Regeneration of auditory synaptic contacts using stem cell based approaches

描述（由申请人提供）：螺旋神经节神经元（SGN）及其与耳蜗核（CN）的突触是听觉系统的重要组成部分，在各种听觉障碍中受损。目前，还没有生物方法可以再生 受损的SGN我们这项提议的长期目标是使用干细胞（SC）衍生的神经元来替代受损的SGN，并再生连接耳蜗和脑干的传入CN突触。在这个建议中，我们将专注于SC衍生的神经元和CN神经元之间的传入突触生成使用在体外和体内模型。再生毛细胞和SC衍生神经元之间的连接将在一个单独的项目中进行研究。最近的一项研究表明，移植的SC衍生细胞改善了动物的诱发听觉反应。然而，植入细胞和宿主CN神经元之间发生突触形成的机制（蛋白质-受体相互作用）仍然未知。我们从小鼠耳蜗-前庭神经节中鉴定出神经干细胞，并诱导这些干细胞分化为神经元。我们观察到，神经营养因子刺激神经突起的延伸从SCN。然而，神经营养因子的补充并没有显着促进SCN和CN神经元之间的突触发生。因此，识别刺激听觉突触再生的机制是听觉功能恢复的主要挑战。最近，我们开发了一种使用SCN和小鼠CN神经元的共培养模型来解决这个问题。我们假设星形胶质细胞释放的血小板反应蛋白-1（TSP 1）和α 2 δ-1（α 2 δ-1）受体对SC为基础的突触发生至关重要。为了验证这一假设，我们提出了以下具体目标：目的1：确定TSP 1是否是体外ACM诱导的突触发生的关键刺激因子;目的2：研究β 2 β 1在共培养中ACM诱导的突触发生中的作用;目的3：研究TSP 1和β 2 β 1在体内CN突触再生中的作用。该提案研究了一种蛋白质和一种受体，它们似乎对SC衍生细胞和CN神经元之间的突触发生的诱导至关重要。当我们的体外和体内模型在这个建议中得到优化时，我们将研究开发和完善突触连接的策略，以确保电路在音调拓扑中正确布线。 在我们未来的研究中。识别对CN突触形成重要的蛋白质和受体不仅对听觉通路再生至关重要，而且还将为其他感觉系统中的突触再生提供线索。

DESCRIPTION (provided by applicant): Spiral ganglion neurons (SGNs) and their synapses with the cochlear nucleus (CN) are important components of the auditory system that are impaired in a variety of auditory disorders. Currently, no biological approach exists to regenerate damaged SGNs. Our long-term aims of this proposal are to use stem cell (SC)-derived neurons to replace injured SGNs and regenerate afferent CN synapses that connect the cochlea to the brainstem. In this proposal, we will focus on afferent synapse generation between SC-derived glutamatergic neurons and CN neurons using in vitro and in vivo models. Regenerating the connections between hair cells and SC-derived neurons will be examined in a separate project. A recent study shows that implanted SC-derived cells improved the evoked auditory responses of deafened animals. However, the mechanism (protein-receptor interaction) whereby synapse formation occurs between implanted cells and host CN neurons remains unknown. We have identified neural SCs from mouse cochlear-vestibular ganglia and induced these SCs to differentiate into glutamatergic neurons (ScNs). We observed that neurotrophins stimulated neurite extensions from ScNs. However, neurotrophin supplementation did not significantly promote synaptogenesis between ScNs and CN neurons. Therefore, identification of mechanisms that stimulate auditory synapse regeneration is a major challenge to the restoration of auditory function. Recently, we developed a co-culture model using ScNs and mouse CN neurons to address this issue. We hypothesize that astrocyte-released thrombospondin-1 (TSP1) and the alpha2delta-1 (�2�-1) receptor are critical for SC-based synaptogenesis. To test this hypothesis, we propose the following specific aims: Aim 1: Determine whether TSP1 is a critical stimulator of ACM-induced synaptogenesis in vitro; Aim 2: Investigate the role of �2�1 in ACM-induced synaptogenesis in co-cultures; Aim 3: Examine the roles of TSP1 and �2�1 in CN synapse regeneration in vivo. This proposal studies a protein and a receptor that appear to be critical for the induction of synaptogenesis between SC-derived cells and CN neurons. When our in vitro and in vivo models are optimized in this proposal, we will study the strategies to develop and refine synaptic connections to ensure that the circuit is properly wired in a tonotopic organization in our future research. Identification of proteins and receptors that are important fo CN synapse formation not only will be fundamental to auditory pathway regeneration, but also will provide cues for synapse regeneration in other sensory systems.