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

项目摘要Julie Ullman 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及其在神经元中的功能知之甚少。有趣的是，爱德华兹实验室最近的工作发现了突触小泡上的一种新的NHE活动。这种活性通过将囊泡的pH梯度(PhipH)转换为膜电位(PhiPsi)来促进兴奋性神经递质谷氨酸进入囊泡。此外，初步工作已将NHE6和NHE9定位于突触前突触，特别是突触小泡，表明这些亚型与观察到的活动有关。不同递质的摄取在不同程度上取决于PhipH或PhiPsi，因此NHE6或NHE9的丢失将对不同的递质产生不同的影响。这一建议的假设是，NHE6和9将PhipH转化为PhiPsi，从而影响神经递质填充突触小泡，具有全局突触效应，这可能是NHE6或9突变患者以及其他形式的自闭症患者观察到的严重表型的基础。这一建议试图从多个生理水平表征NHE6和NHE9的作用：突触小泡功能、神经传递和行为。在突触小泡功能的水平上，我将首先通过免疫分离含有NHE6和9的小鼠脑中的突触小泡，并对各自的突触小泡蛋白进行鉴定，来确定NHE6和9是否针对不同的突触小泡亚群。接下来，从NHE6基因敲除(KO)和NHE9条件性KO(CKO)小鼠脑中分离的突触小泡将被用来评估这两种异构体的功能作用，方法是用吖啶橙测量阳离子(K[+]和Na[+])对囊泡pH的影响，用oxonol V测量膜电位，用放射示踪剂通量分析测量递质摄取的影响。通过原代分离的KO神经元的培养，将在活神经元中测量突触小泡的pH。在突触传递的水平上，分析KO小鼠脑片中自发和诱发的谷氨酸、GABA和多巴胺的释放，将表明NHE6和9在量子大小、递质释放和/或突触后反应中发挥作用。最后，这项建议试图描述由于NHE9基因缺失而导致的具体自闭症行为，重点是自闭症的三个主要诊断标准：社交能力、重复行为和沟通技能。因此，这项工作将检验NHE6和NHE9在突触囊泡充盈、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很大的潜力来阐明自闭症的系统和分子基础，进而将这些机制作为合理的治疗干预的靶点。

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.