Insulin-Like Growth Factors Are Key Regulators of T Helper 17 Regulatory T Cell Balance in Autoimmunity.

Appropriate balance of T helper 17 (Th17) and regulatory T (Treg) cells maintains immune tolerance and host defense. Disruption of Th17-Treg cell balance is implicated in a number of immune-mediated diseases, many of which display dysregulation of the insulin-like growth factor (IGF) system. Here, we show that, among effector T cell subsets, Th17 and Treg cells selectively expressed multiple components of the IGF system. Signaling through IGF receptor (IGF1R) activated the protein kinase B-mammalian target of rapamycin (AKT-mTOR) pathway, increased aerobic glycolysis, favored Th17 cell differentiation over that of Treg cells, and promoted a heightened pro-inflammatory gene expression signature. Group 3 innate lymphoid cells (ILC3s), but not ILC1s or ILC2s, were similarly responsive to IGF signaling. Mice with deficiency of IGF1R targeted to T cells failed to fully develop disease in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Thus, the IGF system represents a previously unappreciated pathway by which type 3 immunity is modulated and immune-mediated pathogenesis controlled.

Metabolic adaptation to tyrosine kinase inhibition in leukemia stem cells.

Tyrosine kinase inhibitors (TKIs) are very effective in treating chronic myelogenous leukemia (CML), but primitive, quiescent leukemia stem cells persist as a barrier to the cure. We performed a comprehensive evaluation of metabolic adaptation to TKI treatment and its role in CML hematopoietic stem and progenitor cell persistence. Using a CML mouse model, we found that glycolysis, glutaminolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS) were initially inhibited by TKI treatment in CML-committed progenitors but were restored with continued treatment, reflecting both selection and metabolic reprogramming of specific subpopulations. TKI treatment selectively enriched primitive CML stem cells with reduced metabolic gene expression. Persistent CML stem cells also showed metabolic adaptation to TKI treatment through altered substrate use and mitochondrial respiration maintenance. Evaluation of transcription factors underlying these changes helped detect increased HIF-1 protein levels and activity in TKI-treated stem cells. Treatment with an HIF-1 inhibitor in combination with TKI treatment depleted murine and human CML stem cells. HIF-1 inhibition increased mitochondrial activity and reactive oxygen species (ROS) levels, reduced quiescence, increased cycling, and reduced the self-renewal and regenerating potential of dormant CML stem cells. We, therefore, identified the HIF-1-mediated inhibition of OXPHOS and ROS and maintenance of CML stem cell dormancy and repopulating potential as a key mechanism of CML stem cell adaptation to TKI treatment. Our results identify a key metabolic dependency in CML stem cells persisting after TKI treatment that can be targeted to enhance their elimination.

ß-hydroxybutyrate administered at reperfusion reduces infarct size and preserves cardiac function by improving mitochondrial function through autophagy in male mice.

Ischemia/reperfusion (I/R) injury after revascularization contributes ∼50% of infarct size and causes heart failure, for which no established clinical treatment exists. ß-hydroxybutyrate (ß-OHB), which serves as both an energy source and a signaling molecule, has recently been reported to be cardioprotective when administered immediately before I/R and continuously after reperfusion. This study aims to determine whether administering ß-OHB at the time of reperfusion with a single dose can alleviate I/R injury and, if so, to define the mechanisms involved. We found plasma ß-OHB levels were elevated during ischemia in STEMI patients, albeit not to myocardial protection level, and decreased after revascularization. In mice, compared with normal saline, ß-OHB administrated at reperfusion reduced infarct size (by 50%) and preserved cardiac function, as well as activated autophagy and preserved mtDNA levels in the border zone. Our treatment with one dose ß-OHB reached a level achievable with fasting and strenuous physical activity. In neonatal rat ventricular myocytes (NRVMs) subjected to I/R, ß-OHB at physiologic level reduced cell death, increased autophagy, preserved mitochondrial mass, function, and membrane potential, in addition to attenuating reactive oxygen species (ROS) levels. ATG7 knockdown/knockout abolished the protective effects of ß-OHB observed both in vitro and in vivo. Mechanistically, ß-OHB's cardioprotective effects were associated with inhibition of mTOR signaling. In conclusion, ß-OHB, when administered at reperfusion, reduces infarct size and maintains mitochondrial homeostasis by increasing autophagic flux (potentially through mTOR inhibition). Since ß-OHB has been safely tested in heart failure patients, it may be a viable therapeutic to reduce infarct size in STEMI patients.

