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

大脑皮层处理更高级的大脑功能，如决策和自主运动。新皮质的兴奋性神经元具有高度精细的树突树，树上有形成突触连接的棘突。树突状细胞及其棘突的成熟缺陷会损害连接和信息处理，并可能导致智能障碍和自闭症谱系障碍等神经发育障碍。我们已经确定了一种由ARHGAP33基因编码的信号蛋白NOMA-GAP，它调节新皮质中的树突状细胞成熟。Noma-Gap的丢失导致简化的树突状结构树具有未成熟的脊椎，并与小鼠的自闭症样行为有关。此外，与临床遗传学家的合作发现，人类ARHGAP33基因首次发生突变，与严重的智力残疾有关。在初步工作中，我们发现了幼年Noma-GAP缺陷小鼠树突的结构变化，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。这些自噬相关的干扰代表了一种以前未知的新皮质病理发育机制。本项目的目标是确定NOMA-GAP在新皮质发育过程中促进树突状细胞成熟的与疾病相关的细胞和分子机制。我们的第一步是确定发育自噬停止的细胞机制。我们将研究这一蛋白质/膜降解过程的不同阶段，以阐明Noma-GAP及其疾病变体的功能。在第二步，我们将在进一步的前期工作基础上再接再厉。通过IP质谱屏，我们已经确定了NOMA-GAP在泛素化调节中的新功能。泛素化是一种蛋白质修饰，可以改变蛋白质的功能或降解，并能促进自噬。我们将研究Noma-GAP及其疾病变体与一个新的参与泛素化的候选分子之间的相互作用，并解决这一途径的去调控对发育自噬和树突状细胞成熟的影响。为此，我们将使用报告分析、实时成像、酶分析和共定位研究，以及(候选和/或Noma-GAP表达缺失的)小鼠品系(候选和/或Noma-GAP表达缺失)，宫内电穿孔和原代神经元转染。此外，我们还将对这些小鼠的蛋白质复合体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。这个项目将加深我们对新皮质发育的理解，并揭示神经发育障碍的新的细胞和分子机制。

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.