Pathogenesis and Treatment of Liver Disease in Transaldolase Deficiency

描述(申请人提供)：尽管氧化应激与急性对乙酰氨基酚诱导的肝功能衰竭和慢性肝病、肝硬变和肝细胞癌有关，但尚未发现共同的潜在代谢途径。初步研究结果表明，磷酸戊糖途径(PPP)中的转醛醇酶(TAL，由TALDO1基因位点编码)的缺失是肝脏氧化应激的重要原因，易导致对乙酰氨基酚诱导的肝功能衰竭和肝癌的发生。最近的病例报告表明，TAL基因的纯合子突变与儿童肝功能衰竭和肝硬变有关。在第一例TAL缺陷患者的成纤维细胞和淋巴母细胞中，我们发现S171缺失导致错误折叠、蛋白酶体介导的降解和酶活性的完全缺乏。TAL缺乏导致7-磷酸七糖(S7P)的积累和6-磷酸葡萄糖(G6P)的耗尽，表明核糖5-磷酸(R5P)不能通过PPP的非氧化分支循环到G6P，从而减少了氧化分支产生NADPH。该患者的细胞表现出线粒体功能障碍、对H2O的敏感性增加以及对Fas凋亡的抵抗力。为了建立TAL缺乏的动物模型，我们在小鼠中灭活了TALDO1基因组位点。肝硬变和结节异型增生在TAL部分缺乏(TAL+/-；62/229，27.0%)或TAL完全缺乏(77/97，79.4%)的小鼠中比野生型(TAL+/+；1/102，1.0%)高。肝硬变总是伴随着微泡和大泡脂滴的形成，表明非酒精性脂肪性肝病(NAFLD)和类似于非酒精性脂肪性肝炎(NASH)的炎性改变。肝癌是TAL+/-(39/229；17%)和TAL-/-小鼠(45/97；46%)的主要死亡原因，而TAL+/+小鼠(1/102；1%)是主要死亡原因。TAL缺乏肝脏的氧化应激特征是S7P积聚，R5P不能循环用于氧化PPP，NADPH和谷胱甘肽水平降低，脂质过氧化氢(LPO)产生增加。肝组织中2-连环素磷酸化水平降低，c-jun表达增强，反映了对氧化应激的适应。TAL-/-肝细胞在体内外对CD95/Fas介导的细胞凋亡均有抵抗作用。值得注意的是，终生给予N-乙酰半胱氨酸(NAC)可阻断TAL缺陷小鼠对乙酰氨基酚的敏感性，恢复Fas依赖的细胞凋亡、2-连环素的磷酸化、c-jun的激活，并阻止NASH、肝硬变和肝癌的发生。我们还发现，在患有肝硬变和肝细胞癌的人类肝脏中，TAL单倍体功能不全的患病率增加。因此，初步研究发现，TAL缺乏是一种新的肝硬变和肝癌的原因，可以通过终身补充有效的抗氧化剂NAC来预防。建议的研究将验证TAL缺乏导致氧化应激的假设，即通过减少NADPH的产生导致继发性GSH耗竭，这是醋氨酚诱导的肝功能衰竭和慢性肝病的易感性的基础，从NAFLD发展到NASH、肝硬化和肝细胞癌。在特定目标1下，我们将确定导致TAL缺陷肝细胞NADPH耗竭的分子机制。在特定的目标2下，我们将验证这样的假设：TAL缺陷小鼠的肝癌发生是由NADPH耗竭诱导的氧化应激触发的，由醛糖还原酶(AR)的过度表达和2-连环蛋白和c-Jun的激活介导，并通过出生后使用N-乙酰半胱氨酸或AR阻断剂来预防。在具体目标3下，我们将确定对乙酰氨基酚诱导的肝功能衰竭、非酒精性脂肪肝、非酒精性脂肪肝、肝硬变和肝细胞癌患者中TAL缺乏的患病率和分子基础。这些研究将在肝脏疾病的发病机制和治疗方面产生基本的新信息。 公共卫生相关性：肝细胞癌是全球第五大常见癌症，也是癌症相关死亡的第三大原因。扑热息痛(也称为泰诺)过量是美国急性肝功能衰竭的主要原因。虽然氧化应激与肝细胞癌和对乙酰氨基酚中毒有关，但使患者易患这些疾病的遗传原因尚不清楚。近年来，转醛醇酶(TAL)缺乏症在肝功能衰竭和肝硬变的儿童中被发现。TAL是一种酶，有助于利用体内某些细胞(如肝细胞)中的糖葡萄糖来产生和维持正常水平的化合物NADPH和谷胱甘肽，这两种化合物保护细胞的完整性免受氧化应激的影响。我们通过在小鼠身上进行定向基因突变，建立了TAL缺乏症的动物模型。尽管部分(TAL+/-)或完全缺乏TAL(TAL-/-)的小鼠发育正常，但它们发生肝硬变和肝癌的可能性是TAL基因和活动正常的兄弟姐妹的27倍和79倍。与携带正常TAL基因的兄弟姐妹相比，TAL缺陷(TAL-/-)小鼠也更容易受到对乙酰氨基酚诱导的肝功能衰竭和死亡的影响。因此，初步研究确定TAL缺乏是导致肝硬变和肝细胞癌以及扑热息痛所致肝功能衰竭的新原因。我们假设NADPH在肝脏肿瘤细胞的存活和增殖中起关键作用。拟议的实验将确定NADPH耗竭的机制，可纠正的缺陷是否导致肝细胞的肿瘤增殖，并检查对乙酰氨基酚诱导的肝功能衰竭、脂肪肝炎症、肝硬变和肝细胞癌患者是否涉及TAL缺乏。这些研究应该会产生关于潜在致命肝脏疾病的病因和治疗的基本新信息。

DESCRIPTION (provided by applicant): Although oxidative stress is implicated in both acute acetaminophen-induced liver failure and chronic liver diseases, cirrhosis and hepatocellular carcinoma (HCC), no common underlying metabolic pathways have been identified. The preliminary results indicate that deficiency of transaldolase (TAL, encoded by the TALDO1 genomic locus), an enzyme of the pentose phosphate pathway (PPP), is an important cause of oxidative stress in the liver that predisposes to acetaminophen-induced liver failure and hepatocarcinogenesis. Recent case reports suggested a link of homozygous mutations in the TAL gene with liver failure and cirrhosis in children. In fibroblasts and lymphoblasts of the first TAL-deficient patient, we found that deletion of S171 caused mis- folding, proteasome-mediated degradation and complete deficiency of enzymatic activity. TAL deficiency resulted in accumulation of sedoheptulose 7-phoshate (S7P) and depletion of glucose 6-phoshate (G6P), indicating a failure to recycle ribose 5-phosphate (R5P) into G6P through the non-oxidative branch of the PPP, thus reducing NADPH production by the oxidative branch. This patient's cells exhibited mitochondrial dysfunction, increased susceptibility to H2O 2 and resistance to Fas apoptosis. To create an animal model of TAL deficiency, we inactivated the TALDO1 genomic locus in the mouse. Cirrhosis and nodular dysplasia were highly prevalent in mice with partial deficiency (TAL+/-; 62/229, 27.0%) or complete deficiency of TAL (TAL-/-; 77/97, 79.4%), relative to wild-type littermates (TAL+/+; 1/102, 1.0%). Cirrhosis invariably followed the formation of microvesicular and macrovesicular lipid droplets indicating nonalcoholic fatty liver disease (NAFLD) and inflammatory changes resembling non-alcoholic steatohepatitis (NASH). HCC was the leading cause of death in both TAL+/- (39/229; 17%) and TAL-/- mice (45/97; 46%) as compared to TAL+/+ littermates (1/102; 1%). Oxidative