A novel approach to measure mitochondrial respiration in frozen biological samples.

Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for freshly isolated mitochondria hinders the feasibility of respirometry in multi-site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90-95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks.

Protein Kinase CK2 Controls CD8+ T Cell Effector and Memory Function during Infection.

Protein kinase CK2 is a serine/threonine kinase composed of two catalytic subunits (CK2α and/or CK2α') and two regulatory subunits (CK2ß). CK2 promotes cancer progression by activating the NF-κB, PI3K/AKT/mTOR, and JAK/STAT pathways, and also is critical for immune cell development and function. The potential involvement of CK2 in CD8+ T cell function has not been explored. We demonstrate that CK2 protein levels and kinase activity are enhanced upon mouse CD8+ T cell activation. CK2α deficiency results in impaired CD8+ T cell activation and proliferation upon TCR stimulation. Furthermore, CK2α is involved in CD8+ T cell metabolic reprogramming through regulating the AKT/mTOR pathway. Lastly, using a mouse Listeria monocytogenes infection model, we demonstrate that CK2α is required for CD8+ T cell expansion, maintenance, and effector function in both primary and memory immune responses. Collectively, our study implicates CK2α as an important regulator of mouse CD8+ T cell activation, metabolic reprogramming, and differentiation both in vitro and in vivo.

Relationships between gene expression and behavior in mice in response to systemic modulation of the O-GlcNAcylation pathway.

Enhancing protein O-GlcNAcylation by pharmacological inhibition of the enzyme O-GlcNAcase (OGA), which removes the O-GlcNAc modification from proteins, has been explored in mouse models of amyloid-beta and tau pathology. However, the O-GlcNAcylation-dependent link between gene expression and neurological behavior remains to be explored. Using chronic administration of Thiamet G (TG, an OGA inhibitor) in vivo, we used a protocol designed to relate behavior with the transcriptome and selected biochemical parameters from the cortex of individual animals. TG-treated mice showed improved working memory as measured using a Y-maze test. RNA sequencing analysis revealed 151 top differentially expressed genes with a Log2fold change >0.33 and adjusted p-value <0.05. Top TG-dependent upregulated genes were related to learning, cognition and behavior, while top downregulated genes were related to IL-17 signaling, inflammatory response and chemotaxis. Additional pathway analysis uncovered 3 pathways, involving gene expression including 14 cytochrome c oxidase subunits/regulatory components, chaperones or assembly factors, and 5 mTOR (mechanistic target of rapamycin) signaling factors. Multivariate Kendall correlation analyses of behavioral tests and the top TG-dependent differentially expressed genes revealed 91 statistically significant correlations in saline-treated mice and 70 statistically significant correlations in TG-treated mice. These analyses provide a network regulation landscape that is important in relating the transcriptome to behavior and the potential impact of the O-GlcNAC pathway.

Mitochondrial haplotype modulates genome expression and mitochondrial structure/function in cardiomyocytes following volume overload.

Mitochondrial DNA (mtDNA) haplotype regulates mitochondrial structure/function and reactive oxygen species in aortocaval fistula (ACF) in mice. Here, we unravel the mitochondrial haplotype effects on cardiomyocyte mitochondrial ultrastructure and transcriptome response to ACF in vivo. Phenotypic responses and quantitative transmission electron microscopy (TEM) and RNA sequence at 3 days were determined after sham surgery or ACF in vivo in cardiomyocytes from wild-type (WT) C57BL/6J (C57n:C57mt) and C3H/HeN (C3Hn:C3Hmt) and mitochondrial nuclear exchange mice (C57n:C3Hmt or C3Hn:C57mt). Quantitative TEM of cardiomyocyte mitochondria C3HWT hearts have more electron-dense compact mitochondrial cristae compared with C57WT. In response to ACF, mitochondrial area and cristae integrity are normal in C3HWT; however, there is mitochondrial swelling, cristae lysis, and disorganization in both C57WT and MNX hearts. Tissue analysis shows that C3HWT hearts have increased autophagy, antioxidant, and glucose fatty acid oxidation-related genes compared with C57WT. Comparative transcriptomic analysis of cardiomyocytes from ACF was dependent upon mtDNA haplotype. C57mtDNA haplotype was associated with increased inflammatory/protein synthesis pathways and downregulation of bioenergetic pathways, whereas C3HmtDNA showed upregulation of autophagy genes. In conclusion, ACF in vivo shows a protective response of C3Hmt haplotype that is in large part driven by mitochondrial nuclear genome interaction.NEW & NOTEWORTHY The results of this study support the effects of mtDNA haplotype on nuclear gene expression in cardiomyocytes. Currently, there is no acceptable therapy for volume overload due to mitral regurgitation. The findings of this study could suggest that mtDNA haplotype activates different pathways after ACF warrants further investigations on human population of heart disease from different ancestry backgrounds.

