The Isolation Of Promoter Regions Of The Parkinson Disease Causitive Gene LRRK2, And Mutation Screening Of The CSP-α, Nrdp1, USP24 Gene In Parkinson Disease Patients

Parkinson's disease (PD) is the second most globally prevalent neurodegenerative disorder. The cause of PD is complex and multifactorial, involving both hereditary and environmental factors. Recent progress in molecular genetic studies of familial PD has led to the identification of 16 susceptible loci and 11 genes responsible for PD. Mutations in LRRK2 are thus far the most prevalent genetic cause associated with autosomal dominant and idiopathic PD. Although the mechanism of how PD-associated LRRK2 mutations cause disease is yet unknown, several studies indicated that certain kinds of mutations including G2019S are likely associated with toxic gain of function. Moreover, to overexpress wildtype LRRK2 was found to be toxic in cultured cells and transgenic fly model. Becides, LRRK2 was found to be associated with abnormal protein deposition in the brains of patients with lowy body disease including PD. To overexpress wildtype LRRK2, G2019S mutant or kinase domain alone accelerate the formation of aggregates caused by a-synuclein A53T mutant. These results suggest that precise regulation of LRRK2 expression and function is necessary for maintaining homeostasis of the organism, and the disruption of the regulation mechanism may cause disease. Therefore, to elucidate the mechanism of LRRK2 transcriptional regulation is helpful to understand PD aetiology. In this study, the promoter activity of the LRRK2 5'-flanking region was analized, which is the first step to understand the transcriptional regulation of LRRK2.Methods:Several on-line softwares were involved in our study to predict the transcriptional start site, the first exon, and the promoter regions. Base on the predictation results,7 fragments whose lengthes was deleted further and further, and constructed the luciferase reporter plasmids. The plasmids were then transfected transiently into the SH-SY5Y and Hela cell lines, and the luciferase activity were analysed to identified the core promoter region.Results:1) The major transcript in the brain-ENST00000298910 was our target transcipt, and we used the'ATG' of the RefSeq:NM₁98578 as the marker to describe the location of the sequences in our study.2) The basal promoter of LRRK2 is located at-232/-143 region that drives the transcription of reporter gene in both neuronal and non-neuronal cells. A 104bp fragment in-530/-426 shows significant enhanced activity, suggesting a positive cis-acting element within region-530/-426. Two fragments in-143/-1 and -659/-530 shows significant decreased activity, suggesting a negative cis-acting element within these two regions. Comparing the structs of LRRK2 2 splice forms (ENST00000298910 and ENST00000343742), they might share the same promoter region. In this region, we also find a fragment in-426/-232 that shows different promoter activity in neuronal and non-neuronal cells. The result is consistent with the fact that LRRK2 is widely expressed while the express level between tissues is not the same. To understand the mechanism behind it needs further research.In summary, we have identified and characterized the promoter of human LRRK2 gene. The results establish the basis for further research on the transcription regulation mechanism of LRRK2. Cysteine string protein-a (CSP-a) is an abundant vesicle protein and molecular chaperone. It is thought to promote synaptic growth and vesicle trafficking/docking, and to participate in the maturation and maintenance of synapses. Genetic inactivation of CSP-a in mice results in severe neurodegeneration and postnatal lethality. Transgenic expression of human/mouse wild-typeα-synuclein rescues the lethality and neurodegeneration induced by CSP-a ablation, indicating the functional interaction ofα-synuclein and CSP-a. a-Synuclein plays a key role in the pathogenesis of Parkinson disease (PD). We therefore hypothesized that CSP-a also contributes to the development of PD via its relationship withα-synuclein. To elucidate the relationship between CSP-a and PD, we screened for potential mutations in the CSP-a gene among Chinese PD patients.Methods:Using direct sequencing, we analyzed the coding regions (exons 2-5) and the exon-intron junctions of CSP-a among 171 PD patients and 273 genetically unrelated control individuals.Results:No missense mutations were found in the coding regions. However,5 variants were identified, including silence variants c.75C＞T (exon 2) and c.144C＞T (exon 3); previously reported polymorphisms c.321+20c>t and c.321+133c>t (both in intron 3); and a rare variant c.107+41c>g (intron 2). Case-control association analysis revealed that the allelic and genotypic distributions of the 4 variants (c.75C>T, c.144C>T, c.321+20c>t and c.321+133c>t) did not significantly differ between patients and controls.Conclusion:we conclude that the CSP-a gene may not affect PD pathogenesis via gene mutations in the Chinese population. Strong evidence has proved that a defect in the Parkin gene is a common, known genetic cause of Parkinson disease (PD). The E3 ubiquitin ligase Nrdp1 is shown to interact with the N terminal of Parkin (the first 76 amino acids) and catalyze degradation of Parkin via the ubiquitin-proteasome pathway, suggesting that Nrdp1 may be involved in PD development via Parkin regulation. For the first time, we screened PD patients for mutations in the Nrdp1 gene to determine the association between these variants and PD.Methods:By direct sequencing, we analysed the entire coding regions and 5' UTR of Nrdp1 in 209 Chinese PD patients and 302 unrelated healthy individuals.Results:No variant was detected in the coding regions (exons 3-7); only 2 variants (c.-206 T>A and c.-208-8 A>G) were identified in the 5' UTR (exon 2) and intron 1. Furthermore, a study of the allelic and genotypic association between patients and controls showed no significant association between the c.-206 T>A polymorphism and PD; c.-208-8 A>G was identified in one PD patient and not in controls.Conclusion:we conclude that the Nrdp1 gene may not affect PD pathogenesis via gene mutations in the Chinese population. Ubiquitin-specific proteases gene (USP24) is a member of the family of the ubiquitin-specific proteases that remove polyubiquitin from target proteins. Ubiquitin-proteasomal pathway is proved to involved in the etiology of PD. Although none gene was cloned in the suspected PD pathogenic PARK 10 locus until now, but USP24 has strong a priori biological plausibility to be involved in PD, also, the evidence has showed that multiple SNPs located in the USP24 gene had significant association with PD risk. Although the association study provided the evidence that USP24 gene might be the suspected PD pathogenic gene, but it is necessary to screen PD patients for mutations in the USP24 gene to determine whether there are the variations contribute to the PD pathogenesis. For the first time, we screened PD patients for mutations in the USP24 gene.Methods:By direct sequencing, we analysed part of the coding regions (exon39-exon68) and exon-intron boundaries in 92 Chinese PD patients.Results:No variant was detected in the coding regions (exons 39-68); only 11 variants were identified in the exon-intron boundaries,3 of them are previously reported polymorphisms (rs6588545, rs12031876 and rs10493176), the variant c.7078+22 a>g only identified in 1 early-onset male patient complicated with rigity, and not in controls.Conclusion:Our data do not support the hypothesized major role of the mutation located in the exon39-exon68 of USP24 gene in PD development in the Chinese population.