Regulation of Mitophagy By O-Linked N-Acetylglucosamine Transferase Is Essential for Hematopoietic Stem Cell Maintenance
Authors: Murakami Koichi , Kurotaki Daisuke , Kawase Wataru , Soma Shunsuke , Fukuchi Yumi , Kunimoto Hiroyoshi , Yoshimi Ryusuke , Koide Shuhei , Oshima Motohiko , Hishiki Takako , Hayakawa Noriyo , Matsuura Tomomi , Ko Minoru , Oda Mayumi , Yanagisawa Kiichi , Kobayashi Hiroshi , Atobe Yoshitoshi , Funakoshi Kengo , Iwama Atsushi , Takubo Keiyo , Okamoto Shinichiro , Tamura Tomohiko , Nakajima Hideaki 
Published: October 2019

Abstract
Hematopoietic stem cells (HSCs) reside in hypoxic niche in the bone marrow (BM), where they are maintained in quiescent state. To adapt to this hypoxic microenvironment, HSCs generate ATP mainly from glycolysis, while suppressing oxidative phosphorylation in mitochondria. Through this metabolic regulation, HSCs protect themselves from oxidative stress caused by reactive oxygen species (ROS). ROS levels are controlled by various mechanisms including autophagy, which is one of the critical mechanisms regulating HSC function mainly through maintaining mitochondrial homeostasis. Glycosylation of serine or threonine residues with O-linked N-acetylglucosamine (O-GlcNAcylation) by O-linked N-acetylglucosamine transferase (OGT) is crucial for regulating various protein functions, and dysregulation of O-GlcNAcylation is critically linked to metabolic or degenerative diseases (i.e. diabetes, Alzheimer disease), tumorigenesis and aging. In hematopoietic system, OGT is essential for differentiation and proliferation of T and B cells. However, a role for OGT in the function of hematopoietic stem and progenitor cells (HSPCs) remains elusive. To elucidate a role for OGT in HSPCs, we analyzed the effect of Ogt loss on HSPCs using Ogt-conditional knockout mice. Conditional disruption of Ogt in adult mice led to pancytopenia and significantly reduced numbers of HSPCs in the BM. In addition, Ogt-deficient HSCs exhibited loss of quiescence, increased apoptosis and impaired long-term repopulation as well as self-renewal capacities by serial competitive repopulation assays. Interestingly, ROS levels were significantly increased in Ogt-deficient HSPCs, and the treatment of mice with N-acetyl cysteine (NAC), a scavenger for ROS, at least partially rescued the phenotype of Ogt-deficient hematopoietic cells in in vivo settings. MitoSOX analysis showed that excessive ROS was generated mainly from mitochondria in Ogt-deficient HSCs. In addition, mitochondrial mass was significantly increased in Ogt-deficient HSCs. Assessment of mitochondrial quality by its membrane potential and extracellular flux analysis revealed that mitochondria of Ogt-deficient HSPCs had significantly lower membrane potential and lower spare respiratory capacity. Morphologically, mitochondria in Ogt-deficient HSPCs were enlarged and swollen with disorganized cristae. These findings suggest that inactivation of Ogt leads to loss of HSPCs and impaired HSC homeostasis, which is due, at least in part, to elevated ROS levels with accumulation of defective mitochondria. Since mitophagy (mitochondria-specific autophagy) plays an important role in quality control of mitochondria, we examined gene expressions of mitophagy regulators by RNA sequencing. This revealed that disruption of Ogt led to down-regulation of key mitophagy inducers, Pink1 and Bnip3. These findings together with the accumulation of defective mitochondria indicated that inactivation of Ogt impairs mitophagy probably due to reduced expression of Pink1 and Bnip3. OGT is known to regulate gene expressions by modifying trimethylation of lysine 4 on the histone H3 (H3K4me3) through proteolytic activation of HCF-1, which is a critical component of SET1/COMPASS complex. Chromatin immunoprecipitation (ChIP) assay showed significantly reduced levels of H3K4me3 at the transcriptional start sites of Pink1 and Bnip3 in Ogt-deficient HSPCs. Next we asked whether defective HSPC homeostasis caused by Ogt disruption could be rescued by restoring mitophagy in Ogt-deficient HSPCs through overexpressing Pink1, a critical initiator of mitophagy. As expected, Pink1 overexpression efficiently restored mitophagy in Ogt-deficient HSCs as shown by normalization of mitochondrial mass. Interestingly, a number and apoptosis of Ogt-deficient HSCs were restored to levels similar to those of wild-type HSCs. These data strongly indicate that Pink1 functions as a key downstream effector of OGT in HSC maintenance. In summary, our results revealed that OGT plays an essential role in HSC maintenance by assuring mitochondrial quality through mitophagy.

Disclosures
No relevant conflicts of interest to declare.