Screening For LRRK2 Interactants By Yeast 2-hybrid Analysis And Association Study In The Promoter Region Of LRRK2 And UCH-L1 With Parkinson's Disease

Parkinson's disease (PD) is the second most common neurogenic disorder after Alzheimer disease (AD). The disease is progressive and mostly occurs during middle or old age, affecting approximately 1% of the population over age 50. In very rare cases, symptoms are developed at young age. The primary symptoms of PD includes resting tremor, muscular rigidity, bradykinesia, and postural instability. Additional features are characteristic postural abnormalities, dysautonomia, dystonic cramps, and dementia. Pathologic features of classic PD include by a loss of dopaminergic neurons in the substantia nigra (SN) and the presence of eosinophilic intracytoplasmic inclusions known as Lewy bodies, in surviving neurons in various areas of the brain,including substantia nigra, locus ceruleus, nucleus basalis, hypothalamus, and cerebral cortex. Autosomal recessive juvenile Parkinson's disease, however, does not have Lewy body pathology. Although the vast majority of PD cases are thought to be sporadic, the role of genetic factors in PD has been valued more and more. Recent progress in molecular genetics studies of families with PD has led to the identification of 12 loci that are linked to certain inherited forms of PD. These includeα-SYNUCLEIN (PARK1), UCH-L1 (PARK5) and LRRK2 (PARK8) leading to autosomal dominant PD, as well as PARKIN (PARK2), PINK1(PARK6), DJ-1 (PARK7), HTRA2(?ARK12) and ATP13A2(PARK9) leading to early onset recessive PD.Mutations in LRRK2 are thus far the most frequent genetic cause associated with autosomal dominant and idiopathic PD. Its mutations are found in approximately 2-13% of patients with familial PD. Importantly, mutations have also been associated with sporadic PD with unprecedented 0.5-3% prevalence. LRRK2 has emerged as, perhaps, the most relevant player in PD pathogenesis identified to date. UCH-L1 is a member of a gene family whose products hydrolyze small C-terminal adducts of ubiquitin to generate the ubiquitin monomer. Expression of UCH-L1 is highly specific to neurons and testis/ovary. Besides its hydrolase activity, UCH-L1 can also associate with monoubiquitin to control ubiquitin levels in the ubiquitin pathway, which has been functionally implicated in various cellular mechanisms, including maintenance of chromatin structurre, transcriptional activation, ribosomal assembly, antigen processing, protein translocation, endosome-lysosome biogenesis, control of the cell cycle and programmed cell death, DNA repair, spermatogenesis, and regulation of viral gene expression et al. Its occurrence in Lewy bodies and its function in the proteasome pathway make it a compelling candidate gene in Parkinson's disease. Therefore, study of LRRK2 and UCH-L1 pathogenic mechanisms relating to neurodegeneration is of crucial importance in understanding and combating PD.The work includes two parts:In part1, we isolated parts of LRRK2 protein and performed a yeast two-hybrid analysis to isolate and identify its potential binding partners in the human fetal brain cDNA library; In part 2, we sequenced the identified minimum promoter region of LRRK2 gene and UCH-L1 gene in PD patients and unrelated control samples to screen for mutations related with PD.Our results show that:1.Potential binding partners of LRRK2 in the human fetal brain cDNA library:Employing a sequence containing full-length of COR domain and part of ROC and MAPKKK domain as bait, we identified STRBP,BAG5,PTPN23,L3MBTL3,RALYL and KIAA1783 as potential binding partners of LRRK2 in the human fetal brain cDNA library through yeast two-hybrid screens; 2.Mutations in the promoter region of LRRK2 gene:In 108 sporadic PD patients and 197 unrelated control samples, we found 4 SNP positions,-838 g/a,-523 a/g,-275 t/c and -221 g/c, in the promoter region of LRRK2 gene and statistical analysis could not completely eliminate the possibility that they are associated with PD. In the region, we also found 2 mutations,-551 a/g and -469 g/t, carried in different PD patients and 1 mutation,-696 c/t,in a healthy control; 3. Mutations in the promoter region of UCH-L1 gene: In 118 sporadic PD patients and 246 unrelated control samples, we found 5 SNP positions,-892 t/c,-307 g/a,-24 g/a,-16 t/c and +247 g/c, in the promoter region of UCH-L1 gene and statistical analysis showed that the last 4 SNP positions may participate in the same haplotype block associated with PD. Besides, we also found-306 g/a,-232 a/g and -150 t/c once in different PD patients and-974 c/t,-957 c/t,-615 t/g,-165 g/c and +52 t/g once in different healthy control.