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

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，四肢精细运动控制和感觉丧失。在常染色体显性2B型charcote - marie Tooth （CMT2B）中，疾病是由内体晚期Rab7-GTPase突变引起的。在神经元中，Rab7-核内体对生长因子的运输很重要：在它们各自的配体激活后，生长因子受体被内噬，随后被分类成Rab7阳性核内体并逆行运输到体细胞，在那里它们调节促存活基因的基因表达。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阳性晚期内体的运输损伤联系起来，但是Rab7-GTPase的突变如何促进CMT2B中生长因子受体的运输仍然难以捉摸。我假设CMT2B-Rab7突变体中的核内体表现出干扰的分选能力，这是由于在分选/运输过程中，受体进入的管状微结构域的形成受到干扰。一般认为，分选发生在早期的核内体上，在那里，受体被分选到回收途径中——要么直接回到质膜，要么通过反式高尔基网络（TGN）。然而，我假设这些后期核内体上的管状微结构域也促进受体分选，可能进入溶酶体，这将是一个与核内体成熟过程分离/平行的过程。因此，这种管状微结构域形成的扰动可能会影响从rab7阳性内体到溶酶体的分选，受体信号传导缺陷，受体降解延迟，并最终下调调节神经支配和/或生存的转录基因。所有这些效应可以解释CMT2B中观察到的几种表型，包括生长因子信号传导延长、生长因子向溶酶体分选延迟以及rab7核内体运输缺陷。该项目的主要目标是通过关注内体分选机制中rab7阳性晚期内体的突变，阐明导致Charcot-Marie-Tooth型2B的致病机制。我将破译CMT2B中rab7突变与细胞分选机制的相互作用，我将使用高分辨率成像来可视化干扰的运输路线，跟踪激活的生长因子受体的亚细胞定位。我将在小鼠DRG神经元以及来自CMT2B患者的ipsc衍生的运动和感觉神经元中解决这个问题。这项工作将揭示CMT2B的分类故障，并通过揭示可能的靶蛋白，有助于评估新的治疗方法。

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.