Table_1_Reduced Expression of GABAA Receptor Alpha2 Subunit Is Associated With Disinhibition of DYT-THAP1 Dystonia Patient-Derived Striatal Medium Spiny Neurons.DOC

DYT-THAP1 dystonia (formerly DYT6) is an adolescent-onset dystonia characterized by involuntary muscle contractions usually involving the upper body. It is caused by mutations in the gene THAP1 encoding for the transcription factor Thanatos-associated protein (THAP) domain containing apoptosis-associated protein 1 and inherited in an autosomal-dominant manner with reduced penetrance. Alterations in the development of striatal neuronal projections and synaptic function are known from transgenic mice models. To investigate pathogenetic mechanisms, human induced pluripotent stem cell (iPSC)-derived medium spiny neurons (MSNs) from two patients and one family member with reduced penetrance carrying a mutation in the gene THAP1 (c.474delA and c.38G > A) were functionally characterized in comparison to healthy controls. Calcium imaging and quantitative PCR analysis revealed significantly lower Ca2+ amplitudes upon GABA applications and a marked downregulation of the gene encoding the GABAA receptor alpha2 subunit in THAP1 MSNs indicating a decreased GABAergic transmission. Whole-cell patch-clamp recordings showed a significantly lower frequency of miniature postsynaptic currents (mPSCs), whereas the frequency of spontaneous action potentials (APs) was elevated in THAP1 MSNs suggesting that decreased synaptic activity might have resulted in enhanced generation of APs. Our molecular and functional data indicate that a reduced expression of GABAA receptor alpha2 subunit could eventually lead to limited GABAergic synaptic transmission, neuronal disinhibition, and hyperexcitability of THAP1 MSNs. These data give pathophysiological insight and may contribute to the development of novel treatment strategies for DYT-THAP1 dystonia.

DYT-THAP1肌张力障碍（原称DYT6）是一种青少年起病型肌张力障碍，以通常累及躯干上部的不自主肌肉收缩为主要特征。该疾病由编码含Thanatos相关蛋白（THAP）结构域的凋亡相关蛋白1的THAP1基因突变所致，以常染色体显性遗传方式传递，且外显率降低。现有转基因小鼠模型研究证实，纹状体神经元投射发育异常与突触功能障碍是该疾病的典型病理特征。为探究其致病机制，本研究对两名携带THAP1基因突变（c.474delA与c.38G>A）的患者及一名外显率降低的家族成员的人类诱导多能干细胞（induced pluripotent stem cell，iPSC）诱导分化而来的中型多棘神经元（medium spiny neuron，MSN）开展功能表征分析，并与健康对照样本进行对比。钙成像及定量PCR检测结果显示，THAP1突变型MSNs在施加γ-氨基丁酸（GABA）后Ca²+振幅显著降低，且编码GABAA受体α2亚基的基因表达显著下调，提示其GABA能递质传递功能受损。全细胞膜片钳记录结果表明，THAP1突变型MSNs的微小突触后电流（miniature postsynaptic current，mPSC）频率显著降低，而自发动作电位（action potential，AP）频率升高，提示突触活动减少可能引发动作电位生成增强。本研究的分子与功能数据表明，GABAA受体α2亚基表达降低最终可导致THAP1突变型MSNs出现GABA能突触传递受限、神经元去抑制及过度兴奋。上述研究结果为该疾病的病理生理学机制提供了新的见解，同时可为DYT-THAP1肌张力障碍的新型治疗策略开发提供理论依据。