Modulation of astrocyte-mediated neurotoxicity by NR1D1 in ALS models

摘要 ALS或Lou Gehrig病是最常见的成人起病运动神经元病，其特征是 脊髓、脑干和运动皮质运动神经元的进行性变性。运动神经元死亡 导致肌肉无力和瘫痪，通常在症状出现后一到五年内导致死亡。这个 肌萎缩侧索硬化症运动神经元变性的分子机制尚不清楚。然而，有几个 一系列证据表明，神经胶质细胞功能障碍在神经退行性变过程中起着重要作用。 星形胶质细胞是中枢神经系统动态平衡的关键调节者，已被证明在 疾病的发展。因此，在细胞培养模型中，从ALS小鼠模型分离的星形胶质细胞或 成纤维细胞诱导的星形胶质细胞分化为星形胶质细胞 肌萎缩侧索硬化症患者，诱发运动神经元死亡。星形胶质细胞在神经元的代谢支持中起着关键作用，并且 缺乏代谢灵活性和线粒体功能障碍是ALS星形胶质细胞的特征。我们最近展示了 NR1D1在星形胶质细胞-运动神经元相互作用的调节中起关键作用。NR1D1是以下组织的成员 核受体超家族，是哺乳动物分子钟的重要组成部分。NR1D1是 也是一种已知的新陈代谢和炎症基因的抑制因子，并已被证明调节线粒体 数量和功能，糖脂代谢，以及正常和病理条件下的炎症。 我们最近报道了NR1D1在有症状的ALS小鼠脊髓中的表达减少。此外， 我们发现，在原代培养的小鼠脊髓星形胶质细胞中，Nr1d1的表达被下调，或者在IPSC-1中。 来源的人类星形胶质细胞，激活核因子-kB信号，并诱导促炎表型，即 不利于共培养运动神经元的存活。此外，我们的初步数据显示，IPSC- 与星形胶质细胞相比，ALS患者来源的星形胶质细胞的NR1D1表达水平较低 来源于健康对照。在这项研究中，我们将研究NR1D1在星形胶质细胞- 肌萎缩侧索硬化症运动神经元相互作用及靶向NR1D1的治疗潜力。我们将不偏不倚地执行 多组学分析概述NR1D1下调诱导神经毒性表型的机制 在人IPSC来源的星形胶质细胞中(目标1)。此外，我们将使用不同的方法来评估 在ALS小鼠模型中调节NR1D1的表达对疾病进展的影响(目标2和3)。这个 所获得的结果将有助于目前对其毒性机制的理解。 肌萎缩侧索硬化症中星形胶质细胞向运动神经元转化。此外，这项提案将确立 靶向NR1D1以减缓或阻止ALS小鼠模型中疾病的进展。

Abstract ALS or Lou Gehrig's disease is the most common adult-onset motor neuron disease, characterized by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Motor neuron death leads to muscle weakness and paralysis, usually causing death in one to five years after symptoms onset. The molecular mechanisms responsible for motor neuron degeneration in ALS remain uncertain. However, several lines of evidence indicate that dysfunction of glial cells significantly contributes to the neurodegenerative process. Astrocytes, key regulators of central nervous system homeostasis, have been shown to play a major role in the progression of the disease. Accordingly, in cell culture models, astrocytes isolated from ALS mouse models or astrocytes differentiated from fibroblast-derived induced pluripotent stem cells (iPSCs) from sporadic and familial ALS patients, induce motor neuron death. Astrocytes have a key role in the metabolic support of neurons, and lack of metabolic flexibility and mitochondrial dysfunction are hallmarks of ALS astrocytes. We recently showed that NR1D1 plays a critical role in the regulation of astrocyte-motor neuron interaction. NR1D1 is a member of the nuclear receptor superfamily and it is an essential component of the molecular clock in mammals. NR1D1 is also a known repressor of metabolic and inflammatory genes, and has been shown to regulate mitochondrial number and function, glucose and lipid metabolism, and inflammation during normal and pathological conditions. We recently reported that NR1D1 expression decreases in the spinal cord of symptomatic ALS mice. Moreover, we showed that the knockdown of Nr1d1 expression in primary mouse spinal cord astrocyte cultures, or in iPSC- derived human astrocytes, activates NF-kB signaling and induces a pro-inflammatory phenotype that is detrimental for the survival of co-cultured motor neurons. In addition, our preliminary data shows that iPSC- derived astrocytes from ALS patients display lower levels of NR1D1 expression when compared to astrocytes derived from healthy controls. In this proposal, we will investigate the role of NR1D1 in the regulation of astrocyte- motor neuron interaction and the therapeutic potential of targeting NR1D1 in ALS. We will perform an unbiased multi-omic analysis to outline the mechanism by which NR1D1 downregulation induces a neurotoxic phenotype in human iPSC-derived astrocytes (Aim 1). In addition, we will use different approaches to evaluate the effect of modulating NR1D1 expression on the progression of the disease in an ALS mouse model (Aims 2 and 3). The results obtained will contribute to the current understanding of the mechanisms involved in the toxicity of astrocytes towards motor neurons in ALS. In addition, this proposal will establish the therapeutic value of targeting NR1D1 to slow or halt the progression of the disease in an ALS mouse model.