The mitochondrial unfolded protein response is activated upon hematopoietic stem cell exit from quiescence

Summary The mitochondrial unfolded protein response (UPR mt), a cellular protective program that ensures proteostasis in the mitochondria, has recently emerged as a regulatory mechanism for adult stem cell maintenance that is conserved across tissues. Despite the emerging genetic evidence implicating the UPR mt in stem cell maintenance, the underlying molecular mechanism is unknown. While it has been speculated that the UPR mt is activated upon stem cell transition from quiescence to proliferation, the direct evidence is lacking. In this study, we devised three experimental approaches that enable us to monitor quiescent and proliferating hematopoietic stem cells (HSCs) and provided the direct evidence that the UPR mt is activated upon HSC transition from quiescence to proliferation, and more broadly, mitochondrial integrity is actively monitored at the restriction point to ensure metabolic fitness before stem cells are committed to proliferation.

Despite the emerging genetic evidence implicating the UPR mt in stem cell maintenance, the underlying molecular mechanism is unknown. The UPR mt is a nascent cellular pathway that is activated when cells experience mitochondrial protein folding stress and retrograde signaling from the mitochondria to the nucleus triggers transcriptional activation of nuclear-encoded mitochondrial chaperones and proteases as well as repression of translation to reestablish proteostasis (Haynes, Fiorese & Lin, 2013;Haynes & Ron, 2010;Mohrin et al., 2015;Munch & Harper, 2016;Zhao et al., 2002). Primarily characterized in C. elegans, the UPR mt is activated during a developmental stage when there is a burst of mitochondrial biogenesis (Houtkooper et al., 2013;Lin et al., 2016;Merkwirth et al., 2016;Nargund, Pellegrino, Fiorese, Baker & Haynes, 2012;Pellegrino et al., 2014;Tian et al., 2016). It is therefore speculated that in stem cells, the UPR mt is activated under a physiological condition when mitochondrial biogenesis is induced. Adult stem cells frequently exit the cell cycle and are predominantly found in the quiescent (G0) state, where the number of mitochondria is low and glycolysis is the primary metabolic pathway to support energy production (Folmes, Dzeja, Nelson & Terzic, 2012;Takubo et al., 2013;Warr & Passegue, 2013;Yu et al., 2013). As stem cells transit from quiescence to proliferation, mitochondrial biogenesis is induced to enable metabolic reprogramming from glycolysis to oxidative phosphorylation to meet increasing energy demands. Because a major event during the transition from quiescence to proliferation is mitochondrial biogenesis, it raises the possibility that the UPR mt is activated during this transition. However, the direct evidence is lacking. In this study, we devised three experimental approaches that enable us to monitor quiescent and proliferating stem cells and directly test this hypothesis.
About 90% of HSCs reside in a quiescent state under homeostatic conditions (Pietras, Warr & Passegue, 2011). We isolated HSCs from mouse bone marrow and stimulated them to exit quiescence ex vivo upon culture with cytokines. We first confirmed that HSCs stimulated with cytokines were actively proliferating ( Figure 1a) and that mitochondrial mass was increased in HSCs upon proliferation (Figure 1b). Compared to freshly isolated quiescent HSCs, proliferating HSCs stimulated with cytokines exhibited increased expression of mitochondrial chaperones and proteases at the transcriptional level ( Figure 1c). Because mitochondrial biogenesis upon HSC transition from quiescence to proliferation is regulated at the translational level mediated by mTOR (Chen et al., 2008;Gan et al., 2010;Gurumurthy et al., 2010;Morita et al., 2013;Nakada, Saunders & Morrison, 2010), increased expression of mitochondrial chaperones and proteases at the transcriptional level reflects de novo activation of the UPR mt .
We further validated these results by stimulating HSCs to exit quiescence in vivo upon transplantation. Compared to HSCs isolated from untransplanted mice, donor HSCs isolated from transplanted recipient mice 2 weeks post-transplant were actively proliferating An alternative approach to model HSC proliferation in vivo is to treat mice with polyinosinic:polycytidylic acid (pIpC), a synthetic double-stranded RNA (dsRNA) mimetic that stimulates the multiple immune signaling pathways that are activated during a viral infection (Walter et al., 2015). Compared to HSCs isolated from untreated mice, HSCs isolated from mice 24 hr after the pIpC treatment These data are consistent with the notion that proliferative HSCs have increased mitochondrial number, experience a metabolic switch from glycolysis to oxidative phosphorylation, and induce the UPR mt to maintain the mitochondrial homeostasis.
Collectively, these results provide direct evidence that the UPR mt is activated upon HSC transition from quiescence to proliferation (Figures 1 and 2), and more broadly, mitochondrial integrity is actively monitored at the restriction point to ensure metabolic fitness before stem cells are committed to proliferation. Stem cell quiescence is a protective mechanism that prevents cell death and the  Table S1.

| mRNA analysis
RNA was isolated from cells using TRIzol reagent (Invitrogen). cDNA was generated using qScript TM cDNA SuperMix (Quanta Biosciences).
Gene expression was determined by real-time PCR using Eva qPCR SuperMix Kit (BioChain Institute) on an ABI StepOnePlus system. All data were normalized to b-actin expression. PCR primer details are provided in Table S2.

| Statistical analysis
The number of mice chosen for each experiment is based on the minimum number of mice necessary to have sufficient statistical power and is comparable to published literature for the same assays performed. Mice were randomized to groups, and analysis of mice and tissue samples was performed by investigators blinded to the treatment of the animals. Statistical analysis was performed with Excel (Microsoft). Means between two groups were compared with Student's t test. Error bars represent standard errors. In all corresponding figures, * represents p < .05, ** represents p < .01, *** represents p < .001, and ns represents p > .05.