Lemur tyrosine kinase-2 and axonal transport of cdk5/p35 and protein phosphatase-1

The abilities of our brains to compute information is linked to the numbers of connections that individual brain neuron cells make with one another. These connections are termed synapses and it is estimated that a human brain contains 100-500 trillion synapses. Loss of synapses occurs as we age and pathogenic synaptic loss is believed be the underlying cause of dementia in Alzheimer's disease. The mechanisms by which synapses are lost in ageing and disease are not properly understood but one suggestion is that it involves, at least in part, defects to the delivery of essential proteins and other components to the synapse. Most synaptic proteins are synthesized in a different part of the neuron cell and so have to be transported to synapses. One type of transport is termed axonal transport and this involves moving protein cargoes through the long axon processes of neurons. This movement involves "molecular motor proteins" and these run on rails called microtubules and utilize a fuel called ATP. Axonal transport is therefore like a train journey with a motor or engine that requires a fuel and runs on rails. Cyclin dependent kinase-5/p35 (Cdk5/p35) and protein phosphatase-1 (PP1) are two major protein complexes that perform a number of essential functions in the synapse. Moreover, disruption to cdk5/p35 and PP1 functions are linked to Alzheimer's and other neurodegenerative diseases. However, the mechanisms by which these important proteins are transported to the synapse are not known. We have shown that cdk5/p35 and PP1 both bind to another protein called lemur tyrosine kinase-2 (LMTK2) and that LMTK2 attaches to a molecular motor called kinesin-1. Thus, cdk5/p35 and PP1 may be transported to synapses on kinesin-1 motors via their attachment to LMTK2. The Aim of this project is to test this possibility. The objectives are:-
1. To finalise our studies demonstrating that LMTK2 scaffolds cdk5/p35 and PP1 to KLCs.
2. To properly characterise axonal transport of LMTK2.
3. To study the role of LMTK2 on axonal transport of cdk5/p35 and PP1.
4. To investigate the mechanisms that regulate axonal transport of the LMTK2-cdk5/p35-PP1 complex

The study will provide important information on fundamental neurophysiological processes. In addition, since damage to axonal transport and to cdk5/p35 and PP1 synaptic functions are all seen in Alzheimer's and related diseases, the work may reveal new targets for therapeutic intervention for these disorders.

Cyclin dependent kinase-5/p35 (cdk5/p35) and protein phosphatase-1 (PP1) are two signaling proteins that function in a number of key processes within the synapse. We and others have shown that both cdk5/p35 and the catalytic subunit of PP1 (PP1C) bind to the serine/threonine kinase lemur tyrosine kinase-2 (LMTK2). In common with most neuronal proteins, cdk5/p35, PP1C and LMTK2 are all believed to be synthesized in cell bodies and then transported through axons to presynaptic termini. However, the mechanisms that mediate this transport are largely unknown. Kinesin-1 is a major neuronal molecular motor that drives anterograde axonal transport and we have obtained evidence that LMTK2 binds to the kinesin-1 light chains (KLC) so as to mediate attachment of LMTK2, cdk5/p35 and PP1C to kinesin-1 motors. The aim of this project is therefore to test the hypothesis that binding of LMTK2 to KLCs mediates kinesin-1 driven axonal transport of a complex containing LMTK2 and associated cdk5/p35 and PP1C to presynaptic termini. The objectives are:-
1. To finalise our studies demonstrating that LMTK2 scaffolds cdk5/p35 and PP1C to KLCs.
2. To properly characterise axonal transport of LMTK2 in transfected living rat neurons using time-lapse microscopy.
3. To study the role of LMTK2 on axonal transport of cdk5/p35 and PP1C.
4. To investigate the mechanisms that regulate axonal transport of the LMTK2-cdk5/p35-PP1C complex and in particular, how phosphorylation of LMTK2 affects its binding to cdk5/p35 and PP1C, and how phosphorylation of KLC1 affects its binding to LMTK2.

Cdk5/p35 and PP1C are key signaling molecules that perform a number of important functions at the synapse. Moreover, perturbation to the functions of these molecules are linked to some neurodegenerative diseases. This project is to gain insight into the molecular mechanisms by which cdk5/p35, PP1C and LMTK2 are transported through axons to presynaptic termini. The beneficiaries of this research and how they will benefit are listed below.

1. Academic scientists and the pharmaceutical industry
Understanding synaptic function and dysfunction are key areas of research. Synapses are lost with ageing and also more acutely in Alzheimer's and related neurodegenerative diseases. Changes in synaptic function are also linked to some neuropsychiatric diseases such as schizophrenia. Likewise, axonal transport is a topical area of research since it too is perturbed in ageing and in Alzheimer's and other neurodegenerative diseases. Indeed, mutations in molecular motor proteins can cause familial forms of some neurodegenerative diseases. The findings from this research will benefit other academic scientists researching into these areas. In addition, since axonal transport is disrupted in neurodegenerative diseases, correcting this defect represents a therapeutic target for the treatment of these disorders. Knowledge of the mechanisms that regulate axonal transport and especially the transport of key synaptic cargoes such as cdk5/p35 and PP1C which are known to have defective functions in disease, may thus lead to the identification of new therapeutic targets. Such information will be beneficial to both academic clinicians and the pharmaceutical industry. CM and WN have excellent links with KCL clinicians and industry. Thus, WNs findings on the therapeutic value of minocycline in Alzheimer's disease models was quickly communicated to KCL psychiatrists and this led to an NIHR funded clinical trial of minocycline in dementia. Likewise, we have collaborated with GlaxoSmithKline, Pfizer, AstraZeneca, Eli Lilly and Trophos. WN currently has two BBSRC Industrial CASE partnership grants with AstraZeneca and Eli Lilly. Thus, we have contacts and collaborations with clinicians and pharmaceutical companies that will facilitate the transfer of our findings.

2. Society and the economy
Alzheimer's disease is the most common form of dementia, affecting approximately 820,000 people in the UK at a cost of £23 billion per year (Alzheimer's Research UK 2012 Dementia Report). There are currently no disease-modifying treatments for Alzheimer's disease and recent failures of clinical trials are likely to stem from an incomplete understanding of disease mechanisms. Research into these mechanisms may pave the way for the development of new therapeutics. In the longer term the results obtained from this project could therefore have considerable socio-economic impact.

3. Junior scientists
The post-doctoral researcher employed on the grant will develop his/her skills in a broad range of scientific techniques including advanced microscopy, molecular and cellular neuroscience and proteomics. They will also develop a number of transferrable skills including time management, data analysis, presentation skills and scientific writing. MSc and BSc students from King's regularly undertake research project in our groups. Some are likely to choose topics that are linked to this project and these students will obtain training in some of the above research areas. Thus the project will facilitate training of junior scientists in important scientific areas and techniques.