Mitochondrial Respiratory Chain Research Articles

The Effect of Neuronal CoQ10 Deficiency and Mitochondrial Dysfunction on a Rotenone-Induced Neuronal Cell Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder currently affecting the ageing population. Although the aetiology of PD has yet to be fully elucidated, environmental factors such as exposure to the naturally occurring neurotoxin rotenone has been associated with an increased risk of developing PD. Rotenone inhibits mitochondrial respiratory chain (MRC) complex I activity as well as induces dopaminergic neuronal death. The aim of the present study was to investigate the underlying mechanisms of rotenone-induced mitochondrial dysfunction and oxidative stress in an in vitro SH-SY5Y neuronal cell model of PD and to assess the ability of pre-treatment with Coenzyme Q10 (CoQ10) to ameliorate oxidative stress in this model. Spectrophotometric determination of the mitochondrial enzyme activities and fluorescence probe studies of reactive oxygen species (ROS) production was assessed. Significant inhibition of MRC complex I and II-III activities was observed, together with a significant loss of neuronal viability, CoQ10 status, and ATP synthesis. Additionally, significant increases were observed in intracellular and mitochondrial ROS production. Remarkably, CoQ10 supplementation was found to reduce ROS formation. These results have indicated mitochondrial dysfunction and increased oxidative stress in a rotenone-induced neuronal cell model of PD that was ameliorated by CoQ10 supplementation.

Mitochondrial biogenesis disorder and oxidative damage promote refractory apical periodontitis in rat and human.

To elucidate whether mitochondrial biogenesis disorder and damage from oxidative stress promote refractory apical periodontitis (RAP) in rat and human. Twenty Enterococcus faecalis-induced RAPs were established in the maxillary first molars of male Wistar rats. Concurrently, 12 periapical lesion specimens from patients presenting with RAP were obtained by apicoectomy. Radiographic examination and histologic analysis were conducted to evaluate periapical bone tissue destruction and morphological changes. The expression of key regulators of mitochondrial biogenesis, PGC-1α and Nrf2, were detected by immunohistochemistry and double immunofluorescence staining, Western blot and real-time PCR were also assayed. Mitochondrial ROS (mtROS) was identified by MitoSOX staining. Mitochondrial function was detected by the quantification of ATP production, mitochondrial DNA (mtDNA) copy number and activities of mitochondrial respiratory chain complexes. Furthermore, mitochondrial oxidative stress was evaluated by the determination of 3-nitrotyrosine (3-NT), 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxy-deoxyguanosine (8-OHdG) expression levels, as well as malondialdehyde (MDA) expression and antioxidant capacity. Student's t-test was performed to determine significance between the groups; p < .05 was considered significant. In the maxilla, significantly more bone resorption, greater number of periapical apoptotic cells and Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells were observed in the RAP group compared with the control group (p < .01). PGC-1α and Nrf2 were significantly reduced in rat and human RAP lesions compared to the control group (p < .01) at both the mRNA and protein levels. Double immunofluorescence analysis of PGC-1α or Nrf2 with TOMM20 also indicated that mitochondrial biogenesis was impaired in RAP group (p < .01). Additionally, mitochondrial dysfunction was observed in RAP group, as reflected by increased mtROS, decreased ATP production, reduced mtDNA copy number and complexes of the mitochondrial respiratory chain. Finally, the expression levels of mitochondrial oxidative stress markers, 3-NT, 4-HNE and 8-OHdG, were significantly increased in the RAP group (p < .01). Consistent with this, systemic oxidative damage was also present in the progression of RAP, including increased MDA expression and decreased antioxidant activity (p < .01). Mitochondrial biogenesis disorder and damage from oxidative stress contribute to the development of RAP.

