Inorganic Polyphosphate in Mitochondrial Energy Metabolism and Pathology.

G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

Gulf War Illness (GWI) corresponds to an array of symptoms that includes chronic fatigue, musculoskeletal pain, cognitive dysfunction, sleep disturbance, gastrointestinal, respiratory, and dermatological symptoms that affect ~250,000 U.S. military veterans that served in Operation Desert Storm/Desert Shield (1990‐1991). Mitochondrial function impairments have been shown in GWI patients. GWI patients report partial amelioration of chronic fatigue and brain fog after medicinal marijuana and CBD oils. Interestingly, cannabidiol (CBD) modulates mitochondrial physiology though this has not been characterized in detail. We hypothesize that GWI mitochondrial pathology can be recapitulated in dermal fibroblasts (DF) from subjects to help define and develop a cell‐based model to study GWI and CBD treatment of DF promotes energy production by improving mitochondrial physiology. GWI patients (gender/age matched to healthy controls) were recruited to collect skin punch biopsy explants that were processed and cultured in DMEM FBS 10% for 30‐days to obtain dermal fibroblasts. DF were treated with serial dilutions of Verséa™ CBD (50mg/mL) lipid formulation (VESIsorb® that increases 4.4‐fold Cmax). Using real time mitochondrial analysis by Seahorse, energy phenotype and mitochondrial function was analyzed in control and GWI DF. Mitochondrial networks and ultrastructure were studied by live‐imaging using MitoTracker™ and transmission electron microscopy, respectively. Energy phenotype studies suggest that GWI DF present with lower mitochondrial metabolism and higher glycolytic activity, compared with controls. Additionally, mitochondrial stress suggests a significant reduction in mitochondrial maximal capacity. Such data establish GWI derived DF as a personalized model system to study mitochondrial pathology in GWI. After 18h treatment with Verséa™ CBD, GWI DF show a significant improvement in mitochondrial and glycolytic metabolism; control patients show no increases in mitochondrial metabolism but improved glycolysis. Verséa™ CBD treatment induced mitochondrial networks re‐organization in DF. These findings suggest that CBD improves GWI DF mitochondrial physiology, thus improving energy homeostasis. Our data provide new evidence that will validate the potential of cannabinoids as a therapeutic strategy to mitigate energy imbalance that may contribute to detrimental symptomatology (i.e., chronic fatigue, brain fog, cognitive dysfunction, etc.) in GWI patients.

Balancing mitochondrial biogenesis and mitophagy to maintain energy metabolism homeostasis.

Osteosarcoma is the most common primary bone malignancy. The crosstalk between osteosarcoma and the surrounding tumour microenvironment (TME) drives key events that lead to metastasization, one of the main obstacles for definitive cure of most malignancies. Extracellular vesicles (EVs), lipid bilayer nanoparticles used by cells for intercellular communication, are emerging as critical biological mediators that permit the interplay between neoplasms and the tumour microenvironment, modulating re-wiring of energy metabolism and redox homeostatic processes. We previously showed that EVs derived from the human osteosarcoma cells influence bone cells, including osteoblasts. We here investigated whether the opposite could also be true, studying how osteoblast-derived EVs (OB-EVs) could alter tumour phenotype, mitochondrial energy metabolism, redox status and oxidative damage in MNNG/HOS osteosarcoma cells.These were treated with EVs obtained from mouse primary osteoblasts, and the following endpoints were investigated: i) cell viability and proliferation; ii) apoptosis; iii) migration and invasive capacity; iv) stemness features; v) mitochondrial function and energy metabolism; vi) redox status, antioxidant capacity and oxidative molecular damage. OB-EVs decreased MNNG/HOS metabolic activity and viability, which however was not accompanied by impaired proliferation nor by increased apoptosis, with respect to control. In addition, OB-EV-treated cells exhibited a significant reduction of motility and in vitro invasion as compared to untreated cells. Although the antioxidant N-acetyl-L-cysteine reverted the cytotoxic effect of OB-EVs, no evidence of oxidative stress was observed in treated cells. However, the redox balance of glutathione was significantly shifted towards a pro-oxidant state, even though the major antioxidant enzymatic protection did not respond to the pro-oxidant challenge. We did not find strong evidence of mitochondrial involvement or major energy metabolic switches induced by OB-EVs, but a trend of reduction in seahorse assay basal respiration was observed, suggesting that OB-EVs could represent a mild metabolic challenge for osteosarcoma cells. In summary, our findings suggest that OB-EVs could serve as important means through which TME and osteosarcoma core cross-communicate. For the first time, we proved that OB-EVs reduced osteosarcoma cells' aggressiveness and viability through redox-dependent signalling pathways, even though mitochondrial dynamics and energy metabolism did not appear as processes critically needed to respond to OB-EVs.

