Beta-catenin inhibition as a novel therapeutic strategy for porphyria

卟啉症是一组由血红素生物合成途径缺陷引起的代谢紊乱。 而肝脏通常是过量卟啉的来源或靶标。治疗大部分脑部疾病 门静脉症是有限的，通常侧重于症状缓解和姑息治疗。因此，基于机械论的 强调治疗发展的研究是迫切需要的。以前的工作已经确定了 Wnt/b-catenin信号通路在3，5-二乙氧基-1，4-二氢碰撞碱中的调控作用 (DDC)饮食诱导的小鼠卟啉中毒模型。抑制b-连环蛋白在DDC所致的小鼠中导致 由于减少了卟啉蓄积，显著减少了肝脏损伤。我们确定了一个关键的网络 血红素生物合成酶，如δ-氨基乙酰丙酸(ALA)合成酶和ALA-脱水酶 在没有b-连环蛋白的情况下被抑制，导致卟啉中间体和 DDC相关蛋白聚集。缺乏b-连环蛋白的小鼠的自噬也会增加，这可能会进一步 有助于保护身体免受伤害。因此，该提案的首要假设是抑制 临床相关的门静脉高压症模型中的WNT/b-连环蛋白信号转导通路将减轻门静脉高压症的损伤和进展 通过减少卟啉中间体的产生和/或增加自噬而引起的疾病。在目标1中，我们 将通过治疗确定受b-连环蛋白抑制影响的通路中最近的一步。 肝细胞与丙氨酸结合，检测卟啉中间体的出现；探讨其作用机制 在计算机研究和染色质研究中，b-连环蛋白通过定点突变调节血红素酶。 免疫沉淀；并证明靶向β-连环蛋白在患者中的治疗意义 免疫组织化学方法检测b-连环蛋白表达程度与血红素酶表达的关系。在……里面 目的研究Wnt/b-catenin抑制后自噬在卟啉症中的作用和调控。 我们将使用RFP-EGFP-LC3小鼠，一个pH依赖的荧光报告菌株，和一个基因敲除 谷氨酰胺合成酶，b-连环蛋白/mTOR途径的一个组成部分，以及体外检测，以 全面阐述这一细胞过程对观察到的受保护表型的贡献。在AIM 3，我们将确定在遗传性卟啉症小鼠模型中抑制b-连环蛋白是否会减少卟啉。 蓄积，改善肝脏病理。两种典型的卟啉症模型：铁络合酶 (Fechm1Pas)突变小鼠，模仿具有显著肝脏的人红细胞生成性原卟啉 以及T1/T2小鼠，它是羟甲基丁烷合成酶和羟甲基丁烷合成酶的复合杂合子。 在苯巴比妥刺激下模拟急性间歇性卟啉症，将被用于确定 B-连环蛋白抑制剂的治疗干预是否可以防止进展或提供保护 在急性发作期间。因此，拟议的研究将进一步加深我们对抑制的潜力的理解。 B-连环蛋白治疗卟啉症，将为治疗和翻译使用提供非常有意义的信息。

The porphyrias are a group of metabolic disorders that are caused by defects in heme biosynthesis pathway enzymes, and liver is commonly either a source or target of excess porphyrins. Treatment for most of the porphyrias is limited, and often focused on symptomatic relief and palliative care. Thus, mechanistic-based studies that emphasize therapeutic development are desperately needed. Previous work has identified the Wnt/b-catenin signaling pathway as a modulatable target in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced mouse model of porphyria. Inhibition of b-catenin in mice subjected to DDC results in a significant reduction in liver injury due to decreased porphyrin accumulation. We identified a network of key heme biosynthesis enzymes such as δ-aminolevulinic acid (ALA) synthetase and ALA-dehydratase that are suppressed in the absence of b-catenin, resulting in decreased production of porphyrin intermediates and DDC-associated protein aggregation. Autophagy is also increased in mice lacking b-catenin, which may further contribute to protection from injury. Thus, the overarching hypothesis of the proposal is that inhibiting Wnt/b-catenin signaling in clinically-relevant models of porphyria will alleviate injury and progression of disease through decreased production of porphyrin intermediates and/or increased autophagy. In aim 1, we will determine the most proximal step in the pathway that is affected by b-catenin inhibition by treating hepatocytes with ALA and measuring the appearance of porphyrin intermediates; investigate the mechanism by which b-catenin regulates heme enzymes through site-directed mutagenesis, in silico studies, and chromatin immunoprecipitation; and demonstrate therapeutic relevance of targeting β-catenin in patients by utilizing immunohistochemistry to correlate the extent of b-catenin expression with expression of heme enzymes. In aim 2, we will characterize the role and regulation of autophagy in porphyria after Wnt/b-catenin inhibition. We will use RFP-EGFP-LC3 mice, a pH-dependent fluorescent reporter strain, and a genetic knockout of glutamine synthetase, a component of the b-catenin/mTOR pathway, as well as in vitro assays, to comprehensively address the contribution of this cellular process to the observed protected phenotype. In aim 3, we will determine whether inhibiting b-catenin in genetic mouse models of porphyria decreases porphyrin accumulation and improves liver pathology. Two well-characterized porphyria models: the ferrochelatase (Fechm1Pas) mutant mice, which mimics human erythropoietic protoporphyria with significant liver involvement; and the T1/T2 mouse, which is compound heterozygous for hydroxymethylbilane synthase and mimics acute intermittent porphyria upon stimulation with phenobarbitol, will be utilized to determine whether therapeutic intervention with a b-catenin inhibitor can prevent progression or provide protection during acute attacks. Thus, the proposed studies will further our understanding of the potential for inhibiting b-catenin to treat porphyria, & will provide highly significant information for therapeutic and translational use.