Regulation Of Bioenergetics Research Articles

Melatonin prevents blood-retinal barrier breakdown and mitochondrial dysfunction in high glucose and hypoxia-induced in vitro diabetic macular edema model

Diabetic macular edema (DME) is a leading cause of blindness in diabetic retinopathy. Prolonged hyperglycemia plus hypoxia contributes to DME pathogenesis. Retinal pigmented epithelial cells comprise the outer blood-retinal barrier and are essential for maintaining physiological functioning of the retina. Melatonin acts as an antioxidant and regulator of mitochondrial bioenergetics and has a protective effect against ocular diseases. However, the role of mitochondrial dysfunction and the therapeutic potential of melatonin in DME remain largely unexplored. Here, we used an in vitro model of DME to investigate blood-retinal barrier integrity and permeability, angiogenesis, mitochondrial dynamics, and apoptosis signaling to evaluate the potential protective efficacy of melatonin in DME. We found that melatonin prevents cell hyper-permeability and outer barrier breakdown by reducing HIF-1α, HIF-1β and VEGF and VEGF receptor gene expression. In addition, melatonin reduced the expression of genes involved in mitochondrial fission (DRP1, hFis1, MIEF2, MFF), mitophagy (PINK, BNip3, NIX), and increased the expression of genes involved in mitochondrial biogenesis (PGC-1α, NRF2, PPAR-γ) to maintain mitochondrial homeostasis. Moreover, melatonin prevented apoptosis of retinal pigmented epithelial cells. Our results suggest that mitochondrial dysfunction may be involved in DME pathology, and melatonin may have therapeutic value in DME, by targeting signaling in mitochondria.

HIF1\u03b1-dependent induction of the mitochondrial chaperone TRAP1 regulates bioenergetic adaptations to hypoxia

The mitochondrial paralog of the Hsp90 chaperone family TRAP1 is often induced in tumors, but the mechanisms controlling its expression, as well as its physiological functions remain poorly understood. Here, we find that TRAP1 is highly expressed in the early stages of Zebrafish development, and its ablation delays embryogenesis while increasing mitochondrial respiration of fish larvae. TRAP1 expression is enhanced by hypoxic conditions both in developing embryos and in cancer models of Zebrafish and mammals. The TRAP1 promoter contains evolutionary conserved hypoxic responsive elements, and HIF1α stabilization increases TRAP1 levels. TRAP1 inhibition by selective compounds or by genetic knock-out maintains a high level of respiration in Zebrafish embryos after exposure to hypoxia. Our data identify TRAP1 as a primary regulator of mitochondrial bioenergetics in highly proliferating cells following reduction in oxygen tension and HIF1α stabilization.

ContactJ: Lipid droplets-mitochondria contacts characterization through fluorescence microscopy and image analysis.

Lipid droplets (LDs) are the major lipid storage organelles of eukaryotic cells and together with mitochondria key regulators of cell bioenergetics. LDs communicate with mitochondria and other organelles forming "metabolic synapse" contacts to ensure that lipid supply occurs where and when necessary. Although transmission electron microscopyanalysis allows an accurate and precise analysis of contacts, the characterization ofa large number ofcells and conditions can become a long-term process.In order to extend contact analysis tohundredsof cellsand multiple conditions,we have combinedconfocalfluorescence microscopy with advanced image analysis methods.In this work, wehave developed the ImageJ macro script ContactJ, a novel and straight image analysis method to identify and quantify contacts between LD and mitochondria in fluorescence microscopy images allowing the automatic analysis. This image analysis workflow combines colocalization and skeletonization methods, enabling the quantification of LD-mitochondria contacts together with a complete characterization of organelles and cellular parameters. The correlation and normalization of these parameters contribute to the complex description of cell behavior under differentexperimentalenergetic states. ContactJ is available here: https://github.com/UB-BioMedMicroscopy/ContactJ/tree/1.0.

