Hepatic Drosha Controls De Novo Lipogenesis And Regulates Neurodegeneration Through The Liver-Brain Axis

Background With the coming of aging society,the incidence of neurodegenerative disorders such as Alzheimer’s disease(AD)is increasing dramatically in our country,exerting enormous economic burden on society.AD is manifested by a chronic and progressive decline in learning and memory.Unfortunately,current therapies for AD are limited.Fully elucidating the pathogenesis mechanisms is essential for developing new therapeutic approaches for AD.Previous studies revealed that microglia hyper-activation and uncontrolled inflammatory response are the most common driving force for neurodegeneration,and insulin resistance recently is proven highly correlated with neuroinflammation.Thus,illustrating how insulin resistance develops may help provide a theoretical basis for effective intervention of AD.Liver is the main organ regulating insulin sensitivity,and hepatic dysfunction such as fatty liver disease often lead to insulin resistance.Emerging evidence suggest that type III RNase Drosha that initiating mi RNA biogenesis may contribute to cellular homeostasis maintaining through RNase-activity independent manner.Currently,it is unclear whether Drosha dysfunction plays a role in the development of fatty liver disease and insulin resistance.Objective Through employing in vitro cell model and animal model,this study is aimed to illustrate the regulatory role of Drosha in lipogenesis and the underlying mechanisms,thus linking Drosha dysfunction to the pathogenesis of insulin resistance and neurodegeneration.We hope that this research could bring new insights for the understanding of liver-brain axis and provided potential therapeutic targets for AD.Methods and results 1.Drosha regulates de novo lipogenesis(DNL)in an RNase activity-independent manner In order to clarify the role of Drosha in DNL,we established AML hepatic cell lines with Drosha or Dicer stably knocked down.Oil red O staining and lipidomics analysis demonstrated that compared with Dicer knockdown,Drosha knockdown dramatically reduced cellular lipids levels.Consistently,q PCR detection revealed that Drosha knockdown markedly inhibited the increase of lipogenesis-related m RNAs upon insulin treatment,whereas Dicer knockdown exerted no significant influence on these m RNAs.Transcriptional factor SREBP is responsible for the expression of most lipogenesis-related m RNAs.Under basal conditions,SREBP is located at endoplasmic reticulum(ER)membrane as its precursor form.SREBP translocates from ER to Golgi stack in a COPII-dependent manner upon insulin exposure.Then SREBP is sequentially cleaved by site-1 protease(S1P)and S2 P at the Golgi to release the transcriptional-active domain.We found that Drosha knockdown rather than Dicer knockdown impaired the cleavage of SREBP1.Through re-introducing transdominant-negative(TN)Drosha or different length of Drosha constructs into Drosha knockdown cells,we confirmed that Drosha controlled SREBP1 maturation via its N-terminus domain instead of the C-terminus region that initiating mi RNA biogenesis.Taken together,these data suggest that Drosha regulates DNL in an RNase activity-independent manner.2.Drosha promotes COPII-dependent SREBP1 tanslocation and processing SREBP1 translocation requires the assistance of its chaperone protein SCAP.SCAP binds with SREBP1 and recruits the COPII components.To elucidate the underlying mechanism through which Drosha controlled SREBP1 cleavage,we examined the translocation GFP-SCAP upon insulin treatment through confocal microscopy detection.The results demonstrated that Drosha knockdown repressed the translocation of SCAP from ER to Golgi.Furthermore,ER isolation,immunofluorescence co-localization and co-immunoprecipitation analysis showed that Drosha can locate at ER and bind with SCAP.Thus,Drosha regulated SREBP1 maturation by controlling its translocation process.Translocation of SREBP1/SCAP requires the COPII budding complex.In order to further clarify how Drosha regulates SREBP1/SCAP movement,we detected the interaction between Drosha and different COPII components.The results showed that Drosha interacts with COPII out-layer protein Sec31.We then co-expressed HA-Sec31 with Drosha constructs lacking different domains and carried out Co-IP assay.This analysis revealed that Drosha bound with Sec31 via its N-terminus RS-rich domain.COPII complex is composed of the inner Sec23-Sec24 heterodimer and outer Sec13-Sec31 heterodimer.We further explored whether Drosha could regulates COPII complex organization by measuring