Vitamin A depletion causes oxidative stress, mitochondrial dysfunction, and PARP‐1‐dependent energy deprivation

Quijano C., Castro L., Peluffo G., Valez V., Radi R.

Hyperglycemic challenge to bovine aortic endothelial cells (BAECs) increases oxidant formation and cell damage that are abolished by MnSOD overexpression, implying mitochondrial superoxide (O2•−) as a central mediator. However, mitochondrial O2•−and its steady-state concentrations have not been measured directly yet. Therefore, we aimed to detect and quantify O2•−through different techniques, along with the oxidants derived from it. Mitochondrial aconitase, a sensitive target of O2•−, was inactivated 60% in BAECs incubated in 30 mM glucose (hyperglycemic condition) with respect to cells incubated in 5 mM glucose (normoglycemic condition). Under hyperglycemic conditions, increased oxidation of the mitochondrially targeted hydroethidine derivative (MitoSOX) to hydroxyethidium, the product of the reaction with O2•−, could be specifically detected. An 8.8-fold increase in mitochondrial O2•−steady-state concentration (to 250 pM) and formation rate (to 6 μM/s) was estimated. Superoxide formation increased the intracellular concentration of both hydrogen peroxide, measured as 3-amino-2,4,5-triazole-mediated inactivation of catalase, and nitric oxide-derived oxidants (i.e., peroxynitrite), evidenced by immunochemical detection of 3-nitrotyrosine. Oxidant formation was further evaluated by chloromethyl dichlorodihydrofluorescein (CM-H2DCF) oxidation. Exposure to hyperglycemic conditions triggered the oxidation of CM-H2DCF and was significantly reduced by pharmacological agents that lower the mitochondrial membrane potential, inhibit electron transport (i.e., myxothiazol), and scavenge mitochondrial oxidants (i.e., MitoQ). In BAECs devoid of mitochondria (rho0cells), hyperglycemic conditions did not increase CM-H2DCF oxidation. Mitochondrial O2•−formation in hyperglycemic conditions was associated with increased glucose metabolization in the Krebs cycle and hyperpolarization of the mitochondrial membrane.

Miwa M., Masutani M.

The polyADP-ribosylation reaction results in a unique post-translational modification involved in various cellular processes and conditions, including DNA repair, transcriptional control, genomic stability, cell death and transformation. The existence of 17 members of the poly(ADP-ribose) polymerase (PARP) family has so far been documented, with overlapping functional consequences. PARP-1 is known to be involved in DNA base excision repair and this explains the susceptibility spectrum of PARP-1 knockout animals to genotoxic carcinogens. The fact that centrosome amplification is induced by a non-genotoxic inhibitor of PARP and in PARP-1 knockout mouse cells, is in line with aneuploidy, which is frequent in cancers. Genetically engineered animal models have revealed that PARP-1 and VPARP impact carcinogenesis. Furthermore, accumulating experimental evidence supports the utility of PARP and PARG inhibitors in cancer therapy and several clinical trials are now ongoing. Increasing NAD(+) levels by pharmacological supplementation with niacin has also been found to exert preventive effects against cancer. In the present review, recent research progress on polyADP-ribosylation related to neoplasia is summarized and discussed.

Bell E.L., Klimova T.A., Eisenbart J., Moraes C.T., Murphy M.P., Budinger G.R., Chandel N.S.

Mammalian cells increase transcription of genes for adaptation to hypoxia through the stabilization of hypoxia-inducible factor 1α (HIF-1α) protein. How cells transduce hypoxic signals to stabilize the HIF-1α protein remains unresolved. We demonstrate that cells deficient in the complex III subunit cytochrome b, which are respiratory incompetent, increase ROS levels and stabilize the HIF-1α protein during hypoxia. RNA interference of the complex III subunit Rieske iron sulfur protein in the cytochrome b–null cells and treatment of wild-type cells with stigmatellin abolished reactive oxygen species (ROS) generation at the Qo site of complex III. These interventions maintained hydroxylation of HIF-1α protein and prevented stabilization of HIF-1α protein during hypoxia. Antioxidants maintained hydroxylation of HIF-1α protein and prevented stabilization of HIF-1α protein during hypoxia. Exogenous hydrogen peroxide under normoxia prevented hydroxylation of HIF-1α protein and stabilized HIF-1α protein. These results provide genetic and pharmacologic evidence that the Qo site of complex III is required for the transduction of hypoxic signal by releasing ROS to stabilize the HIF-1α protein.

Fukuda R., Zhang H., Kim J., Shimoda L., Dang C.V., Semenza G.

