Kinase signaling mechanisms in dendrite, dendritic spine development and synaptogenesis in mice

Lead Research Organisation: MRC National Inst for Medical Research

Abstract

In order to have a properly functioning brain, specialized brain cells called neurons need to differentiate to form input receiving dendritic arbors and output sending axons. Proper synaptic connection between dendrites/ dendritic spines and axons are essential for the wiring of the neural circuits. Failure to form or maintain these synaptic structures lead to neurodevelopmental or neurodegenerative diseases. A large set of cellular tools called proteins, encoded by coded genes, are at play for orchestrating neuron’s differentiation. Among these are kinases, enzymes that regulate all cellular processes via altering the activity of their substrates by phosphorylating them. My lab aims to discover novel kinase signaling networks in the dendritic development and synapse formation process in mice brain.
We use electrophysiological recordings from brain slices, morphological analysis of dendrites and spines, novel chemical genetic methods to identify kinase substrates, cell biological assays and imaging to understand the cellular and molecular functions of kinase cascades in neurons.
Our goal is to achieve a more detailed and broader understanding of molecular mechanisms that are at play in developing dendrites. Kinases are one of the most commonly targeted group of molecules for drug discovery, thus our work on kinases in dendrite development could also lead to identification of novel potential drug targets for therapies of neurological diseases.

Technical Summary

During early postnatal development neurons form elaborate dendritic arbors, which comprise the input receiving domain of neurons where the synapses are made. Kinases regulate most if not all cellular processes. The role of kinases in the development and functioning of neurons is not well understood. In my lab the roles of kinases are investigated using electrophysiology, morphological analysis of dendrites and spines, chemical genetics, biochemistry and imaging methods to determine molecular and cellular mechanisms of kinase function neuronal development. We focus on AAK1/GAK, CDKL5 and MST1/2.
AAK1 and GAK are highly homologous serine threonine kinases which regulate membrane trafficking and receptor signaling. They are both linked to Parkinson’s disease. Our goal is 1) to understand their role by investigating the synaptic function, dendrite morphology and survival of neurons in conditional knockout mice where either or both of these kinases are knocked out in various subtypes of neurons in relevant circuitries to Parkinson’s, thus determining the circuitry for which AAK1/GAK may play a critical role; 2) to determine the signaling events that are regulated by AAK1/GAK by using neuronal culture systems and candidate approach as AAK1/GAK are known to regulate notch, Erbb4 and EGFR signaling; 3) Using chemical genetic methods we will determine the direct downstream targets of AAK1/GAK to gain mechanistic insight on how these kinases regulate receptor signaling. Our findings will have impact on public health as it might reveal novel mechanisms involved in Parkinson’s disease.
CDKL5 when mutated in girls cause severe neurodevelopmental disabilities, which are thought to be similar to Rett syndrome. Downstream effectors of CDKL5 are largely unknown. Our goal is 1) to determine the effectors using chemical genetic methods 2) to understand how CDKL5 regulated neuronal development using conditional CDKL5 knockout mouse that we are generating.
MST1/2 are important regulators of organ size control and are also implicated in cancer. Although role of MST1/2 in organ size/ cell division is extensively studied, their role in neuronal development remains to be investigated. The MST1/2 Drosophila homolog Hippo is important for maintenance of dendrite arbors. Our first goal is to investigate if MST1/2 are necessary for development and function of neurons in hippocampus and cortex. Our second goal is to determine the phosphorylation targets of MST1/2 using chemical genetics methods. Our findings would broaden our understanding of neuronal dendrite and synapse development, while phosphorylation targets of MST1/2 could also be of interest to other areas such as cancer.

Publications

10 25 50
 
Description CDKL5 Research Grant
Amount $50,000 (USD)
Organisation International Foundation for CDKL5 Research 
Sector Charity/Non Profit
Country United States
Start 06/2012 
End 06/2013
 
Description Crick -GSK PhD studentship
Amount £72,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2016 
End 09/2020
 
Description CDKL5's role in sensory neuron development in mice 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution We are collaborating with Dr Simone Di Giovanni's lab at Imperial College London. Their lab is working on the role of CDKL5 kinase on dorsal root ganglion neuron development. They have shown that CDKL5 knockout mice have altered pain sensitivity. We provide knockout mice samples and cultures for their experiments.
Collaborator Contribution Dr. Di Giovanni's group makes the cultures with us and conduct all subsequent experiments.
Impact We found that CDKL5 loss results in deficits in sensory neuron axon development.
Start Year 2016
 
Description Role of Phactr1 in dendritic spines 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution My lab is generating primary neuronal cultures from rat embryos. Our project goal is to investigate the subcellular localization of Phactr1 and its mutant forms in neurons and to test if Phactr1 or its mutants alter spine morphology. I have transfected, stained and imaged neurons.
Collaborator Contribution We are collaborating with Dr Richard Treisman's lab. They have studied Phactr1's role in cell lines extensively. In addition, they have characterized actin and PP1 binding domains of Phactr1 and its regulation by G actin. We are using the plasmid constructs that they have generated in this project.
Impact none
Start Year 2016
 
Description Satb1's role on excitatory synapse development 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution My postdoc Kalbinder Gill has conducted electrophysiology experiments in brain slices from Satb1 knockout mice to test is in LayerV or Layer 4 neurons excitatory synaptic currents are altered.
Collaborator Contribution Vassilis Pachnis's lab have generated and characterised Satb1 knockout mice. They found changes in inhibitory neuron numbers and maturation of neurons.
Impact We have not found any changes in excitatory postsynaptic currents in Satb1 knockout mice. Our findings will be part of a manuscript.
Start Year 2015
 
Description Substrates of Erk5 and Tpl2 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We are collaborating with Steve Ley's lab to identify novel direct substrates of Erk5 and Tpl2. We give advice on using the Shokat method for identifying the substrates of Erk5 and Tpl2.
Collaborator Contribution Steve Ley is an expert in immunology and in particular in TPL2's role in immune response. His lab has generated and purified the analog specific kinase and are labeled substrates from macrophages.
Impact We don't have any publications yet.
Start Year 2014