IRF1 regulates self-renewal and stress responsiveness to support hematopoietic stem cell maintenance.

Hematopoietic stem cells (HSCs) are tightly controlled to maintain a balance between blood cell production and self-renewal. While inflammation-related signaling is a critical regulator of HSC activity, the underlying mechanisms and the precise functions of specific factors under steady-state and stress conditions remain incompletely understood. We investigated the role of interferon regulatory factor 1 (IRF1), a transcription factor that is affected by multiple inflammatory stimuli, in HSC regulation. Our findings demonstrate that the loss of IRF1 from mouse HSCs significantly impairs self-renewal, increases stress-induced proliferation, and confers resistance to apoptosis. In addition, given the frequent abnormal expression of IRF1 in leukemia, we explored the potential of IRF1 expression level as a stratification marker for human acute myeloid leukemia. We show that IRF1-based stratification identifies distinct cancer-related signatures in patient subgroups. These findings establish IRF1 as a pivotal HSC controller and provide previously unknown insights into HSC regulation, with potential implications to IRF1 functions in the context of leukemia.

IRF1 regulates self-renewal and stress-responsiveness to support hematopoietic stem cell maintenance.

Inflammatory mediators induce emergency myelopoiesis and cycling of adult hematopoietic stem cells (HSCs) through incompletely understood mechanisms. To suppress the unwanted effects of inflammation and preserve its beneficial outcomes, the mechanisms by which inflammation affects hematopoiesis need to be fully elucidated. Rather than focusing on specific inflammatory stimuli, we here investigated the role of transcription factor Interferon (IFN) regulatory factor 1 (IRF1), which receives input from several inflammatory signaling pathways. We identify IRF1 as a master HSC regulator. IRF1 loss impairs HSC self-renewal, increases stress-induced cell cycle activation, and confers apoptosis resistance. Transcriptomic analysis revealed an aged, inflammatory signature devoid of IFN signaling with reduced megakaryocytic/erythroid priming and antigen presentation in IRF1-deficient HSCs. Finally, we conducted IRF1-based AML patient stratification to identify groups with distinct proliferative, survival and differentiation features, overlapping with our murine HSC results. Our findings position IRF1 as a pivotal regulator of HSC preservation and stress-induced responses.

Oxidized mitochondrial DNA: a protective signal gone awry.

Despite the emergence of mitochondria as key regulators of innate immunity, the mechanisms underlying the generation and release of immunostimulatory alarmins by stressed mitochondria remains nebulous. We propose that the major mitochondrial alarmin in myeloid cells is oxidized mitochondrial DNA (Ox-mtDNA). Fragmented Ox-mtDNA enters the cytosol where it activates the NLRP3 inflammasome and generates IL-1ß, IL-18, and cGAS-STING to induce type I interferons and interferon-stimulated genes. Inflammasome activation further enables the circulatory release of Ox-mtDNA by opening gasdermin D pores. We summarize new data showing that, in addition to being an autoimmune disease biomarker, Ox-mtDNA converts beneficial transient inflammation into long-lasting immunopathology. We discuss how Ox-mtDNA induces short- and long-term immune activation, and highlight its homeostatic and immunopathogenic functions.

Oxidized DNA fragments exit mitochondria via mPTP- and VDAC-dependent channels to activate NLRP3 inflammasome and interferon signaling.

Mitochondrial DNA (mtDNA) escaping stressed mitochondria provokes inflammation via cGAS-STING pathway activation and, when oxidized (Ox-mtDNA), it binds cytosolic NLRP3, thereby triggering inflammasome activation. However, it is unknown how and in which form Ox-mtDNA exits stressed mitochondria in non-apoptotic macrophages. We found that diverse NLRP3 inflammasome activators rapidly stimulated uniporter-mediated calcium uptake to open mitochondrial permeability transition pores (mPTP) and trigger VDAC oligomerization. This occurred independently of mtDNA or reactive oxygen species, which induce Ox-mtDNA generation. Within mitochondria, Ox-mtDNA was either repaired by DNA glycosylase OGG1 or cleaved by the endonuclease FEN1 to 500-650 bp fragments that exited mitochondria via mPTP- and VDAC-dependent channels to initiate cytosolic NLRP3 inflammasome activation. Ox-mtDNA fragments also activated cGAS-STING signaling and gave rise to pro-inflammatory extracellular DNA. Understanding this process will advance the development of potential treatments for chronic inflammatory diseases, exemplified by FEN1 inhibitors that suppressed interleukin-1ß (IL-1ß) production and mtDNA release in mice.

Metformin inhibition of mitochondrial ATP and DNA synthesis abrogates NLRP3 inflammasome activation and pulmonary inflammation.

