Astrocyte regulation of cortical neurodegeneration in C9orf72 FTD/ALS

项目摘要 C9orf72基因第一内含子的GGGGCC(G4C2)六核苷酸重复序列扩展(HRE)是 与额颞性痴呆(FTD)和肌萎缩侧索硬化症相关的最常见的遗传异常 硬化症(ALS)。该病的发病机制最终导致皮质前脑同时变性。 和脊髓运动神经元，并导致临床运动功能障碍和痴呆。而C9orf72- FTD/ALS疾病的发病机制已被很好地描述在脊髓运动神经元中，并在 观察到的神经退行性变归因于脊髓星形胶质细胞，对此知之甚少 皮质星形胶质细胞的病理生物学及其在皮质神经变性中的作用，这被认为是有贡献的 这些患者群体中的痴呆症症状。在这里，我们假设皮质星形胶质细胞扮演着一个 非细胞自主性疾病在导致皮质退行性变中的整体作用 C9orf72-FTD/ALS的神经元。为了验证这一假设，我们将研究HiPSC衍生的C9orf72-FTD/ALS 皮层星形胶质细胞单独培养和与皮质神经元共培养(目标1)。我们将描述C9orf72- FTD/ALS通过评估星形胶质细胞功能和确定C9orf72 HRE特异性的皮质星形胶质细胞 病理生物学。此外，我们还将建立皮质星形胶质细胞和皮质神经元之间的关系。 用对照细胞和C9orf72-FTD/ALS细胞共培养。将对共同文化的变化进行评估 星形胶质细胞功能、神经元功能和活性，以及C9orf72病的病理生物学。此外，我们还将 检查患病的HiPSC来源的皮质星形胶质细胞的转录改变(目标2)。转录体 在单一培养和共培养条件下，患病的和对照的皮质星形胶质细胞的图谱将是 使用RNA测序产生的。此外，我们还将分析已有的单核rna序列数据。 在实验室中产生，并确定重叠的候选基因，这些基因在大脑皮层中特别失调 C9orf72-FTD/ALS患者和HiPSC系的星形胶质细胞我们将在RNA上验证这些候选基因 分别用RNAScope和免疫组织化学方法检测死后患者组织标本中的蛋白质水平。 选择经过验证的热门歌曲将通过这些基因操作进行初步的机械验证 HiPSC体外模型中的候选基因。这些研究将首次阐明 皮质星形胶质细胞在C9orf72-FTD/ALS皮质神经元退行性变中的作用 这种谱系障碍中痴呆的机制。此外，这项工作将为 药物靶标识别，希望为受影响的患者群体确定新的治疗方法。

PROJECT ABSTRACT The GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the first intron of the gene C9orf72, is the most common genetic abnormality associated with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The disease pathogenesis ultimately leads to the concurrent degeneration of cortical forebrain and spinal motor neurons, and result in the clinical deficits of motor function and dementia. While the C9orf72- FTD/ALS disease pathogenesis has been well characterized in spinal motor neurons and a contribution of the observed neurodegeneration has been attributed to spinal cord astrocytes, there is little known about the pathobiology in cortical astrocytes and their role in cortical neurodegeneration, which is proposed to contribute to the dementia symptoms in this patient population. Here, we hypothesize that cortical astrocytes play an integral role in the non-cell autonomous disease pathology contributing to the degeneration of cortical neurons in C9orf72-FTD/ALS. To test this hypothesis, we will investigate hiPSC-derived C9orf72-FTD/ALS cortical astrocytes in monoculture and in co-culture with cortical neurons (Aim 1). We will characterize C9orf72- FTD/ALS hiPSC-cortical astrocytes by assessing astrocyte function and determine C9orf72 HRE-specific pathobiology. Furthermore, we will establish the relationship between cortical astrocytes and cortical neurons using co-cultures of control and C9orf72-FTD/ALS lines. The co-cultures will be assessed for changes in astrocyte function, neuronal function and viability, and C9orf72-disease pathobiology. In addition, we will examine transcriptomic alterations in the diseased hiPSC-derived cortical astrocytes (Aim 2). Transcriptomic profiles of the diseased and control cortical astrocytes in both monoculture and co-culture conditions will be generated using RNA-sequencing. In addition, we will analyze existing single nuclei RNA seq data already generated in the lab and identify overlapping candidate genes that are specifically dysregulated in cortical astrocytes in C9orf72-FTD/ALS patients and hiPSC lines. We will validate these candidate genes on the RNA and protein level in postmortem patient tissue samples via RNAscope and immunohistochemistry, respectively. Select validated top hits will undergo preliminary mechanistic validation through genetic manipulation of these candidate genes in the hiPSC in vitro model. These studies will for the first time elucidate the contributing role of cortical astrocytes in the neurodegeneration of cortical neurons in C9orf72-FTD/ALS, addressing the disease mechanisms of dementia in this spectrum disorder. Additionally, this work will provide novel opportunities for drug target identification with the hope of identifying novel therapeutics for the affected patient populations.