O(2) is the ultimate electron acceptor for mitochondrial respiration, a process catalyzed by cytochrome c oxidase (COX). In yeast, COX subunit composition is regulated by COX5a and COX5b gene transcription in response to high and low O(2), respectively. Here we demonstrate that in mammalian cells, expression of the COX4-1 and COX4-2 isoforms is O(2) regulated. Under conditions of reduced O(2) availability, hypoxia-inducible factor 1 (HIF-1) reciprocally regulates COX4 subunit expression by activating transcription of the genes encoding COX4-2 and LON, a mitochondrial protease that is required for COX4-1 degradation. The effects of manipulating COX4 subunit expression on COX activity, ATP production, O(2) consumption, and reactive oxygen species generation indicate that the COX4 subunit switch is a homeostatic response that optimizes the efficiency of respiration at different O(2) concentrations. Thus, mammalian cells respond to hypoxia by altering COX subunit composition, as previously observed in yeast, but by a completely different molecular mechanism.

Fossati S., Cipriani G., Moroni F., Chiarugi A.

Poly(ADP-ribose)polymerase-1 (PARP-1) overactivation is a key event in neurodegeneration but the underlying molecular mechanisms wait to be unequivocally identified. Energy failure, transcriptional derangement and deadly nucleus-mitochondria cross-talk have been proposed as mechanisms responsible for PARP-1 neurotoxicity. In this study, we sought to determine how these mechanisms contributes to PARP-1-dependent neuronal death. We report that the PARP-1 activating agent methyl-nitrosoguanidine (MNNG) caused poly(ADP-ribosyl)ation-dependent death of pure mouse cortical neurons in culture. Upon PARP-1 hyperactivation, NAD and ATP storages only partially decreased, neurons rapidly acquired apoptotic morphology, apoptosis inducing factor and cytochrome c were released from mitochondria and caspase activation occurred. No evidence for p53 activation was found, lactate dehydrogenase release occurred only 18h later, and JNK kinase was constitutively activated and not affected by PARP-1 activation. The PARP-1 inhibitors 6-(5)H-phenanthridinone and N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide (PJ-34) prevented nucleotide depletion and cell death, whereas the transcription inhibitor actinomycin D did not affect PARP-1-dependent neurotoxicity. Together, our findings provide the first evidence that neither energy collapse nor transcriptional changes are involved in PARP-1-dependent apoptotic neuronal death, and support the existence of a poly(ADP-ribose)-mediated death signaling targeting mitochondria.

Guzy R.D., Schumacker P.T.

All eukaryotic cells utilize oxidative phosphorylation to maintain their high-energy phosphate stores. Mitochondrial oxygen consumption is required for ATP generation, and cell survival is threatened when cells are deprived of O(2). Consequently, all cells have the ability to sense O(2), and to activate adaptive processes that will enhance the likelihood of survival in anticipation that oxygen availability might become limiting. Mitochondria have long been considered a likely site of oxygen sensing, and we propose that the electron transport chain acts as an O(2) sensor by releasing reactive oxygen species (ROS) in response to hypoxia. The ROS released during hypoxia act as signalling agents that trigger diverse functional responses, including activation of gene expression through the stabilization of the transcription factor hypoxia-inducible factor (HIF)-alpha. The primary site of ROS production during hypoxia appears to be complex III. The paradoxical increase in ROS production during hypoxia may be explained by an effect of O(2) within the mitochondrial inner membrane on: (a) the lifetime of the ubisemiquinone radical in complex III; (b) the relative release of mitochondrial ROS towards the matrix compartment versus the intermembrane space; or (c) the ability of O(2) to access the ubisemiquinone radical in complex III. In summary, the process of oxygen sensing is of fundamental importance in biology. An ability to control the oxygen sensing mechanism in cells, potentially using small molecules that do not disrupt oxygen consumption, would open valuable therapeutic avenues that could have a profound impact on a diverse range of diseases.

Hail N., Kim H.J., Lotan R.

Fenretinide, a synthetic retinoid, has emerged as a promising anticancer agent based on numerous in vitro and animal studies, as well as chemoprevention clinical trials. In vitro observations suggest that the anticancer activity of fenretinide may arise from its ability to induce apoptosis in tumor cells. Diverse signaling molecules including reactive oxygen species, ceramide, and ganglioside GD3 can mediate apoptosis induction by fenretinide in transformed, premalignant, and malignant cells. In many cell types, these signaling intermediates appear to be induced by mechanisms that are independent of retinoic acid receptor activation, and ultimately initiate the intrinsic or mitochondrial-mediated pathway of cell elimination. Numerous investigations conducted during the past 10 years have discovered a great deal about the apoptogenic activity of fenretinide. In this review we explore the mechanisms associated with fenretinide-induced apoptosis and highlight certain mechanistic underpinnings of fenretinide-induced cell death that remain poorly understood and thus warrant further characterization.