Glutamine-dependent effects of nitric oxide on cancer cells subjected to hypoxia-reoxygenation.

Limited O2 availability can decrease essential processes in energy metabolism. However, cancers have developed distinct metabolic adaptations to these conditions. For example, glutaminolysis can maintain energy metabolism and hypoxia signaling. Additionally, it has been observed that nitric oxide (NO) possesses concentration-dependent, biphasic effects in cancer. NO has potent anti-tumor effects through modulating events such as angiogenesis and metastasis at low physiological concentrations and inducing cell death at higher concentrations. In this study, Ewing Sarcoma cells (A-673), MIA PaCa, and SKBR3 cells were treated with DetaNONOate (DetaNO) in a model of hypoxia (1% O2) and reoxygenation (21% O2). All 3 cell types showed NO-dependent inhibition of cellular O2 consumption which was enhanced as O2-tension decreased. L-Gln depletion suppressed the mitochondrial response to decreasing O2 tension in all 3 cell types and resulted in inhibition of Complex I activity. In A-673 cells the O2 tension dependent change in mitochondrial O2 consumption and increase in glycolysis was dependent on the presence of L-Gln. The response to hypoxia and Complex I activity were restored by α-ketoglutarate. NO exposure resulted in the A-673 cells showing greater sensitivity to decreasing O2 tension. Under conditions of L-Gln depletion, NO restored HIF-1α levels and the mitochondrial response to O2 tension possibly through the increase of 2-hydroxyglutarate. NO also resulted in suppression of cellular bioenergetics and further inhibition of Complex I which was not rescued by α-ketoglutarate. Taken together these data suggest that NO modulates the mitochondrial response to O2 differentially in the absence and presence of L-Gln. These data suggest a combination of metabolic strategies targeting glutaminolysis and Complex I in cancer cells.

Counteraction of Myocardial Ferritin Heavy Chain Deficiency by Heme Oxygenase-1.

Given the abundance of heme proteins (cytochromes) in the mitochondrion, it is evident that a meticulously orchestrated iron metabolism is essential for cardiac health. Here, we examined the functional significance of myocardial ferritin heavy chain (FtH) in a model of acute myocardial infarction. We report that FtH deletion did not alter either the mitochondrial regulatory and surveillance pathways (fission and fusion) or mitochondrial bioenergetics in response to injury. Furthermore, deletion of myocardial FtH did not affect cardiac function, assessed by measurement of left ventricular ejection fraction, on days 1, 7, and 21 post injury. To identify the modulated pathways providing cardiomyocyte protection coincident with FtH deletion, we performed unbiased transcriptomic analysis. We found that following injury, FtH deletion was associated with upregulation of several genes with anti-ferroptotic properties, including heme oxygenase-1 (HO-1) and the cystine/glutamate anti-porter (Slc7a11). These results suggested that HO-1 overexpression mitigates ferroptosis via upregulation of Slc7a11. Indeed, using transgenic mice with HO-1 overexpression, we demonstrate that overexpressed HO-1 is coupled with increased Slc7a11 expression. In conclusion, we demonstrate that following injury, myocardial FtH deletion leads to a compensatory upregulation in a number of anti-ferroptotic genes, including HO-1. Such HO-1 induction leads to overexpression of Slc7a11 and protects the heart against ischemia-reperfusion-mediated ferroptosis, preserves mitochondrial function, and overall function of the myocardium.

UAB-UCSD O'Brien Center for Acute Kidney Injury Research.

