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）：神经元之间信息的有效传输发生在专门的细胞触点位点，称为突触。即使是突触强度的微妙变化也会干扰神经元电路，并引起精神病，神经学或神经退行性疾病。有效的突触传播需要Ca2+触发的突触囊泡（SV）融合以及随后有效的SV内吞作用来快速神经递质分泌。从理论上讲，“锥形”脂质可以在SV融合和裂变过程中有助于极端膜曲率。我们假设至少某些P4-ATP酶可以通过局部易位（翻转）特定的脂质从膜的外部移向膜，可以在SV融合和/或裂变过程中有助于膜曲率。我们的初步结果表明，情况确实如此。利用遗传模型系统果蝇，我们已经确定了4个寄生虫人ATP8B1-4 Flippases的果蝇直系同源物（DATP8B）中的突变。 DatP8B的删除会损害生存力，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，我们获得了将DATP8B与E3泛素连接酶UBE3A联系起来的遗传证据，后者的功能障碍会导致Angelman综合征，Angelman综合征是一种遗传性神经系统疾病，导致智力低下。为了获得重要的数据和工具，以获取大规模的联邦资金来测试DATP8B的突触作用，我们建议在AIM 1中生成抗体和标记的转基因，以解决组织特异性的表达模式和DatP8B的突触定位及其推定的COCEFACTOR COCERTOR DCDC50。 AIM 2将在体内和原位“脂质Flippase分析”中确定DatP8B是否介导培养的神经元和突触末端的脂质翻转。确认Flippase活性和DATP8B的突触定位将为稍后测试脂质翻转如何促进SV融合或裂变提供关键基础。 AIM 3将确定DCDC50和P4-ATPASES DATP8A，DATP9，DATP10和DATP11是否需要神经元和/或突触功能。这项“调查”是合理的，因为尽管有些人与阿尔茨海默氏病，自闭症或安吉尔曼综合症有关，但尚不清楚这些P4-ATP酶是否需要神经元功能。这些目标共同提供关键的初步数据和工具，例如抗体和转基因动物，以成功获得大规模的联邦资金，以严格测试脂质Flippases对神经元和突触功能的重要性和作用。对有关突触功能的新组成部分的提议分析不仅会推进我们的基本知识，而且还可以对人脑疾病中同源蛋白的病理学有关键的见解，例如自闭症和安吉尔曼综合症。公共卫生相关性：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。预计该项目的成功完成将显着提高我们对神经细胞通信基础的分子机制的理解，并提供有关这些机制失败如何引起智力低下的见解。这项工作的结果可能与理解Angelman综合征（AS）的功能相关，Angelman综合征是一种遗传性神经系统疾病，其特征是智力低下，言语最少，运动协调的困难和其他缺陷。