Characterization of the Cohen syndrome protein COH1 as a membrane-associated scaffold protein for intracellular transport processes in development and maintenance of neuronal cells

细胞内转运控制着广泛的生物过程，如大脑发育和维持。正常大脑功能的核心决定因素是神经元分化和整合成一个有效的网络。研究遗传性神经系统疾病可能有助于确定这些过程的分子机制。常染色体隐性科恩综合征的主要特征是非进行性精神发育迟滞合并出生后小头畸形，由COH 1（VPS 13 B）基因突变引起。COH 1编码3997个氨基酸（450 kDa）长度的蛋白质，与酵母Vps 13 p具有部分序列同源性。最近，我们确定COH 1作为高尔基体相关基质蛋白，调节高尔基体形态和高尔基体相关膜微管活性。我们的初步数据表明，Coh 1是一个积极的调节神经突，这表明科恩综合征是由缺陷的终端神经元分化和整合。为了进一步研究由COH 1控制的分子机制，我们进行了初步的免疫共沉淀，表明COH 1类似于酵母Vps 13 p与高尔基体相关的小GTdR RAB 6和鸟嘌呤核苷酸交换因子亚基RIC 1相互作用。这一结果暗示了一个由COH 1组成的功能网络，本研究的目的是进一步确定COH 1的功能靶点，以完成我们对迄今为止假定的COH 1-RIC 1/RGP 1-RIC 1-RGP 1-RIC 1-RGP-利用不同的生物化学方法，如酵母双杂交筛选、GST-下拉和免疫共沉淀，对RAB 6蛋白复合物进行了研究。在功能上，我们将通过评估高尔基体相关的运输事件及其对神经突延伸和最终突触整合的影响来补充这些生化分析，使用不同的遗传和细胞生物学操作来干扰COH 1，RAB 6，RIC 1和RGP 1的功能以及先进的成像技术。最后，我们的目标是建立一个条件性基因敲除小鼠模型，以证实我们在体外研究与体内分析Coh 1。在拟议的项目中的研究预计将提供深刻的见解，由COH 1控制的分子机制，以促进高尔基体功能和细胞内运输。结合生物化学，遗传学和细胞生物学的方法，确保我们的研究整合，制定科恩综合征的病理机制模型。同时，我们的研究结果将显着有助于一般理解高尔基体相关的运输过程中的终末神经元分化和整合。

Intracellular transport controls a wide spectrum of biological processes such as brain development and maintenance. Core determinants of regular brain function are neuronal differentiation and integration into an efficient network. Studying inherited neurological disorders may help to define molecular mechanisms underlying these processes. The autosomal recessive Cohen syndrome is mainly characterized by non-progressive mental retardation in combination with postnatal microcephaly and is caused by mutations in the COH1 (VPS13B) gene. COH1 encodes a protein of 3997 amino acids (450 kDa) length with partial sequence homology to yeast Vps13p. Recently, we established COH1 as Golgi-associated matrix protein regulating Golgi morphology and Golgi-associated membrane tubulation activity. Our preliminary data show that Coh1 is a positive regulator of neuritogenesis, suggesting that Cohen syndrome is caused by defective terminal neuronal differentiation and integration. To further investigate the molecular mechanisms controlled by COH1, we performed initial co-immunoprecipitations showing that COH1 similar to yeast Vps13p interacts with the Golgi-associated small GTPase RAB6 and the guanine nucleotide exchange factor subunit RIC1. This result implicates a functional network composed of COH1, RAB6 and the heteromeric complex RIC1/RGP1 in mammalian cells and suggests that disturbed Golgi-associated antero- and retrograde intracellular transport processes underlie defective terminal neuronal differentiation and integration in Cohen syndrome.The proposed project aims to identify further functional targets of COH1 to complete our understanding of the hitherto assumed COH1-RIC1/RGP1-RAB6 protein complex using different biochemical approaches, such as yeast-two-hybrid screens, GST-pull down and co-immunoprecipitations. Functionally, we will complement these biochemical analyses by assessment of Golgi-associated transport events and its impact on neurite extension and final synaptic integration, using different genetic and cell biological manipulations to perturb the function of COH1, RAB6, RIC1, and RGP1 and advanced imaging techniques. Finally, we aim to establish a conditional knockout mouse model to corroborate our in vitro studies with in vivo analyses of Coh1. Research within the proposed project is expected to provide profound insights into the molecular machinery that is controlled by COH1 to facilitate Golgi function and intracellular transport. Combined biochemical, genetic and cell biological approaches ensure the integration of our research to formulate a pathomechanistic model of Cohen syndrome. At the same time, our results will significantly contribute to the general comprehension of Golgi-associated transport processes for terminal neuronal differentiation and integration.