糖体进入位点）翻译，导致更稳定的病变CT（α 1 ACTSCA 6）肽片段，其特异性地在SCA 6患者的胞质中积累，并在较小程度上在核浦肯野细胞（PC）蛋白聚集体中积累。这些患病的CT片段可能潜在地充当显性负突变体以干扰P/Q型钙通道与其底物（结合伴侣）之间的正常相互作用，这最终导致SCA 6疾病状态。过去的工作表明，GABA能药物治疗（即加巴喷丁，普瑞巴林或巴氯芬）的皮质小脑萎缩患者和SCA 1小鼠改善他们的共济失调症状。这些研究共同暗示了GABABR在共济失调中的功能障碍。在这项研究中，我们希望确定a1 ACTSCA 6受损的特定蛋白质靶点和第二信使途径，因此我们可以开发GPCR特异性光遗传学工具来拯救小脑皮层的联合运动学习。我们的初步眨眼条件反射（EBC）数据表明，SCA 6小鼠中GABABR光遗传学工具的光激活使EBC应答部分恢复，表明a1 ACTSCA 6干扰GABABR或其下游效应物的Gi/o-途径。为了证明α 1 ACT SCA 6破坏正常GABABR功能，我们将在GABABR刺激后，在α 1 ACT SCA 6存在下与对照α 1 ACT相比测量Gi/o诱导的GIRK活性。其次，我们希望通过免疫沉淀（IP）测定从表达a1 ACTSCA 6的小鼠的小脑裂解物显示a1 ACTSCA 6和GABABR之间的直接相互作用。然后，我们想通过测量在光激活的GABABR存在下PF-PC突触处的LTD诱导来研究光激活的GABABR是否可以恢复表达a1 ACTSCA 6的PC中受损的LTD。最后，我们将在SCA 6小鼠中用我们的光激活GABABR缓解SCA 6样症状以进行联合运动学习。这些研究可能为SCA 6患者提供有益的治疗工具。

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.