Catherine Corey

PINK1 deficiency impairs mitochondrial homeostasis and promotes lung fibrosis

Although aging is a known risk factor for idiopathic pulmonary fibrosis (IPF), the pathogenic mechanisms that underlie the effects of advancing age remain largely unexplained. Some age-related neurodegenerative diseases have an etiology that is related to mitochondrial dysfunction. Here, we found that alveolar type II cells (AECIIs) in the lungs of IPF patients exhibit marked accumulation of dysmorphic and dysfunctional mitochondria. These mitochondrial abnormalities in AECIIs of IPF lungs were associated with upregulation of ER stress markers and were recapitulated in normal mice with advancing age in response to stimulation of ER stress. We found that impaired mitochondria in IPF and aging lungs were associated with low expression of PTEN-induced putative kinase 1 (PINK1). Knockdown of PINK1 expression in lung epithelial cells resulted in mitochondria depolarization and expression of profibrotic factors. Moreover, young PINK1-deficient mice developed similarly dysmorphic, dysfunctional mitochondria in the AECIIs and were vulnerable to apoptosis and development of lung fibrosis. Our data indicate that PINK1 deficiency results in swollen, dysfunctional mitochondria and defective mitophagy, and promotes fibrosis in the aging lung.

Nitrite activates protein kinase A in normoxia to mediate mitochondrial fusion and tolerance to ischaemia/reperfusion

AIMS Nitrite (NO2(-)), a dietary constituent and nitric oxide (NO) oxidation product, mediates cardioprotection after ischaemia/reperfusion (I/R) in a number of animal models when administered during ischaemia or as a pre-conditioning agent hours to days prior to the ischaemic episode. When present during ischaemia, the reduction of nitrite to bioactive NO by deoxygenated haem proteins accounts for its protective effects. However, the mechanism of nitrite-induced pre-conditioning, a normoxic response which does not appear to require reduction of nitrite to NO, remains unexplored. METHODS AND RESULTS Using a model of hypoxia/reoxygenation (H/R) in cultured rat H9c2 cardiomyocytes, we demonstrate that a transient (30 min) normoxic nitrite treatment significantly attenuates cell death after a hypoxic episode initiated 1 h later. Mechanistically, this protection depends on the activation of protein kinase A, which phosphorylates and inhibits dynamin-related protein 1, the predominant regulator of mitochondrial fission. This results morphologically, in the promotion of mitochondrial fusion and functionally in the augmentation of mitochondrial membrane potential and superoxide production. We identify AMP kinase (AMPK) as a downstream target of the mitochondrial reactive oxygen species (ROS) generated and show that its oxidation and subsequent phosphorylation are essential for cytoprotection, as scavenging of ROS prevents AMPK activation and inhibits nitrite-mediated protection after H/R. The protein kinase A-dependent protection mediated by nitrite is reproduced in an intact isolated rat heart model of I/R. CONCLUSIONS These data are the first to demonstrate nitrite-dependent normoxic modulation of both mitochondrial morphology and function and reveal a novel signalling pathway responsible for nitrite-mediated cardioprotection.

T Cell Receptor–Dependent Activation of mTOR Signaling in T Cells Is Mediated by Carma1 and MALT1, But Not Bcl10

By shifting the constituents of signalosomes, T cells may stimulate distinct pathways in response to antigens. Delineating an Alternative Path to mTOR in T Cells Antigen-dependent stimulation of the T cell receptor (TCR) leads to activation of the kinases PI3K, Akt, and mTOR, which are required for the proliferation of T cells and the increased metabolism that is needed to support their immune function. Hamilton et al. investigated the mechanism through which the TCR activates mTOR and identified a pathway independent of the best known upstream mTOR-activating kinase, Akt. Instead, proteins typically associated with a complex that transmits signals from the TCR to the transcription factor NF-κB were involved. Loss of either the adaptor protein Carma1 or the protease MALT1 in T cell lines blocked their proliferation in response to TCR stimulation. In addition, inhibition of MALT1 activity in primary human T cells inhibited their increased metabolism. Thus, Carma1 and MALT1 may form distinct signaling complexes to transmit signals from the TCR to different downstream effectors. Signaling to the mechanistic target of rapamycin (mTOR) regulates diverse cellular processes, including protein translation, cellular proliferation, metabolism, and autophagy. Most models place Akt upstream of the mTOR complex, mTORC1; however, in T cells, Akt may not be necessary for mTORC1 activation. We found that the adaptor protein Carma1 [caspase recruitment domain (CARD)–containing membrane-associated protein 1] and at least one of its associated proteins, the paracaspase MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), were required for optimal activation of mTOR in T cells in response to stimulation of the T cell receptor (TCR) and the co-receptor CD28. However, Bcl10, which binds to Carma1 and MALT1 to form a complex that mediates signals from the TCR to the transcription factor NF-κB (nuclear factor κB), was not required. The catalytic activity of MALT1 was required for the proliferation of stimulated CD4+ T cells, but not for early TCR-dependent activation events. Consistent with an effect on mTOR, MALT1 activity was required for the increased metabolic flux in activated CD4+ T cells. Together, our data suggest that Carma1 and MALT1 play previously unappreciated roles in the activation of mTOR signaling in T cells after engagement of the TCR.

