Failure in PP2A/mTOR signaling, local protein synthesis and its consequences on functional connectivity in neurons from patients with Opitz BBB/G syndrome

Opitz BBB/G综合征（OS）是一种主要影响腹侧中线结构的单基因疾病。神经系统特征包括智力残疾和精神发育迟缓。OS综合征是由MID 1蛋白的突变引起的，MID 1蛋白已被证明介导蛋白磷酸酶2A（PP 2Ac）的微管相关催化亚基的泛素特异性修饰和降解。在OS患者中发现的突变导致PP 2Ac的积累和微管相关蛋白的低磷酸化。通过抵消mTOR激酶，PP 2A是突触区室中局部蛋白质合成调节的重要参与者，这是长时程增强、学习和记忆的基础。mTOR/PP 2A轴的失调是包括唐氏综合征、脆性X综合征和RETT综合征在内的几种智力残疾综合征的潜在分子机制。OS，MID 1蛋白及其对神经元功能和连接的影响可以教会我们很多关于局部蛋白质合成和mTOR/PP 2A功能的知识，以及它对小鼠和人认知功能的影响。 在携带OS负责基因Mid 1的完全敲除的小鼠模型中，显示出轴突生长的明显缺陷。虽然轴突生长缺陷可以影响OS患者的智力，但OS患者的其他脑缺陷在敲除动物中没有或仅非常模糊地看到。此外，在KO动物中携带人OS突变的Mid 1蛋白的过表达不能挽救表型，但会导致其他缺陷，如发育中的小鼠皮质中的径向迁移缺陷。这使得野生型和突变型MID 1的功能在小鼠和人之间可能不同，并且研究MID 1在人类系统中的作用非常重要。在这个项目中，我们将把一个人类遗传学小组的经验，研究遗传学和生物化学的OS多年和干细胞组，其中有专注于神经干细胞和神经元连接艾德。我们将使用iPS细胞，我们已经从成纤维细胞5 OS患者和同基因对照研究神经发育缺陷OS。我们将分化成神经元前体细胞和2D神经元培养和3D类器官，并将研究这些形态，生化和功能。此外，我们将在神经元培养物中采用基于狂犬病病毒的神经元连接追踪，并在移植iPS衍生神经元后的小鼠皮层中也将使用相同的技术，以研究神经元功能和连接。通过这项工作，我们将能够密切关注mTOR/PP 2A功能障碍对人类神经元形态，功能和连接的影响。这些研究将帮助我们了解mTOR/PP 2A信号如何影响一些基本的大脑功能，如学习和记忆，并导致患者的智力残疾。

Opitz BBB/G syndrome (OS) is a monogenic disorder that mainly affects ventral midline structures. Neurological characteristics include intellectual disability and mental developmental delay. OS syndrome is caused by mutations in the MID1 protein, which has been shown to mediate ubiquitin specific modification and degradation of the microtubule-associated catalytic subunit of protein phosphatase 2A (PP2Ac). Mutations as found in OS patients lead to an accumulation of PP2Ac and a hypophosphorylation of microtubule-associated proteins. By counteracting mTOR kinase PP2A is an important player in the regulation of local protein synthesis in synaptic compartments, which is the basis for long term potentiation, learning and memory. Dysregulation of the mTOR/PP2A axis is the underlying molecular mechanisms of several intellectual disability syndromes including Down syndrome, fragile-X syndrome and RETT syndrome. OS, the MID1 protein and its influence on neuronal function and connectivity can teach us much about local protein synthesis and mTOR/PP2A function on one side and its implications on cognitive (mal-) functions in mouse and man on the other. In a mouse model carrying a full knock-out of the OS responsible gene, Mid1, a pronounced defect in axonal outgrowth was shown. While axonal outgrowth deficiency can influence intellectual abilities in OS patients, other brain defects of OS patients have not or only very vaguely been seen in the knock-out animals. Furthermore, overexpression of Mid1 protein carrying human OS mutations in KO animals does not rescue the phenotype but causes additional defects such as radial migration defects in the developing mouse cortex. This makes it likely that the function of wildtype and mutant MID1 differs between mouse and man and makes it important to study MID1 effects in human systems. In this project we will put together the experience of a human genetics group that has studied genetics and biochemistry of OS for many years and a stem cell group, which has focus¬¬ed on neural stem cells and neuronal connectivity. We will use iPS cells that we have generated from fibroblasts of 5 OS patients and isogenic controls to study neurodevelopmental defects in OS. We will differentiate the cells into neuronal precursors and into 2D neuronal cultures and 3D organoids and will study these morphologically, biochemically and functionally. Furthermore we will employ rabies virus-based tracing of neuronal connectivity in neuronal cultures and will use the same technique also in mouse cortex after transplantation of iPS-derived neurons in order to study neuronal function and connectivity. With this work we will be able to closely look at effects of mTOR/PP2A dysfunction on neuronal morphology, function and connectivity in humans. These studies will help us understand how mTOR/PP2A signalling influences some fundamental brain functions such as learning and memory and causes intellectual disability in patients.