The In Vitro Functional Study Of LRR Domain Of LRRK2-The Disease Gene Of Parkinson's Disease

Objectives: After screening the prey of bookshelf to get the candidates that interact with the LRR domain of LRRK2 by yeast two hybrid system, the candidate molecule was further identified by immunofluorescence staining, which may offer a way to dissect the functions of other domains of LRRK2 protein in vitro to investigate the potential role of LRRK2 in the pathogenesm of Parkinson's disease.Methods: The bait plasmid pGBDU-LRR was constructed by molecular cloning and transformed into yeast to acquired the strain PJ69-4a/pGBDU-LRR. Then the yeast two hybrid system was set up to investigate which prey plasmid (alpha-synuclein,parkin,UCHLl and DJ1)interacted with the bait. There were three groups of HEK293 cells transfected with different combinations of FLAG or HA tagged plasmids persectively: the control (parkin only), the experiment group 1(parkin+wild type LRR) and the experiment group 2 (parkin+mutant LRR). Hoechst staining was done to investigate the cell apoptosis. Immunofluorescent staining was done to show if there is a colocalization of LRR domain and parkin. The western blot was to test whether wild type or mutant LRR domain can coexpress with parkin in HEK293 cells. The immunoprecipitation were performed to further show whether there is an interaction between parkin and LRR(wild type or mutant).Results: It was indicated that protein parkin may interact with wild type LRR domain by yeast two hybrid. Western blot showed that LRR and parkin were co-expressed in HEK293 cells and immunoprecipitation proved that there was an interaction between LRR domain (wild type) and parkin. The immunofluorscence also found out that parkin (green) and wild type LRR domain (Red) were both expressed in cytoplasm and the merged image was orange. It was indicated that LRR domain (wild type) and parkin were colocalized and there was an interaction between parkin and wild type LRR in vitro. However, the mutant LRR domain was expressed in nuclei as aggregates and the merged image was not orange. So mutant LRR domain and parkin were not colocalized and there was no interaction between parkin and mutant LRR. The Hoechst staining showed that the experiment group 2 had a higher rate of cell apoptosis than that of experiment group 1 and control. Moreover, immunostaining also found that there were intranuclear aggregates in experiment group 2 that were similar to Lewy's bodies in patients of Parkinson's disease. Therefore, we had set up the cellular model of Parkinson's disease.Conclusions: The wild type LRR domain colocalized with parkin in the same subcellular compartment and there is an interaction between parkin and wild type LRR, but no evidence of interaction between parkin and mutant LRR domain. The mutant LRR domain can form a intranuclear aggregates that is similar to the structure of Lewy body which is the hallmark of Parkinson's disease.