Acute kidney injury (AKI) remains a significant clinical problem through its diverse etiologies, the challenges of robust measurements of injury and recovery, and its progression to chronic kidney disease (CKD). Bridging the gap in our knowledge of this disorder requires bringing together not only the technical resources for research but also the investigators currently endeavoring to expand our knowledge and those who might bring novel ideas and expertise to this important challenge. The University of Alabama at Birmingham-University of California-San Diego O'Brien Center for Acute Kidney Injury Research brings together technical expertise and programmatic and educational efforts to advance our knowledge in these diverse issues and the required infrastructure to develop areas of novel exploration. Since its inception in 2008, this O'Brien Center has grown its impact by providing state-of-the-art resources in clinical and preclinical modeling of AKI, a bioanalytical core that facilitates measurement of critical biomarkers, including serum creatinine via LC-MS/MS among others, and a biostatistical resource that assists from design to analysis. Through these core resources and with additional educational efforts, our center has grown its investigator base to include >200 members from 51 institutions. Importantly, this center has translated its pilot and catalyst funding program with a $37 return per dollar invested. Over 500 publications have resulted from the support provided with a relative citation ratio of 2.18 ± 0.12 (iCite). Through its efforts, this disease-centric O'Brien Center is providing the infrastructure and focus to help the development of the next generation of researchers in the basic and clinical science of AKI. This center creates the promise of the application at the bedside of the advances in AKI made by current and future investigators.

ZKSCAN3 in severe bacterial lung infection and sepsis-induced immunosuppression.

The mortality rates among patients who initially survive sepsis are, in part, associated with a high risk of secondary lung infections and respiratory failure. Given that phagolysosomes are important for intracellular killing of pathogenic microbes, we investigated how severe lung infections associated with post-sepsis immunosuppression affect phagolysosome biogenesis. In mice with P. aeruginosa-induced pneumonia, we found a depletion of both phagosomes and lysosomes, as evidenced by decreased amounts of microtubule associated protein light chain 3-II (LC3-II) and lysosomal-associated membrane protein (LAMP1). We also found a loss of transcription factor E3 (TFE3) and transcription factor EB (TFEB), which are important activators for transcription of genes encoding autophagy and lysosomal proteins. These events were associated with increased expression of ZKSCAN3, a repressor for transcription of genes encoding autophagy and lysosomal proteins. Zkscan3-/- mice had increased expression of genes involved in the autophagy-lysosomal pathway along with enhanced killing of P. aeruginosa in the lungs, as compared to wild-type mice. These findings highlight the involvement of ZKSCAN3 in response to severe lung infection, including susceptibility to secondary bacterial infections due to immunosuppression.

Bioenergetic maladaptation and release of HMGB1 in calcineurin inhibitor-mediated nephrotoxicity.

Calcineurin inhibitors (CNIs) are potent immunosuppressive agents, universally used following solid organ transplantation to prevent rejection. Although effective, the long-term use of CNIs is associated with nephrotoxicity. The etiology of this adverse effect is complex, and effective therapeutic interventions remain to be determined. Using a combination of in vitro techniques and a mouse model of CNI-mediated nephrotoxicity, we found that the CNIs, cyclosporine A (CsA), and tacrolimus (TAC) share a similar mechanism of tubular epithelial kidney cell injury, including mitochondrial dysfunction and release of High-Mobility Group Box I (HMGB1). CNIs promote bioenergetic reprogramming due to mitochondrial dysfunction and a shift toward glycolytic metabolism. These events were accompanied by diminished cell-to-cell adhesion, loss of the epithelial cell phenotype, and release of HMGB1. Notably, Erk1/2 inhibitors effectively diminished HMGB1 release, and similar inhibitor was observed on inclusion of pan-caspase inhibitor zVAD-FMK. In vivo, while CNIs activate tissue proremodeling signaling pathways, MAPK/Erk1/2 inhibitor prevented nephrotoxicity, including diminished HMGB1 release from kidney epithelial cells and accumulation in urine. In summary, HMGB1 is an early indicator and marker of progressive nephrotoxicity induced by CNIs. We suggest that proremodeling signaling pathway and loss of mitochondrial redox/bioenergetics homeostasis are crucial therapeutic targets to ameliorate CNI-mediated nephrotoxicity.