Acute respiratory distress syndrome (ARDS), an inflammatory condition with high mortality rates, is common in severe COVID-19, whose risk is reduced by metformin rather than other anti-diabetic medications. Detecting of inflammasome assembly in post-mortem COVID-19 lungs, we asked whether and how metformin inhibits inflammasome activation while exerting its anti-inflammatory effect. We show that metformin inhibited NLRP3 inflammasome activation and interleukin (IL)-1ß production in cultured and alveolar macrophages along with inflammasome-independent IL-6 secretion, thus attenuating lipopolysaccharide (LPS)- and SARS-CoV-2-induced ARDS. By targeting electron transport chain complex 1 and independently of AMP-activated protein kinase (AMPK) or NF-κB, metformin blocked LPS-induced and ATP-dependent mitochondrial (mt) DNA synthesis and generation of oxidized mtDNA, an NLRP3 ligand. Myeloid-specific ablation of LPS-induced cytidine monophosphate kinase 2 (CMPK2), which is rate limiting for mtDNA synthesis, reduced ARDS severity without a direct effect on IL-6. Thus, inhibition of ATP and mtDNA synthesis is sufficient for ARDS amelioration.

Functions of outer mitochondrial membrane proteins: mediating the crosstalk between mitochondrial dynamics and mitophagy.

Most cellular stress responses converge on the mitochondria. Consequently, the mitochondria must rapidly respond to maintain cellular homeostasis and physiological demands by fine-tuning a plethora of mitochondria-associated processes. The outer mitochondrial membrane (OMM) proteins are central to mediating mitochondrial dynamics, coupled with continuous fission and fusion. These OMM proteins also have vital roles in controlling mitochondrial quality and serving as mitophagic receptors for autophagosome enclosure during mitophagy. Mitochondrial fission segregates impaired mitochondria in smaller sizes from the mother mitochondria and may favor mitophagy for eliminating damaged mitochondria. Conversely, mitochondrial fusion mixes dysfunctional mitochondria with healthy ones to repair the damage by diluting the impaired components and consequently prevents mitochondrial clearance via mitophagy. Despite extensive research efforts into deciphering the interplay between fission-fusion and mitophagy, it is still not clear whether mitochondrial fission essentially precedes mitophagy. In this review, we summarize recent breakthroughs concerning OMM research, and dissect the functions of these proteins in mitophagy from their traditional roles in fission-fusion dynamics, in response to distinct context, at the intersection of the OMM platform. These insights into the OMM proteins in mechanistic researches would lead to new aspects of mitochondrial quality control and better understanding of mitochondrial homeostasis intimately tied to pathological impacts.

STX17 dynamically regulated by Fis1 induces mitophagy via hierarchical macroautophagic mechanism.

Mitophagy is the selective autophagic targeting and removal of dysfunctional mitochondria. While PINK1/Parkin-dependent mitophagy is well-characterized, PINK1/Parkin-independent route is poorly understood. Using structure illumination microscopy (SR-SIM), we demonstrate that the SNARE protein Syntaxin 17 (STX17) initiates mitophagy upon depletion of outer mitochondrial membrane protein Fis1. With proteomics analysis, we identify the STX17-Fis1 interaction, which controls the dynamic shuffling of STX17 between ER and mitochondria. Fis1 loss results in aberrant STX17 accumulation on mitochondria, which exposes the N terminus and promotes self-oligomerization to trigger mitophagy. Mitochondrial STX17 interacts with ATG14 and recruits core autophagy proteins to form mitophagosome, followed by Rab7-dependent mitophagosome-lysosome fusion. Furthermore, Fis1 loss impairs mitochondrial respiration and potentially sensitizes cells to mitochondrial clearance, which is mediated through canonical autophagy machinery, closely linking non-selective macroautophagy to mitochondrial turnover. Our findings uncover a PINK1/Parkin-independent mitophagic mechanism in which outer mitochondrial membrane protein Fis1 regulates mitochondrial quality control.

Loss of MIEF1/MiD51 confers susceptibility to BAX-mediated cell death and PINK1-PRKN-dependent mitophagy.

