Roles Of Autophagy Related Genes In Zebrafish Bile Acid Metabolism

Autophagy is a cellular degradation pathway which is conserved during evolution.Cytoplasmic materials,such as unfold protein aggregates and damaged organelles,can be delivered into the lysosome for degradation to maintain cellular homeostasis.Recently,autophagy has been identified to involve in many physiological processes,such as aging,immune response,pathogenesis and embryogenesis.Atg7,Atg5 and Beclin 1 are core proteins regulating autophagosome formation.Systemic deletion of Beclin 1 in mouse leads to early embryonic lethality(E8.5)due to the impaired visceral endoderm,and both Atg5^-/-and Atg7^-/-mutant mice die shortly after birth.These data imply that autophagy and autophagy related genes are essential for embryonic and neonatal development.To investigate the biological functions of these autophagy related genes in zebrafish,we knocked out them using CRISPR/Cas9 technology,and harvested stable genetic mutants.Our results showed the deletion of beclin 1 in zebrafish did not affect embryonic development;however,resulted in severe liver defects at larval stage.The loss-of-function of atg5 and atg7 didn't impair the liver structure in larvae,but atg7 mutants showed hepatic phenotypes at about 1 month old,which was similar to beclin 1 mutants.1.Loss-of-function of Beclin 1 leads to bile acid salt aggregates in the liver.Liver is one of the most active metabolic organs in organisms,which can supply energy by carbohydrate and lipid metabolism.The liver in zebrafish is quite similar to those in mammals in both functions and architecture.The liver-specific deficiency of autophagy has been reported to result in the Ubiquitin and P62 positive proteins aggregation and injure the liver function in mice;however,the relationship between autophagy and bile acid metabolism are not well studied.In our study,the beclin 1^-/-mutant displayed the impaired morphological structure of bile duct and hepatocytes.While the bile duct network was relatively completed;however,the bile duct became thicker than that in wild type,and it also lacked dot-like micro-structure(portal area)as in wild type.Most mutant hepatocytes were enlarged and lost the cell-cell adhesion,and bile acid salt was accumulated in cytoplasm showed by TEM.Taken together,the loss-of-function of beclin1 disrupts the bile acid metabolism and leads to the accumulation of bile acid salts in hepatocytes in zebrafish.Bile acid is the major component of bile,which is mainly synthesized in the liver.The homeostasis of bile acid is required for the maintenance of normal physiological function.Disorder of bile acid metabolism will result in various diseases,like cholestasis and bile acid diarrhea.Thus,the bile acid level in organism needs to be precisely controlled.FXR-SHP-Cyp7a1 is one of most important pathways to regulate the bile acid homeostasis.High level of bile acid activates FXR and downregulates the expression of cyp7a1 to reduce bile acid synthesis.We found that the direct target genes of bile acid,FXR and Mafg,were activated in beclin 1 mutants,as a result,cyp7a1 was downregulated.These data agree with the facts that bile acid salts are accumulated in the beclin 1 mutant liver.2.Beclin 1-Uvrag-Vps34-Vps15 regulates the cellular trafficking of BSEP.Mutation in BSEP can lead to Progressive familial intrahepatic cholestasis,type 2 disease(PFIC-2).Our result showed that BSEP was mislocated in hepatocytes in the beclin 1mutant.BSEP was retained in the cytoplasm in the mutant hepatocytes,while it was docked on the apical domain of hepatocytes in wild type.These results suggest that the mislocation of BSEP in hepatocytes is probably responsible for the accumulation of bile acid salts in the beclin 1 mutant.Beclin 1 can form two distinct PI3K complexes with different interactive proteins:Beclin 1-Vps34-Vps15-Atg14(PI3KC3 complex I)for autophagic function;Beclin 1-Vps34-Vps15-Uvrag(PI3KC3 complex II)for endosome trafficking.To explore the possible mechanism for the cellular mislocation of BSEP in the beclin 1 mutant,we applied several compounds to modulate autophagy and examined BSEP distribution in hepatocytes.CQ and Baf1a can suppress the fusion of autophagosome and lysosome;LY294002 and Wortmanin can inhibit the activity of two PI3K complexes.We found that the treatment of CQ and BAf1a had no effect on the cellular distribution of BSEP,but LY294002 and Wortmanin administration in wild type could mimic the BSEP defects in the beclin 1 mutant.These data imply that disruption of Beclin 1-Vps34-Vps15-Uvrag complex in the mutant probably leads to the mislocation of BSEP in beclin 1-deficiency hepatocytes.To test the above hypothesis,we generated uvrag mutant fish by CRISPR/Cas9.Immunofluorescence analysis of BSEP showed that BSEP is mislocated in the uvrag mutant,which is very similar to the phenotype observed in the beclin 1 mutant.The above results suggest that Beclin 1-Vps34-Vps15-Uvrag complex regulates the cellular location of BSEP in zebrafish hepatocytes.Our results have revealed that Beclin 1-Vps34-Vps15-Uvrag complex regulates bile acid salts transport by modulating the subcellular location of BSEP.This may take a new insight for the therapy of the bile acid related diseases.3.The causes of beclin 1 mutant death.At last,we investigated the reason of lethality in the beclin 1 mutants.Our results showed that neither the accumulation of bile acid salts nor ubiquitinated protein aggregates in hepatocytes is the main cause of death in the mutants.This suggests that Beclin 1 plays essential functions in other organs and tissues.