Antibody Preparation, Neuroanatomical Distribution And Subcellular Localization Of LRRK2's Protein Associated With Parkinson's Disease

Parkinson's disease (PD) is the most frequent progressive neurodegenerative movement disorder characterized by age-dependent resting tremor, muscular rigidity, akinesia and relieved temporarily by administration of L-dopa. The prevalence of PD is 0.71% in a German Bavarian population of individuals older than 65 years, and is 0.30% in Singaporeans aged 50 years and older. The pathological hallmarks of PD patients include progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta as well as the presence of ubiquitin-positive Lewy neuritis and Lewy bodies in the remaining neurons. Nine loci in the chromosome have been found to be associated with family PD including PARK1-PARK9 and PARK13 in which PARK1 and PARK4 share the same locus. Mutations in at least seven genes are individually linked to familial forms of PD, including autosomal dominant mutations in a-synuclein, UCH-L1, LRRK2, Omi/HtrA2 and autosomal recessive mutations in parkin, PINK1, and DJ-1.LRRK2 gene has 51 exons. The LRRK2 protein has 2527aa, including ANK, LRR, ROC, COR, Kinase, WD40 six domains. The discovered mutations associated with PD are distributed in all the domains of LRRK2 and the most common one is G2019S. The MW of LRRK2 is 280 KD. It is expressed in brain, other organs and cell line HEK293T with unclear functions. Some research have showed that mutations associated with PD, such as R1441C, G2019S, I2020T, lead to increasing of LRRK2 kinase activity and decreasing of cell activity, indicating that these mutations are toxic to cells. Moreover, LRRK2 plays a role in neurite process morphology and polarity of synaptic vesicles (SV) protein.Anti-LRRK2 antibody is a necessary and important tool of LRRK2 functional research. To prepare anti-LRRK2 antibody, I analyzed LRRK2 protein sequence by DNAstar/Protean software and selected 3 polypeptides which are about 100aa, high antigenicity and good hydrophilicity. Then, the CDS of the selected 3 polypeptides were constructed to pGEX-4T-2 and the GST fusion protein were purified. With the purified GST fusion protein, the rabbits were immunized and a high titer, high specificity anti-LRRK2 rabbit polyclonal antibody was gotten. Functional analysis of anti-LRRK2 showed that it was available in immunohistochemistry, immunostaining and Western Blotting.With the anti-LRRK2 antibody, the distribution of LRRK2 in mouse brain was mapped. It was showed that LRRK2 protein is broadly distributed throughout the mouse brain, but shows important regional differences. Also, the result of Western blotting showed that LRRK2 is expressed in all sections of rat brain. In HEK293, LRRK2 is co-localized with cytochrome C. In addition, the result of alignment showed that amino acids sequences of LRRK2 and LRRK1 are highly homogenous.