Spinal bulbar muscular atrophy (SBMA), the first identified CAG-repeat expansion disorder, is an X-linked neuromuscular disorder involving CAG-repeat-expansion mutations in the androgen receptor (AR) gene. We utilized CRISPR-Cas9 gene editing to engineer novel isogenic human induced pluripotent stem cell (hiPSC) models, consisting of isogenic AR knockout, control, and disease lines expressing mutant AR with distinct repeat lengths, as well as control and disease lines expressing FLAG-tagged wildtype and mutant AR, respectively. Adapting a small-molecule cocktail-directed approach, we differentiate the isogenic hiPSC models into motor neuron-like cells with a highly enriched population to uncover cell-type-specific mechanisms underlying SBMA and to distinguish gain- from loss-of-function properties of mutant AR in disease motor neurons. We demonstrate that ligand-free mutant AR causes drastic mitochondrial dysfunction in neurites of differentiated disease motor neurons due to gain-of-function mechanisms, and such cytotoxicity can be amplified upon ligand (androgens) treatment. We further show that aberrant interaction between ligand-free, mitochondria-localized mutant AR and F-ATP synthase is associated with compromised mitochondrial respiration and multiple other mitochondrial impairments. These findings counter the established notion that androgens are requisite for mutant AR-induced cytotoxicity in SBMA, reveal a compelling mechanistic link between ligand-free mutant AR, F-ATP synthase, and mitochondrial dysfunction, and provide innovative insights into motor neuron-specific therapeutic interventions for SBMA.

中文翻译：

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无配体线粒体定位突变体 AR 诱导的脊髓延髓肌萎缩细胞毒性

脊髓延髓肌萎缩症 (SBMA) 是第一个发现的 CAG 重复扩增障碍，是一种 X 连锁神经肌肉疾病，涉及雄激素受体 (AR) 基因中的 CAG 重复扩增突变。我们利用 CRISPR-Cas9 基因编辑来设计新型同基因人诱导多能干细胞 (hiPSC) 模型，包括同基因 AR 敲除、对照和表达具有不同重复长度的突变 AR 的疾病系，以及表达 FLAG-分别标记了野生型和突变体 AR。采用小分子鸡尾酒导向方法，我们将同基因 hiPSC 模型分化为具有高度富集群体的运动神经元样细胞，以揭示 SBMA 背后的细胞类型特异性机制，并区分获得和丧失功能特性疾病运动神经元中的突变 AR。我们证明，由于功能获得机制，无配体突变体 AR 在分化疾病运动神经元的神经突中引起严重的线粒体功能障碍，并且这种细胞毒性可以在配体（雄激素）处理后放大。我们进一步表明，无配体、定位于线粒体的突变体 AR 和 F-ATP 合酶之间的异常相互作用与受损的线粒体呼吸和多种其他线粒体损伤有关。这些发现反驳了雄激素是突变体 AR 诱导的 SBMA 细胞毒性所必需的既定观念，揭示了无配体突变体 AR、F-ATP 合酶和线粒体功能障碍之间令人信服的机制联系，并为运动神经元特异性治疗提供了创新见解SBMA 的干预措施。