A28 CHARACTERIZING THE ROLE OF NR4A1 IN THE REGULATION OF INTESTINAL SMOOTH MUSCLE CELL PHENOTYPE AND FUNCTION

Abstract Background Intestinal fibrosis and stricture formation are common complications of Crohn’s disease (CD). Recently, smooth muscle hypertrophy/hyperplasia have gained greater recognition as a driver of stricture formation, rather than an increase in fibrosis alone. Despite advances in treatment of CD, current therapies do little to prevent or reverse strictures. NR4A1 is an orphan nuclear receptor that is anti-fibrotic in non-intestinal systems and exhibits anti-proliferative effects in smooth muscle cells (SMCs). NR4A1 gene variants have been associated with increased risk of IBD, however, mechanisms regulating NR4A1 expression and its role in intestinal SMC function have not been investigated. Aims To characterize the role of NR4A1 presence and activation in modulating intestinal SMC phenotype. Methods Primary intestinal SMCs were isolated from Nr4a1+/+and Nr4a1-/-mice. A commercially sourced human primary intestinal SMC line was also used. Mass spectrometry identified proteomic differences between Nr4a1+/+and Nr4a1-/-SMCs. To assess the role of NR4A1 in regulating SMC growth, basal and platelet-derived growth factor-BB (PDGF-BB)-induced proliferation were quantified. NR4A1 activation was induced by selective agonists, cytosporone B (Csn-B) and 6-mercaptopurine (6-MP). Cellular respirometry was used to determine metabolism in Nr4a1+/+and Nr4a1-/-SMCs. Expression of NR4A1 and phenotypic switching mediators were assessed by qPCR. Immunofluorescence was used to assess contractile markers and cell morphology. Results Proteomic analysis revealed increased expression of proteins related to the cell cycle, metabolism, and extracellular matrix synthesis in Nr4a1-/-SMCs. Nr4a1+/+SMCs upregulated proteins involved in smooth muscle contraction and was supported by higher mRNA levels of myocardin and lower Krüppel-like factor 4. Nr4a1-/-cells were more proliferative compared to Nr4a1+/+cells under basal conditions. Treating human intestinal SMCs with Csn-B attenuated proliferation induced by PDGF-BB. Similar effects were observed in Nr4a1+/+SMCs, however, the anti-proliferative effect of Csn-B was absent in Nr4a1-/-cells. Nr4a1-/-SMCs had higher maximal respiration, spare respiratory capacity, and glycolysis. NR4A1 expression was rapidly induced by Csn-B/6-MP and PDGF-BB, the latter suggesting a potential negative feedback mechanism to control mitogen-induced SMC proliferation. Conclusions Our results suggest that NR4A1 is a regulator of intestinal SMC proliferation, bioenergetics, and phenotype. Its induction by mitogens may contribute to a negative feedback loop to control smooth muscle growth. These data support targeting NR4A1 to treat excessive smooth muscle hypertrophy/hyperplasia that contributes to tissue remodelling observed in fibrostenotic CD. Funding Agencies CCC, CIHR

Native Gel Electrophoresis and Immunoblotting to Analyze Electron Transport Chain Complexes.

Native electrophoresis is a powerful tool to analyze the mitochondrial electron transport chain complexes (Cx) I-V and their assembly into supercomplexes. Valuable information regarding the composition and bioenergetic regulation in physiological and pathological conditions can be obtained. This chapter compares different types of native electrophoresis to analyze mitochondrial supercomplexes.

Regulation of neuronal bioenergetics as a therapeutic strategy in neurodegenerative diseases.

Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis are a heterogeneous group of debilitating disorders with multifactorial etiologies and pathogeneses that manifest distinct molecular mechanisms and clinical manifestations with abnormal protein dynamics and impaired bioenergetics. Mitochondrial dysfunction is emerging as an important feature in the etiopathogenesis of these age-related neurodegenerative diseases. The prevalence and incidence of these diseases is on the rise with the increasing global population and average lifespan. Although many therapeutic approaches have been tested, there are currently no effective treatment routes for the prevention or cure of these diseases. We present the current status of our knowledge and understanding of the involvement of mitochondrial dysfunction in these diseases and highlight recent advances in novel therapeutic strategies targeting neuronal bioenergetics as potential approach for treating these diseases.

