Neuronal Role of Lipid Flippases

脂质翻转酶的神经元作用

• 批准号: 7771039
• 负责人: KONRAD ERNST ZINSMAIER
• 金额: $ 7.56万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-03 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7771039
• 关键词: ATP phosphohydrolase; Alzheimer' s Disease; Angelman Syndrome; Antibodies; Autistic Disorder; Behavior; Biological Assay; Biological Models; Brain; Brain Diseases; Cell Communication; Cell Culture Techniques; Cells; Chromaffin granule; Data; Drosophila genus; Endocytosis; Event; Exocytosis; Failure; Family; Foundations; Functional disorder; Funding; Genetic; Genetic Models; Goals; Homologous Protein; Human; In Situ; Inherited; Knowledge; Larva; Lipids; Locomotion; Mediating; Membrane; Mental Retardation; Molecular; Motor; Mutation; Neurodegenerative Disorders; Neurologic; Neurons; Neurotransmitters; Orthologous Gene; Pathology; Pattern; Presynaptic Terminals; Proteins; RNA Interference; Research; Retinal Cone; Role; Shapes; Side; Site; Speech; Surveys; Synapses; Synaptic Transmission; Synaptic Vesicles; Synaptosomes; Testing; Tissues; Transgenes; Transgenic Animals; Work; fly; in vivo; insight; member; mutant; nervous system disorder; overexpression; presynaptic; public health relevance; synaptic function; tool; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): The efficient and stable transfer of information among neurons occurs at specialized cell-cell contact sites, called synapses. Even subtle changes in synaptic strength can disturb neuronal circuits and cause psychiatric, neurological, or neurodegenerative disorders. Effective synaptic transmission requires fast neurotransmitter secretion by Ca2+ triggered synaptic vesicle (SV) fusion and subsequently effective SV endocytosis. It has been theorized that "cone-shaped" lipids may aid the extreme membrane curvatures during SV fusion and fission. We hypothesize that at least some P4-ATPases may aid membrane curvatures during SV fusion and/or fission by locally translocating (flipping) specific lipids from the outer to the inner leaflet of the membrane. Our preliminary results suggest that this may be indeed the case. Taking advantage of the genetic model system Drosophila, we have identified mutations in the Drosophila ortholog (dATP8B) of the 4 paralogous human ATP8B1-4 flippases. Deletion of dATP8B impairs viability, locomotion and SV exo- and endocytosis, suggesting a critical for synaptic function. In addition, we obtained genetic evidence that ties dATP8B to the E3 ubiquitin ligase UBE3A, whose dysfunction causes Angelman Syndrome, an inherited neurological disorder leading to mental retardation. To gain critical data and tools for obtaining large-scale federal funding to test the synaptic role of dATP8B, we suggest in Aim 1 to generate antibodies and tagged transgenes to resolve the tissue-specific expression pattern and synaptic localization of dATP8B and its putative co-factor dCdc50. Aim 2 will establish in vivo and in situ "lipid flippase assays" to determine whether dATP8B mediates lipid flipping in cultured neurons and at synaptic terminals. Confirming flippase activity and a synaptic localization of dATP8B will provide a critical foundation to later test how lipid flipping promotes SV fusion or fission. Aim 3 will determine whether dCdc50 and the P4-ATPases dATP8A, dATP9, dATP10, and dATP11 are required for neuronal and/or synaptic function. This "survey" is justified since it is not known whether these P4-ATPases are required for neuronal function despite the association of some with Alzheimer's disease, Autism or Angelman syndrome. Together, these aims will provide critical preliminary data and tools like antibodies and transgenic animals to successfully obtain large-scale federal funding to rigorously test the significance and role of lipid flippases for neuronal and synaptic function. The proposed analysis of new components governing synaptic function will not only advance our basic knowledge but may also yield critical insights into the pathologies of homologous proteins in human brain disorders, like Autism and Angelman Syndrome. PUBLIC HEALTH RELEVANCE: Transmitting information from one nerve cell to another is critical for brain function. Successful completion of the project is expected to significantly advance our understanding of molecular mechanisms underlying nerve cell communication and provide insights into how failure of these mechanisms causes mental retardation. Results from this work are likely functionally relevant for understanding Angelman Syndrome (AS), an inherited neurological disorder that is characterized by mental retardation, minimal speech, difficulties in motor coordination, and other deficiencies.

描述（由申请人提供）：神经元之间有效且稳定的信息传递发生在称为突触的特殊细胞与细胞接触位点。即使突触强度的细微变化也会扰乱神经元回路并导致精神、神经或神经退行性疾病。有效的突触传递需要 Ca2+ 触发突触小泡 (SV) 融合以及随后有效的 SV 内吞作用来快速分泌神经递质。据推测，“锥形”脂质可能有助于 SV 融合和裂变过程中的极端膜曲率。我们假设至少一些 P4-ATP 酶可能通过局部易位（翻转）特定脂质从膜的外层到内层的方式在 SV 融合和/或裂变过程中帮助膜弯曲。我们的初步结果表明情况可能确实如此。利用果蝇遗传模型系统，我们鉴定了 4 个旁系同源人类 ATP8B1-4 翻转酶的果蝇直系同源物 (dATP8B) 中的突变。 dATP8B 的缺失会损害活力、运动以及 SV 外吞和内吞作用，这表明对突触功能至关重要。此外，我们还获得了将 dATP8B 与 E3 泛素连接酶 UBE3A 联系起来的遗传证据，UBE3A 的功能障碍会导致天使综合征，这是一种导致智力迟钝的遗传性神经系统疾病。为了获得关键数据和工具来获得大规模联邦资金来测试 dATP8B 的突触作用，我们建议在目标 1 中生成抗体和标记转基因，以解决 dATP8B 及其假定的辅因子 dCdc50 的组织特异性表达模式和突触定位。目标 2 将建立体内和原位“脂质翻转酶测定”，以确定 dATP8B 是否介导培养神经元和突触末端的脂质翻转。确认翻转酶活性和 dATP8B 的突触定位将为后续测试脂质翻转如何促进 SV 融合或裂变提供关键基础。目标 3 将确定神经元和/或突触功能是否需要 dCdc50 和 P4-ATP 酶 dATP8A、dATP9、dATP10 和 dATP11。这项“调查”是合理的，因为目前尚不清楚这些 P4-ATP 酶是否是神经元功能所必需的，尽管某些 P4-ATP 酶与阿尔茨海默病、自闭症或天使综合征有关。总之，这些目标将提供关键的初步数据和工具，如抗体和转基因动物，以成功获得大规模联邦资金，以严格测试脂质翻转酶对神经元和突触功能的重要性和作用。对控制突触功能的新成分的分析不仅将增进我们的基础知识，而且还可能对人类大脑疾病（如自闭症和天使综合症）中同源蛋白的病理学产生重要的见解。公共健康相关性：将信息从一个神经细胞传输到另一个神经细胞对于大脑功能至关重要。该项目的成功完成预计将显着增进我们对神经细胞通讯分子机制的理解，并深入了解这些机制的失败如何导致智力低下。这项工作的结果可能在功能上与理解天使综合症（AS）有关，这是一种遗传性神经系统疾病，其特征是智力低下、言语能力低下、运动协调困难和其他缺陷。