Elucidating the role of BDNF/Astrocytic TrkB.T1 signaling on perisynaptic astrocyte process recruitment

项目概要/摘要 突触周围星形胶质细胞突起（perisynaptic astrocyte prostheses，PAPs）是位于突触远端突起的小叶状结构 星形胶质细胞。这些结构包含许多神经递质受体，离子通道，细胞粘附 分子，以及释放突触发生分子的能力，使星形胶质细胞能够促进突触形成。 稳态、发育和稳定。研究表明，PAP包裹或接触突触元件 在成熟的突触上覆盖率增加。此外，最近的文献表明，一旦 存在于突触处，这些PAP是高度运动的结构。然而，迄今为止， 最初将PAP募集到突触仍有待阐明。脑源性神经营养因子（BDNF）是一种重要的 促进神经元成熟、存活和突触发育的因子。RNA测序生成 最近，我们实验室发现了一个新的令人惊讶的发现，即星形胶质细胞表达高水平的BDNF 受体，TrkB，相对于其他细胞群体。对该数据的进一步同种型特异性评价表明， 星形胶质细胞主要表达TrkB的截短同种型TrkB.T1。我们发表的研究表明， BDNF/星形胶质细胞TrkB.T1有助于星形胶质细胞形态发生。全局和星形胶质细胞特异性缺失 TrkB.T1导致星形胶质细胞体积减少，与成熟胶质细胞相关的突触周基因失调， 星形胶质细胞功能，以及星形胶质细胞不能促进体外神经元突触形成和功能。 为该资助产生的初步数据显示，TrkB.T1的整体缺失导致兴奋性神经元的减少。 II/III层皮质中的突触，通过突触前和突触后兴奋性的免疫组织化学评价来评估。 元素此外，TrkB.T1的整体缺失使星形胶质细胞不能对经验依赖性的细胞毒性反应。 通过抑制它们增加细胞体积的能力和它们增加 它们的区域内包含的突触元件的数量。最后，初步的体外数据表明， 相对于野生型（WT）星形胶质细胞，TrkB.T1敲除（KO）星形胶质细胞不能形成PAP或不能包裹突触。 元素总而言之，我们的发现和初步数据已经导致了一个假设，即BDNF信号传导到大脑皮层， 星形胶质细胞TrkB.T1受体是PAP募集到突触所必需的。我们建议 利用体外和体内药理学、遗传学、功能和成像的组合来测试这一假设 技术.成功完成概述的实验可以阐明两者的病理生理学 神经发育和神经精神障碍，这两者都与异常的BDNF/TrkB有关 和突触功能，但从未在BDNF/星形胶质细胞TrkB.T1的背景下进行过研究。的方法 本提案中采用的技术为PI提供了广泛的创新性体外和体内技术。培训 这些技术的专业知识将在申办者的实验室进行。在整个奖学金，职业生涯 专业发展培训将通过弗吉尼亚理工大学的期刊俱乐部、研讨会和协会进行， 以及在国家和地区会议上，为PI作为独立研究员的职业生涯做准备。

PROJECT SUMMARY/ABSTRACT Perisynaptic astrocyte processes (PAPs) are fine, leaflet-like structures that are located on the distal processes of astrocytes. These structures contain numerous neurotransmitter receptors, ion channels, cell-adhesion molecules, and the ability to release synaptogenic molecules, enabling astrocytes to contribute to synapse homeostasis, development, and stabilization. Research indicates that PAPs enwrap or contact synaptic elements with increased coverage at mature, glutamatergic synapses. In addition, recent literature has indicated that once present at a synapse, these PAPs are highly motile structures. However, to date, the signaling mechanisms that initially recruit a PAP to a synapse remain to be elucidated. Brain derived neurotrophic factor (BDNF) is a critical factor that contributes to neuronal maturation, survival, and synapse development. RNA sequencing generated in our lab has recently revealed the novel and surprising finding that astrocytes express high levels of the BDNF receptor, TrkB, relative to other cell populations. Further isoform specific evaluation of this data indicated that astrocytes predominantly express a truncated isoform of TrkB, TrkB.T1. Our published work indicates that BDNF/astrocytic TrkB.T1 contributes to astrocyte morphogenesis. Global and astrocyte specific deletion of TrkB.T1 results in a decrease in astrocytic volumes, dysregulation of perisynaptic genes associated with mature astrocyte function, as well as inability for astrocytes to promote neuronal synapse formation and function in vitro. Preliminary data generated for this grant reveal that global deletion of TrkB.T1 results in a decrease in excitatory synapses in layer II/III cortex, assessed by immunohistochemical evaluation of pre- and post-synaptic excitatory elements. In addition, global deletion of TrkB.T1 disables astrocytes from responding to experience dependent plasticity in the whisker barrel cortex by inhibiting their ability to increase in cell volume and their ability to increase the number of synaptic elements contained within their territories. Finally, preliminary in vitro data indicate that relative to wildtype (WT) astrocytes, TrkB.T1 knockout (KO) astrocytes fail to form PAPs or fail to enwrap synaptic elements. Altogether, our findings and preliminary data have led to the hypothesis that BDNF signaling on to the astrocytic TrkB.T1 receptor is required for the recruitment of PAPs to glutamatergic synapses. We propose to test this hypothesis utilizing a combination of in vitro and in vivo pharmacological, genetic, functional, and imaging techniques. Successful completion of the outlined experiments could shed light on the pathophysiology of both neurodevelopmental and neuropsychiatric disorders, both of which have been implicated by aberrant BDNF/TrkB and synapse function but have never been studied in the context of BDNF/astrocytic TrkB.T1. The approaches employed in this proposal offer a breadth of innovative in vitro and in vivo techniques to the PI. Training for the expertise of these techniques will take place in the sponsor’s laboratory. Throughout the fellowship, career professional development training will occur through journal clubs, seminars, and associations at Virginia Tech, as well as at national and regional conferences to prepare the PI for a career as an independent researcher.