Mitophagy: A New Player in Stem Cell Biology.

Simple SummaryStem cells are required to create all organs and tissues during development, as well as replace or regenerate tissues in adulthood. Stem cells are characterized by two main factors, which are (1) the ability to replenish themselves in order to maintain their population for further use, and (2) the ability to differentiate into specialized cells. These two characteristics are regulated by both external and internal factors. One of these internal factors is mitochondrial function. Mitochondria are organelles that serve an essential role to cells by providing energy and regulating cell survival. These organelles are now known to be important for allowing the stem cell characteristics. Given that proper mitochondrial function is crucial for cells, when they become defective they need to be removed. This process of removal, known as mitophagy or “mitochondrial eating”, is emerging as an important player in stem cells. In this review we discuss the new research that shows the importance of mitophagy in having functional stem cells.The fundamental importance of functional mitochondria in the survival of most eukaryotic cells, through regulation of bioenergetics, cell death, calcium dynamics and reactive oxygen species (ROS) generation, is undisputed. However, with new avenues of research in stem cell biology these organelles have now emerged as signaling entities, actively involved in many aspects of stem cell functions, including self-renewal, commitment and differentiation. With this recent knowledge, it becomes evident that regulatory pathways that would ensure the maintenance of mitochondria with state-specific characteristics and the selective removal of organelles with sub-optimal functions must play a pivotal role in stem cells. As such, mitophagy, as an essential mitochondrial quality control mechanism, is beginning to gain appreciation within the stem cell field. Here we review and discuss recent advances in our knowledge pertaining to the roles of mitophagy in stem cell functions and the potential contributions of this specific quality control process on to the progression of aging and diseases.

Involvement of Sigma Receptor-1 in Lymphatic Endothelial Barrier Integrity and Bioenergetic Regulation.

Background: Lymphatic endothelium plays significant roles in lymph transport and maintaining a barrier between the lymph and interstitial compartments. Lymphatic endothelial dysfunction is suspected to be a key factor in the pathogenesis of lymphatic diseases such as lymphedema. Sigma receptor-1 (σ1) was recently identified to promote endothelial-dependent production of nitric oxide and relaxation of collecting lymphatic vessels. In this study, we investigated the potential role of σ1 in lymphatic endothelial barrier function. Methods and Results: Cultured adult human dermal lymphatic endothelial cells (HDLEC) were grown into confluent monolayers. Transendothelial electrical resistance (TER) served as an index of barrier function. Glycolytic rate of HDLEC was determined with the Agilent Seahorse system. The σ1-selective agonist PRE-084 was used to test the impact of σ1 on HDLEC monolayer barrier function and endothelial bioenergetics, whereas the contribution of basal σ1 activity was assessed with small interfering RNA (siRNA)-mediated knockdown of σ1 expression. The ability of σ1 activation to counteract interleukin (IL)-1β-induced barrier dysfunction was also tested. The results show that PRE-084 increases HDLEC TER in a concentration-dependent manner, whereas reducing σ1 expression with siRNA decreases HDLEC TER. PRE-084 also enhances glycolytic rate parameters in HDLEC. Moreover, PRE-084 treatment partially counteracts IL-1β-induced HDLEC monolayer barrier dysfunction. Conclusions: Collectively, the results suggest that σ1 contributes to basal lymphatic endothelial barrier function, potentially through its ability to enhance glycolytic energy production. Our work also highlights the therapeutic potential of σ1 agonists for preventing lymphatic barrier dysfunction caused by inflammatory mediators.

Protein S-glutathionylation Reactions in the Regulation of Mitochondrial Bioenergetics

Protein S-glutathionylation Reactions in the Regulation of Mitochondrial Bioenergetics

Anticancer Effects of Honokiol via Mitochondrial Dysfunction Are Strongly Enhanced by the Mitochondria-Targeting Carrier Berberine.

