The aim of the project is to gain a better understanding of the molecular mechanisms that underlie on the one hand normal homeostasis that leads to the maintenance of healthy brain function and on the other the biochemical nature and origin of signals that drive the initiation and progression of neurodegeneration.
In this project you will investigate the role played by microglia in these processes. Microglia are specialised immune system cells that are restricted to the nervous system. In the lab we are interested to understand the respective roles of inflammation initiated by the Toll-like receptors and of LRRK2, a protein kinase, variants of which cause a predisposition to Parkinson’s disease in the human. Recent studies suggest that the TLR4 pathway cooperates with LRRK2 to induce neurotoxicity1,2.
You will prepare primary cultures of microglia which will be activated with LPS and other TLR agonists in the presence and absence of the LRRK2 kinase inhibitor, L2In1. Cell lysates will be immunoprecipitated with anti-LRRK2 antibodies. Protein components of the complexes will be separated by SDS-PAGE and identified by tryptic peptide mass analysis using a MALDI-TOF mass spectrometer. The presence of non-protein components of the complexes will also be determined by labeling the microglia cultures with appropriate radiolabelled precursors.
To complement the directed and immunoprecipitation approaches above you will use 2-hybrid techniques to identify binding partners for the LRRK2 leucine rich repeats and WD40 domains. Initially well established yeast 2 hybrid methods will be used. A bait strain will be made from the LRRK2 LRRs or WD40 domain and a cDNA ‘prey library’ from resting and activated microglia. Modern Y2H methods allow the rapid construction of cDNA libraries and use yeast mating to achieve very high transformation efficiencies of the bait strain. As LRRK2 is membrane associated we will also use the ‘split ubiquitin’ approach which can also detect interactions with membrane proteins that are not detected by the standard Y2H technique. In this method protein-protein interactions reconstitute the N and C-terminal portions of ubiquitin and cause the release of a reporter molecule tethered at the ubiquitin C-terminal by ubiquitin specific protease3
- Moehle, M.S., Webber, P.J., Tse, T., Sukar, N., Standaert, D.G., DeSilva, T.M., Cowell, R.M. & West, A.B. LRRK2 inhibition attenuates microglial inflammatory responses. J. Neurosci. 32, 1602-11 (2012).
- Gillardon, F., Schmid, R. & Draheim, H. Parkinson's disease-linked leucine-rich repeat kinase 2(R1441G) mutation increases proinflammatory cytokine release from activated primary microglial cells and resultant neurotoxicity. Neuroscience 208, 41-8 (2012).
- Stagljar, I. Finding partners: emerging protein interaction technologies applied to signaling networks. Science's STKE : signal transduction knowledge environment 2003, pe56 (2003).