stress in TAL-deficient livers was characterized by the accumulation of S7P, failure to recycle R5P for the oxidative PPP, depleted NADPH and glutathione levels, and increased production of lipid hydroperoxides (LPO). Reduced 2-catenin phosphorylation and enhanced c-jun expression in TAL-/- livers reflected adaptation to oxidative stress. TAL-/- hepatocytes were resistant to CD95/Fas-mediated apoptosis in vitro and in vivo. Remarkably, lifelong administration of N-acetylcysteine (NAC) blocked acetaminophen susceptibility, restored Fas-dependent apoptosis, phosphorylation of 2-catenin, activation of c-jun and prevented the development of NASH, cirrhosis, and hepatocarcinogenesis in TAL-deficient mice. We also found an increased prevalence of TAL haploinsufficiency in human livers with cirrhosis and HCC. Thus, the preliminary studies identify TAL deficiency as a novel cause of liver cirrhosis and HCC which are preventable by lifelong supplementation of the potent antioxidant NAC. The proposed studies will test the hypothesis that TAL deficiency leads to oxidative stress, through diminished production of NADPH leading to secondary depletion of GSH that underlies the susceptibility of acetaminophen-induced liver failure and chronic liver disease, progressing from NAFLD to NASH, cirrhosis, and HCC. Under Specific Aim 1, we will determine the molecular mechanisms that cause NADPH depletion in TAL-deficient hepatocytes. Under Specific Aim 2, we will test the hypothesis that hepatocarcinogenesis of TAL-deficient mice is triggered by NADPH depletion- induced oxidative stress, mediated by the over-expression of aldose reductase (AR) and the activation of 2- catenin and c-jun, and prevented by post-natal treatment with N-acetylcysteine or AR blockade. Under Specific Aim 3, we will determine the prevalence and molecular basis of TAL deficiency in patients with acetaminophen-induced liver failure, NAFLD, NASH, cirrhosis and HCC. These studies will generate fundamental new information on the pathogenesis and treatment of liver disease. PUBLIC HEALTH RELEVANCE: Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer- related death worldwide. Acetaminophen (also known as Tylenol) overdose is the leading cause of acute liver failure in the US. Although oxidative stress has been implicated in HCC and acetaminophen toxicity, the genetic causes that predispose patients to these diseases are unknown. Recently, deficiency of transaldolase (TAL) was discovered in children with liver failure and cirrhosis. TAL is an enzyme that helps the utilization of the sugar glucose in certain cells of the body, such as liver cells, to produce and sustain normal levels of the compounds NADPH and glutathione which protect the integrity of the cells against oxidative stress. We created an animal model of TAL deficiency by a targeted genetic mutation in the mouse. Although mice partially (TAL+/-) or completely deficient of TAL (TAL-/-) develop normally, they are 27-fold and 79-fold more likely to develop cirrhosis and HCC than their siblings having normal TAL gene and activity. TAL-deficient (TAL-/-) mice are also more susceptible to acetaminophen-induced liver failure and death than their siblings carrying the normal TAL gene. Thus, the preliminary studies identify TAL deficiency as a novel cause of liver cirrhosis and HCC as well as acetaminophen-induced liver failure. We hypothesize that NADPH plays critical roles in the survival and multiplication of tumor cells in the liver. The proposed experiments will determine the mechanism of NADPH depletion, whether a correctable defect is responsible for the tumor-causing proliferation of liver cells, and examine the involvement of TAL deficiency in patients with acetaminophen- induced liver failure, fatty liver inflammation, cirrhosis and HCC. These studies should generate fundamental new information on the cause and treatment of potentially fatal diseases of the liver.