Roles of SAD kinases in formation and maturation of multiple synaptic types

SAD 激酶在多种突触类型形成和成熟中的作用

• 批准号: 8320871
• 负责人: JOSHUA R SANES
• 金额: $ 25.35万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8320871
• 关键词: Acute; Address; Adult; Affect; Alleles; Animals; Axon; Biological Assay; Birth; Brain; Bypass; Caenorhabditis elegans; Cells; Complex; Defect; Development; Elements; Employee Strikes; Excitatory Synapse; Genes; Genetic; Genetic screening method; Inhibitory Synapse; Lead; Learning; Maintenance; Motor; Motor Neurons; Mus; Mutant Strains Mice; Neonatal; Nerve; Neuromuscular Junction; Neurons; Pathway interactions; Pattern; Peripheral; Phosphotransferases; Physiological; Play; Population; Postsynaptic Membrane; Process; Proteins; Reagent; Role; Sensory; Signal Transduction; Spinal; Staging; Structure; Surface; Sympathetic Ganglia; Synapses; System; Testing; analog; base; in vivo; inhibitor/antagonist; innovation; mutant; neuron development; neurotransmitter release; postsynaptic; presynaptic; response; synaptic function; synaptogenesis

DESCRIPTION (provided by applicant): Synapse formation and maturation require signaling between the synaptic partners and signal transduction within each of them. To learn how assembly of the presynaptic neurotransmitter release apparatus is assembled, we focused on two genes, SAD-A and SAD-B. They are the mammalian orthologues of SAD-1, a gene required for presynaptic differentiation in C. elegans. Because SADs are kinases, we hope they will provide a valuable starting point for elucidating regulatory mechanisms that govern assembly of nerve terminals. Unfortunately, initial genetic tests of this idea gave complex results because the two genes play redundant roles and are involved in multiple steps in neuronal development and because SAD-A/B double mutants die at birth, before most synapses have formed. We therefore developed two genetic strategies to circumvent these limitations of pleitropy and lethality. First, we generated a conditional allele to ablate expression in selected neuronal types. Second, we generated alleles sensitive to a specific inhibitor, which allows precise temporal control of SAD-A/B activity and facilitates substrate identification. Using these new reagents, we have obtained preliminary results indicating that SADs are indeed required for complete presynaptic differentiation of several and perhaps most synaptic types. Here we propose to confirm and extend these results, and to initiate tests of our hypothesis that SAD kinases are critical components of pathways that lead from target-derived synaptic organizing molecules to assembly of nerve terminals. First, we will use the conditional allele to bypass neonatal lethality and characterize presynaptic defects in four peripheral and central excitatory synaptic types. We will also ask whether SADs are also required for development of inhibitory synapses, and whether SADs regulate post- as well as presynaptic development. Second, we will assay synaptic development and function with SAD-A and -B mutant alleles that render the kinases selectively inhibitable by an ATP analog to which unmodified kinases are insensivitive. These alleles provide us with precise and reversible temporal control over SAD kinase activity, both in vivo and in cultures generated from the mutant mice. We can therefore ask when during development SADs are required, whether their activity is required for synaptic maintenance in adults, and whether acute inhibition of the kinases affects the function of synapses that have developed normally. Finally, we will initiate studies aimed at learning how synaptogenic signals activate SADs and how SADs, in turn, coordinate presynaptic differentiation.

描述（由申请人提供）：突触的形成和成熟需要突触配偶体之间的信号传导和它们各自内的信号转导。为了了解突触前神经递质释放装置是如何组装的，我们集中在两个基因上，SAD-A和SAD-B。它们是SAD-1的哺乳动物直向同源物，SAD-1是C.优雅的因为SAD是激酶，我们希望他们将提供一个有价值的出发点，阐明调控机制，控制装配的神经末梢。不幸的是，对这一想法的最初遗传测试给出了复杂的结果，因为这两个基因扮演着冗余的角色，参与了神经元发育的多个步骤，而且因为SAD-A/B双突变体在出生时就死亡了，在大多数突触形成之前。因此，我们开发了两种遗传策略来规避多效性和致死性的这些限制。首先，我们产生了一个条件等位基因，以消除在选定的神经元类型的表达。第二，我们产生了对特定抑制剂敏感的等位基因，这允许精确的时间控制SAD-A/B活性并促进底物鉴定。使用这些新的试剂，我们已经获得了初步的结果表明，SAD确实需要几个，也许是大多数突触类型的完整的突触前分化。在这里，我们建议确认和扩展这些结果，并开始测试我们的假设，SAD激酶是关键组成部分的途径，导致从目标衍生的突触组织分子的组装的神经末梢。首先，我们将使用条件等位基因绕过新生儿的致死性和特点突触前缺陷的四个周边和中央兴奋性突触类型。我们还将询问SAD是否也是抑制性突触发育所必需的，以及SAD是否调节突触后和突触前的发育。第二，我们将检测SAD-A和SAD-B突变等位基因的突触发育和功能，这些突变等位基因使激酶可被ATP类似物选择性地激活，而未修饰的激酶对ATP类似物不敏感。这些等位基因为我们提供了精确和可逆的时间控制SAD激酶活性，无论是在体内和从突变小鼠产生的文化。因此，我们可以问在发育过程中什么时候需要SAD，它们的活性是否是成年人突触维持所必需的，以及激酶的急性抑制是否会影响正常发育的突触的功能。最后，我们将启动研究，旨在了解突触发生信号如何激活SAD，以及SAD如何反过来协调突触前分化。