Mitochondrion is a favorable therapeutic target in cancer, given its regulation of bioenergetics and cell death. Honokiol exhibits antiproliferative effects through mitochondria-mediated death signaling. To enhance its anticancer potential and selectivity, we conjugated honokiol to berberine, a mitochondria-targeting carrier. All designed derivatives displayed 1 order of magnitude increased cytotoxicity compared with the parent compounds, especially with massive cytoplasmic vacuoles. Biological evaluation demonstrated the representative compound 6b localized within the mitochondria, and mitochondrial dilation resulted in vacuolization. 6b induced vacuolation-associated cell death and apoptosis with obvious mitochondrial dysfunction, as demonstrated by booming reactive oxygen species generation, opening mitochondrial permeability transition pore, and reducing mitochondrial membrane potential. The targeting property also conferred 6b with selectivity for tumor cells compared to normal cells. 6b inhibited cancer cell proliferation in the zebrafish xenograft model. These results demonstrate that berberine-linked honokiol derivatives open up a direction for novel mitochondrial-targeting antitumor agents.

Lithium and the Interplay Between Telomeres and Mitochondria in Bipolar Disorder.

Bipolar disorder is a severe psychiatric disorder which affects more than 1% of the world’s population and is a leading cause of disability among young people. For the past 50 years, lithium has been the drug of choice for maintenance treatment of bipolar disorder due to its potent ability to prevent both manic and depressive episodes as well as suicide. However, though lithium has been associated with a multitude of effects within different cellular pathways and biological systems, its specific mechanism of action in stabilizing mood remains largely elusive. Mitochondrial dysfunction and telomere shortening have been implicated in both the pathophysiology of bipolar disorder and as targets of lithium treatment. Interestingly, it has in recent years become clear that these phenomena are intimately linked, partly through reactive oxygen species signaling and the subcellular translocation and non-canonical actions of telomerase reverse transcriptase. In this review, we integrate the current understanding of mitochondrial dysfunction, oxidative stress and telomere shortening in bipolar disorder with documented effects of lithium. Moreover, we propose that lithium’s mechanism of action is intimately connected with the interdependent regulation of mitochondrial bioenergetics and telomere maintenance.

NRF-1 and HIF-1α contribute to modulation of human VDAC1 gene promoter during starvation and hypoxia in HeLa cells

VDAC (Voltage Dependent Anion Channel) is a family of pore forming protein located in the outer mitochondrial membrane. Its channel property ensures metabolites exchange between mitochondria and the rest of the cell resulting in metabolism and bioenergetics regulation, and in cell death and life switch. VDAC1 is the best characterized and most abundant isoform, and is involved in many pathologies, as cancer or neurodegenerative diseases. However, little information is available about its gene expression regulation in normal and/or pathological conditions. In this work, we explored VDAC1 gene expression regulation in normal conditions and in the contest of some metabolic and energetic mitochondrial dysfunction and cell stress as example. The core of the putative promoter region was characterized in terms of transcription factors responsive elements both by bioinformatic studies and promoter activity experiments. In particular, we found an abundant presence of NRF-1 sites, together with other transcription factors binding sites involved in cell growth, proliferation, development, and we studied their prevalence in gene activity. Furthermore, upon depletion of nutrients or controlled hypoxia, as detected in various pathologies, we found that VDAC1 transcripts levels were significantly increased in a time related manner. VDAC1 promoter activity was also validated by gene reporter assays. According to PCR real-time experiments, it was confirmed that VDAC1 promoter activity is further stimulated when cells are exposed to stress. A bioinformatic survey suggested HIF-1α, besides NRF-1, as a most active TFBS. Their validation was obtained by TFBS mutagenesis and TF overexpression experiments. In conclusion, we experimentally demonstrated the involvement of both NRF-1 and HIF-1α in the regulation of VDAC1 promoter activation at basal level and in some peculiar cell stress conditions.