New quantitative approach reveals heterogeneity in mitochondrial structure-function relations in tumor-initiating cells.

Steady-state mitochondrial structure or morphology is primarily maintained by a balance of opposing fission and fusion events between individual mitochondria, which is collectively referred to as mitochondrial dynamics. The details of the bidirectional relationship between the status of mitochondrial dynamics (structure) and energetics (function) require methods to integrate these mitochondrial aspects. To study the quantitative relationship between the status of mitochondrial dynamics (fission, fusion, matrix continuity and diameter) and energetics (ATP and redox), we have developed an analytical approach called mito-SinCe2 After validating and providing proof of principle, we applied mito-SinCe2 on ovarian tumor-initiating cells (ovTICs). Mito-SinCe2 analyses led to the hypothesis that mitochondria-dependent ovTICs interconvert between three states, that have distinct relationships between mitochondrial energetics and dynamics. Interestingly, fusion and ATP increase linearly with each other only once a certain level of fusion is attained. Moreover, mitochondrial dynamics status changes linearly with ATP or with redox, but not simultaneously with both. Furthermore, mito-SinCe2 analyses can potentially predict new quantitative features of the opposing fission versus fusion relationship and classify cells into functional classes based on their mito-SinCe2 states.This article has an associated First Person interview with the first author of the paper.

Poldip2 is an oxygen-sensitive protein that controls PDH and αKGDH lipoylation and activation to support metabolic adaptation in hypoxia and cancer.

Although the addition of the prosthetic group lipoate is essential to the activity of critical mitochondrial catabolic enzymes, its regulation is unknown. Here, we show that lipoylation of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (αKDH) complexes is a dynamically regulated process that is inhibited under hypoxia and in cancer cells to restrain mitochondrial respiration. Mechanistically, we found that the polymerase-δ interacting protein 2 (Poldip2), a nuclear-encoded mitochondrial protein of unknown function, controls the lipoylation of the pyruvate and α-KDH dihydrolipoamide acetyltransferase subunits by a mechanism that involves regulation of the caseinolytic peptidase (Clp)-protease complex and degradation of the lipoate-activating enzyme Ac-CoA synthetase medium-chain family member 1 (ACSM1). ACSM1 is required for the utilization of lipoic acid derived from a salvage pathway, an unacknowledged lipoylation mechanism. In Poldip2-deficient cells, reduced lipoylation represses mitochondrial function and induces the stabilization of hypoxia-inducible factor 1α (HIF-1α) by loss of substrate inhibition of prolyl-4-hydroxylases (PHDs). HIF-1α-mediated retrograde signaling results in a metabolic reprogramming that resembles hypoxic and cancer cell adaptation. Indeed, we observe that Poldip2 expression is down-regulated by hypoxia in a variety of cell types and basally repressed in triple-negative cancer cells, leading to inhibition of lipoylation of the pyruvate and α-KDH complexes and mitochondrial dysfunction. Increasing mitochondrial lipoylation by forced expression of Poldip2 increases respiration and reduces the growth rate of cancer cells. Our work unveils a regulatory mechanism of catabolic enzymes required for metabolic plasticity and highlights the role of Poldip2 as key during hypoxia and cancer cell metabolic adaptation.

Nuclear receptor binding factor 2 (NRBF2) is required for learning and memory.

The mechanisms which underlie defects in learning and memory are a major area of focus with the increasing incidence of Alzheimer's disease in the aging population. The complex genetically-controlled, age-, and environmentally-dependent onset and progression of the cognitive deficits and neuronal pathology call for better understanding of the fundamental biology of the nervous system function. In this study, we focus on nuclear receptor binding factor-2 (NRBF2) which modulates the transcriptional activities of retinoic acid receptor α and retinoid X receptor α, and the autophagic activities of the BECN1-VPS34 complex. Since both transcriptional regulation and autophagic function are important in supporting neuronal function, we hypothesized that NRBF2 deficiency may lead to cognitive deficits. To test this, we developed a new mouse model with nervous system-specific knockout of Nrbf2. In a series of behavioral assessment, we demonstrate that NRBF2 knockout in the nervous system results in profound learning and memory deficits. Interestingly, we did not find deficits in autophagic flux in primary neurons and the autophagy deficits were minimal in the brain. In contrast, RNAseq analyses have identified altered expression of genes that have been shown to impact neuronal function. The observation that NRBF2 is involved in learning and memory suggests a new mechanism regulating cognition involving the role of this protein in regulating networks related to the function of retinoic acid receptors, protein folding, and quality control.

