【作者】 莫晓云；

【导师】 夏昆；

【作者基本信息】 中南大学 ， 医学遗传学， 2010， 博士

【摘要】 帕金森病(Parkinson Disease, PD)是一种常见的神经退行性疾病。PD的发病机制十分复杂,目前认为其是由遗传因素与环境因素的共同作用所导致的。PD遗传致病机制的研究已取得了明显进展,迄今已定位了16个PD相关位点,克隆了11个PD致病基因,其中包括富亮氨酸重复激酶2基因(Leucine-rich repeat kinase 2, LRRK2),该基因不仅是常染色体显性遗传性PD重要致病基因,还与部分原发性PD有关。目前LRRK2基因突变导致PD发生的机制仍不清楚,但已有的研究提示某些LRRK2致病突变(如G2019S)可能引起其功能增强而具有细胞毒性作用,而且过表达野生型LRRK2也可产生细胞毒性作用。此外LRRK2可能参与PD发生过程中异常蛋白沉积。过表达野生型LRRK2、LRRK2的PD相关突变G2019S或LRRK2激酶结构域加速a-Synuclein A53T的聚集。由此可见,对LRRK2的表达和功能的精确调控是维持机体稳态所必需的,调控机制的异常可能导致疾病的发生,因此我们从分离LRRK2基因启动子开始,展开对其转录调控机制的研究。方法：在数据库中查询LRRK2基因相关的序列信息,运用多种在线软件对LRRK2基因的转录起始位点、第一个外显子和启动子进行预测。在预测结果的基础上,设计7个不同长度的缺失片段(它们分别是-941/-1、-659/-1、-659/-143、-530/-143、-426/-143、-332/-143、-232/-143,其中的-941／-1片段包含LRRK2基因启动子的全长预测片段,-659／-1片段将全长片段从5’端向3’端截短至预测CpG岛的5’端上游,-659／-143片段包含LRRK2基因的CpG岛,此后的缺失片段为在此基础上从5’端向3’端每隔100bp左右依次截短),构建荧光素酶报告基因载体,通过在SH-SY5Y细胞和Hela细胞中进行各缺失片段的瞬时转染及荧光素酶活性分析,确定启动子所在区域。并运用MatInspector对LRRK2基因核心启动子可能的顺式元件进行生物信息学分析,为将来顺式元件的鉴定及转录调控研究奠定基础。结果：本研究对LRRK2基因在脑组织中表达的最主要剪切本ENST00000298910进行分析,以LRRK2基因的RefSeq序列NM 198578的蛋白翻译起始位点“ATG”的A碱基为+1位进行描述。结果显示-530／-143间的387bp在2个细胞株中具有最强的启动活性；将片段-530/-143自5’向3’方向截短后,启动活性在两个细胞株中均明显下降,但最短的缺失片段(-232／-143)在2个细胞株中的启动活性仍显著高于pGL3-Basic的基础值(p<0.05),因此-232/-143间的89bp很可能存在LRRK2基因的基础启动子,其在神经细胞及非神经性细胞中均能启动报告基因的表达。将片段-530／-143自5’向3’方向缺失104bp后,启动活性在两个细胞株中均明显减弱(p<0.05),因此推测在-530至-426区间可能存在具有正性调控启动活性的顺式作用元件；将片段-659/-1自3’向5’方向截短142bp得到片段-659／-143,其启动活性明显增强(p<0.05),而片段-659/-143自5’向3’方向截短129bp得到片段-530／-143,其启动活性也增强(p<0.05),因此推测-143至-1区间、-659至-530区间可能存在负性调控启动活性的顺式作用元件。将片段-426/-143自5’向3’方向截短94bp得到片段-332/-143,其启动活性在Hela细胞减弱(p<0.05),而在SH-SY5Y细胞中无明显变化(p>0.05);将片段-332/-143自5’向3’方向截短100bp得到的片段-232／-143,其启动活性在Hela细胞中增强(p<0.05),而在SH-SY5Y细胞中则减弱(p<0.05),因而推测-426至-232区间可能存在一些与不同组织差异性表达有关的顺式作用元件。结论：本研究中,我们确定了LRRK2基因启动子的位置、并对启动子的活性和特征进行了分析,为进一步深入研究LRRK2基因的转录调控机制奠定了基础。α-半胱氨酸串蛋白(Cysteine-string protein-α,C5P-α)是一种突触前囊泡相关蛋白和分子伴侣蛋白,该蛋白参与突触的生长、突触囊泡的运输和对接,并参与大多数突触的成熟、维持及调节。CSP-α基因敲除小鼠出现严重的神经退行性病变和死亡。在CSP-α基因敲除小鼠中过表达人野生型共核蛋白(α-synuclein)和A53T突变型α-synuclein均可减轻CSP-α基因敲除所导致的表型,因此CSP-α与α-synuclein存在功能相关性。共核蛋白基因(SNCA)已被证实是PD的致病基因,它编码的蛋白α-SVnuclein在PD的发病机制中起着非常关键的作用。这些研究结果提示我们CSP-α可能在PD的发病过程中起作用。因此我们在PD患者中对CSP-α基因进行突变筛查,以分析该基因是否参与PD的发病。