Nitrite activates AMP kinase to stimulate mitochondrial biogenesis independent of soluble guanylate cyclase

Nitrite, a dietary constituent and endogenous signaling molecule, mediates a number of physiological responses including modulation of ischemia/reperfusion injury, glucose tolerance, and vascular remodeling. Although the exact molecular mechanisms underlying nitrites actions are unknown, the current paradigm suggests that these effects depend on the hypoxic reduction of nitrite to nitric oxide (NO). Mitochondrial biogenesis is a fundamental mechanism of cellular adaptation and repair. However, the effect of nitrite on mitochondrial number has not been explored. Herein, we report that nitrite stimulates mitochondrial biogenesis through a mechanism distinct from that of NO. We demonstrate that nitrite significantly increases cellular mitochondrial number by augmenting the activity of adenylate kinase, resulting in AMP kinase phosphorylation, downstream activation of sirtuin-1, and deacetylation of PGC1α, the master regulator of mitochondrial biogenesis. Unlike NO, nitrite-mediated biogenesis does not require the activation of soluble guanylate cyclase and results in the synthesis of more functionally efficient mitochondria. Further, we provide evidence that nitrite mediates biogenesis in vivo. In a rat model of carotid injury, 2 weeks of continuous oral nitrite treatment postinjury prevented the hyperproliferative response of smooth muscle cells. This protection was accompanied by a nitrite-dependent upregulation of PGC1α and increased mitochondrial number in the injured artery. These data are the first to demonstrate that nitrite mediates differential signaling compared to NO. They show that nitrite is a versatile regulator of mitochondrial function and number both in vivo and in vitro and suggest that nitrite-mediated biogenesis may play a protective role in the setting of vascular injury.

Platelet bioenergetic screen in sickle cell patients reveals mitochondrial complex V inhibition, which contributes to platelet activation

Bioenergetic dysfunction, although central to the pathogenesis of numerous diseases, remains uncharacterized in many patient populations because of the invasiveness of obtaining tissue for mitochondrial studies. Although platelets are an accessible source of mitochondria, the role of bioenergetics in regulating platelet function remains unclear. Herein, we validate extracellular flux analysis in human platelets and use this technique to screen for mitochondrial dysfunction in sickle cell disease (SCD) patients, a population with aberrant platelet activation of an unknown mechanism and in which mitochondrial function has never been assessed. We identify a bioenergetic alteration in SCD patients characterized by deficient complex V activity, leading to decreased mitochondrial respiration, membrane hyperpolarization, and augmented oxidant production compared with healthy subjects. This dysfunction correlates with platelet activation and hemolysis in vivo and can be recapitulated in vitro by exposing healthy platelets to hemoglobin or a complex V inhibitor. Further, reproduction of this dysfunction in vitro activates healthy platelets, an effect prevented by attenuation of mitochondrial hyperpolarization or by scavenging mitochondrial oxidants. These data identify bioenergetic dysfunction in SCD patients for the first time and establish mitochondrial hyperpolarization and oxidant generation as potential pathogenic mechanism in SCD as well as a modulator of healthy platelet function.

IPF lung fibroblasts have a senescent phenotype

The mechanisms of aging that are involved in the development of idiopathic pulmonary fibrosis (IPF) are still unclear. Although it has been hypothesized that the proliferation and activation of human lung fibroblasts (hLFs) are essential in IPF, no studies have assessed how this process works in an aging lung. Our goal was to elucidate if there were age-related changes on primary hLFs isolated from IPF lungs compared with age-matched controls. We investigated several hallmarks of aging in hLFs from IPF patients and age-matched controls. IPF hLFs have increased cellular senescence with higher expression of β-galactosidase, p21, p16, p53, and cytokines related to the senescence-associated secretory phenotype (SASP) as well as decreased proliferation/apoptosis compared with age-matched controls. Additionally, we observed shorter telomeres, mitochondrial dysfunction, and upon transforming growth factor-β stimulation, increased markers of endoplasmic reticulum stress. Our data suggest that IPF hLFs develop senescence resulting in a decreased apoptosis and that the development of SASP may be an important contributor to the fibrotic process observed in IPF. These results might change the existing paradigm, which describes fibroblasts as aberrantly activated cells, to a cell with a senescence phenotype.

Inorganic nitrite improves components of the metabolic syndrome independent of weight change in a murine model of obesity and insulin resistance

Nitrite acts as an endocrine source of bioavailable nitric oxide that can improve metabolic function. Exogenous administration of nitrite to genetically obese mice enhanced glucose tolerance and increased insulin sensitivity independent of weight change. Exogenous nitrite uncoupled mitochondrial respiration, decreased the generation of ATP, and increased phosphorylation of AMP‐activated protein kinase in skeletal muscle. The weight‐independent improvements in metabolic function from exogenous nitrite were comparable to rosiglitazone, suggesting the potential for therapeutic efficacy of nitrite in obese, insulin‐resistant, or diabetic patients.

