ROLE OF ALDH2--TRANSGENIC MICE CARRYING ASIAN ALDH2-2 VARIANT ALLELE

ALDH2 的作用——携带亚洲 ALDH2-2 变异等位基因的转基因小鼠

• 批准号: 6431367
• 负责人: BYOUNG-JOON SONG
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6431367
• 关键词: alcoholic beverage consumption; alcoholism /alcohol abuse; aldehyde dehydrogenases; alleles; animal breeding; behavioral genetics; enzyme activity; ethanol; gender difference; gene targeting; genetically modified animals; isozymes; laboratory mouse; neurotransmitter metabolism; psychopharmacology

Organ damage caused by chronic alcohol consumption can be initiated, in part, by the accumulation of cytotoxic acetaldehyde in the target tissues, since highly reactive acetaldehyde, produced from ethanol metabolism, interacts with the free amino group of various cellular macromolecules including DNA and proteins, usually altering their physiological functions while initiating auto-immune responses. Accumulation of acetaldehyde can be achieved through inhibition of the major aldehyde metabolizing enzyme, the mitochondrial aldehyde dehydrogenase 2 (ALDH2), by either chemical inhibitors or genetic mutation (G to A nucleotide substitution) with a subsequent change in Glu487Lys in the ALDH2 protein. Individuals with the genetic variation (ALDH2-2) possess reduced ALDH2 activity through dominant inactivation of the enzyme and show aversive reactions and facial flushing, due to acetaldehyde accumulation following alcohol consumption, as observed in many East Asian people. Although chemical inhibitors are frequently used in clinics, they cause many problems due to non-selective interactions with other enzymes and proteins, short duration of action due to rapid metabolism, and numerous side effects. Because of the problems associated with the chemical inhibitors of ALDH2, we have taken genetic approaches using molecular biology techniques. We hypothesized that transgenic mice carrying the dominantly negative human ALDH2-2 (hALDH2-2) transgene or knock-out mice deficient in mouse ALDH2 gene would have reduced ALDH2 activity with elevated levels of acetaldehyde upon alcohol treatment, compared to their wild-type controls. Our results showed that the recombinant human ALDH2-2 variant protein interacted with the mouse ALDH2 protein and dominantly inhibited the activity of the mouse enzyme. In addition, we produced transgenic mice carrying the hALDH2-2 using a pcDNA3 vector containing the full-length cDNA for the hALDH2-2 coding region and the mitochondrial leader sequence under the control of cytomegalovirus promoter with a polyadenylation signal and transcription termination sequences from bovine growth hormone. Expression of the hALDH2-2 transgene in the liver and brain of transgenic mice was detected by RT-PCR of mRNA, enzyme assays and immunoblot analyses using polyclonal antibodies directed against ALDH2. To our surprise, hALDH2-2 protein was expressed at low levels in the liver and brain of transgenic mice. Subsequently, mouse ALDH2 enzyme activity was slightly inhibited in these animals. Although there was no significant difference in liver or brain pathology in mice treated with 20% ethanol (4 g/kg/day) for 2 weeks, hepatic acetaldehyde levels in transgenic mice were increased 50% at 2 h after ethanol injection (p<0.03) relative to control mice while hepatic alcohol concentrations were unchanged. In addition, female transgenic mice, but not males, drank less alcohol than did control mice (p<0.03). These transgenic mice carrying the hALDH2-2 variant can be used as a model to study the role of ALDH in alcohol preference, acetaldehyde accumulation, metabolism of neurotransmitters, and acetaldehyde-mediated tissue damage after long-term alcohol treatment. Furthermore, to have clear results for the physiological roles of ALDH2, we have been trying to prepare knock-out mice deficient in mouse ALDH2 gene by gene disruption technique. During the past year, we were able to produce chimeric mice, which contain our DNA construct specifically designed to delete the mouse ALDH2 gene. We are now identifying the homozygous mice without the mouse ALDH2 gene using restriction analyses and Southern blot analyses.

由慢性酒精消耗引起的器官损伤可以部分地由靶组织中细胞毒性乙醛的积累引发，因为由乙醇代谢产生的高反应性乙醛与包括DNA和蛋白质在内的各种细胞大分子的游离氨基相互作用，通常在引发自身免疫应答的同时改变它们的生理功能。乙醛的蓄积可以通过化学抑制剂或基因突变（G至A核苷酸取代）抑制主要醛代谢酶线粒体醛脱氢酶2（ALDH 2），随后改变ALDH 2蛋白中的Glu 487 Lys来实现。 具有遗传变异（ALDH 2 -2）的个体通过酶的显性失活而具有降低的ALDH 2活性，并且表现出厌恶反应和面部潮红，这是由于饮酒后的乙醛积累，正如在许多东亚人中观察到的那样。虽然化学抑制剂经常用于临床，但由于与其他酶和蛋白质的非选择性相互作用，由于快速代谢导致的作用时间短以及许多副作用，它们会引起许多问题。由于与ALDH 2的化学抑制剂相关的问题，我们采用了分子生物学技术的遗传方法。 我们假设携带显性阴性人ALDH 2 -2（hALDH 2 -2）转基因的转基因小鼠或小鼠ALDH 2基因缺陷的敲除小鼠在酒精处理后，与野生型对照相比，ALDH 2活性降低，乙醛水平升高。我们的结果表明，重组人ALDH 2 -2变体蛋白与小鼠ALDH 2蛋白相互作用，并显性抑制小鼠酶的活性。此外，我们使用含有hALDH 2 -2编码区的全长cDNA和在巨细胞病毒启动子控制下的线粒体前导序列的pcDNA 3载体产生携带hALDH 2 -2的转基因小鼠，所述启动子具有来自牛生长激素的多聚腺苷酸化信号和转录终止序列。通过mRNA的RT-PCR、酶测定和使用针对ALDH 2的多克隆抗体的免疫印迹分析来检测hALDH 2 -2转基因在转基因小鼠的肝脏和脑中的表达。 令人惊讶的是，hALDH 2 -2蛋白在转基因小鼠的肝脏和大脑中以低水平表达。随后，小鼠ALDH 2酶活性在这些动物中受到轻微抑制。 虽然用20%乙醇（4g/kg/天）处理2周的小鼠的肝脏或脑病理学没有显著差异，但相对于对照小鼠，在乙醇注射后2小时，转基因小鼠的肝脏乙醛水平增加了50%（p<0.03），而肝脏乙醇浓度没有变化。此外，雌性转基因小鼠比对照组小鼠喝的酒少，但雄性小鼠没有（p<0.03）。这些携带hALDH 2 -2变体的转基因小鼠可用作研究ALDH在酒精偏好、乙醛积累、神经递质代谢和长期酒精治疗后乙酰丙酮酸介导的组织损伤中的作用的模型。 此外，为了对ALDH 2的生理作用有明确的结果，我们一直试图通过基因破坏技术制备小鼠ALDH 2基因缺陷的敲除小鼠。 在过去的一年里，我们能够生产嵌合小鼠，其中含有我们的DNA构建体，专门设计用于删除小鼠ALDH 2基因。我们现在使用限制性酶切分析和Southern印迹分析鉴定没有小鼠ALDH 2基因的纯合子小鼠。