Kinase signaling pathways in neuronal dendrite development

Lead Research Organisation: The Francis Crick Institute

Abstract

Brain development and neuronal function requires a great number of signalling pathways. Deficiencies in numerous genes expressed in the brain are shown to be associated with neurodevelopmental and neurodegenerative disorders. Unravelling of genetics of neurological diseases leads to redefining subtypes of previously not-stratified disorders some of which are of rare diseases. These new disease definitions is bringing about the important possibility of studying neuronal development and function mechanisms in close conjunction with human diseases caused by defects in these pathways.
Functional significance of numerous kinases expressed in brain is not well-understood. We use a unique combination of methods including chemical genetics, mass spectrometry, biochemistry, cell biology, knockout mouse and human IPSC methods to understand the function of a subset of kinases in the brain. We use imaging and physiological assays to determine neuronal growth and function, in mice and in cultured neurons. Our work will help uncover mechanisms of synapse formation and plasticity, cellular correlates of learning and memory.
One of the kinases we are working on, CDKL5, is an X-linked gene, mutations in which causes a rare neurodevelopmental disorder with early onset seizures. We have identified molecular mechanisms that are altered when CDKL5 is mutated. Our finding can help pave the path for designing more therapies for patients with this rare neurological disease.

Technical Summary

This work was supported by the Francis Crick Institute which receives its core funding from the UK Medical Research Council (FC001000), the Wellcome Trust (FC001000),and Cancer Research UK (FC001000)

Wiring of the brain circuitry relies on formation of elaborate dendritic arbors, the input receiving regions of neurons. Excitatory neurons in the cortex and hippocampus contain thousands of postsynaptic specializations called dendritic spines. Dendritic spines are actin-rich structures mostly containing a single excitatory synapse harbouring glutamate receptors responsible for conducting synaptic current. Spines not only acquire and lose receptors but also form and retract throughout adulthood reflecting learning-induced changes in mammals. Mechanisms of dendrite arborisation, spine synapse formation and plasticity is an active research area in neuroscience.
Our overall aim is to determine how kinases regulate neuronal dendrite and synapse development and plasticity in pyramidal neurons. We are particularly interested in processes governing membrane and receptor trafficking as well as cytoskeletal dynamics in dendrites. We apply a diverse set of methodologies to address this aim. In general, we aim to find kinase substrates using chemical genetics/ mass spectrometry and validate substrates in vitro using biochemistry and in knockout mice using phosphospecific antibodies. Next, by expressing phosphomutant substrates and shRNA in primary neurons we determine how substrates affect dendrites, spine development, membrane trafficking and cytoskeletal dynamics using electrophysiology and imaging methods. Third, we determine which aspects of kinase loss of function phenotypes in neurons may be mediated by the identified substrates using rescue experiments in primary neurons in culture.
Nuclear Dbf2 related kinases NDR1/2 and LATS1/2 (Large tumour suppressor 1/2) are shown to regulate dendrite development and maintenance in Drosophila. We found that NDR1/2 is required dendrite and synapse formation in mice and identified its substrates to be several membrane trafficking components (Ultanir et al, Neuron 2012). One of our aims is to determine how phosphorylation of NDR kinases’ substrates alter their function and regulate membrane trafficking.
Cyclin-dependent kinase like 5 (CDKL5) mutations in humans cause a very rare neurodevelopmental disorder with the same name. CDKL5 patients have refractory seizures by 6 months of age, sensory- motor problems and sleep disturbances. We identified CDKL5 substrates and we aim to discover the cellular mechanisms downstream of CDKL5 by studying these substrates. Our findings may have translational implications such as identification of biomarkers of CDKL5 activity and developing assays to screen for molecules that may counteract the deficit in CDKL5 neurons.
Cyclin G associated kinase (GAK), also called Auxilin 2, is linked to Parkinson’s disease via GWAS studies. GAK has a kinase domain and a DNAJ domain that functions as a co-chaperone for HSP70. GAK and its homolog without the kinase domain (Auxilin 1) are required for uncoating of clathrin after clathrin mediated vesicle formation. Kinase domain of GAK belongs to Numb associated kinase (NAK) family including a brain expressed kinase adaptor associated kinase 1 (AAK1), which is also a NDR1/2 substrate. Our aim in this project is to determine how GAK and AAK1 (which share substrates) function control membrane trafficking and cytoskeletal dynamics in neurons. Our findings can have implications on understanding pathological features of Parkinson’s disease.

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