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连锁的脊柱和鳞茎肌肉萎缩（SBMA，肯尼迪氏病）是一种遗传性神经肌肉 疾病的特征是较低的运动神经元变性。 SBMA是由CAG/聚谷氨酰胺重复引起的 人类雄激素受体基因的膨胀，是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 疾病发病机理。为了实现这些目标，我们创建了SBMA的新型鼠标模型，包括BAC 含有flox的第一外显子（即BAC FXAR121系）的转基因小鼠允许细胞类型的特异性切除 AR转基因。我们将BAC FXAR121小鼠与人骨骼肌（HSA）-CRE小鼠越过 记录了从骨骼肌中切除AR转基因的切除，以阻止两种全身性发展 和神经肌肉SBMA表型，揭示了突变polyq-ar肌肉表达至关重要的作用 SBMA运动神经元变性。我们生产了针对AR的反义寡核苷酸（ASO），然后 外围递送后，我们证明了对Polyq-AR救出运动缺陷的外围抑制 肌肉基因表达的改变，并在SBMA小鼠中明显延长了寿命。这些 挑衅性的发现暗示骨骼肌是SBMA病发病机理的关键部位。确定 运动神经元（MN）对SBMA的贡献，我们用vChat-c​​re小鼠越过BAC FXAR121小鼠，然后 注意到临后小鼠的运动性能有所改善，但显着改善。因此，SBMA病 发病机理涉及骨骼之间病理相互作用的改变的融合 肌肉和MN。我们还发现自噬失调是SBMA MN疾病的定义特征 分析体内和体外模型，包括人类SBMA干细胞模型。这些研究揭示了 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的发作联系起来。描绘 这种效果的基础，我们考虑了自噬的转录调控，发现了一种相互作用 在AR和转录因子EB（TFEB）之间，并确定TFEB失调解释 SBMA中的自噬缺陷。在这个项目中，我们将定义骨骼肌和 通过在我们的各种条件缺失SBMA小鼠模型中执行转录组分析，通过转录组分析； 通过开发从骨骼肌和sBMA的MN模型来描述MN灭亡的细胞基础 患者iPSC，并确定在这些干细胞模型中是否可以概括非细胞自主毒性； 并通过识别其共同激活和polyQ-ar抑制TFEB的基础 在PolyQ-AR存在下，与AR和TFEB的相互作用和功能改变了。