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; 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.

项目摘要朱莉·乌尔曼 最近的人类遗传分析发现，阳离子/质子交换器NHE6和NHE9中的突变都导致自闭症。关于这两种密切相关的蛋白质或它们在神经元中的功能知之甚少。有趣的是，爱德华兹实验室的最新工作已经确定了对突触囊泡的新活性。该活性通过将囊泡pH梯度（PHIPH）转化为膜电位（PHIPSI），促进兴奋性神经递质谷氨酸谷氨酸的摄取。此外，初步工作将NHE6和NHE9定位于突触前的胸子，特别是突触囊泡，这表明这些同工型是造成观察到的活性的原因。不同发射器的摄取取决于对Phiph或Phipsi的变化，因此预计NHE6或NHE9的损失将对不同的发射器产生不同的影响。该提案的假设是NHE6和9将Phiph转化为Phipsi，因此影响了用神经递质填充突触囊泡，其全球突触作用可能是NHE6或9中突变患者在NHE6或9的突变患者中观察到的严重表型的基础。该建议旨在表征NHE6和NHE9在多个生理水平上的作用：突触囊泡功能，神经传播和行为。在突触囊泡功能的水平上，我将首先确定NHE6和9的目标是否通过对含有小鼠脑的NHE6和9的突触囊泡进行免疫共糖化以及相应的突触囊泡蛋白的表征，以通过免疫共糖化为突触囊泡的不同子集。接下来，将使用从NHE6敲除（KO）和NHE9条件KO（CKO）小鼠中分离出来的突触囊泡，用于评估这两种同工型的功能作用，通过使用Actridine Orange对囊泡py的阳离子（K [+]和Na [+]）对膜元素的阳离子的效果来测量阳离子（K [+]和Na [+]）对使用膜元素的元素进行跨氧化管的效果。突触囊泡的pH值将通过原发性分离的KO神经元培养在活神经元中测量。在突触传播的水平上，对谷氨酸，GABA和多巴胺在SLICES中的自发和诱发释放的分析将表明NHE6和9在量子大小，发射器释放和/或共抗抗反应后的作用。最后，该建议旨在表征由于NHE9丧失而导致的特定自闭症行为，重点介绍了自闭症的三个主要诊断标准：社交，重复性行为和沟通技巧。因此，这项工作将检验以下假设：NHE6和9在突触囊泡填充，神经递质释放和突触传递中的作用会产生对行为的影响。最终，生理缺陷与自闭症行为的相关性具有巨大的潜力，可以阐明系统以及自闭症的分子基础，这反过来又将这些机制视为理性，治疗干预的目标。