Post-translational modifications of tomosyn and synaptic plasticity

描述(由申请人提供)：通过突触的神经回路传递信息是认知、学习、记忆和运动功能的基本要求。突触传递不是固定的，而是动态可修改的，这一过程被称为突触可塑性。突触小泡的胞吐是由SNARE家族蛋白介导的，这些蛋白在突触小泡和突触前质膜之间形成反式SNARE复合体。这些SNARE复合体的形成是突触传递功效的中枢调控分子机制。Tomosyn是一种独特的突触前R-SNARE蛋白，因为它是胞浆的，并且是一种强大的胞吐负调节因子。Tomosyn在神经末梢的可用性和活性状态通过负向调节囊泡的启动来影响释放概率。目前尚不清楚的是调节Tomosyn活性的分子机制和信号通路。最近的证据表明，泛素(Ub)和小泛素样修饰物(SUMO)通路在突触强度的调节中发挥着特别重要的影响。值得注意的是，我的初步证据表明，Tomosyn蛋白的水平和活性受到这些不同的翻译后修饰的调节。因此，我假设Tomosyn的影响 突触可塑性受到这些系统活动的动态调节。我建议通过结合遗传操作、生物化学、荧光成像和电生理测量来验证这一假说，以描绘：1)Tomosyn是否通过泛素蛋白酶体系统(UPS)受到调节的降解；2)Tomosyn的SUMOM化如何调节其亚细胞定位、蛋白质-蛋白质相互作用和功能活性。具体地说，这些目的将决定通过Ub和SUMO对Tomosyn进行翻译后修改的突触活动状态，定义这些修改与Tomosyn活动机制变化之间的关系，并将这些活动变化与突触前介导的可塑性诱导的功能变化联系起来。这些目标的成功完成将揭示神经可塑性是如何诱导和调节的，包括Tomosyn参与神经活动诱导的突触前可塑性和内稳态变化的影响和程度。收集的数据将超越基础科学研究，为涉及神经传递的疾病的预防和治疗方法提供潜在的机械目标，包括最近与Tomosyn和UPS介导的神经传递和人类中枢神经系统功能和健康改变(例如自闭症谱系障碍和阿尔茨海默病)有关的疾病。

DESCRIPTION (provided by applicant): Transmission of information through neural circuits at synapses is an essential requirement for cognition, learning, memory, and motor function. Synaptic transmission is not fixed but dynamically modifiable, a process referred to as synaptic plasticity. Exocytosis of synaptic vesicles is mediated by the SNARE family proteins which form trans-SNARE complexes between the vesicle and presynaptic plasma membrane. Formation of these SNARE complexes serves as a central regulated molecular mechanism underlying the efficacy of synaptic transmission. Tomosyn is a unique presynaptic R-SNARE protein in that it is cytosolic and serves as a potent negative regulator of exocytosis. The availability and activity-state of tomosyn in nerve terminals affects release probability by negatively regulating the priming of vesicles for release. What remains unknown are the molecular mechanisms and signaling pathways by which tomosyn activity is modulated. Provocative recent evidence suggests that the ubiquitin (Ub) and small ubiquitin-like modifier (SUMO) pathways exert particularly important influences on the modulation of synaptic strength. Remarkably, my preliminary evidence indicates that tomosyn protein levels and activity are subject to regulation by these distinct post-translational modifications. Therefore, I hypothesize that tomosyn's affects on synaptic plasticity are under dynamic modulation of the activity of these systems. I propose to test this hypothesis by employing a combination of genetic manipulations, biochemistry, fluorescence imaging, and electrophysiological measurements to delineate: 1) if tomosyn is subject to regulated degradation via the ubiquitin proteasome system (UPS), and 2) how sumoylation of tomosyn modulates its sub-cellular localization, protein-protein interactions, and functional activity. Specifically, these aims will determine the synaptic activity states that indue post-translational modification of tomosyn via Ub and SUMO, define the relationship between these modifications and changes in tomosyn's mechanism of activity, and link these activity alterations to functional changes in presynaptically- mediated plasticity induction. Successful completion of these aims will reveal novel insights into how neural plasticity is induced and regulated, including the effects by which, and extent of, tomosyn involvement in neural activity-induced changes in presynaptic plasticity and homeostasis. Data gathered will extend beyond basic science research by providing potential mechanistic targets for preventative and therapeutic approaches for diseases involving neurotransmission, including those recently linked to tomosyn- and UPS-mediated alterations in neurotransmission and human central nervous system function and health (e.g. autism-spectrum disorders and Alzheimer's disease).