Kim J., Tchernyshyov I., Semenza G.L., Dang C.V.

Activation of glycolytic genes by HIF-1 is considered critical for metabolic adaptation to hypoxia through increased conversion of glucose to pyruvate and subsequently to lactate. We found that HIF-1 also actively suppresses metabolism through the tricarboxylic acid cycle (TCA) by directly trans-activating the gene encoding pyruvate dehydrogenase kinase 1 (PDK1). PDK1 inactivates the TCA cycle enzyme, pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Forced PDK1 expression in hypoxic HIF-1alpha null cells increases ATP levels, attenuates hypoxic ROS generation, and rescues these cells from hypoxia-induced apoptosis. These studies reveal a hypoxia-induced metabolic switch that shunts glucose metabolites from the mitochondria to glycolysis to maintain ATP production and to prevent toxic ROS production.

Papandreou I., Cairns R.A., Fontana L., Lim A.L., Denko N.C.

Summary The HIF-1 transcription factor drives hypoxic gene expression changes that are thought to be adaptive for cells exposed to a reduced-oxygen environment. For example, HIF-1 induces the expression of glycolytic genes. It is presumed that increased glycolysis is necessary to produce energy when low oxygen will not support oxidative phosphorylation at the mitochondria. However, we find that while HIF-1 stimulates glycolysis, it also actively represses mitochondrial function and oxygen consumption by inducing pyruvate dehydrogenase kinase 1 (PDK1). PDK1 phosphorylates and inhibits pyruvate dehydrogenase from using pyruvate to fuel the mitochondrial TCA cycle. This causes a drop in mitochondrial oxygen consumption and results in a relative increase in intracellular oxygen tension. We show by genetic means that HIF-1-dependent block to oxygen utilization results in increased oxygen availability, decreased cell death when total oxygen is limiting, and reduced cell death in response to the hypoxic cytotoxin tirapazamine.

WANG H., SHIMOJI M., YU S., DAWSON T.M., DAWSON V.L.

Experimental intoxication models are used to study the more common sporadic form of Parkinson's disease (PD). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine (MPTP) animal models of PD provide a valuable and predictive tool to probe the molecular mechanisms of dopamine neuronal cell death in PD. MPTP is a powerful neurotoxin that induces neuronal degeneration in the substantia nigra pars compacta and produces PD-like symptoms in several mammalian species tested, a feat not yet accomplished in genetically engineered mice expressing human genetic mutations. The mechanisms of MPTP-induced neurotoxicity are not yet fully understood but involve activation of N-methyl-D-aspartate (NMDA) receptors by glutamate, production of NO by nNOS and iNOS, oxidative injury to DNA, and activation of the DNA damage-sensing enzyme poly (ADP-ribose) polymerase (PARP). Recent experiments indicate that translocation of a mitochondrial protein apoptosis inducing factor (AIF) from mitochondria to the nucleus depends on PARP activation and plays an important role in excitotoxicity-induced cell death. This article briefly reviews the experimental findings regarding excitotoxicity, PARP activation, and AIF translocation in MPTP toxicity and dopaminergic neuronal cell death.

Celeste Simon M.

Multicellular organisms initiate adaptive responses when oxygen (O2) availability decreases. The underlying mechanisms of O2 sensing remain unclear. Mitochondria have been implicated in many hypoxia-inducible factor (HIF) -dependent and -independent hypoxic responses. However, the role of mitochondria in mammalian cellular O2 sensing has remained controversial, particularly regarding the use pharmacologic agents to effect hypoxic HIFα stabilization, which has produced conflicting data in the literature. Using murine embryonic cells lacking cytochrome c, we show that mitochondrial reactive O2 species (ROS) are essential for O2 sensing and subsequent HIFα stabilization at 1.5% O2. In the absence of this signal, HIFα subunits continue to be hydroxylated and degraded via the proteasome. Importantly, exogenous treatment with H2O2 and severe O2 deprivation is sufficient to stabilize HIFα even in the absence of functional mitochondrial. These results demonstrate that mitochondria function as O2 sensors and signal hypoxic HIFα stabilization by releasing ROS to the cytoplasm. The cytochrome c mutant embryonic cells provide a unique reagent to further dissect the role of mitochondria in O2 mediated-intracellular events.

Farivar A.S., McCourtie A.S., MacKinnon-Patterson B.C., Woolley S.M., Barnes A.D., Chen M., Jagtap P., Szabó C., Salerno C.T., Mulligan M.S.