Adaptation to chronic exposure to hypoxia alters energy metabolism in the heart, particularly in the left ventricle, which undergoes a loss in oxidative capacity. Highly lipophilic local anesthetics interfere with mitochondrial energy metabolism. The purpose of this study was to compare the effects of bupivacaine on mitochondrial energy metabolism in heart of rats subjected to normoxic or hypoxic environments. Male Wistar rats (n = 10) were subjected to hypobaric hypoxia (simulated altitude = 5,000 m, 380 mmHg) for 2 weeks. Control rats (n = 10) were maintained in an ambient normoxic environment. Mitochondrial metabolism (oxygen consumption and adenosine triphosphate synthesis) was assessed using saponin-skinned ventricular fibers. Bupivacaine (0-5 mM) was tested on both left and right ventricles of normoxic or hypoxic heart. In animals exposed to hypobaric hypoxia for 14 days, cardiac mass significantly increased, and the right-to-left ventricular ratio was approximately twofold (0.48 +/- 0.11 vs. 0.22 +/- 0.04, P < 0.05). Oxygen consumption and adenosine triphosphate synthesis were significantly lower in the hypoxic left ventricles but not in the right ones. The uncoupling effect of bupivacaine was more pronounced in the left ventricle from hypoxic heart than in the right ventricle; the bupivacaine-induced decrease in the adenosine triphosphate synthesis rate and in the adenosine triphosphate-to-oxygen ratio was significantly greater in the hypoxic left ventricle than in the normoxic one. Chronic hypoxia impairs cardiac energy metabolism in left ventricles and enhances the depressant effects of bupivacaine on mitochondrial functions.

A wide range of clinical findings have been associated with mutations in Syntaxin Binding Protein 1 (STXBP1), including multiple forms of epilepsy, nonsyndromic intellectual disability, and movement disorders. STXBP1 mutations have recently been associated with mitochondrial pathology, although it remains unclear if this phenotype is a part of the core feature for this gene disorder. We report a 7-year-old boy who presented for diagnostic evaluation of intractable epilepsy, episodic ataxia, resting tremor, and speech regression following a period of apparently normal early development. Mild lactic acidemia was detected on one occasion at the time of an intercurrent illness. Due to the concern for mitochondrial disease, ophthalmologic evaluation was performed that revealed bilateral midperiphery pigmentary mottling. Optical coherence tomography (OCT) testing demonstrated a bilaterally thickened ganglion cell layer in the perifovea. Skeletal muscle biopsy analysis showed no mitochondrial abnormalities or respiratory chain dysfunction. Exome sequencing identified a de novo c.1651C>T (p.R551C) mutation in STXBP1. Although mitochondrial dysfunction has been reported in some individuals, our proband had only mild lactic acidemia and no skeletal muscle tissue evidence of mitochondrial disease pathology. Thus, mitochondrial dysfunction is not an obligate feature of STXBP1 disease.

Inorganic polyphosphate (polyP) is an ancient polymer that is well-conserved throughout evolution. It is formed by multiple subunits of orthophosphates linked together by phosphoanhydride bonds. The presence of these bonds, which are structurally similar to those found in ATP, and the high abundance of polyP in mammalian mitochondria, suggest that polyP could be involved in the regulation of the physiology of the organelle, especially in the energy metabolism. In fact, the scientific literature shows an unequivocal role for polyP not only in directly regulating oxidative a phosphorylation; but also in the regulation of reactive oxygen species metabolism, mitochondrial free calcium homeostasis, and the formation and opening of mitochondrial permeability transitions pore. All these processes are closely interconnected with the status of mitochondrial bioenergetics and therefore play a crucial role in maintaining mitochondrial and cell physiology. In this invited review, we discuss the main scientific literature regarding the regulatory role of polyP in mammalian mitochondrial physiology, placing a particular emphasis on its impact on energy metabolism. Although the effects of polyP on the physiology of the organelle are evident; numerous aspects, particularly within mammalian cells, remain unclear and require further investigation. These aspects encompass, for example, advancing the development of more precise analytical methods, unraveling the mechanism responsible for sensing polyP levels, and understanding the exact molecular mechanism that underlies the effects of polyP on mitochondrial physiology. By increasing our understanding of the biology of this ancient and understudied polymer, we could unravel new pharmacological targets in diseases where mitochondrial dysfunction, including energy metabolism dysregulation, has been broadly described.

Electroacupuncture Relieves Hippocampal Injury by Heme Oxygenase-1 to Improve Mitochondrial Function.

