Objective/Rationale:
Several years after the discovery of mutations in the LRRK2 gene as an important cause of Parkinson’s disease, relatively little is known regarding the basic cellular function of the protein (LRRK2) and its role in the pathologic events leading to disease. One major obstacle impeding LRRK2 research is the lack of reliable antibodies. Many available LRRK2 antibodies lack specificity/sensitivity and are limited in their applicability to different immunological techniques. Reasons for the difficulties in raising sensitive and specific LRRK2 antibodies is the large size of the LRRK2 protein, its complex domain structure, and most importantly, the high homology of the protein between different species. We aim to overcome these obstacles by using several complimentary approaches to generate antibodies in models which do not have the gene encoding LRRK2.
Project Description:
We propose to generate LRRK2-specific antibodies in models devoid of the murine homologue of LRRK2. The lack of endogenous LRRK2 protein renders the models much more susceptible to the antigen response caused by human LRRK2 fragments and stimulates the immune system to produce highly specific and sensitive antibodies. Our first experiment has been designed to test (i) five LRRK2 peptides, (ii) the recombinant LRRK2 C-terminus and (iii) cDNA encoding the LRRK2 C-terminus, to examine their respective ability to induce the generation of LRRK2 specific antibodies. After this pre-screening the best antigen will then be selected for the generation of monoclonal antibodies.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Elucidation of the molecular mechanisms leading to disease generally precedes the development of novel therapeutic approaches. Highly sensitive and specific antibodies are essential for the elucidation of the pathways involving LRRK2, the identification of LRRK2-interacting partners and to visualize LRRK2 localization in healthy and diseased brain tissue. The generated antibodies will also help determine whether the LRRK2 protein is present in the pathological hallmark Lewy bodies, thereby serving as a further diagnostic protein marker.
Anticipated Outcome:
This project will generate LRRK2 antibodies with a high degree of specificity and sensitivity allowing for the investigation of LRRK2’s cellular biology. It will also help to visualize LRRK2-related changes, identify disease mechanisms, and promote the discovery of potential targets for future therapeutic intervention.