Genetic dissection of peripheral glia and glial sheath development

外周胶质细胞和胶质鞘发育的遗传解剖

• 批准号: RGPIN-2019-04929
• 负责人: Auld, Vanessa
• 金额: $ 3.06万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=766117
• 关键词: Genetic; dissection; peripheral; glia; glial

The long-term goal of my research program is to understand how glia ensheath and protect the peripheral nerve. We study the mechanisms that underlie glia development in Drosophila melanogaster, where each peripheral nerve is insulated by layers of glia and insulation is essential for nervous system function. Little is known about the mechanisms that trigger glia to migrate along and wrap axons or surround and support the nerve. The formation of the glial wrap involves adhesion and communication between glia-glia, glia-axon and glia-extracellular matrix (ECM) paired with extensive rearrangements of the cytoskeleton. In Drosophila three distinct glial layers contribute to the mature peripheral nerve and the glial sheath must be maintained and expand to match animal growth. Loss of glia-glia adhesion disrupts the glial sheath leading to paralysis and lethality due to loss of the blood-brain barrier (BBB), yet how the sheath forms and is maintained is not well understood. Loss of the glia-ECM interaction leads to disruption of the structure of the CNS and peripheral nerve integrity. The aim of my research is to identify the mechanisms underlying glial sheath formation and test the functional consequences that disruption of these highly conserved processes have on nervous system function. Our NSERC funded research identified protein complexes critical to establish the glial sheath. We found that integrins are key to glia-glia and glia-ECM communication in the peripheral nervous system and maintain the glial sheath and the BBB. We found Basigin (Bsg) regulates integrin-mediated glial adhesion to the ECM. We determined that Cadherins ensure the formation of the glial wrap and BBB. Of interest, Cadherins and Integrins function together in this process and understanding how these complexes control glial ensheathment is the next step in our research program. The two short term goals of the current grant are to determine the mechanisms that: Aim 1) Underly the convergence of integrin and cadherin function in sheath and BBB formation. Aim 2) Link integrins and Basigin to modulate glia-ECM interactions and ensure the structural integrity of the peripheral nerve. My laboratory has extensive experience studying peripheral glia development and sheath formation. We have generated a suite of genetic tools that allow us to manipulate the genes expressed in each of the three peripheral glia layers and then assess the results on nervous system function and morphology. We utilize a multi-faceted approach that incorporates genetics (including RNAi, CRISPR genome editing), cell biology (including fluorophore-tagged endogenous proteins, FRET based tension sensors), biochemistry (including proximity ligation assays), optogenetics to regulate axonal activity, super-resolution imaging and behaviourial studies.  Thus we use the Drosophila model to characterize the fundamental mechanisms that underlie glial cell development and sheath formation in all animals.

我的研究计划的长期目标是了解神经胶质神经如何保护周围神经系统。我们研究了果蝇果蝇中神经胶质发育的基础的机制，在果蝇中，每种周围神经都被神经胶质层隔离，并且绝缘是神经系统功能必不可少的。关于触发神经胶质的机制几乎不知道，沿着轴突迁移并包裹轴突或周围并支撑神经系统。神经胶质包裹的形成涉及胶质细胞，神经胶质轴和神经胶质细胞基质（ECM）之间的粘附和通信，并与细胞骨架的广泛重排配对。在果蝇中，三个不同的神经胶质层有助于成熟的周围神经，并且必须保持神经胶质鞘并扩展以匹配动物的生长。神经胶质胶质粘合剂的丧失破坏了由于血脑屏障（BBB）的丧失而导致瘫痪和致死性的神经胶质鞘，但是尚不清楚鞘形的形式和维持的鞘形。胶质ECM相互作用的丧失导致中枢神经系统结构和周围神经完整性的破坏。我的研究的目的是确定神经胶质鞘形成的机制，并测试这些高度保守过程对神经系统功能的破坏的功能后果。我们的NSERC资助研究确定了建立神经胶质鞘至关重要的蛋白质复合物。我们发现整联蛋白是周围神经系统中神经胶质细胞和胶质ECM通信的关键，并保持神经胶质鞘和BBB。我们发现Basgin（BSG）调节整联蛋白介导的神经胶质粘合剂对ECM。我们确定钙粘蛋白确保形成神经胶质包裹和BBB。感兴趣的是，钙粘蛋白和整合素在这一过程中共同发挥作用，并了解这些复合物如何控制神经胶质宿内是我们研究计划的下一步。当前赠款的两个短期目标是确定以下机制：目标1）在护套和BBB形成中整联蛋白和钙粘蛋白功能的融合基础。目标2）将整联蛋白和BASIGIN连接起来调节胶质ECM相互作用，并确保周围神经的结构完整性。我的实验室具有研究周围神经胶质发育和鞘形成的丰富经验。我们已经生成了一套遗传工具，使我们能够操纵在三个外周胶质层中表达的基因，然后评估神经系统功能和形态的结果。我们利用一种多面方法，该方法结合了遗传学（包括RNAi，CRISPR基因组编辑），细胞生物学（包括荧光团标签的内源性蛋白质，基于FRET的张力传感器），生物化学（包括近距离结 - 结扎测定），选择遗传学，以调节轴突活性，超级差异，超级差异和超级差异研究。我们使用果蝇模型来表征所有动物的神经胶质细胞发育和鞘形成的基本机制。