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; 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）：突触形成和成熟需要在每个人内部的突触伙伴和信号转导之间的信号传导。为了了解如何组装突触前神经递质释放器的组装，我们专注于两个基因SAD-A和SAD-B。它们是SAD-1的哺乳动物直系同源物，这是秀丽隐杆线虫中突触前分化所需的基因。由于SADS是激酶，我们希望它们将为阐明管理神经终端组装的调节机制提供一个宝贵的起点。不幸的是，这一想法的初始基因检测给出了复杂的结果，因为两个基因在神经元发育中扮演多余的角色，并且在大多数突触形成之前，都参与了神经元发育的多个步骤，并且SAD-A/B双突变体在出生时死亡。因此，我们开发了两种遗传策略来规避这些胸膜疾病和致死性的局限性。首先，我们在选定的神经元类型中生成了有条件的等位基因。其次，我们生成对特定抑制剂敏感的等位基因，该等位基因可以精确地控制SAD-A/B活性并促进底物鉴定。使用这些新试剂，我们获得了初步结果，表明确实需要SADS才能完全突触前分化几种，甚至大多数突触类型。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检验，即SAD激酶是途径的关键成分，这些途径是从目标衍生的突触组织分子到神经末端组装的途径。首先，我们将使用条件等位基因绕过新生儿致死性，并表征四种外围和中央兴奋性突触类型中突触前缺陷。我们还将询问是否还需要SAD来发展抑制性突触，以及SADS是否调节后和突触前发育。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通过ATP类似物选择性抑制，而未修饰的激酶不敏化。这些等位基因为我们提供了对SAD激酶活性的精确和可逆的时间控制，无论是在体内和突变小鼠产生的培养物中。因此，我们可以询问何时需要开发SAD，是否需要其活动才能进行成人突触维持的活动，以及对激酶的急性抑制是否会影响正常发展的突触功能。最后，我们将启动旨在学习突触源性信号如何激活SAD的研究以及SAD如何协调突触前分化。