BackgroundEndometriosis is the growth of uterine lining (endometrium) outside of the uterus. In other chronic inflammatory diseases, mitochondrial dysfunction is suspected of playing a role in disease pathogenesis. However, little is known about endometriosis mitochondrial function or its effects on tissue metabolism. The objectives of this study were to analyze mitochondrial function in nonhuman primate (NHP) endometrium and endometriosis tissue and to identify the metabolic features of these tissues that may contribute to disease.MethodsMitochondrial function in endometriosis tissue and endometrium was measured using mitochondrial respirometry analysis to determine if changes in oxidative phosphorylation exist in endometrium and endometriosis tissue compared to control endometrium from clinically healthy NHPs. Targeted metabolomics and multidimensional statistical analysis were applied to quantify key metabolites in energy and amino acid biosynthesis pathways.ResultsMitochondrial respirometry assays showed endometrium from NHPs with endometriosis had reduced complex II-mediated oxygen consumption rates (OCR) across all energy states (basal, p = 0.01; state 3, p = 0.02; state 3u, p = 0.04; state 4o, p = 0.008) and endometriosis tissue had reduced state 3, complex I-mediated OCR (p = 0.02) and respiratory control rates (p = 0.01) compared to normal endometrium. Targeted metabolomics performed on tissue revealed carnitine (p = 0.001), creatine phosphate (p = 0.01), NADH (p = 0.0001), FAD (p = 0.001), tryptophan (p = 0.0009), and malic acid (p = 0.005) were decreased in endometriosis tissue compared to normal endometrium samples. FAD (p = 0.004), tryptophan (p = 0.0004) and malic acid (p = 0.03) were significantly decreased in endometrium from NHPs with endometriosis compared to normal endometrium. Significant metabolites identified in endometriosis and endometrium samples from animals with endometriosis were part of amino acid biosynthesis or energy metabolism pathways.ConclusionsHere, endometrial mitochondrial energy production and metabolism were decreased in endometrium and endometriosis tissue. Decreased mitochondrial energy production may be due to oxidative stress-induced damage to mitochondrial DNA or membranes, a shift in cell metabolism, or decreased energy substrate; however, the exact cause remains unknown. Additional research is needed to determine the implications of reduced mitochondrial energy production and metabolism on endometriosis and endometrium.

Powering the brain in health and disease.

Estrogen-related receptor gamma (ERRg) is a crucial orphan nuclear receptor that governs cardiac metabolism, mitochondrial function, and contractility. It regulates the expression of genes involved in oxidative phosphorylation, fatty acid b-oxidation and ATP production, providing a sustained energy supply essential for cardiac function. Dysregulation of ERRg has been implicated in pathological cardiac remodelling, including metabolic dysfunctions, hypertrophy and heart failure, highlighting its potential as a therapeutic target for cardiovascular diseases. The purpose of this study is to investigate the regulatory mechanism of ERRg and a membrane protein of gut bacterium Akkermansia muciniphila (Amuc_1100) in cardiac mitochondrial dynamics and energy metabolism. Methods: Two groups of wild-type (WT) and cardiac ERRg-specific knockout (KO) mice underwent a 12-week high-fat diet (HFD) followed by cardiac function assessments using echocardiography and gene expression analysis using q-RT-PCR and immunoblotting analysis. A membrane protein of Akkermansia muciniphila (Amuc_1100) was expressed in human cardiomyocytes (CM) AC16 to determine its interaction with ERRg in cardiac health. Results: Echocardiographic analysis revealed that cardiac-specific depletion of ERRg significantly reduced stroke volume and cardiac output, despite left ventricular (LV) mass remaining unchanged between WT and KO mice. Analysis of key genes involved in mitochondrial energy metabolism revealed that oxidative phosphorylation and fatty acid β-oxidation, including PPARa, CPT1a, ACOX1 and PGC1β, were markedly downregulated in the cardiac tissue of KO mice. Furthermore, regulators of mitochondrial dynamics, such as MFN1 and MFN2 that mediate mitochondrial fusion, were also suppressed in the KO mouse hearts, indicating mitochondrial stress and impaired energy homeostasis. Following a 12-week HFD, both WT and KO mice exhibited a reduction in LV mass, accompanied by a decline in cardiac output. Notably, HFD significantly suppressed the expression of ERRg in the WT mouse hearts and induced the expression of fibrogenic genes, including Stat3, KIF5, BMP4, and TGFβ1/β2, to a more severe extent in KO mice compared to their WT counterparts, suggesting an increased susceptibility of KO mice to cardiac fibrosis. In vitro, expression of Amuc_1100 in human CMs (AC16) upregulated ERRg which was accompanied by increased expression of MFN1, MFN2 and fatty acid b-oxidation genes, indicating the cardioprotective effect of this gut bacterial protein. Conclusion: ERRg interacted with a membrane protein of A. muciniphila to regulate cardiac mitochondrial dynamics and energy metabolism, which provides potential support for a therapeutic strategy targeting cardiovascular disease by manipulating the interaction between host genes i.e., ERRg and bioactive components of gut bacteria, i.e., membrane protein of A. muciniphila, Amuc_1100.ERRg and Amuc_1100 in Cardiac Health

