Evolution and diversity of synaptic transmission: Roles for voltage-gated calcium channels and PDZ-domain mediated scaffolding

突触传递是神经系统功能的核心。在我的研究中，我试图了解远亲动物之间突触结构和功能的不同，以及突触是如何进化的。事实上，深入了解突触及其进化的系统发生学将有助于拓宽我们对这种复杂而动态的细胞-细胞信号装置的理解，这是一门基础科学，可以帮助指导未来治疗涉及突触传递缺陷的神经系统疾病的努力。*有趣的是，最近的基因组测序研究表明，早期分化的缺乏神经系统的“原始”动物，如小型海洋无脊椎动物粘性毛藻，拥有突触传递所需的大部分基因。同样有趣的是，已知的最早分化的动物，如雷氏沼虾，具有神经系统和突触传递，似乎是独立进化的。我在分子生物学、电生理学和基因组学/转录学方面的广泛背景使我和我的人员能够利用这些动物来洞察突触多样性和进化。在这笔为期5年的NSERC拨款中，我和我的团队将通过完成以下目标来探索这些主题：*1)目标1：电压门控钙(Cav)通道的比较结构-功能和生理学研究，它在突触传递中发挥关键作用，并与许多人类疾病有关。我们将比较人类和其他无脊椎动物Cav通道与Trichoplax和Mnformopsis的高度不同的同源物/同源物，以获得对每种Cav通道类型保守和定义的生物物理特征的一般见解，并确定Cav通道在Trichoplax和Mnymopsis中所起的作用是否类似于其他已知的CAV通道在其他已有研究的生物中的突触功能。*2)目标2：高通量蛋白质组学，以评估在突触支架下的关键蛋白质-蛋白质相互作用，包括Cav通道与突触前神经递质分泌的联系，是否在Trichoplax中缺失，与其缺乏突触相一致，并确定Mnformopsis独立进化和鲜为人知的突触的分子结构。*3)目标3：Mrichoplax和独立进化的突触的缺失应该反映在关键突触蛋白-蛋白质相互作用的缺失/差异上。我们将使用从目标1和2收集的信息来指导果蝇的活体救援实验，使用来自Trichoplax和Mnymopsis的野生型与“体外进化”的突触基因以及引入的蛋白质相互作用区域，来测试关于可能促进突触进化的关键分子创新的假说。

Synaptic transmission lies at the very core of nervous system function. In my research, I seek to understand how synaptic structure and function differs between distantly-related animals, and how the synapse evolved. Indeed, gaining a deep, phylogenetic understanding of the synapse and its evolution will help broaden our understanding of this complex and dynamic cell-cell signaling apparatus, basic science that can help guide future efforts to treat diseases of the nervous system involving deficiencies in synaptic transmission. ***Interestingly, recent genome sequencing studies have revealed that early-diverging “primitive” animals that lack nervous systems, such as the small marine invertebrate Trichoplax adhaerens, harbour a majority of genes required for synaptic transmission. Also interesting is that the most early-diverging animals known, ctenophores such as Mnemiopsis leidyi, bear nervous systems and synaptic transmission that appears to have evolved independently. My broad background in molecular biology, electrophysiology and genomics/transcriptomics uniquely positions me and my personnel to exploit these animals for gaining insights into synaptic diversity and evolution. Over this 5 year NSERC grant, my team and I will explore these subjects by completing the following objectives: ***1) Objective 1: Comparative structure-function and physiology studies of voltage-gated calcium (Cav) channels, which play crucial roles in synaptic transmission and are implicated in numerous human diseases. We will compare human and other invertebrate Cav channels with highly divergent orthologues/homologues from Trichoplax and Mnemiopsis, to gain general insights into conserved and defining biophysical features of each Cav channel type, and to determine whether the roles that Cav channels play in Trichoplax and Mnemiopsis resemble known synaptic functions of Cav channels in other well-studied organisms. ***2) Objective 2: High-throughput proteomics to evaluate if key protein-protein interactions that underlie synaptic scaffolding, including the association of Cav channels with pre-synaptic neurotransmitter secretion, are absent in Trichoplax, in accordance with its lack of synapses, and to define the molecular architecture of the independently evolved and poorly understood synapses of Mnemiopsis. ***3) Objective 3: The absence of synapses in Trichoplax, and the independently evolved ones of Mnemiopsis, should be reflected by absences/differences in key synaptic protein-protein interactions. We will use information gathered from objectives 1 and 2 to inform in vivo rescue experiments in the fruit fly Drosophila, using wildtype vs. “in vitro evolved” synaptic genes from Trichoplax and Mnemiopsis with introduced protein interaction domains, to test hypotheses about key molecular innovations that might have facilitated synapse evolution.**