Heme Deficiency-triggered Bach1b/Nrf2a Signaling Contributes Lipid Droplet Accumulation In Acute Intermittent Porphyria Zebrafish

In mammals,heme(iron protoporphyrin IX)can act as a component of many hemoproteins,such as hemoglobin,myoglobin,cytochrome and so on.Meanwhile,heme can also take part in the regulation of the expressions of some related genes,peroxidase,nitric oxide synthase,guanylate cyclase,for instance,to regulate metabolism.Heme biosynthesis is catalyzed by a cascade of eight enzymes.The first step is the condensation of glycine and succinylcoenzyme A to form ALA(5-Aminolaevulinic acid)under the catalysis of ALA synthetase.The other seven enzymes are ALAD(Aminolevulinic Acid Dehydratase),HMBS(hydroxymethyl porphyrin deaminase),UROS(uroporphyrinogen three synthetase),UROD(uroporphyrinogen decarboxylase),CPOX(coproporphyrinogen oxidase),PPOX(Polyepoxypropane oxidase),FECH(ferrochelatase).Deficiencies in these heme biosynthesis enzymes can lead to a series of syndromes called porphyria.The clinical symptoms of porphyria are complicated and the current diagnosis and treatment methods are relatively limited.Hence the model organisms have been employed as the disease models to carry out the treatment research.As an emerging animal model,zebrafish have unique advantages.At present,the mutations of alas2,urod,ppox,fech in zebrafish have been established for the disease models as human congenital sideroblastic anemia(CSA),hepatic erythropoietic porphyria(HEP),heterochromic porphyria(VP)and Erythropoietic protoporphyria(EPP).However,there is still no zebrafish model with the gene mutations encoding for the third enzyme HMBS of heme synthesis.Phylogenetic analysis shows that zebrafish hmbsa and hmbsb are co-orthologs of human HMBS.Since we already have hmbsa-/-mutant,the hmbsb-/-mutant is generated by the CRISPR-Cas9 technique.The double knockout mutants of hmbsa and hmbsb will be generateded to further study the specific functions of these two genes.The results showed that the homozygous mutants hmbsa-/-/hmbsb-/-were lethal and survived for only approximately 10 days while the single hmbs-/-mutant fish are viable.However,both hmbsa-/-and hmbsb-/-mutants display anemia with the significantly reduced heme contents,elevated ALA and PBG levels,and anxious and hyperactive behaviors.Intriguingly,all these symptoms of hmbsa-/-and hmbsb-/-mutants can be rescued by the other zebrafish hmbs gene and human HMBS,and heme treatment,demonstrating that hmbsa-/-and hmbsb-/-mutants can be used as human acute intermittent porphyrin(AIP)model.Because transplantation of the liver was commonly used in the clinical treatment of human AIP and the accumulation of ALA and PBG was reported in the liver of AIP mouse model.Our previous data also found that the expression of hmbs genes is highly expressed in the liver of adult zebrafish.Therefore,we focus on the liver function of hmbsb-/-mutants.In particular,we observed accumulation of lipid droplets in hmbs-/-mutants,in both the larval liver and adult fish liver organ sections.To further analyze the underlying mechanism,we conducted the transcriptome analysis of the hmbs-/-mutant and wild-type livers.The results showed that the expression of many genes involved in fat acid methbolism,such as fabp10 a,is altered in hmbs-/-mutants,which was further confirmed by q RT-PCR.Luciferase reporter and Ch IP assays show that heme can activate the expression of fabp10 a through the Bach1b/Nrf2a-Mafk pathway,and the hemin treament can inhibit the lipid droplet accumulation and upregulate the expression of fabp10 a.Together,our findings suggest that hmbsa-/-and hmbsb-/-mutants display heme deficiency and can be used to model human AIP,which allows for studying the pathogensis of of this human disease.We also find that heme can regulate fabp10 a expression via Bach1b/Nrf2a-Mafk pathway and contribute to lipid droplet accumulation.