Neuronal Role of Lipid Flippases

描述(申请人提供)：神经元之间高效而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、神经或神经退行性疾病。有效的突触传递需要通过钙离子触发的突触小泡(SV)融合和随后有效的SV内吞来快速分泌神经递质。已有理论认为，在SV融合和分裂过程中，“锥形”脂质可能有助于极大的膜曲率。我们推测，在SV融合和/或分裂过程中，至少有一些P4-ATPase可能通过局部将特定的脂类从膜的外部转移(翻转)到膜的内部小叶来帮助膜的曲率。我们的初步结果表明，情况可能的确如此。利用遗传模型系统果蝇，我们已经鉴定了4个近缘人类ATP8B1-4触发器的果蝇直系同源基因(DATP8B)的突变。DATP8B的缺失会损害细胞的活力、运动和SV的内外吞作用，提示这是突触功能的关键。此外，我们还获得了将dATP8B与E3泛素连接酶UBE3A联系在一起的遗传证据，UBE3A的功能障碍会导致Angelman综合征，这是一种导致智力低下的遗传性神经疾病。为了获得关键的数据和工具，以获得大规模的联邦资金来测试dATP8B的突触作用，我们在目标1中建议产生抗体和标记的转基因来解决dATP8B及其假定的辅助因子dCDC50的组织特异性表达模式和突触定位。目的建立体内和原位的“脂质翻转酶实验”，以确定dATP8B是否在培养的神经元和突触终末介导脂质翻转。确认dATP8B的Flippase活性和突触定位将为稍后测试脂质翻转如何促进SV融合或分裂提供关键基础。AIM 3将确定dCDC50和P4-ATPase dATP8A、dATP9、dATP10和dATP11是否是神经元和/或突触功能所必需的。这项“调查”是合理的，因为目前尚不清楚这些P4-ATPase是否是神经元功能所必需的，尽管有些P4-ATPase与阿尔茨海默病、自闭症或安杰曼综合症有关。总而言之，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。对支配突触功能的新成分的拟议分析不仅将增进我们的基本知识，还可能对自闭症和安杰曼综合征等人类大脑疾病中同源蛋白的病理机制产生关键的见解。与公共健康相关：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极大地促进我们对神经细胞通讯背后的分子机制的理解，并为这些机制的失败如何导致智力低下提供见解。这项工作的结果可能在功能上与理解Angelman综合征(AS)有关，AS是一种遗传性神经疾病，其特征是智力低下、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.