Expression of voltage-dependent anion channels in endometrial cancer and its potential prognostic significance.

The exchange of metabolites between mitochondria and cytosol occurs through pores formed by voltage-dependent anion channel proteins. Voltage-dependent anion channels appear to be master regulators of mitochondrial bioenergetics and the intracellular flow of energy. Deregulation of voltage-dependent anion channels expression is thought to be related to mitochondrial dysfunction in cancer. The aim of this study was to investigate the mRNA and protein expression levels of VDAC1, VDAC2, and VDAC3 in relation to clinicopathological characteristics of endometrial cancer as well as the prognostic significance of voltage-dependent anion channels expression for overall survival. VDAC1 and VDAC3 expressions were significantly higher in cancer compared to normal tissues. Kaplan-Meier analysis indicated that high expression of all VDAC genes or high VDAC2 protein level predicted poor overall survival. Multivariate analysis identified the VDAC1 and VDAC2 mRNA levels as well as VDAC2 protein level as independent prognostic factors. Our results suggest that increased expression of voltage-dependent anion channels correlates with tumor progression and may serve as a potential prognostic biomarker in endometrial cancer.

Role of Mitochondria-Cytoskeleton Interactions in the Regulation of Mitochondrial Structure and Function in Cancer Stem Cells.

Despite the promise of cancer medicine, major challenges currently confronting the treatment of cancer patients include chemoresistance and recurrence. The existence of subpopulations of cancer cells, known as cancer stem cells (CSCs), contributes to the failure of cancer therapies and is associated with poor clinical outcomes. Of note, one of the recently characterized features of CSCs is augmented mitochondrial function. The cytoskeleton network is essential in regulating mitochondrial morphology and rearrangement, which are inextricably linked to its functions, such as oxidative phosphorylation (OXPHOS). The interaction between the cytoskeleton and mitochondria can enable CSCs to adapt to challenging conditions, such as a lack of energy sources, and to maintain their stemness. Cytoskeleton-mediated mitochondrial trafficking and relocating to the high energy requirement region are crucial steps in epithelial-to-mesenchymal transition (EMT). In addition, the cytoskeleton itself interplays with and blocks the voltage-dependent anion channel (VDAC) to directly regulate bioenergetics. In this review, we describe the regulation of cellular bioenergetics in CSCs, focusing on the cytoskeleton-mediated dynamic control of mitochondrial structure and function.

Berberine protects against diabetic kidney disease via promoting PGC-1α-regulated mitochondrial energy homeostasis.

Background and PurposeDisordered lipid metabolism and disturbed mitochondrial bioenergetics play pivotal roles in the initiation and development of diabetic kidney disease (DKD). Berberine is a plant alkaloid, used in Chinese herbal medicine. It has multiple therapeutic actions on diabetes mellitus and its complications, including regulation of glucose and lipid metabolism, improvement of insulin sensitivity, and alleviation of oxidative damage. Here, we investigated the reno‐protective effects of berberine.Experimental ApproachWe used samples from DKD patients and experiments with models of DKD (db/db mice) and cultured podocytes, to characterize energy metabolism profiles using metabolomics. Molecular targets and mechanisms involved in the regulation of mitochondrial function and bioenergetics by berberine were investigated, along with its effects on metabolic alterations in DKD mice.Key ResultsMetabolomic analysis suggested altered mitochondrial fuel usage and generalized mitochondrial dysfunction in patients with DKD. In db/db mice, berberine treatment reversed the disordered metabolism, podocyte damage and glomerulosclerosis. Lipid accumulation, excessive generation of mitochondrial ROS, mitochondrial dysfunction, and deficient fatty acid oxidation in DKD mouse models and in cultured podocytes were suppressed by berberine. These protective effects of berberine were accompanied by activation of the peroxisome proliferator‐activated receptor γ coactivator‐1α (PGC‐1α) signalling pathway, which promoted mitochondrial energy homeostasis and fatty acid oxidation in podocytes.Conclusion and ImplicationsPGC‐1α‐mediated mitochondrial bioenergetics could play a key role in lipid disorder‐induced podocyte damage and development of DKD in mice. Restoration of PGC‐1α activity and the energy homeostasis by berberine might be a potential therapeutic strategy against DKD.

