Role of ATAD3A in Lysosomal Homeostasis and Neurogenesis

项目总结/摘要 ATAD 3A（ATP家族AAA-结构域包含蛋白3A）是一种线粒体膜蛋白， 在线粒体膜动力学中。我们发现ATAD 3A的显性突变导致人类 以早发性周围神经病变、视神经萎缩和轻度脑损伤为特征的神经系统综合征 畸形ATAD 3A功能缺失突变患者继续被鉴定，表现为 严重的神经发育缺陷，支持这种蛋白质在人类健康中的重要性。但 在细胞和分子水平上的这种综合征的根本原因，以及改善症状的策略 仍然是未解决的问题。 我们的长期目标是确定ATAD 3A在发育和代谢稳态中的作用 作为治疗患有ATAD 3A相关疾病的患者的疗法的基础。我们的目标是 一项提案是揭示ATAD 3A控制营养感应（即mTORC 1）、溶酶体和细胞的机制。 使用果蝇和ATAD 3A患者衍生的诱导多能干细胞的生物发生和神经元发育 细胞我们的中心假设是ATAD 3A在mTORC 1信号传导和溶酶体生物发生中起关键作用 通过Rag GT3调节，ATAD 3A依赖的营养传感和溶酶体稳态 是正常神经发生和发育所必需的，基于以下令人信服的证据。简言之， 使用IP-质谱和co-IP，我们鉴定了ATAD 3A的内源性结合伴侣，包括溶酶体 RagD蛋白，一种激活mTORC 1所需的GT3，和MiT-TFE蛋白，一种转录因子， 溶酶体生物发生我们发现ATAD 3A与活性Rag GTP酶和MiT-TFE蛋白形成复合物。 这一发现有助于解释我们的发现，携带显性负性ATAD 3A突变的果蝇 （R528 W）表现出营养感测缺陷（涉及Rag/mTORC 1），以及异常升高的溶酶体 发育中的神经元中的含量（涉及MITF）。在果蝇中，我们发现ATAD 3A无效突变导致 胚胎致死，中枢和外周神经元的模式和形态异常。另外我们 发现从患者iPSC中获得的脑类器官的大小明显小于从患者iPSC中获得的脑类器官的大小。 与同基因对照相比我们将通过执行以下具体目标来检验我们的中心假设：（1） 确定ATAD 3A如何调节mTORC 1信号传导;（2）确定ATAD 3A突变如何导致 神经元中溶酶体生物发生异常;（3）确定ATAD 3A缺失如何导致神经发生缺陷。 这些研究将表征一种新的α-溶酶体-mTORC 1信号传导轴， 揭示了患者神经元中细胞缺陷的新分子见解，这些细胞缺陷是ATAD 3A相关的 神经系统疾病。我们期待着神经系统疾病的潜在治疗靶点的确定 这不仅与ATAD 3A突变有关，而且与线粒体和溶酶体内稳态的缺陷有关。

PROJECT SUMMARY/ABSTRACT ATAD3A (ATP family AAA-domain containing protein 3A) is a mitochondrial membrane protein that is implicated in mitochondrial membrane dynamics. We discovered that dominant mutations in ATAD3A cause a human neurological syndrome characterized by early-onset peripheral neuropathy, optic atrophy and mild brain malformation. Patients with loss of function mutations in ATAD3A continue to be identified, presenting with severe neurodevelopmental defects, supporting the importance of this protein in human health. However, the root cause of this syndrome at the cellular and molecular levels, as well as strategies to ameliorate the symptoms remain unsolved issues. Our long-term goal is to determine the roles of ATAD3A in development and in metabolic homeostasis as the basis for therapies to treat patients suffering from ATAD3A-associated diseases. The objective of our proposal is to uncover the mechanisms by which ATAD3A controls nutrition sensing (i.e. mTORC1), lysosomal biogenesis and neuronal development using Drosophila and ATAD3A patient-derived induced pluripotent stem cells. Our Central Hypothesis is that ATAD3A plays a key role in mTORC1 signaling and lysosomal biogenesis through Rag GTPase modulation, and that ATAD3A-dependent nutrition sensing and lysosomal homeostasis are required for proper neurogenesis and development based on the following compelling evidence. Briefly, using IP-mass spec and co-IP, we identified endogenous binding partners of ATAD3A, including the lysosomal proteins RagD, a GTPase required for activating mTORC1, and MiT-TFE proteins, transcriptional factors for lysosomal biogenesis. We found that ATAD3A forms a complex with active Rag GTPases and MiT-TFE proteins. This finding helped explain our discovery that Drosophila bearing a dominant negative ATAD3A mutation (R528W) exhibit defects in nutrition sensing (implicating Rag/mTORC1), and aberrantly elevated lysosomal content in developing neurons (implicating MITF). In Drosophila, we found that ATAD3A null mutations caused embryonic lethality with abnormal patterning and morphology of central and peripheral neurons. In addition, we found that the sizes of brain organoids derived from the patient iPSCs are significantly smaller than those derived from isogenic controls. We will test our central hypothesis by performing the following Specific Aims: (1) to determine how ATAD3A regulates mTORC1 signaling; (2) to determine how ATAD3A mutations lead to abnormal lysosomal biogenesis in neurons; (3) to determine how ATAD3A loss causes neurogenesis defects. These studies will characterize a novel axis of mitochondria-lysosomal-mTORC1 signaling that should reveal novel molecular insights into the cellular defects in patient neurons that underlie ATAD3A-associated neurological diseases. We anticipate the identification of potential therapeutic targets for neurological diseases associated not only with ATAD3A mutations, but also with defects in mitochondrial and lysosomal homeostasis.