Astrocyte-microglial communication in developmental synapse formation

项目总结/摘要 突触稳态的调节对于正常的大脑发育和功能是必不可少的， 依赖于星形胶质细胞和小胶质细胞--大脑的支持细胞。星形胶质细胞接触成千上万的 突触和促进发育突触的形成。小胶质细胞是大脑中的免疫细胞 越来越多地参与突触的形成和修剪。我们发现了一种新的信号电路 在发育中的中枢神经系统中促进突触消除的星形胶质细胞和小胶质细胞之间 （CNS）。我们发现星形胶质细胞表达免疫信号白细胞介素-33（IL-33），而小胶质细胞表达免疫信号白细胞介素-33（IL-33）。 IL-33受体（IL 1 RL 1.）我们随后发现，在脊髓中， 发育中的星形胶质细胞导致突触过多，IL-33向小胶质细胞发出信号以驱动突触 吞噬并导致突触耗竭。我们的中心假设是星形胶质细胞表达并释放 IL-33对神经元来源的信号作出反应，而IL-33反过来又驱动小胶质细胞突触 淘汰我们将在三个不同但相互关联的目标中测试这一假设，重点是一个定义明确的， 在腹侧基底感觉丘脑（VB）中实验可及的回路，其中IL-33高度表达 在突触细化过程中。在目标一，我们将确定星形胶质细胞IL-33如何调节丘脑突触 子类型和电路功能。我们以前的研究表明，IL-33的缺失会导致心肌细胞的过度兴奋。 VB电路和多余的突触。在这里，我们将有条件地删除星形胶质细胞中的IL-33，并探索这些细胞的功能。 更详细的表型，量化传入兴奋性和抑制性突触的亚型，以了解 电路的不同组成部分是如何改变的。在目标二中，我们将确定 调节小胶质细胞突触吞噬。我们先前发现IL-33促进突触后神经元的吞噬， 小胶质细胞的蛋白质。在这里，使用标准和高分辨率技术（扩展显微镜），我们 将量化突触前和突触后兴奋性元件以及抑制性突触的吞噬。我们 将测试通过其受体的条件性缺失直接向小胶质细胞发出信号的要求。在目标3中，我们 鉴定诱导星形胶质细胞表达和释放IL-33的神经元分子。我们的初步数据 表明去甲肾上腺素是一种神经元源性的线索，促进灰质中IL-33的表达 星形胶质细胞在这里，我们将进一步测试星形胶质细胞上的去甲肾上腺素能受体介导体内这种作用。我们 还将通过以下方式检验IL-33的细胞外释放依赖于神经元突触活性的假设： 在体外和体内调节活性。总之，这三个目标探讨了一种新的神经胶质细胞的作用， 调节突触稳态的回路。我们预测，对神经胶质如何通过网络进行交流的更广泛了解 调节突触的免疫分子将从根本上影响我们对神经回路 学习和发展以及神经发育疾病的变化。

PROJECT SUMMARY/ ABSTRACT Regulation of synapse homeostasis is essential for normal brain development and function, and intimately dependent on astrocytes and microglia -- the support cells of the brain. Astrocytes contact thousands of synapses and promote developmental synapse formation. Microglia are brain-resident immune cells increasingly implicated in both synapse formation and pruning. We have discovered a novel signaling circuit between astrocytes and microglia that promotes synapse elimination in the developing central nervous system (CNS). We found that astrocytes express the immune signal Interleukin-33 (IL-33) whereas microglia express the IL-33 receptor (IL1RL1.) We subsequently showed that in the spinal cord, eliminating IL-33 from developing astrocytes leads to excess synapses, and that IL-33 signals to microglia to drive synapse engulfment and lead to synapse depletion. Our central hypothesis is that astrocytes express and release IL-33 in response to neuron-derived signals, and that IL-33 in turn drives microglial synapse elimination. We will test this hypothesis in three distinct but interrelated aims, focusing on a well-defined and experimentally accessible circuit in the ventrobasal sensory thalamus (VB), where IL-33 is highly expressed during synapse refinement. In Aim One we will determine how astrocytic IL-33 regulates thalamic synapse subtypes and circuit function. We previously showed that global deletion of IL-33 lead to hyperexcitability of the VB circuit and excess synapses. Here we will conditionally delete IL-33 from astrocytes and explore these phenotypes in more detail, quantifying subtypes of afferent excitatory and inhibitory synapses to understand how different components of the circuit are altered. In Aim Two, we will determine the molecular mechanisms regulating microglial synapse engulfment. We previously found that IL-33 promotes engulfment of postsynaptic proteins by microglia. Here, using both standard and high resolution techniques (expansion microscopy), we will quantify engulfment of both pre- and postsynaptic excitatory elements, as well as inhibitory synapses. We will test the requirement for direct signaling to microglia via conditional deletion of its receptor. In Aim 3, we will identify neuronal molecules that induce astrocyte expression and release of IL-33. Our preliminary data demonstrates that norepinephrine is a neuron-derived cue that promotes expression of IL-33 in gray matter astrocytes. Here we will further test which noradrenergic receptors on astrocytes mediate this effect in vivo. We will also test the hypothesis that extracellular release of IL-33 is dependent on neuronal synaptic activity, by modulating activity in vitro and in vivo. Together, these three aims explore the role of a novel glial-neuronal circuit mediating synapse homeostasis. We predict that a broader understanding of how glia communicate via immune molecules to regulate synapses will fundamentally impact our understanding of how neural circuits change in learning and development as well as in neurodevelopmental diseases.