Brain Distribution And Cellular Localization Of LRRK2 And Prokaryotic Expression Of Its Functional Domains

Parkinson's disease (PD) is a common neurodegeneration diseasecharacterized by tremor, rigidity, bradycinesia and postural instability. MostPD cases are sporadic, but 5%-15% PD cases have shown to be inherited.a-synuclein, parkin, PINK1, DJ-1, LRRK2, UCH-L1, and ATP13A2 arelinked to inherited form of PD. LRRK2 locate in chromosome 12, coding akinase with 2573 amino acid. At least 9 pathogenic mutations in LRRK2have been identified so far. G2019S, probably the most common mutation ininherited PD patients, could also be found in sporadic PD cases. LRRK2protein belongs to a Roco family, containing 6 domain: ANK, LRR, ROC,COR, MAPKKK and WD40. The protein is highly conserved through outthe evolution.Objects: To study the expression pattern of LRRK2 in mouse brain; Toobserve whether LRRK2 co-localize with mitochondria; To express andpurify the LRR and COR domains of LRRK2.Methods: We expressed and purified LRRK2 recombinant proteins inEcoli.BL21. With the antigens, we developed polyclonal antibody againstLRRK2 through immunizing New Zealand rabbits. With the antibody, wedetermined the distribution of LRRK2 in the mouse brain throughimmunohistochemistry and localized LRRK2 in HEK293 cells byimmunofluorescence. Through GST affinity purification, we further purified the recombinant LRR and COR domains of LRRK2 protein in Ecoli.BL21.Results: LRRK2 were widely expressed through out the mouse brain,but showed significant differences in different region of the brain. LRRK2showed highest expression level in cortex, hippocampus, striatum andamygdale; with modest signal in substantia nigra, thalamus, some parts ofhypothalamus and Purkinge cell of cerebellum. LRRK2 is endogenouslyexpressed in HEK293 cells. It is basically a cytoplasm protein and couldco-localize with mitochondria. Finally, we purified LRR and COR-GSTfusion protein.Conclusion: We systematically studied the distribution pattern ofLRK2 in the mouse brain and find that LRRK2 co-localize withmitochondria. In addition, we purified LRR and COR-GST fusion protein.Our study provids important foundation for the further research aboutphysiological function of LRRK2 and its role in the PD pathogenesis...