Synergistic Interaction of FTD Genes in Neuroinflammation and Neurodegeneration

项目摘要 额颞性痴呆(FTD)是一种早期发病的神经退行性疾病，也是第二常见的 60岁或以下患者患痴呆症的原因。大多数家族性FTD是由内含子引起的 染色体9开放阅读框72(C9orf72)基因中六核苷酸(CCCCGG)重复序列的扩增 原颗粒蛋白(GRN)基因的显性突变，导致两个基因单倍性不足，异常蛋白 神经元中的聚集体形成。一些功能和转录学研究表明，Null基因缺失的小鼠 C9orf72或Grn突变显示小胶质细胞(常驻中枢神经系统免疫细胞)异常激活 FTD神经退行性变的发病机制。而C9orf72和原颗粒蛋白(PGRN)的确切功能 [蛋白质])仍然不清楚，几项研究已经涉及到自噬和内溶酶体途径。 神经元和小胶质细胞，以及同时发生的突变导致患者脑萎缩加剧。这些结果 提示C9orf72和PGRN在脑老化过程中可能存在神经退行性变的相互作用 我的项目的目标是研究C9orf72和Grn基因在神经胶质细胞内稳态中的协同作用 以及使用小鼠模型的神经元退化。为了支持这一点，我的初步数据显示C9orf72-/- ；Grn-/-DKO小鼠的寿命显著缩短，远短于C9orf72-/-小鼠、Grn-/-小鼠和对照组 老鼠。C9orf72-/-；Grn-/-DKO小鼠脑病理检查显示年龄依赖性胶质细胞增生症 神经元TDP-43聚集体比C9orf72-/-或Grn-/-小鼠更明显和分布更广。 这些结果支持我的假设，并进一步表明C9orf72和Grn的丢失协同破坏 胶质细胞-神经元动态平衡，并导致更明显的神经退行性变表型。对于F00阶段， 我建议通过单细胞来揭示C9和PGRN在衰老脑神经退行性变中的作用机制 7个月和12个月龄对照组、C9orf72-/-、Grn-/-和C9orf72-/-；Grn-/-DKO脑 确定这两个FTD基因的丢失如何破坏神经胶质细胞-神经元相互作用中的动态平衡(目标2a)。这些 转录数据将使用原位杂交、免疫组织化学和免疫印迹(AIM)进行验证 2B)。最后，我建议发展体外培养，包括纯神经元培养和神经胶质细胞-神经元联合培养， 这将为C9orf72和PGRN在自噬中的协同作用提供更多的见解- 溶酶体途径和神经胶质细胞介导的神经元毒性(目标2c)。 在这个奖学金的K00阶段，我计划开发诱导多能干细胞(IPSC)来源的3D大脑 有机化合物作为模型系统，研究神经退行性变的发病机制和确定治疗方法。 为了确定可能受疾病影响的信号通路，我计划在 患者IPSC获得神经胶质细胞器质，随后基于CRISPR/Cas9的操作策略 信号通路在这些脑有机体中失调，以阐明疾病的进展机制。

Project Summary Frontotemporal dementia (FTD) is an early onset neurodegenerative disease, and the second most common cause of dementia in patients 60 years or younger. The majority of familial FTD are caused by intronic hexanucleotide (CCCCGG) repeat expansion in chromosome 9 open reading frame 72 (C9orf72) gene and by dominant mutations in the Progranulin (GRN) gene, causing haploinsufficiency in both genes, abnormal protein aggregate formation in neurons. Several functional and transcriptomic studies have shown that mice with null mutation in C9orf72 or Grn show abnormal microglia (resident CNS immune cells) activation mediated pathogenesis of neurodegeneration in FTD. While the exact functions for C9orf72 and Progranulin (PGRN [protein]) are still unclear, several studies have implicated both in autophagy and endolysosomal pathways in neurons and microglia, and concurrent mutations resulting in increased brain atrophy in patients. These results suggest a possible interaction between C9orf72 and PGRN in neurodegeneration during brain aging The goal of my project is to investigate the synergistic interaction of C9orf72 and Grn genes in glial homeostasis and neuronal degeneration using mouse models. In support of this, my preliminary data showed that C9orf72-/- ;Grn-/- DKO mice have significantly shortened lifespan, much shorter than C9orf72-/- mice, Grn-/- mice and control mice. Brain pathology examination in C9orf72-/-;Grn-/- DKO mice showed age-dependent gliosis as well as neuronal TDP-43 aggregates that are more pronounced and wide-spread than those in C9orf72-/- or Grn-/- mice. These results support my hypothesis and further indicate that loss of C9orf72 and Grn synergistically disrupt glia-neuron homeostasis and lead to more pronounced neurodegeneration phenotype. For the F00 phase, I propose to uncover the mechanism of C9 and PGRN in neurodegeneration in the aging brain via single-cell and bulk RNA-sequencing in 7 and 12 months old control, C9orf72-/-, Grn-/- and C9orf72-/-;Grn-/- DKO brain to determine how loss of these two FTD genes disrupts the homeostasis in glia-neuron interaction (Aim 2a). These transcriptomic data will be validated using in situ hybridization, immunohistochemistry and western blots (Aim 2b). Finally, I propose to develop in vitro cultures, including neuron-only cultures and glia-neuron co-cultures, which will provide more insights into the synergistic interaction between C9orf72 and PGRN in the autophagy- lysosome pathways and in glia-mediated toxicity to neurons (Aim 2c). For the K00 phase of this fellowship, I plan to develop induced pluripotent stem cells (IPSC)-derived 3D brain organoids as model systems to investigate disease mechanism and identify therapeutics for neurodegeneration. To identify signaling pathways that could be affected by diseases, I plan on using single-cell transcriptomics on patient IPSC derived glia-neuron organoids, followed by CRISPR/Cas9-based manipulation strategies on the signaling pathways dysregulated in these brain organoids to elucidate the diseases progression mechanisms.