Heart transplantation is an accepted treatment modality for end-stage heart failure. However, acute cellular rejection (ACR) continues to be a morbid complication. Recently a novel mechanism of inflammatory allograft injury has been characterized which involves overactivation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). In the present studies, we compared the efficacy of INO-1001, a novel, potent PARP inhibitor, in limiting ACR with and without adjuvant low-dose cyclosporine (CSA).Heterotopic heart transplantation was performed utilizing Brown-Norway strains as donors and Lewis rats as recipients. Groups received daily intraperitoneal injections of: vehicle, low-dose CSA, low-dose INO-1001, high-dose INO-1001, and low-dose CSA combined with high-dose INO-1001. Additional animals were sacrificed on postoperative Day 5 for histologic assessments of allograft inflammation, including immunohistochemistry for nitrotyrosine and poly (ADP-ribose) (the product of PARP) staining.PARP inhibition significantly prolonged allograft survival relative to vehicle controls. The combination of low-dose CSA and INO-1001 resulted in a marked increase in allograft survival and significant reductions in allograft rejection scores. This was associated with decreased nitrotyrosine and PAR staining in transplanted cardiac allografts.Pharmacologic inhibition of INO-1001 prolongs allograft survival in a dose-dependent fashion in a rodent model of heart transplantation. PARP inhibitors may permit reductions in the dose of CSA needed for adequate immunosuppression after heart transplantation.

Thiel M., Chouker A., Ohta A., Jackson E., Caldwell C., Smith P., Lukashev D., Bittmann I., Sitkovsky M.V.

Acute respiratory distress syndrome (ARDS) usually requires symptomatic supportive therapy by intubation and mechanical ventilation with the supplemental use of high oxygen concentrations. Although oxygen therapy represents a life-saving measure, the recent discovery of a critical tissue-protecting mechanism predicts that administration of oxygen to ARDS patients with uncontrolled pulmonary inflammation also may have dangerous side effects. Oxygenation may weaken the local tissue hypoxia-driven and adenosine A2A receptor (A2AR)-mediated anti-inflammatory mechanism and thereby further exacerbate lung injury. Here we report experiments with wild-type and adenosine A2AR-deficient mice that confirm the predicted effects of oxygen. These results also suggest the possibility of iatrogenic exacerbation of acute lung injury upon oxygen administration due to the oxygenation-associated elimination of A2AR-mediated lung tissue-protecting pathway. We show that this potential complication of clinically widely used oxygenation procedures could be completely prevented by intratracheal injection of a selective A2AR agonist to compensate for the oxygenation-related loss of the lung tissue-protecting endogenous adenosine. The identification of a major iatrogenic complication of oxygen therapy in conditions of acute lung inflammation attracts attention to the need for clinical and epidemiological studies of ARDS patients who require oxygen therapy. It is proposed that oxygen therapy in patients with ARDS and other causes of lung inflammation should be combined with anti-inflammatory measures, e.g., with inhalative application of A2AR agonists. The reported observations may also answer the long-standing question as to why the lungs are the most susceptible to inflammatory injury and why lung failure usually precedes multiple organ failure.

Cipriani G., Rapizzi E., Vannacci A., Rizzuto R., Moroni F., Chiarugi A.

To obtain further information on time course and mechanisms of cell death after poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, we used HeLa cells exposed for 1 h to the DNA alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine. This treatment activated PARP-1 and caused a rapid drop of cellular NAD(H) and ATP contents, culminating 8–12 h later in cell death. PARP-1 antagonists fully prevented nucleotide depletion and death. Interestingly, in the early 60 min after challenge with N-methyl-N′-nitro-N-nitrosoguanidine, mitochondrial membrane potential and superoxide production significantly increased, whereas cellular ADP contents decreased. Again, these events were prevented by PARP-1 inhibitors, suggesting that PARP-1 hyperactivity leads to mitochondrial state 4 respiration. Mitochondrial membrane potential collapsed at later time points (3 h), when mitochondria released apoptosis-inducing factor and cytochrome c. Using immunocytochemistry and targeted luciferase transfection, we found that, despite an exclusive localization of PARP-1 and poly(ADP-ribose) in the nucleus, ATP levels first decreased in mitochondria and then in the cytoplasm of cells undergoing PARP-1 activation. PARP-1 inhibitors rescued ATP (but not NAD(H) levels) in cells undergoing hyper-poly(ADP-ribosyl)ation. Glycolysis played a central role in the energy recovery, whereas mitochondria consumed ATP in the early recovery phase and produced ATP in the late phase after PARP-1 inhibition, further indicating that nuclear poly(ADP-ribosyl)ation rapidly modulates mitochondrial functioning. Together, our data provide evidence for rapid nucleus-mitochondria cross-talk during hyper-poly(ADP-ribosyl)ation-dependent cell death.

