Kinase signalling in neuronal membrane trafficking

神经元膜运输中的激酶信号传导

• 批准号: 1809605
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1809605
• 关键词: Kinase; signalling; neuronal; membrane; trafficking

Development and maintenance of neuronal structure requires numerous signalling mechanisms acting in concert. Neuronal dendrites are input receiving regions which contain postsynaptic specializations contacting incoming axons. Membrane trafficking from intracellular compartments is shown to be essential for maintenance of dendritic spines, sites for more than ninety percent of excitatory synapses in mammalian brain. Synaptic structures can be modified by turn-over of transmembrane proteins. Our lab studies how kinases regulate cellular processes to affect neuronal structure and function. Malfunctioning and loss of dopaminergic neurons in the midbrain substantia nigra region causes Parkinson's disease (PD), a common neurodegenerative disorder. Cellular mechanisms that lead to the degeneration remain an active research area. Important clues originated from clinical genetic studies which implicated several genes in PD. Among these are two kinases: cyclin G-associated kinase (GAK) and Leucine Rich Repeat Kinase 2 (LRRK2). GAK is identified as a PD risk factor via multiple genome wide association studies [1]. GAK is ubiquitously expressed in many tissues including the brain. It is present in Golgi and is known to regulate clathrin mediated endocytosis. LRRK2 mutations cause familial forms of Parkinson's disease and polymorphisms in LRRK2 are risk factors for non-familial PD [2]. LRRK2 is a large multi-domain protein which has been associated with several cellular processes; yet its cellular role still remains unclear. LRRK2's downstream phosphorylation targets have been an intense area of study in the past decades. Recently a membrane trafficking regulator Rab8 is shown to be a LRRK2 substrate [3]. Interestingly, an unbiased biochemical screen identified GAK as a physical interactor of LRRK2 [4].We confirmed that GAK and LRRK2 can associate, indicating that they may be parts of a protein complex. Whether or not GAK and LRRK2 participate in a common cellular mechanism such as membrane trafficking or how they may affect each other's function are unknown. We will use knockout and knock-in mouse models of LRRK2 and GAK, confocal imaging and biochemistry methods to address these questions. With this project we hope to gain understanding of the cellular pathways that are regulated by GAK and LRRK2, which may raise possibilities about pathological changes in PD.Our specific aims are:1) Determine the protein domains required for GAK- LRRK2 association and test if GAK and LRRK2 associate endogenously in mouse brain. 2) Determine if GAK and LRRK2 regulate each other's localization in neurons. We generated GAK conditional knockout mice in which GAK is deleted in excitatory neurons in the cortex and hippocampus. LRRK2 knockout mice, LRRK2 kinase dead knock-in mice and LRRK2 G2019S knock-in mice, with increased activity, are available from GSK partners. 3) Test if GAK and LRRK2 could regulate functional output of each other. We will use phospho-specific antibodies towards substrates of GAK and LRRK2 and cellular assays in neuronal cultures to investigate this question.Development and maintenance of neuronal structure requires numerous signalling mechanisms acting in concert. Neuronal dendrites are input receiving regions which contain postsynaptic specializations contacting incoming axons. Membrane trafficking from intracellular compartments is shown to be essential for maintenance of dendritic spines, sites for more than ninety percent of excitatory synapses in mammalian brain. Synaptic structures can be modified by turn-over of transmembrane proteins. Our lab studies how kinases regulate cellular processes to affect neuronal structure and function. Malfunctioning and loss of dopaminergic neurons in the midbrain substantia nigra region causes Parkinson's disease (PD), a common neurodegenerative disorder. Cellular mechanisms that lead to the degeneration remain an active research area. Important clues

神经元结构的发育和维持需要许多信号传导机制协同作用。神经元树突是输入接受区域，其包含接触传入轴突的突触后特化。从细胞内室的膜运输被证明是必不可少的树突棘，网站超过百分之九十的兴奋性突触在哺乳动物大脑的维护。突触结构可以通过跨膜蛋白的翻转来修饰。我们的实验室研究激酶如何调节细胞过程以影响神经元的结构和功能。中脑黑质区域中多巴胺能神经元的功能障碍和丧失导致帕金森病（PD），一种常见的神经退行性疾病。导致退变的细胞机制仍然是一个活跃的研究领域。重要的线索来自临床遗传学研究，涉及PD的几个基因。其中有两种激酶：细胞周期蛋白G相关激酶（GAK）和富含亮氨酸重复序列激酶2（LRRK 2）。GAK通过多个全基因组关联研究被确定为PD风险因素[1]。GAK在包括脑在内的许多组织中普遍表达。它存在于高尔基体中，并且已知调节网格蛋白介导的内吞作用。LRRK 2突变导致家族性帕金森病，LRRK 2多态性是非家族性PD的风险因素[2]。LRRK 2是一个大的多结构域蛋白，与几个细胞过程相关;但其细胞作用仍不清楚。LRRK 2的下游磷酸化靶点在过去几十年中一直是一个激烈的研究领域。最近，膜运输调节剂Rab 8被证明是LRRK 2底物[3]。有趣的是，无偏的生化筛选将GAK鉴定为LRRK 2的物理相互作用物[4]。我们证实GAK和LRRK 2可以缔合，表明它们可能是蛋白质复合物的一部分。GAK和LRRK 2是否参与共同的细胞机制，如膜运输或它们如何影响彼此的功能尚不清楚。我们将使用LRRK 2和GAK的敲除和敲入小鼠模型，共聚焦成像和生物化学方法来解决这些问题。本研究的主要目的是：1）确定GAK与LRRK 2结合所需的蛋白结构域，并检测GAK与LRRK 2在小鼠脑内是否存在内源性结合。2)确定GAK和LRRK 2是否调节彼此在神经元中的定位。我们产生了GAK条件性敲除小鼠，其中GAK在皮质和海马的兴奋性神经元中缺失。活性增加的LRRK 2敲除小鼠、LRRK 2激酶死亡敲入小鼠和LRRK 2 G2019 S敲入小鼠可从GSK合作伙伴获得。3)测试GAK和LRRK 2是否可以相互调节功能输出。我们将使用磷酸化特异性抗体对基板的GAK和LRRK 2和细胞测定在神经元culture.Development和维护的神经元结构需要许多信号机制的一致行动，以调查这个问题。神经元树突是输入接受区域，其包含接触传入轴突的突触后特化。从细胞内室的膜运输被证明是必不可少的树突棘，网站超过百分之九十的兴奋性突触在哺乳动物大脑的维护。突触结构可以通过跨膜蛋白的翻转来修饰。我们的实验室研究激酶如何调节细胞过程以影响神经元的结构和功能。中脑黑质区域中多巴胺能神经元的功能障碍和丧失导致帕金森病（PD），一种常见的神经退行性疾病。导致退变的细胞机制仍然是一个活跃的研究领域。重要线索