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

项目摘要/摘要 突触周围星形细胞突起是位于突起远端的细小的叶状结构。 星形胶质细胞。这些结构含有大量的神经递质受体、离子通道、细胞黏附 分子，以及释放突触分子的能力，使星形胶质细胞能够为突触做出贡献 动态平衡、发展和稳定。研究表明，PAPs包裹或接触突触元件 随着成熟的谷氨酸能突触覆盖率的增加。此外，最近的文献表明，一旦 存在于突触中，这些脑页是高度能动的结构。然而，到目前为止， 最初招募PAP到突触仍有待阐明。脑源性神经营养因子(BDNF)是一种 促进神经元成熟、存活和突触发育的因子。生成的RNA测序 我们实验室最近发现了一个新的令人惊讶的发现，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。对该数据的进一步的亚型特异性评估表明 星形胶质细胞主要表达TrkB的截短亚型TrkB.T1。我们发表的研究表明， BDNF/星形细胞TrkB.T1参与星形胶质细胞的形态发生。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，与成熟相关的突触周围基因调控失调 星形胶质细胞的功能，以及星形胶质细胞在体外不能促进神经元突触的形成和功能。 为这项研究产生的初步数据显示，TrkB.T1的全局删除导致兴奋性减少 通过对突触前和突触后兴奋性的免疫组织化学评价来评估II/III层皮质中的突触 元素。此外，TrkB.T1的全局缺失使星形胶质细胞无法对经验依赖做出反应 通过抑制胡须桶皮质细胞体积的增加和细胞体积的增加来实现可塑性 包含在其领土内的突触元素的数量。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基因敲除(KO)星形胶质细胞不能形成PAPs或不能包裹突触 元素。综上所述，我们的发现和初步数据导致了BDNF信号传导到 星形细胞TrkB.T1受体是PAPs向谷氨酸能突触募集所必需的。我们建议 利用体外和体内药理学、遗传学、功能性和成像相结合的方法来验证这一假设 技巧。上述实验的成功完成将有助于揭示两者的病理生理学。 神经发育和神经精神障碍，两者都与BDNF/TrkB异常有关 和突触功能，但从未在脑源性神经营养因子/星形细胞TrkB.T1的背景下进行研究。这些方法 这项提案中采用的技术为PI提供了广泛的体外和体内创新技术。培训： 这些技术的专业知识将在赞助商的实验室进行。在整个团契、职业生涯中 职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， 以及在国家和区域会议上为独立研究人员的职业生涯做好准备。

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.