the binding level of Sec23-Sec31.The results showed that Drosha knockdown largely impaired the Sec23-Sec31 interaction under insulin treatment.By contrast,Dicer knockdown exerted no significant effect on Sec23-Sec31 interaction.By re-introducing wild type or TN Drosha constructs into Drosha knockdown cell,we demonstrated that Drosha also regulated COPII complex assembly in a RNase activity-independent manner,consistent with the previous results showing that Drosha promoted SREBP1 cleavage regardless of its RNase activity.3.Phosphorylation of Ser237 of Drosha by Akt links Drosha to lipogenesis regulation As Drosha is required for insulin-stimulated lipogenesis and its protein level and ER distribution was not significantly altered by insulin treatment,we speculated that insulin may regulated the function of Drosha through post-translational modifications.As insulin mainly activates the PI3K/Akt signaling,we examined whether Akt could phosphorylate Drosha.By employing in vitro kinase assay,constitutive active Akt construct and specific Akt inhibitor,we confirmed that Drosha was a new substrate of Akt kinase.Furthermore,Co-IP analysis revealed that Akt could bind with Drosha,and this interaction was evidently enhanced by insulin.Domain mapping showed that Akt phosphorylation site was located at the N-terminus RS-rich domain(220-390aa)of Drosha.Combined sequence analysis and site mutation demonstrated that Ser237 was the main site phosphorylated by Akt.To delineate the role of Ser237 phosphorylation in lipogenesis regulation,we re-introduced wild type or phospho-resistant Drosha construct(S237A)into Drosha knockdown cell.Co-IP assay showed that S237 A mutation largely abrogated Drosha-Sec31 interaction and COPII assembly upon insulin treatment,compared with cells expressing wild type Drosha.The immunoblot analysis showed that S237 A blocked SREBP1 maturation.Finally,through PCR detection and oil red O staining,we confirmed that cells expressing S237 A Drosha displayed decreased expression of lipogenesis-related m RNAs and lipids content upon insulin treatment.Collectively,these results suggest that Drosha Ser237 phosphorylation was a prerequisite for cell to initiate DNL,and the Akt-Drosha-SREBP1 axis represented an important regulatory mechanism for DNL.4.Hepatic Drosha expression regulates insulin resistance and neurodegeneration In high fat diet-induced mouse obesity models,hepatic phosphorylation level of akt and Drosha dramatically increased,which indicated hyper-activation of the Insulin-Akt-Drosha axis.Consistently,q PCR detection and oil red O staining showed that the level of lipogenesis-related genes and lipids content in liver also increased.In order to further elucidate the regulatory role of Drosha on liver lipogenesis and insulin resistance,we specifically knock out Drosha expression in liver by injecting virus expressing Cre recombinase driven by hepatic TBG promoter to Droshaflox/flox mouse.The results demonstrated that Drosha depletion significantly decreased SREBP1 maturation and its down-stream m RNAs.Meanwhile,biochemical analysis showed that Drosha depletion lowered the levels of cholesterol,triglyceride and fatty acid in both liver and serum.Glucose tolerance test revealed that hepatic Drosha knockout could alleviate the degree of insulin resistance.Insulin resistance is highly correlated with microglia activation and neurodegeneration.Consistently,the m RNA levels of multiple cytokines and chemokine ligands were increased in obese mouse.We further tested whether liver Drosha knockout could influence these pathological processes.Iba1 immunofluorescence revealed that microglia was activated in obese mouse brains,and Drosha depletion largely repressed microglia activation.Moreover,β-galactosidase staining confirmed that chronic insulin resistance induced neuronal senescence,which was markedly alleviated by Drosha depletion.Conclusion In summary,this study identifies a new non-canonical function of Drosha.We demonstrated that Drosha regulated lipogenesis in a RNase-independent manner.Mechanically,Drosha promoted SREBP1 maturation and transcription of lipogenesis-related genes,by controlling COPII-mediated translocation of SREBP1 from ER to Golgi.Moreover,Ser 237 of Drosha could be phosphorylated by Akt,and this phosphorylation is required for insulin-stimulated lipogenesis.Therefore,the Akt-Drosha-SREBP1 axis was an important regulatory mechanism for lipogenesis.The research not only broadens our understanding of the role of Drosha in cellular homeostasis maintaining,but also adding new evidence to the liver-brain regulatory axis.