Structural variability, coordination and adaptation of a native photosynthetic machinery.

Cyanobacterial thylakoid membranes represent the active sites for both photosynthetic and respiratory electron transport. We used high-resolution atomic force microscopy to visualize the native organization and interactions of photosynthetic complexes within the thylakoid membranes from the model cyanobacterium Synechococcus elongatus PCC 7942. The thylakoid membranes are heterogeneous and assemble photosynthetic complexes into functional domains to enhance their coordination and regulation. Under high light, the chlorophyll-binding proteins IsiA are strongly expressed and associate with Photosystem I (PSI), forming highly variable IsiA-PSI supercomplexes to increase the absorption cross-section of PSI. There are also tight interactions of PSI with Photosystem II (PSII), cytochrome b6f, ATP synthase and NAD(P)H dehydrogenase complexes. The organizational variability of these photosynthetic supercomplexes permits efficient linear and cyclic electron transport as well as bioenergetic regulation. Understanding the organizational landscape and environmental adaptation of cyanobacterial thylakoid membranes may help inform strategies for engineering efficient photosynthetic systems and photo-biofactories.

Test–retest variability and reference region-based quantification of 18F-BCPP-EF for imaging mitochondrial complex I in the human brain

Mitochondrial complex I (MC-I) is an essential regulator of brain bioenergetics and can be quantified in the brain using PET radioligand 18F-BCPP-EF. Here we evaluate the test–retest reproducibility of 18F-BCPP-EF in humans, and assess the use of a non-invasive quantification method (standardised uptake value ratio – SUVR). Thirty healthy volunteers had a 90-min dynamic 18F-BCPP-EF scan with arterial blood sampling, five of which received a second scan to be included in the test–retest analysis. Time-activity curves (TAC) were analysed using multilinear analysis 1 (MA1) and the two-tissue compartment model (2TC) to estimate volumes of distribution (VT). Regional SUVR-1 values were calculated from the 70 to 90-min TAC data using the centrum semiovale as a pseudo reference region, and compared to kinetic analysis-derived outcome measures. The mean absolute test–retest variability of VT ranged from 12% to 18% across regions. Both DVR-1and SUVR-1 had improved test–retest variability in the range 2%–7%. SUVR-1 was highly correlated with DVR-1 (r2 = 0.97, n = 30). In conclusion, 18F-BCPP-EF has suitable test–retest reproducibility and can be used to quantify MC-I in clinical studies.

322-OR: Impairment of Alpha-Cell Bioenergetics Contributes to Increased Basal Yet Decreased Glucose Stimulated Oxygen Consumption Rates of Isolated Human Pancreatic Islets in T1DM and T2DM