Вплив кверцетину на показники оксидативнонітрозативного стресу в тканинах нирок щурів за умов гострого десинхронозу та ліпополісахаридіндукованої системної запальної відповіді

The relevance of this study stems from the growing interest in the impact of acute desynchronosis (AD) on oxidative disorders and the insufficiently studied effect of flavonoids on these processes in kidney tissue. This study investigates the effect of quercetin on oxidative and nitrosative stress in rat kidney homogenate under AD and 3 lipopolysaccharide (LPS)-induced systemic inflammatory response (SIR). The animals were randomly divided into five groups: group 1 served as the control, group 2 underwent LPS-induced SIR modeling for 8 weeks, group 3 experienced AD reproduction by shifting the light-dark cycle 6 hours earlier for 7 days, group 4 underwent AD modeling during the last week of LPS-induced SIR, and rats in group 5 were administered a water-soluble form of quercetin (corvitin) intragastrically was administered at a dose of 200 mg/kg (20 mg/kg in terms of quercetin) daily during the AD modeling against LPS-induced SIR. It was found that the modeling of LPS-induced oxidative stress and AD significantly increased the rate of superoxide anion radical production in kidney homogenate by microsomal monooxygenases and NO synthase (NOS), mitochondrial respiratory chain, leukocyte NADPH oxidase, inducible NOS activity, and decreased the activity of the constitutive NOS isoform and its coupling index. Under AD modeling, the rate of superoxide anion radical production by leukocyte NADPH oxidase, total and inducible NOS activity was significantly lower compared with LPS-induced SIR. When modeling AD against the background of LPS-induced SIR, the production of superoxide anion radical by microsomes, mitochondria and leukocyte NADPH oxidase exceeded the respective results of group 2 by 15.9%, 16.0% and 38.5%, and group 3 by 24.4%, 17.5% and 74.6%. The inducible NOS activity was 22.9% higher compared to the value of group 2 and 93.4% higher in group 3, the concentration of peroxynitrites of alkali and alkaline earth metals was 35.5% higher than the respective parameters of group 2 and 57.9% higher than in group 3. Administration of quercetin during AD modeling and LPS-induced oxidative stress significantly reduced the rate of superoxide anion radical production by microsomal monooxygenases by 17.1%, mitochondrial respiratory chain by 31.2%, leukocyte NADPH oxidase by 19.4%, total and inducible NOS activity by 50.4% and 55.5%, respectively; the concentration of peroxynitrites of alkali and alkaline earth metals diminished by 41.4% compared to the value of group 4. It has been concluded that the administration of quercetin significantly reduces the signs of oxidative-nitrosative stress in the kidney homogenate of rats.

The possible association of mitochondrial fusion and fission in copper deficiency-induced oxidative damage and mitochondrial dysfunction of the heart

IntroductionAs an essential trace element, Copper (Cu) participates in numerous physiological and biological reactions in the body. Cu is closely related to heart health, and an imbalance of Cu will cause cardiac dysfunction. The research aims to examine how Cu deficiency affects the heart, assess mitochondrial function in the hearts, and disclose possible mechanisms of its influence. MethodsWeaned mice were fed Cu-deficient diets and intraperitoneally given copper sulfate (CuSO4) to correct the Cu deficiency. The pathological change of the heart was assessed using histological inspection. Cardiac function and oxidative stress levels were evaluated by biochemical assay kits. ELISA and ATP detection kits were used to detect the levels of complexes I-IV in the mitochondrial respiratory chain (MRC) and ATP, respectively. Real time PCR was utilized to determine mRNA expressions, and Western blotting was adopted to determine protein expressions, of molecules related to mitochondrial fission and fusion. ResultsCu deficiency gave rise to elevated heart index, cardiac histological alterations and oxidation injury, increased serum levels of creatine kinase (CK), lactic dehydrogenase (LDH), and creatine kinase isoenzyme MB (CK-MB) together with increased malondialdehyde (MDA) production, decreased the glutathione (GSH), Superoxide Dismutase (SOD), and Catalase (CAT) activities or contents. Besides, Cu deficiency caused mitochondrial damage characterized by decreased contents of complexes I-IV in the MRC and ATP in the heart. In the meantime, Cu deficiency also reduced protein and mRNA expressions of factors associated with mitochondrial fusion, including Mfn1 and Mfn2, while significantly increased factors Drip1 and Fis1 related to mitochondrial fission. However, adding CuSO4 improved the above changes significantly. ConclusionAccording to research results, Cu deficiency can cause heart damage in mice, along with oxidative damage and mitochondrial dysfunction, which are closely related to mitochondrial fusion and fission disorders.

