TRAK-mediated neuronal mitochondrial trafficking mechanisms: regulation and impact on neuronal function

Information in our brains is processed by a network of specialized cells, neurons. Neurons are asymmetric. Each has a cell body from which processes known as axons and dendrites emerge. Axons transmit information to other neurons by forming connections (synapses) onto cell bodies or dendrites. Dendrites are extensive processes that receive information from axons. Synaptic transmission, information transfer from axon terminal to dendrites, requires particular proteins and energy in the form of the molecule, ATP. Further the transfer of information from dendrites to cell bodies also requires special proteins and energy. New proteins are mostly synthesized in cell bodies so they have to be trafficked to their correct location. This is transport. It is important for movement of newly synthesized proteins and trafficking of organelles such as mitochondria, the ATP supply of cells. The components of transport are kinesins (motor proteins) which travel along the microtubular network ferrying their cargoes, proteins and organelles. The kinesins do not bind directly to their cargoes but employ adaptor proteins.

At any one time, ~ 30% of neuronal mitochondria are mobile moving towards (anterograde) and away from (retrograde) synapses. Mitochondria need to be able to translocate rapidly to areas of high energy demand (synapses) and once there, need to be anchored or "parked". Until recently, we did not know the identity of any of the players that are involved in moving mitochondria. Significant advances have now been made. The Stephenson group identified a family of proteins, the TRAKs, which are the major adaptors involved in their anterograde transport. She used advanced imaging methods to visualize moving mitochondria in axons in cultured neuronal cells and showed that TRAKs are important for mitochondrial movement. TRAKs are present in dendrites too where they are likely to be involved in mitochondrial motility. Having discovered the function of this important family of proteins, we now want to determine the importance of this for proper neuronal function (synaptic transmission and dendritic information processing) in the brain. We plan to investigate this using acute brain slices obtained from adult rodent brain where neuronal network activity is retained together with state-of the art techniques such as electrophysiological recordings from single cells and two photon imaging.

We also want to understand further how TRAK-mediated mitochondrial transport is regulated. We have found that an enzyme, N-acetylglucosamine transferase (OGT), is another component of the TRAK/kinesin mitochondrial trafficking complex. OGT is a nutrient sensor. This means that its activity is regulated by metabolic demands. It modifies proteins by addition of a sugar, N-acetylglucosamine. Since mitochondria supply energy it is not unreasonable to hypothesize that the activity of this enzyme may regulate the formation of the kinesin/TRAK/mitochondrial complex to enable mitochondria to traffic to respond to local energy requirements.

These are important questions since many neurodegenerative diseases, for example Alzheimer's disease, motor neuron disease and Huntington's disease, for which there are currently no effective treatments have deficits with respect to mitochondrial distribution and function. Deficits in mitochondrial trafficking i.e. appropriate availability of energy sources may be early events compromising neuronal function eventually contributing or even being causal to these diseases. Understanding these basic trafficking mechanisms may contribute towards development of innovative therapies for their treatment.

Neuronal mitochondria are mobile utilising kinesin motor proteins and the microtubule network. They undergo fusion and fission and are immobilized in an activity-dependent manner. Evidence suggests that their movement is essential for some forms of synaptic release and plasticity. Regulated trafficking mechanisms must thus exist to facilitate transport and docking. Proteins implicated in these have recently been identified but how they interact can only now be studied. Mitochondrial trafficking proteins include the TRAK family of kinesin adaptors which were discovered by the Stephenson group. They developed a kinesin/TRAK/mitochondrial trafficking complex model and demonstrated its functional role in axonal mitochondrial transport in cultured hippocampal pyramidal neurons.

It is unknown whether TRAK-mediated transport plays a role in synaptic transmission and plasticity. We will thus infect, using viral technology, a previously validated TRAK2 dominant negative (DN) construct and TRAK1/TRAK2 shRNAs, into hippocampal neurons. Electrophysiological recordings and two photon- imaging will be used to determine whether the arrest of TRAK-mediated mitochondrial movement alters glutamate release from axons and how TRAK-mediated mitochondrial motility affects post-synaptic dendritic excitability.

We will continue to understand further the regulation of the TRAK/kinesin complex, focusing on the post-translational modification, nutrient regulated enzyme, N-acetylglucosamine transferase (OGT) which the Stephenson group showed is an integral component of the complex. We will investigate whether the post-translational modification of the complex regulates protein-protein interactions that determine formation or dissociation and thereby mitochondrial transport using biochemical techniques and live imaging of hippocampal neurons.

This proposal will enhance understanding of the regulation of mitochondrial movement as well as its significance for neuronal function.

Potential beneficiaries of this proposal include researchers investigating neurodegenerative disorders, the pharmaceutical industry, junior scientists, the public and ultimately, should we be successful, the economy. Aberrant distribution of mitochondria is a feature of debilitating neurodegenerative diseases which include Alzheimer's disease. Dementia is a global problem. In the UK, currently, ~ 820 000 people live with dementia. The current cost to the UK economy of looking after dementia patients is £23 billion per year. This is largely due to care costs since although some drugs namely the anticholinesterases, are available for the treatment of mild cognitive impairment, their efficacy is questioned. Although advances are being made with regard to the underlying cell biology of dementias this has not yet led to new therapies. The idea that defects in mitochondrial transport may contribute or indeed be causative to disease processes opens up a new area for investigation in this field. We will contribute reagents to facilitate these investigations in animal models of disease as well as in human postmortem tissue. Our speculation is that defects in mitochondrial transport may contribute or indeed be causative to disease processes. We will be producing new information and research reagents which will allow this hypothesis to be tested. This may lead to new medicines and in the long term, enhance the quality of life and health in the UK.

Our research teams are basic science groups so we need to ensure that we secure engagement with the
neurodegenerative research community, with industry, with our colleagues and with the public. This will be achieved by:-

(i) presenting our work at national and international meetings in the form of oral and poster presentations. We attend a wide range of workshops and learned society meetings (see Academic Beneficiaries). We also have experience in organising symposia at national and international levels. Forthcoming meetings include a symposium to be held at the 2012 FENS meeting in Barcelona (Shah) and The Biochemical Society and the European Society for Neurochemistry, (2013, Bath, UK, Stephenson). Through attendance at these meetings we will network with researchers in the basic sciences in addition to clinical colleagues, the latter being seen as important for future exploitation. As appropriate, work will be published in high
profile journals.

(ii) We already engage with industry having industrial collaborations in place, i.e. Shah has an active collaboration with Merck Research Laboratories (New Jersey, USA); Stephenson with the Millipore Corporation, California, and Abcam in Cambridge, UK for the commercial production and marketing of specific antibody reagents. Should we need further advice we will utilise the UCL Business Office which can advise on intellectual property rights and collaborative industrial ventures.

(iii) For engagement with junior scientists, we have a track record but we seek to maintain and extend this. Previous activities include, Shah and Stephenson organized in 2010 the Inaugural Neuroscience Retreat for SoP neuroscientists. All PhD students presented posters and post-doctoral research fellows gave talks, giving both an opportunity to develop communication skills. Dr Shah co-organised a SoP seminar programme open to researchers at all levels. We both train our own PhD students. MSc students and MPharm students carry out research projects in our respective laboratories. Stephenson teaches research skills on the PhD Training Course; Stephenson and Shah teach development skills for MRes
students. Stephenson has also recently been appointed one of 50 appointees in the UCL "Future Fifty Mentoring Scheme' to mentor a younger colleague. Shah was recently appointed a mentor by The Physiological Society.