Small GTPase activity and astrocyte morphology as determinants of astrocyte Ca2+ signalling

小 GTP 酶活性和星形胶质细胞形态作为星形胶质细胞 Ca2 信号传导的决定因素

• 批准号: 284079634
• 负责人: Professor Dr. Christian Henneberger
• 金额: --
• 依托单位: Institut für Neurophysiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/284079634?language=en
• 关键词: Small; GTPase; activity; astrocyte; morphology

Astrocytes, an abundant type of glia in the brain, play important roles in maintaining neuronal network function through multiple mechanisms, e.g. energy supply and neurotransmitter clearance. Their unique morphology enables single astrocytes to fulfil these functions at thousands of synapses. In addition, it has become evident that astrocytes are able to directly sense and modulate signalling and plasticity at these synapses. Astrocyte Ca2+ signalling is thereby heavily implicated. The properties and propagation of the largely store-dependent Ca2+ signals depend on diffusion and therefore on astrocyte morphology, which is itself dynamic. However, which signalling cascades are critical in controlling astrocyte morphology and, in turn, Ca2+ signalling remains poorly understood. We therefore propose to uncover the mechanisms by which small GTPases of the Rho family control astrocyte morphology and astrocyte Ca2+ signalling. Our central hypothesis thereby is that activity of small GTPases of the Rho family dynamically determine astrocyte morphology and consequently the Ca2+ signals implicated in neuron-glia interactions. Our preliminary experiments confirmed that astrocytes Ca2+ dynamics follow power law. Interestingly, we found the power law exponent to depend on astrocyte morphology, in line with our main hypothesis. Transient expression of active and dominant negative form of small GTPases of the Rho family revealed their profound impact on astrocyte morphology. These suggest that, indeed, small GTPase activity modifies astrocyte Ca2+ signalling through structural changes. To complement these in vitro studies, we have developed a novel procedure to study astrocyte morphology and its changes in situ using two-photon excitation fluorescence imaging. Preliminary data obtained in situ suggest that epileptiform activity, for instance, is a potent trigger of astrocyte morphology changes requiring intact RhoA signalling. Together, these data indicate that small GTPases could be a critical determinant of astrocyte morphology and Ca2+ signalling in vitro and in situ and may be of pathophysiological relevance. Thus, the goal of this proposal is to uncover the specific mechanisms that govern this relationship. To this end, we will manipulate small GTPase by molecular, pharmacological and optogenetic tools in vitro and in situ. We will then visualize and quantify Ca2+ signalling in parallel to astrocyte morphology changes. What triggers or controls small GTPase activity is also of physiological importance. Therefore, we have already established biosensor monitoring of GTPase activity, which will allow us to test what neuronal activity recruits GTPase activity. Finally, we apply the newly gained insight to reveal how GTPase-mediated astrocyte restructuring is involved in epileptogenesis. In summary, the proposed work will provide novel insights into the fundamental mechanisms that shape astrocyte Ca2+ signaling in physiological and pathological conditions.

星形胶质细胞是脑内数量丰富的胶质细胞，通过能量供应和神经递质清除等多种机制在维持神经网络功能中发挥重要作用。它们独特的形态使单个星形胶质细胞能够在数千个突触中履行这些功能。此外，它已成为明显的是，星形胶质细胞能够直接感知和调节这些突触的信号和可塑性。星形胶质细胞Ca 2+信号转导因此受到严重影响。的性质和传播的很大程度上存储依赖的Ca 2+信号依赖于扩散，因此对星形胶质细胞的形态，这本身是动态的。然而，哪些信号级联在控制星形胶质细胞形态中至关重要，反过来，Ca 2+信号仍然知之甚少。因此，我们建议揭示Rho家族的小GTP酶控制星形胶质细胞形态和星形胶质细胞Ca 2+信号传导的机制。因此，我们的中心假设是，Rho家族的小GTP酶的活性动态地决定星形胶质细胞的形态，从而影响神经元-胶质细胞相互作用的Ca 2+信号。我们的初步实验证实星形胶质细胞Ca ~（2+）动力学遵循幂律。有趣的是，我们发现幂律指数依赖于星形胶质细胞的形态，与我们的主要假设一致。Rho家族的小GTP酶的活性和显性负性形式的瞬时表达揭示了它们对星形胶质细胞形态的深远影响。这些表明，确实，小GTdR活性通过结构变化改变星形胶质细胞Ca 2+信号。为了补充这些体外研究，我们已经开发了一种新的程序，研究星形胶质细胞的形态和原位变化，使用双光子激发荧光成像。原位获得的初步数据表明，癫痫样活动，例如，是一个强大的触发星形胶质细胞形态的变化，需要完整的RhoA信号。总之，这些数据表明，小GTP酶可能是星形胶质细胞形态和钙信号在体外和原位的一个关键决定因素，并可能是病理生理学的相关性。因此，本提案的目标是揭示管理这种关系的具体机制。为此，我们将在体外和原位通过分子，药理学和光遗传学工具操纵小GTbR。然后，我们将可视化和定量的钙离子信号在平行星形胶质细胞形态的变化。触发或控制小GT3活性的物质也具有生理重要性。因此，我们已经建立了生物传感器监测GT3活性，这将使我们能够测试什么神经元活动招募GT3活性。最后，我们应用新获得的见解，揭示如何GTP酶介导的星形胶质细胞重组参与癫痫发生。总之，所提出的工作将提供新的见解的基本机制，形状星形胶质细胞Ca 2+信号在生理和病理条件。