Regulation of autophagy and ubiquitination by ARHGAP33/NOMA-GAP during normal and pathological development of the mammalian neocortex.

ARHGAP33/NOMA-GAP 在哺乳动物新皮质正常和病理发育过程中对自噬和泛素化的调节。

• 批准号: 246199359
• 负责人: Professorin Dr. Marta de Rocha Rosário
• 金额: --
• 依托单位: Institut für Zell- und Neurobiologie (CCM)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/246199359?language=en
• 关键词: Regulation; autophagy; ubiquitination; ARHGAP33; NOMA

The cerebral neocortex processes higher brain functions such as decision-making and voluntary movement. The excitatory neurons of the neocortex possess highly elaborate dendritic trees with spines that form synaptic connections. Defects in the maturation of dendritic trees and their spines impair connectivity and information processing and can lead to neurodevelopmental disorders such as intellectual disability and autism spectrum disorders.We have identified a signalling protein, NOMA-GAP, encoded by the ARHGAP33 gene, that regulates dendritic maturation in the neocortex. Loss of NOMA-GAP results in simplified dendritic trees with immature spines and is associated with autism-like behavior in mice. Furthermore, a collaboration with clinical geneticists, has revealed first mutations in the human ARHGAP33 gene that are associated with severe intellectual disability. In preliminary work we have uncovered structural alterations in the dendrites of young NOMA-GAP-deficient mice previously only observed in aged rodents or in degenerative conditions such as Alzheimer’s disease. These autophagy-linked disturbances represent a previously unrecognized mechanism for pathological development of the neocortex. The goal of the current project is to identify disease-relevant cellular and molecular mechanisms by which NOMA-GAP promotes dendritic maturation during neocortex development.Our first step is to identify the cellular mechanism underlying developmental autophagy arrest. We will investigate the different stages of this protein/membrane degradation process to elucidate the function of NOMA-GAP and its disease variants.In a second step, we will build on further preliminary work. Through IP mass spectrometry screens, we have identified a novel function for NOMA-GAP in the regulation of ubiquitination. Ubiquitination is a protein modification that alters protein function or degradation and can promote autophagy. We will investigate the interaction between NOMA-GAP and its disease-variants with a new candidate molecule involved in ubiquitination and address the consequences of de-regulation of this pathway on developmental autophagy and dendritic maturation.To this end, we will use reporter assays, live imaging, enzymatic assays and co-localization studies, together with knockout mouse lines (deficient in expression of the candidate and/or NOMA-GAP), in utero electroporation and transfection of primary neurons. In addition, we will perform IP mass spectrometric analysis of changes in protein complex formation and in the ubiquitome of in these mice to reveal the functional and molecular significance. This project will deepen our understanding of neocortex development and reveal a new cellular and molecular mechanism for neurodevelopmental disorders.

大脑新皮层负责处理高级大脑功能，如决策和自主运动。新皮质的兴奋性神经元拥有高度复杂的树突树，树突树的棘形成突触连接。树突树及其棘成熟过程中的缺陷会损害连通性和信息处理，并可能导致神经发育障碍，如智力残疾和自闭症谱系障碍。我们已经确定了一个信号蛋白，NOMA-GAP，由ARHGAP33基因编码，调节新皮层树突成熟。NOMA-GAP的缺失导致树突树的简化和未成熟的棘，并与小鼠的自闭症样行为有关。此外，与临床遗传学家合作发现了人类ARHGAP33基因的首次突变，该突变与严重的智力残疾有关。在初步工作中，我们发现了年轻的noma - gap缺陷小鼠树突的结构改变，以前只在老年啮齿动物或阿尔茨海默病等退行性疾病中观察到。这些自噬相关的紊乱代表了一种以前未被认识到的新皮层病理发育机制。当前项目的目标是确定NOMA-GAP在新皮层发育过程中促进树突成熟的疾病相关细胞和分子机制。我们的第一步是确定发育性自噬阻滞的细胞机制。我们将研究这种蛋白质/膜降解过程的不同阶段，以阐明NOMA-GAP及其疾病变体的功能。第二步，我们将以进一步的初步工作为基础。通过IP质谱筛选，我们已经确定了NOMA-GAP在泛素化调控中的新功能。泛素化是一种改变蛋白质功能或降解的蛋白质修饰，可以促进自噬。我们将研究NOMA-GAP及其疾病变体与泛素化的新候选分子之间的相互作用，并解决该途径的去调控对发育性自噬和树突成熟的影响。为此，我们将使用报告基因分析、活体成像、酶促分析和共定位研究，以及敲除小鼠系（缺乏候选基因和/或NOMA-GAP的表达），在子宫内电穿孔和转染初级神经元。此外，我们将对这些小鼠中蛋白质复合物形成和泛素组的变化进行IP质谱分析，以揭示其功能和分子意义。该项目将加深我们对新皮层发育的理解，揭示神经发育障碍的新的细胞和分子机制。