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: 9535519
• 负责人: ALBERT R LA SPADA
• 金额: $ 49.48万
• 依托单位: DUKE UNIVERSITY
• 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/9535519
• 关键词: Actins; Androgen Antagonists; Androgen Receptor; Antisense Oligonucleotides; Autophagocytosis; Autophagosome; Bacterial Artificial Chromosomes; Bioinformatics; Biological; Cataloging; Catalogs; Cell Culture Techniques; Cell model; Cells; Coculture Techniques; Complex; Defect; Development; Disease; Excision; Exons; Functional disorder; Gene Expression; Genetic Transcription; Goals; Human; Inherited; Investigation; Kennedy Syndrome; Knock-in Mouse; Ligands; Link; Longevity; LoxP-flanked allele; Lumbar spinal cord structure; Lysosomes; Modeling; Molecular; Motor; Motor Neuron Disease; Motor Neurons; Mus; Muscle; Muscle Fibers; Nerve Degeneration; Neurodegenerative Disorders; Neuromuscular Diseases; Nuclear; Pathogenesis; Pathologic; Pathway interactions; Patients; Performance; Peripheral; Phenotype; Process; Proteins; Receptor Gene; Role; Site; Skeletal Muscle; Stem cells; Toxic effect; Transcriptional Activation; Transcriptional Regulation; Transgenes; Transgenic Mice; Validation; Work; cell type; disease phenotype; gene repression; in vitro Model; in vivo; induced pluripotent stem cell; molecular pathology; motor control; motor deficit; motor neuron degeneration; mouse model; mutant; nerve stem cell; neuromuscular; novel; polyglutamine; prevent; quadriceps muscle; receptor; receptor expression; skeletal; spinal and bulbar muscular atrophy; stem; transcription factor; transcriptome; transcriptome sequencing; transgene expression

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， 以下运动神经元退化为特征的疾病。SBMA由CAG/多聚谷氨酰胺重复序列引起 人类雄激素受体基因扩增，是导致人类雄激素受体基因扩增的九种神经退行性疾病之一。 聚谷氨酰胺（polyQ）蛋白。我们着手确定SBMA的细胞和分子基础 发病机理为了实现这些目标，我们创建了新的SBMA小鼠模型，包括BAC 含有floxed第一外显子的转基因小鼠（即BAC fxAR 121系），以允许细胞类型特异性切除 AR转基因我们将BAC fxAR 121小鼠与人骨骼肌肌动蛋白（HSA）-Cre小鼠杂交， 证明了从骨骼肌中切除AR转基因可以阻止全身性 和神经肌肉SBMA表型，揭示了肌肉表达的突变polyQ-AR的关键作用， 运动神经元变性我们生产了针对AR的反义寡核苷酸（ASO）， 在外周递送时，我们证明了多聚Q-AR的外周抑制挽救了运动缺陷， 逆转肌肉基因表达的改变，并显着延长SBMA小鼠的寿命。这些 一个有争议的发现暗示骨骼肌是SBMA疾病发病机制的关键部位。确定 为了研究运动神经元（MN）对SBMA的贡献，我们将BAC fxAR 121小鼠与vChAT-Cre小鼠杂交， 注意到在双基因小鼠的运动表现中有适度但显著的改善。因此，SBMA疾病 发病机制涉及骨骼肌之间的病理相互作用引起的变化的集合， 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 因此，本发明涉及从iPSC衍生的祖细胞，从而将自噬功能障碍与SBMA的发作联系起来。划定 这种效应的基础，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失调解释了 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， MNs到SBMA通过在我们的各种条件性缺失SBMA小鼠模型中进行转录组分析; 通过开发SBMA的骨骼肌和MN模型，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; 并通过鉴定共调节子来确定TFEB的AR共激活和polyQ-AR抑制的基础， 与复合物中的AR和TFEB的相互作用和功能在polyQ-AR的存在下改变。