Mitochondrial dynamics is highly implicated in a plethora of cellular processes including apoptosis and mitophagy. However, little is known about the scope and precise functions of mitochondrial dynamics proteins for mitochondrial quality control and cellular homeostasis. Whether mitochondrial dynamics proteins serve in cellular processes reliant on mitochondrial fission-fusion is still not fully explored. MIEF1/MiD51 (mitochondrial elongation factor 1) is known to promote mitochondrial fission via the recruitment of GTPase protein DNM1L/DRP1 (dynamin 1 like), but the fundamental understandings of MIEF1 for mitochondrial-dependent cellular processes are largely elusive. Here, we report novel roles of MIEF1 in responding to apoptotic stimuli and mitochondrial damage. Given our result that staurosporine (STS) treatment induced the degradation of MIEF1 via the ubiquitin-proteasome system (UPS), we are motivated to explore the role of MIEF1 in apoptosis. MIEF1 loss triggered the imbalance of BCL2 family members on the mitochondria, consequently initiating the translocation of BAX onto the mitochondria, catalyzing the decrease of mitochondrial membrane potential and promoting the release of DIABLO/SMAC (diablo IAP-binding mitochondrial protein) and CYCS (cytochrome c, somatic). We further demonstrate that MIEF1 deficiency impaired mitochondrial respiration and induced mitochondrial oxidative stress, sensitizing cells to PINK1-PRKN-mediated mitophagy. The recruitment of PRKN to depolarized mitochondria modulated the UPS-dependent degradation of MFN2 (mitofusin 2) and FIS1 (fission, mitochondrial 1) specifically, to further promote mitophagy. Our findings uncover a bridging role of MIEF1 integrating cell death and mitophagy, unlikely dependent on mitochondrial dynamics, implying new insights to mechanisms determining cellular fate.Abbreviations: ActD: actinomycin D; BAX: BCL2 associated X, apoptosis regulator; BAK1: BCL2 antagonist/killer 1; BCL2L1: BCL2 like 1; BMH: 1,6-bismaleimidohexane; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CHX: cycloheximide; CQ: chloroquine; CYCS: cytochrome c, somatic; DIABLO: diablo IAP-binding mitochondrial protein; DKO: double knockout; DNM1L/DRP1: dynamin 1 like; FIS1: fission, mitochondrial 1; GFP: green fluorescent protein; IP: immunoprecipitation; MFN1: mitofusin 1; MFN2: mitofusin 2; MG132: carbobenzoxy-Leu-Leu-leucinal; MIEF1/MiD51: mitochondrial elongation factor 1; MIEF2/MiD49: mitochondrial elongation factor 2; MOMP: mitochondrial outer membrane permeabilization; MTR: MitoTracker Red; OA: oligomycin plus antimycin A; OCR: oxygen consumption rate; OMM: outer mitochondrial membrane; PARP: poly(ADP-ribose) polymerase; PI: propidium iodide; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; SD: standard deviation; STS: staurosporine; TNF: tumor necrosis factor; UPS: ubiquitin-proteasome system; VDAC1: voltage dependent anion channel 1.

Reactive oxygen species trigger Parkin/PINK1 pathway-dependent mitophagy by inducing mitochondrial recruitment of Parkin.

Defective mitophagy linked to dysfunction in the proteins Parkin and PTEN-induced putative kinase 1 (PINK1) is implicated in the pathogenesis of Parkinson's disease. Although the mechanism by which Parkin mediates mitophagy in a PINK1-dependent manner is becoming clearer, the triggers for this mitophagy pathway remain elusive. Reactive oxygen species (ROS) have been suggested as such triggers, but this proposal remains controversial because ROS scavengers fail to retard mitophagy. Here we demonstrate that the role of ROS in mitophagy has been underappreciated as a result of the inefficiency of ROS scavengers to control ROS bursts after high-dose treatment with carbonyl cyanide m-chlorophenylhydrazone. Supporting this, combinatorial treatment with N-acetyl-l-cysteine and catalase substantially inhibited the ROS upsurge and PINK1-dependent Parkin translocation to mitochondria in response to carbonyl cyanide m-chlorophenylhydrazone treatment. In addition to the chemical mitophagy inducer, overexpression of voltage-dependent anion channel 1 (VDAC1) induced Parkin translocation to mitochondria, presumably by stimulating ROS generation. Similarly, combined N-acetyl-l-cysteine and catalase treatment also suppressed VDAC1-induced redistribution of Parkin. Alongside these observations, we also found that the elevated protein level of PINK1 was not necessary for Parkin translocation to mitochondria. Thus, our data suggest that ROS may act as a trigger for the induction of Parkin/PINK1-dependent mitophagy. In addition, our study casts doubt on the importance of protein quantity of PINK1 in the recruitment of Parkin to mitochondria.

Death-associated Protein 3 Regulates Mitochondrial-encoded Protein Synthesis and Mitochondrial Dynamics.

Mitochondrial morphologies change over time and are tightly regulated by dynamic machinery proteins such as dynamin-related protein 1 (Drp1), mitofusion 1/2, and optic atrophy 1 (OPA1). However, the detailed mechanisms of how these molecules cooperate to mediate fission and fusion remain elusive. DAP3 is a mitochondrial ribosomal protein that involves in apoptosis, but its biological function has not been well characterized. Here, we demonstrate that DAP3 specifically localizes in the mitochondrial matrix. Knockdown of DAP3 in mitochondria leads to defects in mitochondrial-encoded protein synthesis and abnormal mitochondrial dynamics. Moreover, depletion of DAP3 dramatically decreases the phosphorylation of Drp1 at Ser-637 on mitochondria, enhancing the retention time of Drp1 puncta on mitochondria during the fission process. Furthermore, autophagy is inhibited in the DAP3-depleted cells, which sensitizes cells to different types of death stimuli. Together, our results suggest that DAP3 plays important roles in mitochondrial function and dynamics, providing new insights into the mechanism of a mitochondrial ribosomal protein function in cell death.