MICA: Role of D1R-D3R heteromers on striatal function in L-DOPA-induced dyskinesias

Many people with Parkinson's disease do not respond well to therapy with the most widely-used drug called L-DOPA, having uncontrollable body movements that make them feel ashamed or even fall and get injured. We and other doctors think that this occurs because proteins called dopamine receptors associate in unusual structures called heteromers in nerve cells. However, no one knows how and where heteromers are formed, whether they affect other receptors and if it actually makes people feel bad. We have shown that there really are many more heteromers in brains of the animals with experimental Parkinson's disease put on human therapy. Now, we want to use special animals that will enable us to see where heteromers are formed. Further, we made contacts with companies to make smaller proteins of our design that will break heteromers apart, which could then be used to prevent side effects of drugs.
A part of the brain that is essential for manifestations of Parkinson's disease is called striatum. It is controlled by inflow of impulses that release a substance called dopamine. The main problem of Parkinson's disease is that the source of dopamine is lost. In therapy, L-DOPA serves to compensate for the lack of dopamine.
Importantly, not all nerve cells in striatum are the same: there are two subtypes with contrasting properties, particularly how they react to dopamine.
Nerve cells communicate and transmit information across structures called synapses. The sending nerve cell (presynaptic) relays the information by releasing chemical transmitters. The receiving cell (postsynaptic) detects that signal by specialized receptor proteins present on its body or fine extensions called dendrites. At the points of contact, dendrites have bud-like protrusions called dendritic spines that possess molecular machinery necessary to process the signal. Different types of dopamine receptors in striatal spines respond to dopamine differently, transmitting the signal in a specific way. Normally they stand apart, but can also aggregate into heteromers, which will transmit the signal in a different way.
Among other specialized receptor proteins in spines are AMPA and NMDA receptors, responsible for nearly all of the fast communication between neurones in the brain.
A lot is known about interplay between AMPA, NMDA and dopamine receptors, both in health and Parkinson's disease. For example, we know many ways how they affect each other to work more or less strongly or how the signals from one receptor make other receptors to incorporate into the synapse or completely leave it, thus modulating the overall synaptic function. Almost nothing is, however, known whether the same rules apply when dopamine receptor-heteromers are present, or about the consequences they may have in Parkinson's disease.
This is important because it could tell us why patient's brains make wrong calculations and send wrong signals that result in unwanted movements.
To answer all these questions, we will use special animals that allow us to tell between subtypes of nerve cells in striatum, even allowing us to see when heteromers are present in them, because they become fluorescent. We will apply chemicals that cause Parkinson's disease-like condition in these animals. Then, using special methods, we will be able to track the fluorescent dopamine receptor heteromers and see them within the living cells. To achieve this, we will use a powerful confocal microscopy to see tiny details within nerve cells. We are good in applying this methodology, so we can minimize the number of animals used.
Understanding what heteromers do and how they themselves are regulated will help us try to find the way to prevent them from overtaking control over striatum. This will help us devise a new strategy in fight against Parkinson's disease and the deleterious side effects of its treatment.

Disturbances in striatal function in Parkinson's disease are associated with a loss of dopaminergic signalling. Widely used in therapy, L-DOPA typically leads to dyskinesias which strongly impair patients' quality of life. The cause of dyskinesias is unknown, but is believed to be associated with increased presence of D3 dopamine receptors. Postsynaptic dopamine receptor subtypes D1Rs and D3R are able to aggregate in D1R/D3R heteromers (D1/3-Hets) with altered signalling and trafficking properties. It is believed that D1/3-Hets trigger the events leading to L-DOPA-induced dyskinesia. Previous results show that D1/3-Het striatal expression is significantly higher in Parkinson's disease model animals on chronic L-DOPA therapy. However, the molecular mechanisms underlying D1/3-Hets' contribution to altered striatal synaptic function are not well understood.
Extending these findings, I intend to develop a transgenic strain which allows D1/3-Het visualization in living neurones within striatal circuitry. It will serve as a research model to test how the interplay between postsynaptic D1/3-Hets and AMPAR/NMDAR trafficking and function affects the synaptic output from striatum, which is a key determinant for expression of dyskinesias.
Further, I will develop antibodies specific to D1/3-Hets, as well as small peptide fragments able to disrupt D1/3-Het assemblage.
This project is timely and relevant and it will fundamentally contribute to our understanding of pathogenesis of dyskinesias in Parkinson's disease therapy. Establishing D1-D3 heteromers as relevant factors will inform future research on making them potentially amenable to pharmacological modulation.

Parkinson's disease patients: Every year, there are 10,000 new Parkinson's patients in the UK. In less than five years after diagnosis, half of them will severely suffer from therapy-induced adverse effects. My ultimate goal is to facilitate the translation of the project results into solid therapeutically useful reagents that will treat, prevent and cure PD. This will lead to major improvements in the patients' quality of life.
Patients with other brain disorders: Identifying the roles and possible protective modulations of dopaminergic-glutamatergic interaction on synaptic plasticity will be relevant to many psychiatric and neurodegenerative disorders, ranging from addiction to dementias.

Members of Academia: When starting a new lab, one of the key objectives is to ensure effective and timely dissemination of results. In that, I intend to develop a good track record by making knowledge, reagents and resources freely available. I will strive for regular participation in and organization of national and international workshops and conferences. After accumulating appropriate data, I intend to continue publishing in high-impact journals. Progress summaries and links to original publications will be posted on the Web site of the School of Pharmacy and Biomedical Sciences.

Non-academic beneficiaries: Dissemination of findings of my future group will be facilitated by CLOK - Central Lancashire Online Knowledge, the innovative institutional repository for research outputs that has a proven track-record in promoting and implementing collaboration with the wider community.
Industrial beneficiaries: Abcam UK will benefit from our research through the transfer of specific technical and experimental knowledge related to the behavioural and electrophysiological studies. There will also be the chance to work directly with researchers and policy makers at Abcam in developing specific future collaborative research projects.
Commercial benefits: The in vivo model for visualization of D1R-D3R heteromers will impact Pharma by serving as a first-order, mid-to-high-throughput screening of candidate antiparkinsonic drugs. In addition, our results will be sufficient to justify a large-scale industrial effort for developing a new generation of small molecule anti-parkinsonic drugs that target heteromeric receptors.
The antibody against heteromeric D1R-D3Rs will be commercialized via collaboration of Research and Innovation Office at UCLan and Abcam. Working with team from Luigs&Neumann, I will develop a system providing improvement in visualization of neurones in the slices with features important for the actual end-user (patch-clamp electrophysiologist). The product will have direct commercial impact even prior to the end of the project.
NHS: I hope that preclinical data I will generate will be incorporated into NHS research and the wider health system for translation, exploitation and application. By helping to develop effective preventative strategies, this project may lead to substantial reduction of healthcare costs.
The lab members (PhD students and postdocs) will benefit from multidisciplinary training in wide range of electrophysiological, as well as molecular, biochemical and imaging techniques for studying synaptic function in PD (a well-documented gap in the current UK and international knowledge base). They will also develop transferable skills including: knowledge of, and experience in Home Office legislation concerning the ethical treatment of animals in research; time and project management; communication skills training; team working and networking.
General public: Brain Awareness Week - In 2011, I have initiated and organized the first participation of Belgrade School of Medicine in DANA Foundation- and IBRO-organized event and later succeeded in obtaining funds for this type of activity (FENS grant, 2012). I intend to promote this global event at the University of Central Lancashire and in the surrounding area.