Department of Life Science and Graduate Institute of Biotechnology, National Dong Hwa University, Hualien 974, Taiwan.
Everfront Biotech Inc., New Taipei City 221, Taiwan.
Int J Mol Sci. 2022 Jan 26;23(3):1391. doi: 10.3390/ijms23031391.
Spinocerebellar ataxia type 3 (SCA3) is characterized by the over-repetitive CAG codon in the ataxin-3 gene (), which encodes the mutant ATXN3 protein. The pathological defects of SCA3 such as the impaired aggresomes, autophagy, and the proteasome have been reported previously. To date, no effective treatment is available for SCA3 disease. This study aimed to study anti-excitotoxic effects of n-butylidenephthalide by chemically insulted Purkinje progenitor cells derived from SCA3 iPSCs. We successfully generated Purkinje progenitor cells (PPs) from SCA3 patient-derived iPSCs. The PPs, expressing both neural and Purkinje progenitor's markers, were acquired after 35 days of differentiation. In comparison with the PPs derived from control iPSCs, SCA3 iPSCs-derived PPs were more sensitive to the excitotoxicity induced by quinolinic acid (QA). The observations of QA-treated SCA3 PPs showing neural degeneration including neurite shrinkage and cell number decrease could be used to quickly and efficiently identify drug candidates. Given that the QA-induced neural cell death of SCA3 PPs was established, the activity of calpain in SCA3 PPs was revealed. Furthermore, the expression of cleaved poly (ADP-ribose) polymerase 1 (PARP1), a marker of apoptotic pathway, and the accumulation of ATXN3 proteolytic fragments were observed. When SCA3 PPs were treated with n-butylidenephthalide (n-BP), upregulated expression of calpain 2 and concurrent decreased level of calpastatin could be reversed, and the overall calpain activity was accordingly suppressed. Such findings reveal that n-BP could not only inhibit the cleavage of ATXN3 but also protect the QA-induced excitotoxicity from the Purkinje progenitor loss.
脊髓小脑性共济失调 3 型（SCA3）的特征是在 ataxin-3 基因（）中出现过度重复的 CAG 密码子，该基因编码突变的 ATXN3 蛋白。先前已经报道了 SCA3 的病理性缺陷，如受损的聚集物、自噬和蛋白酶体。迄今为止，尚无有效的 SCA3 疾病治疗方法。本研究旨在通过化学刺激来自 SCA3 iPSC 的浦肯野前体细胞来研究丁烯基邻苯二甲酰亚胺的抗兴奋毒性作用。我们成功地从 SCA3 患者来源的 iPSC 中生成了浦肯野前体细胞（PPs）。在分化 35 天后，获得表达神经和浦肯野前体细胞标记物的 PPs。与来自对照 iPSC 的 PPs 相比，SCA3 iPSC 衍生的 PPs 对喹啉酸（QA）诱导的兴奋毒性更为敏感。观察到 QA 处理的 SCA3 PPs 表现出神经退行性变，包括神经突收缩和细胞数量减少，可用于快速有效地鉴定候选药物。鉴于已经建立了 SCA3 PPs 的 QA 诱导的神经细胞死亡，研究了 SCA3 PPs 中的钙蛋白酶活性。此外，还观察到裂解多聚（ADP-核糖）聚合酶 1（PARP1）的表达，这是凋亡途径的标志物，以及 ATXN3 蛋白水解片段的积累。当 SCA3 PPs 用丁烯基邻苯二甲酰亚胺（n-BP）处理时，上调的钙蛋白酶 2 的表达和钙蛋白酶抑制剂 1 的下调水平可以逆转，并且整体钙蛋白酶活性相应地受到抑制。这些发现表明，n-BP 不仅可以抑制 ATXN3 的切割，还可以保护浦肯野前体细胞免受 QA 诱导的兴奋毒性的损失。
