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

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病 以较低的运动神经元退化为特征的疾病。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增，是导致九种神经退行性疾病之一 来自多聚谷氨酰胺(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，我们创造了包括BAC在内的SBMA的新型小鼠模型 含有第一外显子的转基因小鼠(即BAC fxAR121系)允许特定细胞类型的切除 AR转基因。我们将BAC fxAR121小鼠与人骨骼肌动蛋白(HSA)-Cre小鼠杂交，并 有文献证明，从骨骼肌中切除AR转基因可以阻止两种系统性疾病的发展 和神经肌肉SBMA表型，揭示了突变的PolyQ-AR在肌肉表达中的关键作用 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 在外周分娩时，我们证明了外周抑制PolyQ-AR可以挽救运动障碍， 逆转了肌肉基因表达的变化，并显著延长了SBMA小鼠的寿命。这些 挑衅性的发现暗示骨骼肌是SBMA病发病的关键部位。要确定 运动神经元(MNS)对SBMA的贡献，我们将BAC fxAR121小鼠与vChAT-CRE小鼠杂交，并 注意到大基因小鼠的运动能力有了适度但显著的改善。因此，SBMA病 发病机制涉及由骨骼之间的病理相互作用引起的改变的汇聚 肌肉和MNS。我们还发现自噬失调是SBMA MN病的一个明显特征 分析体内和体外模型，包括人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小体成熟和溶酶体融合的异常 来自ipscs的祖细胞，从而将自噬功能障碍与SBMA的发病联系起来。描绘，描绘 在这种效应的基础上，我们考虑了自噬的转录调控，揭示了一种相互作用 AR和转录因子EB(TFEB)之间的关系，并确定TFEB调节失调是 SBMA中的自噬缺陷。在这个项目中，我们将定义骨骼肌的分子贡献和 通过在我们的各种条件缺失SBMA小鼠模型中进行转录组分析，将MNS转化为SBMA； 通过建立SBMA骨骼肌和MN模型阐明MN死亡的细胞学基础 患者IPSCs，并确定非细胞自主毒性是否可以在这些干细胞模型中概括； 并确定了AR共激活和多聚Q-AR抑制TFEB的基础，方法是识别其 在多聚Q-AR的存在下，复合体与AR和TFEB的相互作用和功能发生了变化。

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.