Colonocyte keratins stabilize mitochondria and contribute to mitochondrial energy metabolism.

Keratin intermediate filaments form dynamic filamentous networks, which provide mechanical stability, scaffolding, and protection against stress to epithelial cells. Keratins and other intermediate filaments have been increasingly linked to the regulation of mitochondrial function and homeostasis in different tissues and cell types. While deletion of keratin 8 (K8-/-) in mouse colon elicits a colitis-like phenotype, epithelial hyperproliferation, and blunted mitochondrial ketogenesis, the role of K8 in colonocyte mitochondrial function and energy metabolism is unknown. We used two K8 knockout mouse models and CRISPR/Cas9 K8-/- colorectal adenocarcinoma Caco-2 cells to answer this question. The results show that K8-/- colonocyte mitochondria in vivo are smaller and rounder and that mitochondrial motility is increased in K8-/- Caco-2 cells. Furthermore, K8-/- Caco-2 cells displayed diminished mitochondrial respiration and decreased mitochondrial membrane potential compared with controls, whereas glycolysis was not affected. The levels of mitochondrial respiratory chain complex proteins and mitochondrial regulatory proteins mitofusin-2 and prohibitin were decreased both in vitro in K8-/- Caco-2 cells and in vivo in K8-/- mouse colonocytes, and reexpression of K8 into K8-/- Caco-2 cells normalizes the mitofusin-2 levels. Mitochondrial Ca2+ is an important regulator of mitochondrial energy metabolism and homeostasis, and Caco-2 cells lacking K8 displayed decreased levels and altered dynamics of mitochondrial matrix and cytoplasmic Ca2+. In summary, these novel findings attribute an important role for colonocyte K8 in stabilizing mitochondrial shape and movement and maintaining mitochondrial respiration and Ca2+ signaling. Further, how these metabolically compromised colonocytes are capable of hyperproliferating presents an intriguing question for future studies.NEW & NOTEWORTHY In this study, we show that colonocyte intermediate filament protein keratin 8 is important for stabilizing mitochondria and maintaining mitochondrial energy metabolism, as keratin 8-deficient colonocytes display smaller, rounder, and more motile mitochondria, diminished mitochondrial respiration, and altered Ca2+ dynamics. Changes in fusion-regulating proteins are rescued with reexpression of keratin 8. These alterations in colonocyte mitochondrial homeostasis contribute to keratin 8-associated colitis pathophysiology.

Unveiling the mechanism of photothermal therapy in acne man-agement: targeting sebaceous gland ferroptosis via umbilical cord mesenchymal stem cell membrane-encapsulated Au-Ag-PDA.

Branched gold and silver nanoparticles coated with polydopamine (Au-Ag-PDA) demonstrate high photothermal conversion efficiency. Utilizing umbilical cord mesenchymal stem cell membranes (MSCM) as an effective drug delivery system, our preliminary studies investigated the suppression of sebum secretion in sebaceous glands using MSCM-coated Au-Ag-PDA nano-particles (Au-Ag-PDA@MSCM) combined with 808nm laser irradiation, showing potential for dermatological applications in acne treatment. This study employs proteomic analysis, complemented by subsequent techniques such as Western blotting (WB), small interfering RNA (siRNA), and transmission electron microscopy, to further investigate the differential mechanisms by which Au-Ag-PDA and Au-Ag-PDA@MSCM-mediated photothermal therapy (PTT) suppress sebum secretion. Our proteomic analysis indicated mitochondrial respiratory chain damage in sebaceous gland tissues post-PTT, with further validation revealing ferroptosis in sebaceous cells and tissues. Acyl-CoA Synthetase Long-Chain Family Member 4 (Acsl4) has been identified as a critical target, with Au-Ag-PDA@MSCM demonstrating enhanced ferroptotic effects. These findings significantly advance our understanding of how PTT mediated by Au-Ag-PDA@MSCM nanoparticles reduces sebum secretion and underscore the pivotal role of MSCM in inducing ferroptosis in sebaceous glands, thus providing a robust theoretical foundation for employing PTT via specific molecular pathways in acne treatment.

