Deconstructing the cellular and molecular basis of SBMA motor neuron disease: From mechanism to therapy

解构 SBMA 运动神经元疾病的细胞和分子基础：从机制到治疗

• 批准号: 10355757
• 负责人: ALBERT R LA SPADA
• 金额: $ 29.18万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-30 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10355757
• 关键词: Deconstructing; cellular; molecular; basis; SBMA

X-linked spinal and bulbar muscular atrophy (SBMA, Kennedy's disease) is an inherited neuromuscular disorder characterized by lower motor neuron degeneration. SBMA is caused by CAG/polyglutamine repeat expansions in the human androgen receptor gene, and is one of nine neurodegenerative disorders that result from polyglutamine (polyQ) proteins. We set out to determine the cellular and molecular basis of SBMA disease pathogenesis. To achieve these goals, we created novel mouse models of SBMA, including BAC transgenic mice containing a floxed first exon (i.e. the BAC fxAR121 line) to permit cell-type specific excision of the AR transgene. We crossed BAC fxAR121 mice with Human Skeletal Actin (HSA)-Cre mice, and documented that excision of the AR transgene from skeletal muscle prevented development of both systemic and neuromuscular SBMA phenotypes, revealing a crucial role for muscle expression of mutant polyQ-AR in SBMA motor neuron degeneration. We produced antisense oligonucleotides (ASOs) directed against AR, and upon peripheral delivery, we demonstrated that peripheral suppression of polyQ-AR rescued motor deficits, reversed alterations in muscle gene expression, and markedly extended lifespan in SBMA mice. These provocative findings implicate skeletal muscle as a key site for SBMA disease pathogenesis. To determine the contribution of motor neurons (MNs) to SBMA, we crossed BAC fxAR121 mice with vChAT-Cre mice, and noted a modest, but significant improvement in motor performance in bigenic mice. Hence, SBMA disease pathogenesis involves a convergence of alterations stemming from pathological interactions between skeletal muscle and MNs. We also uncovered autophagy dysregulation as a defining feature of SBMA MN disease by analyzing in vivo and in vitro models, including a human SBMA stem cell model. These studies revealed abnormalities of autophagosome maturation and lysosome fusion in SBMA cell models, mice, and neuronal progenitor cells derived from iPSCs, thereby linking autophagy dysfunction to the onset of SBMA. To delineate the basis of this effect, we considered the transcriptional regulation of autophagy, uncovered an interaction between AR and transcription factor EB (TFEB), and determined that TFEB dysregulation accounts for autophagy defects in SBMA. In this project, we will define the molecular contributions of skeletal muscle and MNs to SBMA by performing transcriptome analysis in our various conditional deletion SBMA mouse models; delineate the cellular basis for MN demise by developing skeletal muscle and MN models of SBMA from patient iPSCs, and determining if non-cell autonomous toxicity can be recapitulated in these stem cell models; and define the basis for AR co-activation and polyQ-AR repression of TFEB by identifying co-regulators whose interactions and functions with AR and TFEB in complex are altered in the presence of polyQ-AR.

X连锁脊髓和延髓肌萎缩症（SBMA，肯尼迪病）是一种遗传性神经肌肉萎缩症 以下运动神经元变性为特征的疾病。 SBMA 是由 CAG/聚谷氨酰胺重复序列引起的 人类雄激素受体基因的扩展，是导致的九种神经退行性疾病之一 来自聚谷氨酰胺 (polyQ) 蛋白。我们着手确定 SBMA 的细胞和分子基础 疾病发病机制。为了实现这些目标，我们创建了新型 SBMA 小鼠模型，包括 BAC 含有 floxed 第一个外显子（即 BAC fxAR121 系）的转基因小鼠，允许细胞类型特异性切除 AR转基因。我们将 BAC fxAR121 小鼠与人类骨骼肌动蛋白 (HSA)-Cre 小鼠杂交，并且 据记载，从骨骼肌中切除 AR 转基因可阻止全身肌肉的发育 和神经肌肉 SBMA 表型，揭示了突变体 PolyQ-AR 的肌肉表达的关键作用 SBMA 运动神经元变性。我们生产了针对 AR 的反义寡核苷酸 (ASO)，并且 在外周传递后，我们证明了外周抑制polyQ-AR可以挽救运动缺陷， 逆转了 SBMA 小鼠肌肉基因表达的改变，并显着延长了寿命。这些 令人兴奋的发现表明骨骼肌是 SBMA 疾病发病机制的关键部位。确定 运动神经元 (MN) 对 SBMA 的贡献，我们将 BAC fxAR121 小鼠与 vChAT-Cre 小鼠杂交，并且 注意到双基因小鼠的运动表现有适度但显着的改善。因此，SBMA 病 发病机制涉及骨骼之间病理相互作用引起的变化的汇集 肌肉和 MN。我们还发现自噬失调是 SBMA MN 疾病的一个定义特征 分析体内和体外模型，包括人类 SBMA 干细胞模型。这些研究揭示 SBMA 细胞模型、小鼠和神经元中自噬体成熟和溶酶体融合的异常 源自 iPSC 的祖细胞，从而将自噬功能障碍与 SBMA 的发生联系起来。划定 基于这种效应，我们考虑了自噬的转录调控，发现了一种相互作用 AR 和转录因子 EB (TFEB) 之间的关系，并确定 TFEB 失调是造成 SBMA 中的自噬缺陷。在这个项目中，我们将定义骨骼肌和骨骼肌的分子贡献 通过在我们的各种条件删除 SBMA 小鼠模型中进行转录组分析，将 MN 转化为 SBMA； 通过开发 SBMA 的骨骼肌和 MN 模型来描绘 MN 死亡的细胞基础 患者 iPSC，并确定非细胞自主毒性是否可以在这些干细胞模型中重现； 并通过识别协同调节剂来定义 TFEB 的 AR 共激活和 polyQ-AR 抑制的基础 PolyQ-AR 的存在会改变复合物中 AR 和 TFEB 的相互作用和功能。