Beta-catenin inhibition as a novel therapeutic strategy for porphyria

卟啉症是由于血红素生物合成途径的缺陷而引起的一类代谢性疾病 酶，肝脏通常是过量卟啉的来源或目标。治疗大多数 卟啉症是有限的，往往侧重于症状缓解和姑息治疗。因此，基于机械的 迫切需要强调治疗发展的研究。以前的工作已经确定了 Wnt/β-catenin信号通路作为3，5-二乙氧羰基-1，4-二氢可力丁的可调控靶点 (DDC)饮食诱导的小鼠卟啉病模型。在DDC小鼠中抑制b-连环蛋白导致 由于卟啉积累减少，肝损伤显著减少。我们发现了一个关键网络 血红素生物合成酶如δ-氨基乙酰丙酸（ALA）合成酶和ALA-乙酰丙酸酶， 在缺乏β-连环蛋白的情况下受到抑制，导致卟啉中间体的产生减少， DDC相关蛋白聚集。自噬在缺乏b-连环蛋白的小鼠中也增加，这可能进一步 有助于保护免受伤害。因此，该提案的首要假设是， 在卟啉症的临床相关模型中，Wnt/β-连环蛋白信号传导将减轻损伤和进展， 疾病通过减少卟啉中间体的产生和/或增加自噬。在目标1中，我们 将确定通路中受β-连环蛋白抑制影响的最近步骤 用ALA处理肝细胞并测量卟啉中间体的出现;研究机制 β-连环蛋白通过定点诱变、计算机模拟研究和染色质 免疫沉淀;并通过利用靶向β-连环蛋白来证明患者中的治疗相关性 免疫组化以将b-连环蛋白表达的程度与血红素酶的表达相关联。在 目的2：研究Wnt/β-catenin抑制后自噬在卟啉病中的作用和调控。 我们将使用RFP-EGFP-LC 3小鼠，一种pH依赖性荧光报告菌株，和一种基因敲除的 谷氨酰胺合成酶，β-连环蛋白/mTOR途径的组分，以及体外试验， 全面解决这一细胞过程对观察到的受保护表型的贡献。在aim中 3，我们将确定在卟啉症的遗传小鼠模型中抑制b-连环蛋白是否会减少卟啉 积累并改善肝脏病理。两种特征良好的卟啉病模型：亚铁螯合酶 （Fechm 1 Pas）突变小鼠，模拟人类红细胞生成性原卟啉症，肝脏明显 参与;和T1/T2小鼠，其是羟甲基胆烷合酶的复合杂合型， 模拟急性间歇性卟啉病，在用苯巴比妥刺激后，将用于确定 用β-连环蛋白抑制剂进行治疗性干预是否可以预防疾病进展或提供保护 急性发作时。因此，拟议的研究将进一步了解抑制的潜力， β-连环蛋白治疗卟啉症，并将提供高度重要的信息，为治疗和翻译使用。

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.