方法：对CSP-α基因的外显子2-5设计引物进行PCR扩增,采用PCR产物直接测序法在173例中国汉族PD患者中对该基因的编码区(外显子2-5)及外显子-内含子交界区进行突变筛查；由于在患者中,位于CSP-α基因的外显子2和外显子3存在基因变异,因此我们还在273例无神经系统疾病的对照组受试者中对该基因的外显子2、3进行突变筛查。结果：在CSP-α基因编码区中,我们未检测到错义突变,但检测出5个变异,其中2个是位于编码区的同义突变(exon2的c.75C>T和exon3的c.144C>T)；其他3个位于内含子,分别是位于intron2的c.107+41c>g及位于intron3的c.321+20c>t和c.321+133a>g,其中c.321+20c>t和c.321+133a>g为已知多态,c.107+41c>g为一罕见变异,仅在一例36岁以震颤为主要临床表现的女性散发患者中发现该变异,在273例正常人中未能发现该变异的存在。4种多态(c.75C>T,c.144C>T,c.321+20c>t和c.321+133a>g)的基因型和等位基因频率在173例PD患者和273例对照组受试者之间不存在差别(p值均>0.05)。结论：在中国汉族PD患者中,未发现CSP-α基因编码区及剪切位点存在致病突变,CSP-α基因突变可能不是中国PD患者的主要致病原因。神经调节蛋白受体降解蛋白1(neuregulin receptor degradation protein 1, Nrdp1)是一种泛素E3连接酶,属RBCC (N-terminal RING finger/B-box/coiled coil)蛋白亚家族成员。Nrdp1与受体酪氨酸激酶家族成员-神经调节蛋白受体ErbB3及ErbB4互作,通过调节神经调节蛋白受体在细胞表面的稳定性,而影响细胞的生长、增殖及分化过程,并与肿瘤的发生有关。Zhong等通过酵母双杂交实验,在果蝇pACT2 cDNA中鉴定出Nrdp1为Parkin的互作蛋白。Nrdp1通过泛素-蛋白酶体系统降解Parkin,即Parkin是Nrdp1的一个泛素化底物,并且Nrdp1可能通过作用于Parkin而影响线粒体功能。Parkin是遗传性PD最为重要的致病基因之一,在早发性和散发性PD的发病机制中也起着重要的作用。Parkin蛋白是一种E3泛素连接酶,通过降解特定易聚集且具有神经毒性的底物,抑制神经元凋亡,并参与维持线粒体正常功能以及介导细胞对异常线粒体的清除,在PD的发病机制中起着关键作用。由此,我们推测Nrdp1可能在PD发生过程中起作用,因此我们在PD患者中对Nrdp1基因进行突变筛查,以分析该基因是否参与PD的发病。方法：对Nrdp1基因的外显子3-7设计引物进行PCR扩增,采用PCR产物直接测序法在209例中国汉族PD患者和302例健康对照者中对该基因的5’UTR区(外显子1-2)、蛋白编码区(外显子3-7)及外显子-内含子交界区进行突变筛查。结果：在Nrdp1基因编码区(外显子3-7)中未发现序列变异,在5’UTR区和第一内含子区中发现2个未报道的变异：c.-206 T>A与c.-208-8A>G。我们进一步在无神经系统疾病的对照组受试者中对这2个变异进行检测,并进行病例-对照相关分析,结果显示c.-206 T>A不论是等位基因频率还是基因型频率在PD患者与对照组间均不存在显著差异(p>0.05)；而c.-208-8 A>G变异仅在1例PD患者中检测到,并未在对照组中检测到。结论：在中国汉族PD患者中,未发现Nrdp1基因编码区及剪切位点存在致病突变,Nrdp1基因突变可能不是中国PD患者的主要致病原因。泛素-特异性蛋白酶24(USP24)是泛素-特异性蛋白酶家族的成员,其功能是将多聚泛素从靶蛋白上移走,防止靶蛋白被蛋白酶体降解,而蛋白酶体降解系统异常及蛋白聚集是参与帕金森病(PD)发病的关键机制；此外,在1p32已定位了PD遗传易感位点PARK10,至今未在该位点克隆出PD致病基因,而USP24基因位于PARK10上。尽管有学者用单核苷酸多态(SNP)在患者与正常人间进行了相关分析并得出USP24基因与PD相关的结论,但他们并未在PD患者中对USP24基因进行突变筛查,本研究首次在PD患者中对USP24基因的部分外显子,外显子39-68进行突变筛查,以探讨该基因是否参与PD的发病。方法：对USP24基因的外显子39-68设计引物进行PCR扩增,采用PCR产物直接测序法在92例中国汉族PD患者中对该基因的外显子39-68及外显子-内含子交界区进行突变筛查。结果：在USP24基因的exon39-68中未发现任何碱基变异,但在外显子-内含子交界区检测出11种变异,其中3种为已知多态(rs6588545,rs12031876和rs10493176),位于exon 59的c.7078+22 a>g变异仅在1例以强直为主要临床症状的男性早发性PD患者中检测到,在95例正常人中不存在此变异。结论：在中国汉族PD患者中,未发现USP24基因编码区及剪切位点存在致病突变,位于USP24基因exon 39-exon 68的基因突变可能不是中国PD患者的主要致病原因。更多还原

【Abstract】 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.更多还原