Receptor sorting through tubular microdomains of Rab7-positive endosomes in Charcot- Marie-Tooth disease 2B

夏科-玛丽-图斯(Charcot-Marie-Tooth，CMT)病是一种遗传性神经病变，影响周围神经系统，导致神经变性。疾病通常发生在青春期或成年期，表现为肌肉无力、失去精细运动控制和四肢感觉。在常染色体显性遗传的2B型Charcot-Marie Tooth(CMT2B)中，疾病是由晚期内体Rab7-GTP酶突变引起的。在神经元中，Rab7-内体对生长因子的运输很重要：在被各自的配体激活后，生长因子受体被内吞，随后被分成Rab7阳性的内体，并逆行运输到胞体，在那里它们调节促生存基因的基因表达。最后，受体通过溶酶体途径降解。有趣的是，最近的研究已经将CMT2B与Rab7阳性晚期内体运输的损害联系在一起，但Rab7-GTPase突变如何促进CMT2B中生长因子受体的运输仍然难以捉摸。我推测，CMT2B-Rab7突变体中的内体表现出扰乱的分选能力，这是由于在分选/运输过程中受体被路由到的管状微区的干扰形成。通常认为，分选发生在早期的内体上，在那里受体被分选到循环途径中-要么直接回到质膜，要么通过反式高尔基网络(TGN)。然而，我推测，晚期内体上的这些管状微域也有助于受体的分离，潜在地进入溶酶体，这将是一个与内体成熟过程解偶联/平行的过程。因此，形成这种管状微区的扰动可能会影响从Rab7阳性的内体到溶酶体的分选，受体信号的缺陷，受体降解的延迟，以及最终调节神经支配和/或生存的转录基因的下调。所有这些效应可以解释CMT2B中观察到的几种表型，包括生长因子信号转导延长，生长因子分选到溶酶体的延迟，以及Rab7内体运输的缺陷。本项目的主要目标是通过重点研究Rab7阳性的晚期内体分选机制中的突变，阐明导致Charcot-Marie-Tooth 2B型的致病机制。我将破译CMT2B中Rab7-突变与细胞分选机器的相互作用，并将使用高分辨率成像跟踪激活的生长因子受体的亚细胞定位，以可视化受干扰的运输路线。我将在小鼠背根神经节神经元以及来自CMT2B患者的IPSC衍生的运动神经元和感觉神经元中解决这个问题。这项拟议的工作将揭开CMT2B中的分类故障，并通过揭示可能的靶蛋白来帮助评估新的治疗方法。

Charcot-Marie-Tooth (CMT) disease is an inherited neuropathy which affects the peripheral nervous system, leading to neurodegeneration. Disease onset occurs generally during adolescence or adulthood and is manifested by muscle weakness, loss of fine motor control and sensation in the extremities. In autosomal dominant Charcot-Marie Tooth type 2B (CMT2B), disease is caused by mutations in the late endosomal Rab7-GTPase. In neurons, Rab7- endosomes are important for the trafficking of growth-factors: Following activation by their respective ligand, growth-factor receptors are endocytosed, subsequently sorted into Rab7-positive endosomes and transported retrogradely to the soma where they regulate gene expression of pro-survival genes. Finally, receptors are degraded through the lysosomal pathway. Intriguingly, recent studies have linked CMT2B to impairments in trafficking of Rab7-positive late endosomes, but how mutations in Rab7-GTPase contribute to growth- factor receptors trafficking in CMT2B remains elusive. I hypothesize that endosomes in CMT2B-Rab7 mutants show perturbed sorting abilities due to disturbed formation of tubular microdomains into which receptors are routed during the sorting/trafficking process. Generally, it is considered that sorting occurs on early endosomes where receptors are sorted into the recycling pathways- either directly back to the plasma membrae or through the trans-golgi network (TGN). However, I hypothesize that these tubular microdomains on late endosomes also facilitate receptor sorting, potentially into lysosomes which would be a process uncoupled/in parallel from the process of endosomal maturation. The perturbation in forming such tubular microdomains could therefore affect sorting from Rab7-positive endosomes into lysosomes, defective receptor signalling, a delay in receptor degradation and ultimately downregulation of transcription genes which regulate innervation and/ or survival. All of these effects would explain several of the observed phenotypes in CMT2B, which includes prolonged growth-factor signaling, delay in growth factor sorting into lysosomes and defects in Rab7-endosome trafficking.The main goal of this project is to elucidate the pathogenic mechanisms leading to Charcot-Marie-Tooth type 2B, by focusing on mutations in Rab7-positive late endosomes within the endosomal sorting machinery. I will decipher interactions of Rab7-mutations in CMT2B with the cellular sorting machinery and I will follow subcellular localization of activated growth-factor receptors using high resolution imaging to visualize disturbed trafficking routes. I will address this in mouse DRG neurons as well as iPSC-derived motor- and sensory neurons from CMT2B patients. This proposed work will unravel sorting malfunctions in CMT2B and contribute to the evaluation of new therapeutic approaches by revealing possible target proteins.