Presynaptic regulation of quantal size by the cation/H+ exchangers NHE6 & NHE9

阳离子/H 交换器 NHE6 对量子大小的突触前调节

• 批准号: 8256121
• 负责人: Julie C Ullman
• 金额: $ 3.35万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2013-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8256121
• 关键词: Acridine Orange; Address; Adult; Affect; Angelman Syndrome; Anions; Attention; Autistic Disorder; Behavior; Behavioral; Biochemical; Biological Assay; Brain; Brain region; Cations; Cell physiology; Chemicals; Clinical; Complement; Corpus striatum structure; Defect; Development; Developmental Delay Disorders; Diagnostic; Disease; Dopamine; Exhibits; Family; Functional disorder; Gene Mutation; Genes; Genetic; Glutamates; Heterogeneity; Hippocampus (Brain); Human; Human Genetics; Individual; Knock-out; Knockout Mice; Life; Measures; Membrane Potentials; Molecular; Mus; Mutation; Neurobiology; Neurons; Neurotransmitters; Pathway interactions; Patients; Phenotype; Physiological; Process; Protein Isoforms; Proteins; Protons; Rattus; Regulation; Role; Seizures; Slice; Societies; Synapses; Synaptic Transmission; Synaptic Vesicles; Syndrome; System; Testing; Therapeutic Intervention; Time; United States; Vesicle; Work; autism spectrum disorder; autistic behaviour; base; communication behavior; driving force; gamma-Aminobutyric Acid; genetic analysis; monoamine; neurotransmission; neurotransmitter release; neurotransmitter uptake; novel; pH gradient; postsynaptic; presynaptic; programs; radiotracer; response; skills; uptake

Project Summary Julie Ullman Recent human genetic analysis has found that mutations in the cation/proton exchangers NHE6 and NHE9 both result in autism. Little is known about these two closely related proteins or their function in neurons. Interestingly, recent work from the Edwards lab has identified a novel NHE activity on synaptic vesicles. This activity promotes the uptake of excitatory neurotransmitter glutamate into vesicles by converting the vesicle pH gradient (PhipH) into membrane potential (PhiPsi). Furthermore, preliminary work has localized NHE6 and NHE9 to presynaptic boutons and in particular synaptic vesicles, suggesting that these isoforms are responsible for the observed activity. The uptake of different transmitters depends to varying extents on either PhipH or PhiPsi, and hence loss of NHE6 or NHE9 would be predicted to have different effects on different transmitters. The hypothesis of this proposal is that NHE6 and 9 convert PhipH into PhiPsi and thus influence the filling of synaptic vesicles with neurotransmitter, with global synaptic effects that might underlie the severe phenotype observed in patients with mutations in NHE6 or 9 and perhaps other forms of autism. This proposal seeks to characterize the role of NHE6 and NHE9 at multiple physiological levels: synaptic vesicle function, neural transmission and behavior. At the level of synaptic vesicle function, I will first determine whether NHE6 and 9 target to distinct subsets of synaptic vesicles through immunoisolation of synaptic vesicles containing NHE6 and 9 from mouse brain, and characterization of the respective synaptic vesicle proteins. Next, synaptic vesicles isolated from the brains of NHE6 knock-out (KO) and NHE9 conditional KO (cKO) mice will be used to assess the functional role of these two isoforms by measuring the effect of cations (K[+] and Na[+]) on vesicle pH using acridine orange, on membrane potential using oxonol V and on transmitter uptake by radiotracer flux assay. The pH of synaptic vesicles will be measured in live neurons through the culture of primary dissociated KO neurons. At the level of synaptic transmission, analysis of the spontaneous and evoked release of glutamate, GABA and dopamine in slices from KO mice will indicate the role of NHE6 and 9 in quantal size, transmitter release and/or the postsynaptic response. Finally, this proposal seeks to characterize the specific autistic behaviors that result from loss of NHE9, focusing on the three main diagnostic criteria for autism: sociability, repetitive behaviors and communication skills. This work will thus test the hypothesis that the roles of NHE6 and 9 in synaptic vesicle filling, neurotransmitter release and synaptic transmission give rise to the effects on behavior. Ultimately, the correlation of physiological defects with autistic behavior has great potential to elucidate the systems as well as molecular basis for autism, which would in turn implicate these mechanisms as targets for rational, therapeutic intervention.

项目概要 朱莉·乌尔曼 最近的人类基因分析发现，阳离子/质子交换器 NHE6 和 NHE9 的突变都会导致自闭症。人们对这两种密切相关的蛋白质或其在神经元中的功能知之甚少。有趣的是，Edwards 实验室最近的工作发现了突触小泡上的一种新的 NHE 活性。该活性通过将囊泡 pH 梯度 (PhipH) 转化为膜电位 (PhiPsi)，促进囊泡摄取兴奋性神经递质谷氨酸。此外，初步工作已将 NHE6 和 NHE9 定位于突触前小泡，特别是突触小泡，表明这些亚型是观察到的活动的原因。不同递质的吸收在不同程度上取决于 PhipH 或 PhiPsi，因此 NHE6 或 NHE9 的损失预计会对不同递质产生不同的影响。该提议的假设是，NHE6 和 9 将 PhipH 转化为 PhiPsi，从而影响突触小泡的神经递质填充，整体突触效应可能是在 NHE6 或 9 突变以及其他形式的自闭症患者中观察到的严重表型的基础。该提案旨在描述 NHE6 和 NHE9 在多个生理水平上的作用：突触小泡功能、神经传递和行为。在突触小泡功能水平上，我将首先通过免疫分离小鼠大脑中含有 NHE6 和 9 的突触小泡，并表征各自的突触小泡蛋白，确定 NHE6 和 9 是否靶向不同的突触小泡子集。接下来，将从 NHE6 敲除 (KO) 和 NHE9 条件 KO (cKO) 小鼠大脑中分离的突触小泡用于通过测量阳离子（K[+] 和 Na[ +]) 使用吖啶橙测量囊泡 pH，使用 oxonol V 测量膜电位，以及通过放射性示踪剂通量测定测量递质摄取。通过培养原代分离的 KO 神经元，测量活神经元中突触小泡的 pH 值。在突触传递水平上，对 KO 小鼠切片中谷氨酸、GABA 和多巴胺的自发和诱发释放的分析将表明 NHE6 和 9 在量子大小、递质释放和/或突触后反应中的作用。最后，该提案旨在描述因 NHE9 缺失而导致的特定自闭症行为的特征，重点关注自闭症的三个主要诊断标准：社交性、重复行为和沟通技巧。因此，这项工作将检验 NHE6 和 9 在突触小泡填充、神经递质释放和突触传递中的作用对行为产生影响的假设。最终，生理缺陷与自闭症行为的相关性具有阐明自闭症系统和分子基础的巨大潜力，这反过来又将这些机制暗示为合理的治疗干预的目标。