Platelets from Asthmatic Individuals Show Less Reliance on Glycolysis

Asthma, a chronic inflammatory airway disease, is typified by high levels of TH2-cytokines and excessive generation of reactive nitrogen and oxygen species, which contribute to bronchial epithelial injury and airway remodeling. While immune function plays a major role in the pathogenesis of the disease, accumulating evidence suggests that altered cellular metabolism is a key determinant in the predisposition and disease progression of asthma. Further, several studies demonstrate altered mitochondrial function in asthmatic airways and suggest that these changes may be systemic. However, it is unknown whether systemic metabolic changes can be detected in circulating cells in asthmatic patients. Platelets are easily accessible blood cells that are known to propagate airway inflammation in asthma. Here we perform a bioenergetic screen of platelets from asthmatic and healthy individuals and demonstrate that asthmatic platelets show a decreased reliance on glycolytic processes and have increased tricarboxylic acid cycle activity. These data demonstrate a systemic alteration in asthma and are consistent with prior reports suggesting that oxidative phosphorylation is more efficient asthmatic individuals. The implications for this potential metabolic shift will be discussed in the context of increased oxidative stress and hypoxic adaptation of asthmatic patients. Further, these data suggest that platelets are potentially a good model for the monitoring of bioenergetic changes in asthma.

The Proapoptotic F-box Protein Fbxl7 Regulates Mitochondrial Function by Mediating the Ubiquitylation and Proteasomal Degradation of Survivin

Background: The SCF ubiquitin E3 ligase component Fbxl7 possesses proapoptotic activity. Results: Fbxl7 targets the antiapoptotic protein survivin for polyubiquitylation and proteasomal degradation. Conclusion: Survivin protects mitochondria from damage induced by Fbxl7. Significance: Understanding how F-box proteins regulate survivin might impact therapies to preserve cellular bioenergetics. Fbxl7, a component of the Skp1·Cul1·F-box protein type ubiquitin E3 ligase, regulates mitotic cell cycle progression. Here we demonstrate that overexpression of Fbxl7 in lung epithelia decreases the protein abundance of survivin, a member of the inhibitor of apoptosis family. Fbxl7 mediates polyubiquitylation and proteasomal degradation of survivin by interacting with Glu-126 within its carboxyl-terminal α helix. Furthermore, both Lys-90 and Lys-91 within survivin serve as ubiquitin acceptor sites. Ectopically expressed Fbxl7 impairs mitochondrial function, whereas depletion of Fbxl7 protects mitochondria from actions of carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of oxidative phosphorylation. Compared with wild-type survivin, cellular expression of a survivin mutant protein deficient in its ability to interact with Fbxl7 (E126A) and a ubiquitylation-resistant double point mutant (KK90RR/KK91RR) rescued mitochondria to a larger extent from damage induced by overexpression of Fbxl7. Therefore, these data suggest that the Skp1·Cul1·F-box protein complex subunit Fbxl7 modulates mitochondrial function by controlling the cellular abundance of survivin. The results raise opportunities for F-box protein targeting to preserve mitochondrial function.

Minnelide/Triptolide Impairs Mitochondrial Function by Regulating SIRT3 in P53-Dependent Manner in Non-Small Cell Lung Cancer.

Minnelide/Triptolide (TL) has recently emerged as a potent anticancer drug in non-small cell lung cancer (NSCLC). However, the precise mechanism of its action remains ambiguous. In this study, we elucidated the molecular basis for TL-induced cell death in context to p53 status. Cell death was attributed to dysfunction of mitochondrial bioenergetics in p53-deficient cells, which was characterized by decreased mitochondrial respiration, steady-state ATP level and membrane potential, but augmented reactive oxygen species (ROS). Increased ROS production resulted in oxidative stress in TL-treated cells. This was exhibited by elevated nuclear levels of a redox-sensitive transcriptional factor, NF-E2-related factor-2 (NRF2), along with diminished cellular glutathione (GSH) content. We further demonstrated that in the absence of p53, TL blunted the expression of mitochondrial SIRT3 triggering increased acetylation of NDUAF9 and succinate dehydrogenase, components of complexes I and II of the electron transport chain (ETC). TL-mediated hyperacetylation of complexes I and II proteins and these complexes displayed decreased enzymatic activities. We also provide the evidence that P53 regulate steady-state level of SIRT3 through Proteasome-Pathway. Finally, forced overexpression of Sirt3, but not deacetylase-deficient mutant of Sirt3 (H243Y), restored the deleterious effect of TL on p53-deficient cells by rescuing mitochondrial bioenergetics. On contrary, Sirt3 deficiency in the background of wild-type p53 triggered TL-induced mitochondrial impairment that echoed TL effect in p53-deficeint cells. These findings illustrate a novel mechanism by which TL exerts its potent effects on mitochondrial function and ultimately the viability of NSCLC tumor.