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伙伴之间的信号传导以及每个突触伙伴内的信号转导。为了了解突触前神经递质释放装置的组装方式，我们重点关注两个基因：SAD-A 和 SAD-B。它们是 SAD-1 的哺乳动物直系同源物，SAD-1 是秀丽隐杆线虫突触前分化所需的基因。由于 SAD 是激酶，我们希望它们能够为阐明神经末梢组装的调控机制提供一个有价值的起点。不幸的是，这个想法的最初基因测试给出了复杂的结果，因为这两个基因发挥着冗余作用，并参与神经元发育的多个步骤，而且因为 SAD-A/B 双突变体在大多数突触形成之前就在出生时死亡。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，我们生成了一个条件等位基因来消除选定神经元类型中的表达。其次，我们生成了对特定抑制剂敏感的等位基因，这可以精确控制 SAD-A/B 活性并促进底物识别。使用这些新试剂，我们获得了初步结果，表明 SAD 确实是几种甚至大多数突触类型的完全突触前分化所必需的。在这里，我们建议确认和扩展这些结果，并开始对我们的假设进行测试，即 SAD 激酶是从靶标衍生的突触组织分子到神经末梢组装的途径的关键组成部分。首先，我们将使用条件等位基因来绕过新生儿致死率并表征四种外周和中枢兴奋性突触类型的突触前缺陷。我们还将询问抑制性突触的发育是否也需要 SAD，以及 SAD 是否调节突触后和突触前的发育。其次，我们将用 SAD-A 和 -B 突变等位基因测定突触发育和功能，这些突变等位基因使激酶可被 ATP 类似物选择性抑制，而未修饰的激酶对其不敏感。这些等位基因为我们提供了对 SAD 激酶活性的精确且可逆的时间控制，无论是在体内还是在突变小鼠产生的培养物中。因此，我们可以问，在发育过程中何时需要 SAD，它们的活性是否是成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功能。最后，我们将启动旨在了解突触信号如何激活 SAD 以及 SAD 如何协调突触前分化的研究。