Hoyos B., Jiang S., Hammerling U.

Redox activations of serine/threonine kinases represent alternate pathways in which vitamin A plays a crucial co-factor role. Vitamin A binds the zinc finger domain of c-Raf with nanomolar affinity. The retinoid-binding site has been mapped within this structure by scanning mutagenesis. The deduced contact sites were found anchored on Phe-8, counting from the 1st conserved histidine of the zinc finger. These sites agreed with contact amino acids identified by computational docking. The boundaries of a related binding pocket were identified by mutagenesis and partially confirmed by docking trials in the protein kinase C-α C1A zinc finger. They comprised Phe-7, Phe-8, and Trp-22. This trio was absent from the αC1B domain, explaining why the latter did not bind retinol. Reconfiguring at a minimum the two corresponding amino acids of αC1B, Thr-7 and Tyr-22, to conform to αC1A converted this domain to a binder. Deletion of the predicted retinoid-binding site in the full-length molecule created a mutant c-Raf that was deficient in retinol-dependent redox activation but fully responsive to epidermal growth factor. Our findings indicate that ligation of retinol to a specific site embedded in the regulatory domain is an important feature of c-Raf regulation in the redox pathway.

Hagiu B.

Lim S.M., Ng Y.L., Majeed A.B., Tan M.P., Khor H.M., Kamaruzzaman S.B., Ramasamy K.

The present study explored for the first time the blood-based proteomic signature that could potentially distinguish older adults with and without cognitive frailty (CF). The participants were recruited under the Malaysian Elders Longitudinal Research (MELoR) study. Cognition and physical frailty were determined using the Montreal Cognitive Assessment (MoCA) and Fried’s criteria, respectively. The differential protein expression in the blood samples (38 CF vs 40 robust) were then determined using the Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH) analysis. A total of 294 proteins were found to be differentially expressed in the CF group as opposed to the robust group. Considering proteins with fold change (FC) ≥  ± 2 and p-values < 0.05, 13 proteins were significantly upregulated and nine proteins significantly downregulated in the CF group when compared to the robust group. Subsequent correlation analysis identified nine dysregulated proteins, namely APOA1, APOA2, APOA4, APOC1, APOE, GPX3, RBP4, SERPINC1 and TTR, to exhibit significantly and moderately strong correlations with parameters of cognitive and/or frailty assessments. These proteins could potentially serve as useful proteomic signature of CF given their sensitivity > 78%, specificity > 75%, accuracy > 80% and area under the curve (AUC) > 0.8. The major biological pathways that could be potentially dysregulated by the nine proteins were associated with lipid metabolism and the retinoid system. The present findings warrant further validation in future studies that involve a larger cohort.

Roy S., Hore M., Bhattacharyya S.

The delicate balance between oxidants and antioxidants is a dynamic process, and when it hampers, oxidative stress occurs. Oxidative stress is now suggested to have a direct correlation with a viral infection, which in turn induces several oxidants like nitric oxide radicals, superoxide anions, hydroxyl radicals and their by-products (viz. hydrogen peroxide). All of these oxidants and their by-products contribute to viral pathogenesis and ultimately cause infectious diseases. The consequences of viral diseases account for considerable economic loss worldwide. In response to this, the scientific fraternity throughout the world is investigating the basic mechanisms underlying such diseases, as well as identifying novel therapeutic strategies for the prevention and treatment of such maladies. Over the last few decades, scientists oriented their research aims mostly towards elucidating the immunological basis of viral replication and pathogenesis, but a little is written about the implications of such research for drug development, which provides the impetus behind the creation of the present chapter enabling the readers to have a comprehensive overview on the involvement of free radicals in viral diseases along with latest updates towards developing novel therapeutic strategies against these diseases. The present chapter summarizes the relationship between oxidative stress, viral infection, and a variety of therapeutic strategies conferred by antioxidants. Antiviral therapeutic strategies based on antioxidants are considered to be a promising area of research against viral infections.

Harshithkumar R., Shah P., Jadaun P., Mukherjee A.