Differential effects of REV-ERBα/ß agonism on cardiac gene expression, metabolism, and contractile function in a mouse model of circadian disruption.

Cell-autonomous circadian clocks have emerged as temporal orchestrators of numerous biological processes. For example, the cardiomyocyte circadian clock modulates transcription, translation, posttranslational modifications, ion homeostasis, signaling cascades, metabolism, and contractility of the heart over the course of the day. Circadian clocks are composed of more than 10 interconnected transcriptional modulators, all of which have the potential to influence the cardiac transcriptome (and ultimately cardiac processes). These transcriptional modulators include BMAL1 and REV-ERBα/ß; BMAL1 induces REV-ERBα/ß, which in turn feeds back to inhibit BMAL1. Previous studies indicate that cardiomyocyte-specific BMAL1-knockout (CBK) mice exhibit a dysfunctional circadian clock (including decreased REV-ERBα/ß expression) in the heart associated with abnormalities in cardiac mitochondrial function, metabolism, signaling, and contractile function. Here, we hypothesized that decreased REV-ERBα/ß activity is responsible for distinct phenotypical alterations observed in CBK hearts. To test this hypothesis, CBK (and littermate control) mice were administered with the selective REV-ERBα/ß agonist SR-9009 (100 mg·kg-1·day-1 for 8 days). SR-9009 administration was sufficient to normalize cardiac glycogen synthesis rates, cardiomyocyte size, interstitial fibrosis, and contractility in CBK hearts (without influencing mitochondrial complex activities, nor normalizing substrate oxidation and Akt/mTOR/GSK3ß signaling). Collectively, these observations highlight a role for REV-ERBα/ß as a mediator of a subset of circadian clock-controlled processes in the heart.

AMPK-ACC signaling modulates platelet phospholipids and potentiates thrombus formation.

AMP-activated protein kinase (AMPK) α1 is activated in platelets on thrombin or collagen stimulation, and as a consequence, phosphorylates and inhibits acetyl-CoA carboxylase (ACC). Because ACC is crucial for the synthesis of fatty acids, which are essential for platelet activation, we hypothesized that this enzyme plays a central regulatory role in platelet function. To investigate this, we used a double knock-in (DKI) mouse model in which the AMPK phosphorylation sites Ser79 on ACC1 and Ser212 on ACC2 were mutated to prevent AMPK signaling to ACC. Suppression of ACC phosphorylation promoted injury-induced arterial thrombosis in vivo and enhanced thrombus growth ex vivo on collagen-coated surfaces under flow. After collagen stimulation, loss of AMPK-ACC signaling was associated with amplified thromboxane generation and dense granule secretion. ACC DKI platelets had increased arachidonic acid-containing phosphatidylethanolamine plasmalogen lipids. In conclusion, AMPK-ACC signaling is coupled to the control of thrombosis by specifically modulating thromboxane and granule release in response to collagen. It appears to achieve this by increasing platelet phospholipid content required for the generation of arachidonic acid, a key mediator of platelet activation.

Precisely Control Mitochondria with Light to Manipulate Cell Fate Decision.

Mitochondrial dysfunction has been implicated in many pathological conditions and diseases. The normal functioning of mitochondria relies on maintaining the inner mitochondrial membrane potential (also known as ΔΨm) that is essential for ATP synthesis, Ca2+ homeostasis, redox balance, and regulation of other key signaling pathways such as mitophagy and apoptosis. However, the detailed mechanisms by which ΔΨm regulates cellular function remain incompletely understood, partially because of the difficulty of manipulating ΔΨm with spatiotemporal resolution, reversibility, or cell type specificity. To address this need, we have developed a next generation optogenetic-based technique for controllable mitochondrial depolarization with light. We demonstrate successful targeting of the heterologous channelrhodopsin-2 fusion protein to the inner mitochondrial membrane and formation of functional cationic channels capable of light-induced selective ΔΨm depolarization and mitochondrial autophagy. Importantly, we for the first time, to our knowledge, show that optogenetic-mediated mitochondrial depolarization can be well controlled to differentially influence the fate of cells expressing mitochondrial channelrhodopsin-2; whereas sustained moderate light illumination induces substantial apoptotic cell death, transient mild light illumination elicits cytoprotection via mitochondrial preconditioning. Finally, we show that Parkin overexpression exacerbates, instead of ameliorating, mitochondrial depolarization-mediated cell death in HeLa cells. In summary, we provide evidence that the described mitochondrial-targeted optogenetics may have a broad application for studying the role of mitochondria in regulating cell function and fate decision.