Fuel stimulated hormone release (HR) and the oxygen consumption rates (OCR) of isolated normal pancreatic islets are positively correlated illustrating that cellular energy state is a critical determinant of stimulus-secretion coupling. We investigated the relationship between hormone release and OCR in islets from donors with T1DM and T2DM, using unique oxygen measurement technology and optimized perifusion protocols. HR and OCR were quantified in 20 human donor islet preparations obtained from the Human Islet Resource Center at the University of Pennsylvania within 3-4 days after isolation (5 each of nondiabetic, T1DM, T2DM and anti-islet AutoAntiBody positive (AAB+). A physiological amino acid mixture (4mM) was used to stimulate glucagon release, then 3 and 16.7mM glucose were added to stimulate insulin release and inhibit glucagon release. Respiration was then uncoupled with FCCP and finally blocked by NaN3. Basal OCR was increased in islets from AAB+, T1DM and T2DM donors compared to controls. Low glucose failed to increase OCR in T1DM and T2DM islets while high glucose-stimulated OCR was both decreased and delayed. Glucose stimulated insulin release much less effectively and failed to suppress glucagon release in both T1DM and T2DM islets. AAB+ islets showed normal insulin release but lacked glucose suppression of glucagon release. The observation of increased basal-OCR of islets in all diabetes related conditions and reduced glucose-stimulated-OCR in T1DM and T2DM islets unrelated to hormone release rates is striking and indicates a profound alteration of bioenergetics regulation in these conditions hitherto unrecognized. Common to this pathophysiology are an increase in relative alpha-cell mass (T1DM and T2DM) or a functional defect of these cells (AAB+) providing a plausible explanation for this phenomenology and implying impaired alpha-cell energy metabolism. Disclosure N.M. Doliba: None. A.V. Rozo: None. W. Qin: None. J. Roman: None. C. Liu: None. A. Naji: None. K.H. Kaestner: None. D.A. Stoffers: None. F. Matschinsky: None. Funding National Institutes of Health (UC4DK112217)

Distinct regulation of bioenergetics and translation by group I mGluR and NMDAR.

Neuronal activity is responsible for the high energy consumption in the brain. However, the cellular mechanisms draining ATP upon the arrival of a stimulus are yet to be explored systematically at the post-synapse. Here, we provide evidence that a significant fraction of ATP is consumed upon glutamate stimulation to energize mGluR-induced protein synthesis. We find that both mGluR and NMDAR alter protein synthesis and ATP consumption with distinct kinetics at the synaptic-dendritic compartments. While mGluR activation leads to a rapid and sustained reduction in neuronal ATP levels, NMDAR activation has no immediate impact on the same. ATP consumption correlates inversely with the kinetics of protein synthesis for both receptors. We observe a persistent elevation in protein synthesis within 5minutes of mGluR activation and a robust inhibition of the same within 2minutes of NMDAR activation, assessed by the phosphorylation status of eEF2 and metabolic labeling. However, a delayed protein synthesis-dependent ATP expenditure ensues after 15minutes of NMDAR stimulation. We identify a central role for AMPK in the correlation between protein synthesis and ATP consumption. AMPK is dephosphorylated and inhibited upon mGluR activation, while it is phosphorylated upon NMDAR activation. Perturbing AMPK activity disrupts receptor-specific modulations of eEF2 phosphorylation and protein synthesis. Our observations, therefore, demonstrate that the regulation of the AMPK-eEF2 signaling axis by glutamate receptors alters neuronal protein synthesis and bioenergetics.

Role of 3-Mercaptopyruvate Sulfurtransferase in the Regulation of Proliferation and Cellular Bioenergetics in Human Down Syndrome Fibroblasts.

Down syndrome (trisomy of human chromosome 21) is a common genetic disorder. Overproduction of the gaseous mediator hydrogen sulfide (H2S) has been implicated in the pathogenesis of neurological and metabolic deficits associated with Down syndrome. Several lines of data indicate that an important enzyme responsible for H2S overproduction in Down syndrome is cystathionine-β-synthase (CBS), an enzyme localized on chromosome 21. The current study explored the possibility that a second H2S-producing enzyme, 3-mercaptopyruvate sulfurtransferase (3-MST), may also contribute to the development of functional deficits of Down syndrome cells. Western blotting analysis demonstrated a significantly higher level of 3-MST protein expression in human Down syndrome fibroblasts compared to cells from healthy control individuals; the excess 3-MST was mainly localized to the mitochondrial compartment. Pharmacological inhibition of 3-MST activity improved mitochondrial electron transport and oxidative phosphorylation parameters (but did not affect the suppressed glycolytic parameters) and enhanced cell proliferation in Down syndrome cells (but not in healthy control cells). The findings presented in the current report suggest that in addition to the indisputable role of CBS, H2S produced from 3-MST may also contribute to the development of mitochondrial metabolic and functional impairments in Down syndrome cells.