Role of ATAD3A in Lysosomal Homeostasis and Neurogenesis

项目摘要/摘要 ATAD3A(ATP家族aaa域包含蛋白3A)是一种与线粒体有关的膜蛋白 在线粒体膜动力学中。我们发现ATAD3A的显性突变导致人类 以早发性周围神经病、视神经萎缩和轻度脑为特征的神经综合征 畸形。ATAD3A功能缺失突变的患者继续被发现，表现为 严重的神经发育缺陷，支持这种蛋白质对人类健康的重要性。然而， 在细胞和分子水平上导致这种综合征的根本原因以及改善症状的策略 仍然是悬而未决的问题。 我们的长期目标是确定ATAD3A在发育和代谢稳态中的作用 作为治疗ATAD3A相关疾病患者的基础。我们的目标是 建议揭示ATAD3A控制营养感知(即mTORC1)、溶酶体的机制 果蝇和ATAD3A患者来源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ATAD3A在mTORC1信号转导和溶酶体生物发生中起关键作用 通过RAG GTP酶的调节，ATAD3A依赖的营养感知和溶酶体动态平衡 是正常的神经发生和发育所必需的，基于以下令人信服的证据。简单地说， 利用IP-MS和co-IP，我们鉴定了ATAD3A的内源性结合伙伴，包括溶酶体 RagD蛋白，激活mTORC1所需的GTP酶，以及MIT-TFE蛋白，转录因子 溶酶体的生物发生。我们发现ATAD3A与活性的Rag GTP酶和MIT-TFE蛋白形成了一个复合体。 这一发现有助于解释我们的发现，果蝇携带显性负ATAD3A突变 (R528W)表现出营养感知缺陷(牵涉RAG/mTORC1)，并异常升高溶酶体 发育中神经元的含量(与MITF有关)。在果蝇中，我们发现ATAD3A零突变导致 胚胎致死，中枢和外周神经元图案和形态异常。此外，我们 研究发现，从患者的ipscs中提取的脑器官的大小明显小于从患者中提取的。 来自同基因对照。我们将通过执行以下具体目标来验证我们的中心假设：(1) 确定ATAD3A如何调节mTORC1信号；(2)确定ATAD3A突变如何导致 神经元中溶酶体生物发生异常；(3)确定ATAD3A缺失如何导致神经发生缺陷。 这些研究将表征一种新的线粒体-溶酶体-mTORC1信号轴，它应该 揭示构成ATAD3A相关的患者神经元细胞缺陷的新分子见解 神经系统疾病。我们期待着神经疾病潜在治疗靶点的确定。 不仅与ATAD3A突变有关，而且与线粒体和溶酶体稳态缺陷有关。

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.