Reactive oxygen species (ROS) are widely regarded as signaling molecules and play essential roles in various cellular processes, but when present in excess, they can lead to oxidative stress (OS). Growing evidence suggests that the OS plays a critical role in the pathogenesis of HIV infection and is associated with several comorbidities in HIV-infected individuals. ROS, generated both naturally during mitochondrial oxidative metabolism and as a response to various cellular processes, can trigger host antiviral responses but can also promote viral replication. While the multifaceted roles of ROS in HIV pathophysiology clearly need more investigation, this review paper unravels the mechanisms of OS generation in the context of HIV infections, offering insights into HIV viral protein-mediated and antiretroviral therapy-generated OS. Though the viral protein Tat is significantly attributed to the endogenous cellular increase in ROS post HIV infection, this paper sums up the contribution of other viral proteins in HIV-mediated elicitation of ROS. Given the investigations recognizing the significant role of ROS in the onset and progression of diverse pathologies, the paper also explores the critical function of ROS in the mediation of an of array of pathologies associated with HIV infection and retroviral therapy. HIV patients are observed with disruption to the antioxidant defense system, the antioxidant therapy is gaining focus as a potential therapeutic intervention and is well discussed. While ROS play a significant role in the HIV scenario, further exploratory studies are imperative to identifying alternative therapeutic strategies that could mitigate the toxicities and pathologies associated with ART-induced OS.

Li P., Xu J., Guo Y., Ma X., Zhou S., Zhang C., Yu H., Wang Y., Wang X., Yuan L.

Recent studies indicated that vitamin A (VA) might be involved in the pathology of type 2 diabetes mellitus (T2DM). This cross-sectional study was conducted to explore the association between circulating VA level and T2DM. A total of 1818 subjects aged 50-year-old and above were recruited from the community. Binomial logistic regression and restricted cubic spline (RCS) were applied to analyze the association of plasma VA level with the risk of T2DM. Serum VA and lipid-adjusted VA levels of T2DM patients were significantly higher than that of non-T2DM subjects (P < 0.05). The ratios of plasma VA/TC, VA/HDL-c and VA/LDL-c were positively associated with the risk of T2DM in the aging population (P < 0.05). Compared with the Q1 level, subjects with Q2 to Q3 levels of plasma VA/TG have decreased risk of T2DM (ORQ2 = 0.68, PQ2 = 0.021; ORQ3 = 0.59, PQ3 < 0.01). Our results indicated that the imbalance of circulating lipids and VA might affect the relationship between VA and T2DM. The middle and aging subjects with higher ratios of plasma VA/TC, VA/HDL-c, and VA/LDL-c displayed increased risk for T2DM, but the moderate ratio of VA/TG might protect against risk of T2DM.

Brandis A., Roy D., Das I., Sheves M., Eisenbach M.

AbstractIn recent years it became apparent that, in mammals, rhodopsin and other opsins, known to act as photosensors in the visual system, are also present in spermatozoa, where they function as highly sensitive thermosensors for thermotaxis. The intriguing question how a well-conserved protein functions as a photosensor in one type of cells and as a thermosensor in another type of cells is unresolved. Since the moiety that confers photosensitivity on opsins is the chromophore retinal, we examined whether retinal is substituted in spermatozoa with a thermosensitive molecule. We found by both functional assays and mass spectrometry that retinal is present in spermatozoa and required for thermotaxis. Thus, starvation of mice for vitamin A (a precursor of retinal) resulted in loss of sperm thermotaxis, without affecting motility and the physiological state of the spermatozoa. Thermotaxis was restored after replenishment of vitamin A. Using reversed-phase ultra-performance liquid chromatography mass spectrometry, we detected the presence of retinal in extracts of mouse and human spermatozoa. By employing UltraPerformance convergence chromatography, we identified a unique retinal isomer in the sperm extracts—tri-cis retinal, different from the photosensitive 11-cis isomer in the visual system. The facts (a) that opsins are thermosensors for sperm thermotaxis, (b) that retinal is essential for thermotaxis, and (c) that tri-cis retinal isomer uniquely resides in spermatozoa and is relatively thermally unstable, suggest that tri-cis retinal is involved in the thermosensing activity of spermatozoa.

Huang Y., Huang D., Klochkov V., Chan C., Chen Y., Lin W.

The activation of microglia and the production of cytokines are key factors contributing to progressive neurodegeneration. Despite the well-recognized neuronal programmed cell death regulated by microglial activation, the death of microglia themselves is less investigated. Nucleotide-binding oligomerization domain, leucine-rich repeat-containing X1 (NLRX1) functions as a scaffolding protein and is involved in various central nervous system diseases. In this study, we used the SM826 microglial cells to understand the role of NLRX1 in lipopolysaccharide (LPS)-induced cell death. We found LPS-induced cell death is blocked by necrostatin-1 and zVAD. Meanwhile, LPS can activate poly (ADP-ribose) polymerase-1 (PARP-1) to reduce DNA damage and induce heme oxygenase (HO)-1 expression to counteract cell death. NLRX1 silencing and PARP-1 inhibition by olaparib enhance LPS-induced SM826 microglial cell death in an additive manner. Less PARylation and higher DNA damage are observed in NLRX1-silencing cells. Moreover, LPS-induced HO-1 gene and protein expression through the p62-Keap1-Nrf2 axis are attenuated by NLRX1 silencing. In addition, the Nrf2-mediated positive feedback regulation of p62 is accordingly reduced by NLRX1 silencing. Of note, NLRX1 silencing does not affect LPS-induced cellular reactive oxygen species (ROS) production but increases mixed lineage kinase domain-like pseudokinase (MLKL) activation and cell necroptosis. In addition, NLRX1 silencing blocks bafilomycin A1-induced PARP-1 activation. Taken together, for the first time, we demonstrate the role of NLRX1 in protecting microglia from LPS-induced cell death. The underlying protective mechanisms of NLRX1 include upregulating LPS-induced HO-1 expression via Nrf2-dependent p62 expression and downstream Keap1-Nrf2 axis, mediating PARP-1 activation for DNA repair via ROS- and autophagy-independent pathway, and reducing MLKL activation.

