Mechanisms of synaptic loss by the classical complement pathway in motor circuit development and disease

项目摘要/摘要 这是一个多样性补充的申请，根据FOA：促进研究补充 我实验室的博士生Tatiana M.Gonzalez女士的健康相关研究多样性(PA-21-071)。女士。 冈萨雷斯将为她的博士研究工作进行实验，她将获得目前正在使用的R01奖学金 (AA027079，题为《经典补体通路在运动回路中突触丢失的机制》) 发展与疾病“，PI：G.Z.Mentis)。 这项拨款的实质是解开导致 通过突触消除破坏神经元网络，导致运动输出受损，两者 在正常发育和运动神经元疾病中，脊髓性肌萎缩症(SMA)。这些 在早期正常发育和发育过程中，未成熟的感觉-运动回路的精炼是以机制为基础的 是SMA严重表型缺陷的主要原因。我们目前正在进行的赠款的具体目标是 在目标1中，我们将确定经典补体途径的异常激活是否会诱导 SMA中脆弱的突触丢失。在目标2中，我们将研究经典补语的要求 感觉-运动神经回路正常发育的途径。在目标3中，我们将确定MEGF10和 MerTK在调节C1q和C3(经典补体蛋白)标记和消除感觉中的作用 突触。 Tatiana Gonzalez女士将调查的研究将涉及目标2和目标3的各个方面。 具体地说，她将调查在多大程度上多余和不适当的本体感觉 突触是通过C1q和C3参与形成的。为了解决这个问题，冈萨雷斯女士将用一本小说 逆行病毒介导的神经回路示踪法选择性标记和定量后肢肌肉特异性 不同发育阶段小鼠肌肉脊髓运动神经元上的感觉本体感觉突触 年龄。这项检测依赖于本体受体对小白蛋白(PV)的表达；以启动荧光报告 在本体受体中的表达，小白蛋白导向的FlpO重组酶(PV：：FlpO)小鼠系被交叉 与依赖Cre和FLP的tdTomato报告小鼠(Ai65)。将注射PV：：FlpO；Ai65新生小鼠 出生时肌肉注射CAV2-Cre病毒进入胫骨前(TA)激活td番茄表达 支配TA的本体感觉神经元。标记支配拮抗剂的运动神经元 这些小鼠将同时注射scAAV6-GFP病毒以表达GFP。 在Gs的运动神经元。以这种方式，本体感受性TA纤维将被标记为TdTomato(红色)和Gs 绿色荧光蛋白运动神经元(绿色)。使用免疫组织化学和共聚焦显微镜，兴奋性 谷氨酸能本体感觉突触将使用抗VGluT1抗体和存在的标记 通过VGluT1和tdTomato在GFP Gs上的共同定位，将检测到不适当的突触 运动神经元。关于AIM 3，冈萨雷斯将调查MEGF10和MERTK是否参与其中 在SMA的脊髓突触过度修剪中。为此，她将使用适当的敲除(KO)鼠标 杂交如下：她将产生MEGF10KO：：SMA和MERTKKO：：SMA小鼠并执行翻正时间 用行为学方法评估缺乏MEGF10或MERTK的SMA小鼠是否有任何改善的运动 与正常的SMA小鼠相比，其功能更佳。她将在MEGF10KO：：SMA上进行生理实验 并与MERTKKO：：SMA小鼠进行体外脊髓制备，建立运动神经元的波幅 并将它们与正常SMA小鼠的突触电位进行比较，确定是否取消 MEGF10或MERTK对SMA小鼠有任何功能上的好处。最后，她将评估废除 MEGF10和MERTK通过免疫组织化学和共聚焦显微镜破坏C1q和C3标记。

Project Summary/Abstract This is an application for a diversity supplement according to FOA: Research Supplements to Promote Diversity in Health-Related Research (PA-21-071) for Ms. Tatiana M. Gonzalez, a PhD student in my lab. Ms. Gonzalez will be working on experiments for her PhD studies on the awarded and currently active R01 grant (AA027079, entitled: “Mechanisms of synaptic loss by the classical complement pathway in motor circuit development and disease”, PI: G.Z. Mentis). The essence of this grant is to unravel the molecular and cellular mechanisms responsible for the disruption of neuronal networks through synaptic elimination which leads to compromised motor output, both during normal development and in the motor neuron disease, spinal muscular atrophy (SMA). These mechanisms underlie the refinement of immature sensory-motor circuits during early normal development and are the main cause for the severe phenotypic deficits in SMA. The Specific Aims of our currently active grant are as follows: In Aim 1, we will determine whether aberrant activation of the classical complement pathway induces loss of vulnerable synapses in SMA. In Aim 2, we will investigate the requirement of the classical complement pathway for normal development of sensory-motor circuits. In Aim 3, we will determine the role of MEGF10 and MERTK in mediating C1q and C3 (proteins of the classical complement) in tagging and eliminating sensory synapses. The studies that will be investigated by Ms. Tatiana Gonzalez, will involve aspects of Aim 2 and Aim 3. Specifically, she will investigate to which extent supernumerary and inappropriate proprioceptive sensory synapses are formed through C1q and C3 involvement. To address this, Ms. Gonzalez will utilize a novel retrograde viral-mediated neuronal circuit tracing assay to selectively label and quantify hindlimb muscle-specific sensory proprioceptive synapses on muscle-identified spinal motor neurons in mice at different developmental ages. This assay relies on the expression of parvalbumin (PV) by proprioceptors; to prime fluorescent reporter expression in the proprioceptors, a parvalbumin-directed FlpO recombinase (PV::FlpO) mouse line is crossed with Cre- and Flp-dependent tdTomato reporter mice (Ai65). PV::FlpO;Ai65 neonatal mice will be injected intramuscularly at birth with CAV2-CRE virus into the tibialis anterior (TA) to activate tdTomato expression in proprioceptive sensory neurons innervating the TA. To label motor neurons innervating the antagonistic Gastrocnemius (Gs) muscle, these mice will concomitantly be injected with scAAV6-GFP virus to express GFP in Gs motor neurons. In this manner, proprioceptive TA fibers will be labelled with TdTomato (in red) and Gs motor neurons with GFP (in green). Using immunohistochemistry and confocal microscopy, excitatory glutamatergic proprioceptive sensory synapses will be labeled using an anti-VGluT1 antibody and the presence of inappropriate synapses will be detected through the co-localization of VGluT1 with tdTomato on GFP+ Gs motor neurons. With respect to Aim 3, Ms Gonzalez will investigate whether MEGF10 and MERTK are involved in the excessive pruning of spinal synapses in SMA. To do so, she will use the appropriate knock out (KO) mouse crosses as follows: she will generate MEGF10KO::SMA and MERTKKO::SMA mice and perform the righting time behavioral assay to assess whether the SMA mice lacking MEGF10 or MERTK have any improved motor function in comparison to regular SMA mice. She will perform physiological experiments on MEGF10KO::SMA and MERTKKO::SMA mice using the ex vivo spinal cord preparation, to establish the amplitude of motor neuron synaptic potentials and compared them to those from regular SMA mice and determine whether abolition of MEGF10 or MERTK confers any functional benefit in SMA mice. Lastly, she will assess whether abolition of MEGF10 and MERTK disrupts C1q and C3 tagging by utilizing immunohistochemistry and confocal microscopy.