Kinase signalling in neuronal membrane trafficking
Lead Research Organisation:
University College London
Department Name: Neuroscience Physiology and Pharmacology
Abstract
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
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
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/P504531/1 | 01/10/2016 | 31/03/2021 | |||
| 1809605 | Studentship | BB/P504531/1 | 01/10/2016 | 30/09/2020 | Flavia Rosianu |