Function of profilins in the tripartite synapse- from structural plasticity to functional modulation

长期以来，细胞生物学家一直致力于了解细胞自组织过程是如何产生动态的、健壮的和复杂的组织细胞的结构的。特别是，我们对单个分子的理解与我们对这些分子如何共同形成活神经元的理解之间存在着根本的差距。在不同的长度尺度和时间尺度上建立秩序的生物学重要性，以及理解自组织分子系统如何执行细胞功能的挑战，可能最好地通过细胞骨架的研究来说明。这里肌动蛋白细胞骨架是最重要的。它在所有动物细胞的运动中都起着至关重要的作用，特别是在神经元和星形胶质细胞中，它在运动过程中是必不可少的。神经元对不同的活动水平作出反应，其结构变化如神经元分化、树突和轴突的维持以及树突、轴突和突触的可塑性适应。微丝系统的调节剂介导信号从突触前或突触后膜到肌动蛋白细胞骨架，并通过这种方式改变特定神经元室的功能和结构。在这种情况下，肌动蛋白结合蛋白谱可能是一个主要的参与者。在中枢神经系统中，profilin的两种亚型pfn1和pfn2a共表达。最近关于profilin 1和2a在神经元和神经胶质细胞中的细胞作用的研究表明，这两种亚型都具有重叠和亚型特异性功能。此外，通过敲除其中一种异构体获得的发现不能排除每一种异构体都可以补偿另一种异构体的损失。这一假设得到了RNA干扰对每种亚型的急性下调的支持，这导致了与敲除研究不完全一致的结果。因此，我们将在这里通过其急性失活来解决profins在星形胶质细胞和神经元中的功能作用。通过这种方法，我们将研究三方突触的形成和调节，从而研究它们对神经元功能的影响。为了避免彼此谱的代偿效应，我们将通过CRISPR/Cas9介导的方法以细胞类型特异性的方式同时敲除这两种亚型。我们将通过现代成像和电生理方法研究profilin亚型在神经元活动转化为细胞骨架重组中的功能，特别是在结构和功能突触可塑性的过程中。此外，我们将使用行为测试来探索profilin亚型在海马体依赖性学习任务中的可能作用。

For a long time cell biologists have been motivated to understand how the process of cellular self-organization generates dynamic, robust and elaborate structures that organize cells. In particular there is a fundamental gap between our understanding of individual molecules and our understanding of how these molecules function collectively to form living neurons. The biological importance of establishing order over diverse length scales and timescales, as well as the challenges of understanding how systems of self-organizing molecules carry out cellular functions, is perhaps best illustrated by studies of the cytoskeleton. And here the actin cytoskeleton is of upmost important. It is crucially involved in motility in all animal cells and particular in neurons and astrocytes, where it is mandatory in processes of motility. Neurons react to different activity levels with structural changes e.g. neuronal differentiation, dendritic and axonal maintenance and plastic adaption of dendrites, axons and synapses. Modulators of the microfilament system mediate signals from the pre- or postsynaptic membrane to the actin cytoskeleton and by this means change function and structure of specific neuronal compartments. In this context the actin binding protein profilin could be a mayor player. In the central nervous system, two isoforms of profilin, PFN 1 and PFN 2a, are co-expressed. Recent studies focusing on the cellular role of profilin 1 and 2a in neurons and glia cells are showing that both isoforms possess overlapping as well as isoform specific functions. Furthermore, the findings obtained by knocking out only one of the isoforms could not exclude that each isoform could compensate the loss of the other one. This hypothesis was supported via the acute downregulation of each isoform by RNA interference which leads to results that are not completely in line with the knock out studies. Therefore we will here address the functional role of profilins in astrocytes and in neurons by its acute inactivation. By this means we will study the formation and modulation of the tripartite synapse and thereby their impact on neuronal function. To avoid compensatory effects of each other profilin we will knock out both isoforms simultaneously in a cell-type specific manner by a CRISPR/Cas9 mediated approach. We will investigate the function of the profilin isoforms in the translation of neuronal activity into cytoskeletal reorganization, specifically in processes of structural and functional synaptic plasticity via modern imaging and electrophysiological methods. We will in addition use behavioral tests in order to explore the possible role of profilin isoforms in hippocampus dependent learning tasks.