Longitudinal Analysis of Mitochondrial Function in a Choline-Deficient L-Amino Acid-Defined High-Fat Diet-Induced Metabolic Dysfunction-Associated Steatohepatitis Mouse Model.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is one of the most common chronic liver diseases worldwide. Some patients with MAFLD develop metabolic dysfunction-associated steatohepatitis (MASH), which can lead to severe liver fibrosis. However, the molecular mechanisms underlying this progression remain unknown, and no effective treatment for MASH has been developed so far. In this study, we performed a longitudinal detailed analysis of mitochondria in the livers of choline-deficient, methionine-defined, high-fat-diet (CDAHFD)-fed mice, which exhibited a MASH-like pathology. We found that FoF1-ATPase activity began to decrease in the mitochondria of CDAHFD-fed mice prior to alterations in the activity of mitochondrial respiratory chain complex, almost at the time of onset of liver fibrosis. In addition, the decrease in FoF1-ATPase activity coincided with the accelerated opening of the mitochondrial permeability transition pore (PTP), for which FoF1-ATPase might be a major component or regulator. As fibrosis progressed, mitochondrial permeability transition (PT) induced in CDAHFD-fed mice became less sensitive to cyclosporine A, a specific PT inhibitor. These results suggest that episodes of fibrosis might be related to the disruption of mitochondrial function via PTP opening, which is triggered by functional changes in FoF1-ATPase. These novel findings could help elucidate the pathogenesis of MASH and lead to the development of new therapeutic strategies.

Photobiomodulation Therapy on Brain: Pioneering an Innovative Approach to Revolutionize Cognitive Dynamics.

Photobiomodulation (PBM) therapy on the brain employs red to near-infrared (NIR) light to treat various neurological and psychological disorders. The mechanism involves the activation of cytochrome c oxidase in the mitochondrial respiratory chain, thereby enhancing ATP synthesis. Additionally, light absorption by ion channels triggers the release of calcium ions, instigating the activation of transcription factors and subsequent gene expression. This cascade of events not only augments neuronal metabolic capacity but also orchestrates anti-oxidant, anti-inflammatory, and anti-apoptotic responses, fostering neurogenesis and synaptogenesis. It shows promise for treating conditions like dementia, stroke, brain trauma, Parkinson's disease, and depression, even enhancing cognitive functions in healthy individuals and eliciting growing interest within the medical community. However, delivering sufficient light to the brain through transcranial approaches poses a significant challenge due to its limited penetration into tissue, prompting an exploration of alternative delivery methods such as intracranial and intranasal approaches. This comprehensive review aims to explore the mechanisms through which PBM exerts its effects on the brain and provide a summary of notable preclinical investigations and clinical trials conducted on various brain disorders, highlighting PBM's potential as a therapeutic modality capable of effectively impeding disease progression within the organism-a task often elusive with conventional pharmacological interventions.

Membrane-mediated modulation of mitochondrial physiology by terahertz waves.

Extensive studies have demonstrated the diverse impacts of electromagnetic waves at gigahertz and terahertz (THz) frequencies on cytoplasmic membrane properties. However, there is little evidence of these impacts on intracellular membranes, particularly mitochondrial membranes crucial for mitochondrial physiology. In this study, human neuroblast-like cells were exposed to continuous 0.1 THz radiation at an average power density of 33 mW/cm2. The analysis revealed that THz exposure significantly altered the mitochondrial ultrastructure. THz waves enhanced the enzymatic activity of the mitochondrial respiratory chain but disrupted supercomplex assembly, compromising mitochondrial respiration. Molecular dynamics simulations revealed altered rates of change in the quantity of hydrogen bonds and infiltration of water molecules in lipid bilayers containing cardiolipin, indicating the specific behavior of cardiolipin, a signature phospholipid in mitochondria, under THz exposure. These findings suggest that THz radiation can significantly alter mitochondrial membrane properties, impacting mitochondrial physiology through a mechanism related to mitochondrial membrane, and provide deeper insight into the bioeffects of THz radiation.

APE1 regulates mitochondrial DNA damage repair after experimental subarachnoid haemorrhage in vivo and in vitro

BackgroundSubarachnoid haemorrhage (SAH) can result in a highly unfavourable prognosis. In recent years, the study of SAH has focused on early brain injury (EBI), which is a crucial progress that...