Dysregulation of mitochondrial energy metabolism is a major hallmark of mammalian aging. Calorie restriction (CR) has been shown to effectively increase lifespan and is associated with a metabolic shift toward a decrease in glycolysis and an increase in fatty acid oxidation. This shift in fuel utilization that occurs with CR relies heavily on mitochondrial energy metabolism. Thus, a diet that can mimic the metabolic reprogramming that occurs with CR may reduce age‐related decline in mitochondrial function and energy metabolism, and contribute to healthy aging and longevity. A ketogenic diet, which is depleted in carbohydrates, would drive the utilization of fat as a fuel and increase mitochondrial fatty acid oxidation. By chronically stimulating pathways in mitochondrial oxidative metabolism, the ketogenic diet could influence mitochondrial function and biogenesis, and may prevent age‐related decreases in mitochondrial content. In this study, mice were fed an isocaloric control (CLT), low carbohydrate (low‐CHO), or ketogenic diet (KD) from the age of 12 months. Tissues were collected after 1 month and 14 months of dietary treatment for evaluation of mitochondrial content. The results showed that the low‐CHO and KD increase markers of mitochondrial content in skeletal muscle in old mice (age of 26 months). The activities of both Complex I and IV were increased (p&lt;0.05) in the low CHO and KD diet compared to the CTL diet. Old mice fed a KD also maintained activity of Complex I compared to the young mice, while mice fed the CLT and low‐CHO diets showed a decline (p&lt;0.05) in Complex I activity with aging. Mice fed a KD also showed increased muscle citrate synthase activity with age. However, these changes are tissue specific with liver showing only an increase in Complex IV activity with aging in the KD group. In summary, the study demonstrates that long‐term ketogenic diet may prevent age related decline in mitochondrial content in some tissues in adult mice.Support or Funding InformationNIH Grant PO1AGO25532

The role of mitochondria in the recovery of neurons after injury.

Objective To observe the effect of mild hypothermia on mitochondrial reactive oxygen species (ROS) generation and energy metabolism in acute phase of spontaneous subarachnoid hemorrhage (SAH) in rats,and to explore the relative signaling pathway in this process.Methods SD rats were randomly divided into three groups:sham-operated group,normothermic SAH group (SAH-N),and hypothermic SAH group (SAH-H).The SAH models were induced by injection of autologous arterial blood into the prechiasmatic cistem.After the injection,rats in SAH-H group were subjected to mild hypothermia intervention (32.5-33.5 ℃).All animals were sacrificed by decapitation two hours after SAH.Ultrastructures of CA1 section of the hippocampus were observed under transmission electron microscopy.The enzyme activities of mitochondrial manganese superoxide dismutase (MnSOD),glutathione peroxidase (GPx) and catalase (CAT),and the malonaldehyde (MDA) level were measured with colorimetry method.Mitochondrial respiratory function was measured with polarography method.Mitochondrial ROS generation was determined using dichlorofluorescein (DCF) method.Membrane potent was determined using JC-1 fluorescence labeling.Adenodine triphospate (ATP) synthesis capacity was determined using bioluminescence technique.The Sirtuin-3 (SIRT3) mRNA expression in the brain tissues was detected by real-time quantitative PCR.The mitochondrial SIRT3 protein expression was detected by Western blotting.Results As compared with those in the SAH-N group,mitochondrial SIRT3 mRNA and protein expressions,ATP synthetase activity,membrane potent,state 3 respiratory rate,respiratory control ratio,ratio of ADP to oxygen,and activities of MnSOD,GPx and CAT significantly increased in the SAH-H group (P＜0.05); and state 4 respiratory rate,mitochondrial ROS generation and MDA level significantly decreased in SAH-H group (P＜0.05).Conclusion Mild hypothermia could efficiently promote mitochondrial energy metabolism and ROS scavenging capacity,and in turn improve SAH tolerance in brain mitochondria. Key words: Subarachnoid hemorrhage; Mild hypothermia; Mitochondrial energy metabolism; Oxidative stress; Sirtuin-3

Hydroxysafflor yellow A alleviates ischemic myocardial injury by targeting SF3A1 to improve mitochondrial energy metabolism.