single-cell RNA sequencing on Drosophila melanogaster brain in an ALS/FTD model

The mutation in the C9orf72 gene with a hexanucleotide repeat has been reported multiple times to be the most common genetic cause of FTD and ALS (Frontotemporal Dementia and Amyotrophic Lateral Sclerosis), both of which are devastating neurodegenerative diseases having no cures currently. Our lab previously created fruit fly models expressing 36(C4G2) repeats, these are highly toxic to adult neurons of fruit flies. This is one of the most commonly used fly models of disease. Like many neurodegenerative diseases, FTD and ALS display selective neuronal vulnerability: only some neuronal populations succumb to disease, even though the toxic species are ubiquitously expressed. Our lab proposes to identify which neuronal populations are selectively depleted in response to the expression of the repeats and analyse which pathways are activated in vulnerable and resistant neuronal populations using our fly model of disease. This is done by scRNA sequencing across multiple time points, tracking disease development. The workflow was first having the flies ready and their brains being dissected. The brains were then dissociated by collagenase and dispase, and the cell suspensions were passed through a 10um cell strainer. The single-cell suspensions were checked for viability and the single-cell libraries were prepared with 10X Chromium 3' platform.

C9orf72基因的六核苷酸重复突变已被多次报道为额颞叶痴呆（Frontotemporal Dementia，简称FTD）与肌萎缩侧索硬化（Amyotrophic Lateral Sclerosis，简称ALS）最常见的遗传病因，二者均为目前尚无治愈方案的毁灭性神经退行性疾病。本实验室此前已构建表达36(C4G2)重复序列的果蝇模型，该模型对果蝇成年神经元具有高度毒性，是目前最常用的疾病果蝇模型之一。与多数神经退行性疾病类似，FTD与ALS呈现选择性神经元易感性：尽管毒性物质在机体中广泛表达，仍仅有部分神经元群体受累发病。本实验室拟依托该疾病果蝇模型，鉴定因重复序列表达而发生选择性丢失的神经元群体，并分析易损与耐受神经元群体中激活的信号通路。本研究将通过多时间点单细胞RNA测序（scRNA sequencing）追踪疾病进展，具体实验流程为：首先制备果蝇样本并解剖其大脑；随后通过胶原酶与分散酶（Dispase）解离脑组织，将细胞悬液经10μm细胞筛过滤；对单细胞悬液进行活性检测，并采用10X Chromium 3'平台制备单细胞测序文库。