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

运动回路发育和疾病中经典补体途径突触损失的机制

• 批准号: 10517958
• 负责人: George Z Mentis
• 金额: $ 3万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-15 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10517958
• 关键词: Address; Affect; Age; Antibodies; Award; Behavior; Behavioral Assay; Biological Assay; Birth; CAV2 gene; Cause of Death; Classical Complement Pathway; Complement; Complement 1q; Confocal Microscopy; Development; Disease; Doctor of Philosophy; Excision; Fiber; Gastrocnemius Muscle; Genetic; Glutamates; Grant; Health; Hindlimb; Human; Immunohistochemistry; Intramuscular; Knockout Mice; Label; MERTK gene; Mediating; Molecular; Motor; Motor Neuron Disease; Motor Neurons; Motor output; Movement; Mus; Muscle; Neurodegenerative Disorders; Neurons; Parvalbumins; Pathologic; Phenotype; Physiological; Preparation; Proprioceptor; Proteins; Reporter; Research; Role; Sensory; Spinal; Spinal Cord; Spinal Muscular Atrophy; Synapses; Synaptic Potentials; System; Time; Viral; Virus; doctoral student; experimental study; human disease; infancy; motor function improvement; multidisciplinary; neonatal mice; neuronal circuitry; novel; recombinase; tibialis anterior muscle

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.

项目总结/摘要 这是根据FOA的多样性补充申请：研究补充，以促进 健康相关研究的多样性（PA-21-071），Tatiana M女士。冈萨雷斯，我实验室的博士生。女士 冈萨雷斯将致力于她的博士研究的实验授予和目前活跃的R 01赠款 （AA 027079，标题为：“运动回路中经典补体途径引起的突触丧失机制”， 发展和疾病”，PI：G. Z. Mentis）。 这项资助的实质是要解开负责的分子和细胞机制， 通过突触消除破坏神经网络，导致运动输出受损， 在正常发育和运动神经元疾病中，脊髓性肌萎缩症（SMA）。这些 在早期正常发育过程中，未成熟的感觉-运动回路的精细化机制是基础， 是SMA严重表型缺陷的主要原因。我们目前积极资助的具体目标是 如下：在目标1中，我们将确定经典补体途径的异常激活是否诱导 SMA中脆弱突触的丧失。在目标2中，我们将研究经典补语的要求 感觉运动回路正常发育的途径。在目标3中，我们将确定MEGF 10的作用， MERTK介导C1 q和C3（经典补体的蛋白质）标记和消除感觉 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。 具体来说，她将调查在何种程度上多余的和不适当的本体感觉 突触通过C1 q和C3参与形成。为了解决这个问题，冈萨雷斯女士将利用一本小说 选择性标记和定量后肢肌肉特异性的逆行病毒介导的神经元回路追踪测定 不同发育阶段小鼠脊髓运动神经元上的感觉本体感受突触 年龄该测定依赖于本体感受器对小清蛋白（PV）的表达;以引发荧光报告基因 为了在本体感受器中表达，将小清蛋白指导的FlpO重组酶（PV：：FlpO）小鼠系杂交 用Cre和Flp依赖性tdTomato报告基因小鼠（Ai 65）。将注射PV：：FlpO; Ai 65新生小鼠 在出生时肌内注射CAV 2-CRE病毒进入胫骨前肌（TA），以激活tdTomato表达， 支配TA的本体感觉神经元。为了标记支配拮抗神经元的运动神经元， 腓肠肌（Gs）肌肉，这些小鼠将伴随注射scAAV 6-GFP病毒以表达GFP 在Gs运动神经元中。以这种方式，本体感受TA纤维将用TdTomato（红色）和Gs标记。 带有GFP的运动神经元（绿色）。使用免疫组织化学和共聚焦显微镜， 将使用抗VGluT 1抗体标记突触能本体感受感觉突触， 通过VGluT 1与tdTomato在GFP+ Gs上的共定位， 运动神经元关于目标3，Gonzalez女士将调查MEGF 10和MERTK是否涉及 过度修剪SMA的脊髓突触为此，她将使用适当的敲除（KO）小鼠 她将产生MEGF 10 KO：：SMA和MERTKKO：：SMA小鼠，并进行翻正时间 行为测定以评估缺乏MEGF 10或MERTK的SMA小鼠是否具有任何改善的运动 与常规SMA小鼠相比，她将在MEGF 10 KO：：SMA上进行生理实验 和MERTKKO：：SMA小鼠使用离体脊髓制备，以建立运动神经元的振幅 突触电位，并将其与常规SMA小鼠的突触电位进行比较， MEGF 10或MERTK在SMA小鼠中赋予任何功能益处。最后，她将评估是否废除 MEGF 10和MERTK通过利用免疫组织化学和共聚焦显微镜破坏C1 q和C3标记。