Translation Rescue by Targeting Ppp1r15a through Its Upstream Open Reading Frame in Sepsis-Induced Acute Kidney Injury in a Murine Model
Authors: Kidwell Ashley , Yadav Shiv Pratap Singh , Maier Bernhard , Zollman Amy , Ni Kevin , Halim Arvin , Janosevic Danielle , Myslinski Jered , Syed Farooq , Zeng Lifan , Waffo Alain Bopda , Banno Kimihiko , Xuei Xiaoling , Doud Emma H. , Dagher Pierre C. , Hato Takashi 
Published: October 2022

Significance Statement
Extreme stress, such as life-threatening sepsis, triggers the integrated stress response and causes translation shutdown, a hallmark of late-phase, sepsis-induced kidney injury. Although a brief period of translation shutdown could be cytoprotective, prolonged translation repression can have negative consequences and has been shown to contribute to sepsis-induced kidney failure. Using a murine model of endotoxemia, the authors show that the duration of stress-induced translation shutdown in the kidney can be shortened by overexpressing protein phosphatase 1 regulatory subunit 15A (Ppp1r15a, also known as GADD34), a key regulator of the translation initiation complex. They achieved overexpression of Ppp1r15a with genetic and oligonucleotide approaches, targeting its upstream open reading frame (uORF). Altering Ppp1r15a expression through its uORF to counter translation shutdown offers a potential strategy for the treatment of sepsis-induced kidney failure.

Background
Translation shutdown is a hallmark of late-phase, sepsis-induced kidney injury. Methods for controlling protein synthesis in the kidney are limited. Reversing translation shutdown requires dephosphorylation of the eukaryotic initiation factor 2 (eIF2) subunit eIF2α; this is mediated by a key regulatory molecule, protein phosphatase 1 regulatory subunit 15A (Ppp1r15a), also known as GADD34.

Methods
To study protein synthesis in the kidney in a murine endotoxemia model and investigate the feasibility of translation control in vivo by boosting the protein expression of Ppp1r15a, we combined multiple tools, including ribosome profiling (Ribo-seq), proteomics, polyribosome profiling, and antisense oligonucleotides, and a newly generated Ppp1r15a knock-in mouse model and multiple mutant cell lines.

Results
We report that translation shutdown in established sepsis-induced kidney injury is brought about by excessive eIF2α phosphorylation and sustained by blunted expression of the counter-regulatory phosphatase Ppp1r15a. We determined the blunted Ppp1r15a expression persists because of the presence of an upstream open reading frame (uORF). Overcoming this barrier with genetic and antisense oligonucleotide approaches enabled the overexpression of Ppp1r15a, which salvaged translation and improved kidney function in an endotoxemia model. Loss of this uORF also had broad effects on the composition and phosphorylation status of the immunopeptidome—peptides associated with the MHC—that extended beyond the eIF2α axis.

Conclusions
We found Ppp1r15a is translationally repressed during late-phase sepsis because of the existence of an uORF, which is a prime therapeutic candidate for this strategic rescue of translation in late-phase sepsis. The ability to accurately control translation dynamics during sepsis may offer new paths for the development of therapies at codon-level precision.