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

描述(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递和每个突触伙伴之间的信号转导。为了了解突触前神经递质释放装置的组装是如何组装的，我们关注了两个基因，SAD-A和SAD-B。它们是哺乳动物中SAD-1的同源基因，SAD-1是线虫突触前分化所必需的基因。因为SADS是一种激酶，我们希望它们能为阐明支配神经末梢组装的调节机制提供一个有价值的起点。不幸的是，对这一想法的初步基因测试得出了复杂的结果，因为这两个基因扮演着多余的角色，参与了神经元发育的多个步骤，而且SAD-A/B双突变体在出生时就死亡了，大多数突触还没有形成。因此，我们开发了两种遗传策略来绕过这些多发性和致命性的限制。首先，我们产生了一个条件等位基因来抑制选定的神经元类型中的表达。其次，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，并有助于底物识别。使用这些新试剂，我们获得了初步结果，表明SADS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，我们建议证实和推广这些结果，并开始测试我们的假设，即SAD激酶是从靶来源的突触组织分子到神经末梢组装的通路的关键组成部分。首先，我们将使用条件等位基因绕过新生儿致死性，并表征四种外周和中枢兴奋性突触类型的突触前缺陷。我们还将询问SADS是否也是抑制性突触发育所必需的，以及SADS是否调节突触后和突触前的发育。其次，我们将用SAD-A和-B突变的等位基因来分析突触的发育和功能，这些突变的等位基因使得这些激酶可以被一种未修饰的激酶不敏感的ATP类似物选择性地抑制。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，无论是在体内还是在突变小鼠的培养中都是如此。因此，我们可以问在发育过程中何时需要SADS，它们的活动是否对成人突触的维持是必需的，以及对这些激酶的急性抑制是否会影响已正常发育的突触的功能。最后，我们将启动旨在了解突触信号如何激活SADS以及SADS如何反过来协调突触前分化的研究。

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.