Acute increases in O-GlcNAc indirectly impair mitochondrial bioenergetics through dysregulation of LonP1-mediated mitochondrial protein complex turnover.

The attachment of O-linked ß-N-acetylglucosamine (O-GlcNAc) to the serine and threonine residues of proteins in distinct cellular compartments is increasingly recognized as an important mechanism regulating cellular function. Importantly, the O-GlcNAc modification of mitochondrial proteins has been identified as a potential mechanism to modulate metabolism under stress with both potentially beneficial and detrimental effects. This suggests that temporal and dose-dependent changes in O-GlcNAcylation may have different effects on mitochondrial function. In the current study, we found that acutely augmenting O-GlcNAc levels by inhibiting O-GlcNAcase with Thiamet-G for up to 6 h resulted in a time-dependent decrease in cellular bioenergetics and decreased mitochondrial complex I, II, and IV activities. Under these conditions, mitochondrial number was unchanged, whereas an increase in the protein levels of the subunits of several electron transport complex proteins was observed. However, the observed bioenergetic changes appeared not to be due to direct increased O-GlcNAc modification of complex subunit proteins. Increases in O-GlcNAc were also associated with an accumulation of mitochondrial ubiquitinated proteins; phosphatase and tensin homolog induced kinase 1 (PINK1) and p62 protein levels were also significantly increased. Interestingly, the increase in O-GlcNAc levels was associated with a decrease in the protein levels of the mitochondrial Lon protease homolog 1 (LonP1), which is known to target complex IV subunits and PINK1, in addition to other mitochondrial proteins. These data suggest that impaired bioenergetics associated with short-term increases in O-GlcNAc levels could be due to impaired, LonP1-dependent, mitochondrial complex protein turnover.

Glutaminolysis is required for transforming growth factor-ß1-induced myofibroblast differentiation and activation.

Myofibroblasts participate in physiological wound healing and pathological fibrosis. Myofibroblast differentiation is characterized by the expression of α-smooth muscle actin and extracellular matrix proteins and is dependent on metabolic reprogramming. In this study, we explored the role of glutaminolysis and metabolites of TCA in supporting myofibroblast differentiation. Glutaminolysis converts Gln into α-ketoglutarate (α-KG), a critical intermediate in the TCA cycle. Increases in the steady-state concentrations of TCA cycle metabolites including α-KG, succinate, fumarate, malate, and citrate were observed in TGF-ß1-differentiated myofibroblasts. The concentration of glutamate was also increased in TGF-ß1-differentiated myofibroblasts compared with controls, whereas glutamine levels were decreased, suggesting enhanced glutaminolysis. This was associated with TGF-ß1-induced expression of the glutaminase (GLS) isoform, GLS1, which converts Gln into glutamate, at both the mRNA and protein levels. The stimulation of GLS1 expression by TGF-ß1 was dependent on both SMAD3 and p38 mitogen-activated protein kinase activation. Depletion of extracellular Gln prevented TGF-ß1-induced myofibroblast differentiation. The removal of extracellular Gln postmyofibroblast differentiation decreased the expression of the profibrotic markers fibronectin and hypoxia-inducible factor-1α and reversed TGF-ß1-induced metabolic reprogramming. Silencing of GLS1 expression, in the presence of Gln, abrogated TGF-ß1-induced expression of profibrotic markers. Treatment of GLS1-deficient myofibroblasts with exogenous glutamate or α-KG restored TGF-ß1-induced expression of profibrotic markers in GLS1-deficient myofibroblasts. Together, these data demonstrate that glutaminolysis is a critical component of myofibroblast metabolic reprogramming that regulates myofibroblast differentiation.