Kim S.H., Kim J.H., Choi Y.M., Seo S.M., Jang E.Y., Lee S.J., Cho S., Jeong D.H., Lee K.H.

AbstractBackgroundCurrent methods for evaluating efficacy of cosmetics have limitations because they cannot accurately measure changes in the dermis. Skin sampling using microneedles allows identification of skin‐type biomarkers, monitoring treatment for skin inflammatory diseases, and evaluating efficacy of anti‐aging and anti‐pigmentation products.Materials and methodsTwo studies were conducted: First, 20 participants received anti‐aging treatment; second, 20 participants received anti‐pigmentation treatment. Non‐invasive devices measured skin aging (using high‐resolution 3D‐imaging in the anti‐aging study) or pigmentation (using spectrophotometry in the anti‐pigmentation study) at weeks 0 and 4, and adverse skin reactions were monitored. Skin samples were collected with biocompatible microneedle patches. Changes in expression of biomarkers for skin aging and pigmentation were analyzed using qRT‐PCR.ResultsNo adverse events were reported. In the anti‐aging study, after 4 weeks, skin roughness significantly improved in 17 out of 20 participants. qRT‐PCR showed significantly increased expression of skin‐aging related biomarkers: PINK1 in 16/20 participants, COL1A1 in 17/20 participants, and MSN in 16/20 participants. In the anti‐pigmentation study, after 4 weeks, skin lightness significantly improved in 16/20 participants. qRT‐PCR showed significantly increased expression of skin‐pigmentation‐related biomarkers: SOD1 in 15/20 participants and Vitamin D Receptor (VDR) in 15/20 participants. No significant change in TFAP2A was observed.ConclusionSkin sampling and mRNA analysis for biomarkers provides a novel, objective, quantitative method for measuring changes in the dermis and evaluating the efficacy of cosmetics. This approach complements existing evaluation methods and has potential application in assessing the effectiveness of medical devices, medications, cosmeceuticals, healthy foods, and beauty devices.

Qiao L., Dou X., Song X., Chang J., Yi H., Xu C.

Post-weaning diarrhea (PWD) in piglets poses a significant challenge and presents a grave threat to the global swine industry, resulting in considerable financial losses and compromising the welfare of animals. PWD is commonly associated with gut homeostatic imbalance, including oxidative stress, excessive inflammation, and microbiota dysbiosis. Antibiotic use has historically been a common initiative to combat PWD, but concerns about the development of antibiotic resistance have led to increased interest in alternative strategies. Mitochondria are key players in maintaining cellular homeostasis, and their dysfunction is intricately linked to the onset and progression of PWD. Accumulating evidence suggests that targeting mitochondrial function using antioxidant nutrients, such as vitamins, minerals and polyphenolic compounds, may represent a promising approach for preventing and treating PWD. Moreover, nutrients based on antioxidant strategies have been shown to improve mitochondrial function, restore intestinal redox balance, and reduce oxidative damage, which is a key driver of PWD. The present review begins with an overview of the potential interplay between mitochondria and gut homeostasis in the pathogenesis of PWD in piglets. Subsequently, alternative strategies to prevent and treat PWD using antioxidant nutrients to target mitochondria are described and discussed. Ultimately, we delve into potential limitations and suggest future research directions in this field for further advancement. Overall, targeting mitochondria using antioxidant nutrients may be a promising approach to combat PWD and provides a potential nutrition intervention strategy for regulating gut homeostasis of weaned piglets.

Hoffman S.S., Liang D., Hood R.B., Tan Y., Terrell M.L., Marder M.E., Barton H., Pearson M.A., Walker D.I., Barr D.B., Jones D.P., Marcus M.

Shastak Y., Pelletier W.

Jakaria M., Belaidi A.A., Bush A.I., Ayton S.

