Enhancement of hepatic autophagy increases ureagenesis and protects against hyperammonemia

Leandro R. Soriaa, Gabriella Allegrib, Dominique Melckc, Nunzia Pastored,e, Patrizia Annunziataa, Debora Parisc, Elena Polishchuka, Edoardo Nuscoa, Beat Thönyb, Andrea Mottac, Johannes Häberleb, Andrea Ballabioa,d,e,f, and Nicola Brunetti-Pierria,f,1

aTelethon Institute of Genetics and Medicine, 80078 Pozzuoli, Italy; bDivision of , University Children’s Hospital Zurich and Children’s Research Center, 8032 Zurich, Switzerland; cInstitute of Biomolecular , National Research Council, 80078 Pozzuoli, Italy; dDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030; eJan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX 77030; and fDepartment of Translational Medicine, Federico II University, 80131 Naples, Italy

Edited by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved November 29, 2017 (received for review August 21, 2017)

Ammonia is a potent neurotoxin that is detoxified mainly by the (12). Increased autophagy was previously found in the skeletal cycle in the . Hyperammonemia is a common complication muscle of mice with hyperammonemia and may contribute to of a wide variety of both inherited and acquired liver diseases. If not sarcopenia in cirrhosis (13). Nevertheless, the role of - treated early and thoroughly, it results in encephalopathy and death. induced autophagy in liver has not been evaluated so far. In this Here, we found that hepatic autophagy is critically involved in study, we investigated the role of hepatic autophagy in ammonia systemic ammonia by providing key urea-cycle inter- detoxification. mediates and ATP. Hepatic autophagy is triggered in vivo by Results hyperammonemia through an α-ketoglutarate–dependent inhibition of the mammalian target of rapamycin complex 1, and deficiency of Acute Hyperammonemia Activates Hepatic Autophagy. To interro- autophagy impairs ammonia detoxification. In contrast, autophagy gate the relevance of liver autophagy during hyperammonemia, enhancement by means of hepatic gene transfer of the master reg- we used an established in vivo mouse model of acute hyper- ulator of autophagy transcription factor EB or treatments with the ammonemia (14) in which acute and transient elevations of autophagy enhancers rapamycin and Tat-Beclin-1 increased ureagen- and hepatic ammonia and urea and increased blood glutamine are induced by i.p. injections of 10 mmol/kg of ammonium chloride in esis and protected against hyperammonemia in a variety of acute C57BL/6 wild-type (WT) mice (SI Appendix,Fig.S1A–E). and chronic hyperammonemia models, including acute liver harvested at 0.5 and 2.0 h postammonia challenge showed an in- failure and ornithine transcarbamylase deficiency, the most frequent creased conversion of LC3I to the autophagosome-associated urea-cycle disorder. In conclusion, hepatic autophagy is an important lipidated form LC3II and reduced levels of two main autophagy mechanism for ammonia detoxification because of its support of substrates (p62/SQSTM1 and NBR1) (Fig. 1 A and B). In GFP- urea synthesis, and its enhancement has potential for therapy of LC3 transgenic mice (15), i.p. injections of ammonium chloride both primary and secondary causes of hyperammonemia. increased hepatic GFP-LC3 puncta (Fig. 1C). By electron mi- croscopy (EM), compared with saline-injected controls, livers autophagy | mTORC1 | ornithine transcarbamylase deficiency | of WT mice injected with ammonium chloride showed an in- hyperammonemia | ureagenesis c