Neuronal Role of Lipid Flippases

脂质翻转酶的神经元作用

• 批准号: 7996028
• 负责人: KONRAD ERNST ZINSMAIER
• 金额: $ 7.58万
• 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/7996028
• 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; Health; Homologous Protein; Human; In Situ; Inherited; Knowledge; Larva; Lipids; Locomotion; Mediating; Membrane; Mental Retardation; Mental disorders; Molecular; Motor; Mutation; Neurodegenerative Disorders; 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; 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）：神经元之间的有效和稳定的信息传递发生在专门的细胞-细胞接触部位，称为突触。即使是突触强度的细微变化也会扰乱神经元回路，导致精神病、神经病或神经退行性疾病。有效的突触传递需要通过Ca 2+触发的突触囊泡（SV）融合和随后有效的SV内吞作用的快速神经递质分泌。理论上，“锥形”脂质可能有助于SV融合和分裂期间的极端膜曲率。我们推测，至少有一些P4-ATP酶可能有助于SV融合和/或分裂过程中的膜曲率局部易位（翻转）特定的脂质从外到内小叶的膜。我们的初步结果表明，情况可能确实如此。利用果蝇的遗传模型系统，我们已经确定了4个旁系同源人类ATP 8B 1 -4翻转酶的果蝇直系同源物（dATP 8B）中的突变。dATP 8B的缺失损害活力、运动和SV胞吞和胞吞，这表明对突触功能至关重要。此外，我们获得了将dATP 8B与E3泛素连接酶UBE 3A联系在一起的遗传证据，UBE 3A的功能障碍导致安格尔曼综合征，这是一种导致智力迟钝的遗传性神经系统疾病。为了获得大规模联邦资助以测试dATP 8 B的突触作用的关键数据和工具，我们在目标1中建议产生抗体和标记的转基因以解析dATP 8 B及其推定的辅因子dCdc 50的组织特异性表达模式和突触定位。目的2建立体内和原位的脂质翻转酶实验，以确定dATP 8B是否介导了培养神经元和突触终末的脂质翻转。明确翻转酶活性和dATP 8B的突触定位将为以后测试脂质翻转如何促进SV融合或分裂提供关键基础。目的3将确定dCdc 50和P4-ATP酶dATP 8A、dATP 9、dATP 10和dATP 11是否是神经元和/或突触功能所必需的。这项“调查”是合理的，因为它是不知道是否需要这些P4-ATP酶的神经功能，尽管一些与阿尔茨海默氏症，自闭症或安格尔曼综合征。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，以成功获得大规模的联邦资金，严格测试脂质翻转酶对神经元和突触功能的意义和作用。对控制突触功能的新成分的拟议分析不仅将推进我们的基础知识，而且还可能对人类大脑疾病（如自闭症和安格尔曼综合征）中同源蛋白质的病理学产生重要的见解。公共卫生相关性：将信息从一个神经细胞传递到另一个神经细胞对大脑功能至关重要。该项目的成功完成预计将显着推进我们对神经细胞通信的分子机制的理解，并提供对这些机制的失败如何导致精神发育迟滞的见解。这项工作的结果可能与理解Angelman综合征（AS）功能相关，这是一种遗传性神经系统疾病，其特征是精神发育迟滞，语言障碍，运动协调困难和其他缺陷。