Vitamin A (retinol) is a lipid-soluble vitamin that acts as a precursor for several bioactive compounds, such as retinaldehyde (retinal) and isomers of retinoic acid. Retinol and all-trans-retinoic acid (atRA) penetrate the blood-brain barrier and are reported to be neuroprotective in several animal models. We characterised the impact of retinol and its metabolites, all-trans-retinal (atRAL) and atRA, on ferroptosis-a programmed cell death caused by iron-dependent phospholipid peroxidation. Ferroptosis was induced by erastin, buthionine sulfoximine or RSL3 in neuronal and non-neuronal cell lines. We found that retinol, atRAL and atRA inhibited ferroptosis with a potency superior to α-tocopherol, the canonical anti-ferroptotic vitamin. In contrast, we found that antagonism of endogenous retinol with anhydroretinol sensitises ferroptosis induced in neuronal and non-neuronal cell lines. Retinol and its metabolites atRAL and atRA directly interdict lipid radicals in ferroptosis since these compounds displayed radical trapping properties in a cell-free assay. Vitamin A, therefore, complements other anti-ferroptotic vitamins, E and K; metabolites of vitamin A, or agents that alter their levels, may be potential therapeutics for diseases where ferroptosis is implicated.

Zhou L., Ma Z., Gao X.

Parkinson's disease (PD), the second-most prevalent neurodegenerative disorder, is characterized by the aberrant deposition of α-synuclein (α-Syn) aggregation in neurons. Recent reports have shown that retinoic acid (RA) ameliorates motor deficits. However, the underlying molecular mechanisms remain unclear. In this article, we investigated the effects of RA on cellular and animal models of PD. We found that RA is beneficial for neuronal survival in PD-associated models. In α-Syn preformed fibrils-treated mice, RA administration relieved the formation of intracellular inclusions, dopaminergic neuronal loss, and behavioral deficits. α-Syn preformed fibrils-treated SH-SY5Y cells manifested decreased cell viability, apoptosis, α-Syn aggregation, and autophagy defects. All these negative phenomena were alleviated by RA. More importantly, RA could inhibit the neurotoxicity via inhibiting α-Syn preformed fibrils-induced STAT1-PARP1 signaling, which could also be antagonized by IFN-γ. In conclusion, RA could hinder α-Syn preformed fibrils-induced toxicity by inhibiting STAT1-PARP1 signaling. Thus, we present new insight into RA in PD management.

Hammerling U., Kim Y., Quadro L.

AbstractThis Perspective discusses how retinol catalyzes resonance energy transfer (RET) reactions pivotally important for mitochondrial energy homeostasis by protein kinase C δ (PKCδ). PKCδ signals to the pyruvate dehydrogenase complex, controlling oxidative phosphorylation. The PKCδ-retinol complex reversibly responds to the redox potential of cytochrome c, that changes with the electron transfer chain workload. In contrast, the natural retinoid anhydroretinol irreversibly activates PKCδ. Its elongated conjugated-double-bond system limits the energy quantum absorbed by RET. Consequently, while capable of triggering the exergonic activating pathway, anhydroretinol fails to activate the endergonic silencing path, trapping PKCδ in the ON position and causing harmful levels of reactive oxygen species. However, physiological retinol levels displace anhydroretinol, buffer cyotoxicity and potentially render anhydroretinol useful for rapid energy generation. Intriguingly, apocarotenoids, the primary products of the mitochondrial β-carotene,9'-10'-oxygenase, have all the anhydroretinol-like features, including modulation of energy homeostasis. We predict significant conceptual advances to stem from further understanding of the retinoid-catalyzed RET.

Brown G.

All-trans retinoic acid is a morphogen during embryogenesis and a teratogen. Cancer is an error of development, and the retinoic acid receptors (RAR) for all-trans retinoic acid play a role in cancer. Expression of the cytosolic aldehyde dehydrogenases, which mediate the last step to the synthesis of all-trans retinoic acid, is deregulated in various human cancers. Inhibiting these enzymes using a variety of agents reduced the proliferation of lung cancer cells, reduced the proliferation and induced apoptosis of ovarian, prostate, squamous, and uterine cancer cells, and sensitised breast, colorectal and ovarian cancer cells to chemotherapeutic agents. RARγ is an oncogene within some cases of AML, cholangiocarcinoma, colorectal cancer, clear cell renal cell carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, prostate cancer, and ovarian cancer. Pan-RAR and RARγ antagonist inhibition of the action of RARγ led to necroptosis of human prostate and pediatric brain tumour cancer stem cells. Treatment of hepatocellular carcinoma cells with the flavenoid acacetin, which interferes with the action of RARγ, decreased cell growth and induced apoptosis. Targeting the retinoic acid pathway is promising regarding the development of new drugs to eradicate cancer stem cells.