Neuronal Role of Lipid Flippases

描述（由申请人提供）：神经元之间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回路，导致精神、神经或神经退行性疾病。有效的突触传递需要通过Ca2+触发的突触囊泡（SV）融合和随后有效的SV内吞作用快速分泌神经递质。理论上，“锥形”脂质可能有助于SV聚变和裂变过程中的极端膜曲率。我们假设在SV融合和/或裂变过程中，至少一些p4 - atp酶可能通过局部将特定的脂质从膜的外层转移到膜的内部小叶来帮助膜曲率。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个相似的人类ATP8B1-4翻转酶在果蝇同源（dATP8B）上的突变。dATP8B的缺失会损害生存能力、运动能力和SV的外显和内吞作用，这表明它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，该酶的功能障碍导致Angelman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，以获得大规模的联邦资助来测试dATP8B的突触作用，我们在Aim 1中建议产生抗体和标记转基因来解决组织特异性表达模式和dATP8B及其推定的辅助因子dCdc50的突触定位。目的2将在体内和原位建立“脂质翻转酶实验”，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的突触定位将为随后测试脂质翻转如何促进SV融合或裂变提供关键基础。目的3将确定dCdc50和P4-ATPases dATP8A、dATP9、dATP10和dATP11是否为神经元和/或突触功能所必需。这项“调查”是合理的，因为目前尚不清楚这些p4 - atp酶是否与神经元功能有关，尽管它们与阿尔茨海默病、自闭症或天使综合症有关。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，以成功获得大规模的联邦资金，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推进我们的基础知识，而且可能对人类大脑疾病（如自闭症和天使综合症）中同源蛋白的病理产生关键的见解。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。该项目的成功完成有望大大提高我们对神经细胞通讯的分子机制的理解，并为这些机制的失败如何导致智力迟钝提供见解。这项工作的结果可能在功能上与理解Angelman综合征（AS）有关，这是一种遗传性神经系统疾病，其特征是智力迟钝，言语障碍，运动协调困难和其他缺陷。

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.