Effect of Rotenone on the Neurodegeneration among Different Models.

Rotenone is a naturally occurring plant product used as an insecticide, pesticide and piscicide. It is lipophilic in nature and can cross the blood-brain barrier and induce the degeneration of neurons. It inhibits the mitochondrial respiratory chain complex I and stops the transfer of electrons. It induces ROS generation, which impairs mitochondrial activity. Rotenone is a toxic agent which causes the death of neurons. The present review describes the effect of rotenone on neurodegeneration with an emphasis on behavioral, pathological and neuropathological components carried out on various experimental models such as cell lines, Drosophila melanogaster, mice and rats.

Empagliflozin Improves Diastolic Function in HFpEF by Restabilizing the Mitochondrial Respiratory Chain.

Clinical studies demonstrated beneficial effects of sodium-glucose-transporter 2 inhibitors on the risk of cardiovascular death in patients with heart failure with preserved ejection fraction (HFpEF). However, underlying processes for cardioprotection remain unclear. The present study focused on the impact of empagliflozin (Empa) on myocardial function in a rat model with established HFpEF and analyzed underlying molecular mechanisms. Obese ZSF1 (Zucker fatty and spontaneously hypertensive) rats were randomized to standard care (HFpEF, n=18) or Empa (HFpEF/Empa, n=18). ZSF1 lean rats (con, n=18) served as healthy controls. Echocardiography was performed at baseline and after 4 and 8 weeks, respectively. After 8 weeks of treatment, hemodynamics were measured invasively, mitochondrial function was assessed and myocardial tissue was collected for either molecular and histological analyses or transmission electron microscopy. In HFpEF Empa significantly improved diastolic function (E/é: con: 17.5±2.8; HFpEF: 24.4±4.6; P<0.001 versus con; HFpEF/Empa: 19.4±3.2; P<0.001 versus HFpEF). This was accompanied by improved hemodynamics and calcium handling and by reduced inflammation, hypertrophy, and fibrosis. Proteomic analysis demonstrated major changes in proteins involved in mitochondrial oxidative phosphorylation. Cardiac mitochondrial respiration was significantly impaired in HFpEF but restored by Empa (Vmax complex IV: con: 0.18±0.07 mmol O2/s/mg; HFpEF: 0.13±0.05 mmol O2/s/mg; P<0.041 versus con; HFpEF/Empa: 0.21±0.05 mmol O2/s/mg; P=0.012 versus HFpEF) without alterations of mitochondrial content. The expression of cardiolipin, an essential stability/functionality-mediating phospholipid of the respiratory chain, was significantly decreased in HFpEF but reverted by Empa (con: 15.9±1.7 nmol/mg protein; HFpEF: 12.5±1.8 nmol/mg protein; P=0.002 versus con; HFpEF/Empa: 14.5±1.8 nmol/mg protein; P=0.03 versus HFpEF). Transmission electron microscopy revealed a reduced size of mitochondria in HFpEF, which was restored by Empa. The study demonstrates beneficial effects of Empa on diastolic function, hemodynamics, inflammation, and cardiac remodeling in a rat model of HFpEF. These effects were mediated by improved mitochondrial respiratory capacity due to modulated cardiolipin and improved calcium handling.

A zebrafish tufm mutant model for the COXPD4 syndrome of aberrant mitochondrial function

Mitochondrial dysfunction is a critical factor leading to a wide range of clinically heterogeneous and often severe disorders due to its central role in generating cellular energy. Mutations in the TUFM gene are known to cause combined oxidative phosphorylation deficiency 4 (COXPD4), a rare mitochondrial disorder characterized by a comprehensive quantitative deficiency in mitochondrial respiratory chain (MRC) complexes. The development of a reliable animal model for COXPD4 is crucial for elucidating the roles and mechanisms of TUFM in disease pathogenesis and benefiting its medical management. In this study, we construct a zebrafish tufm−/− mutant that closely resembles the COXPD4 syndrome, exhibiting compromised mitochondrial protein translation, dysfunctional mitochondria with oxidative phosphorylation defects, and significant metabolic suppression of the tricarboxylic acid cycle. Leveraging this COXPD4 zebrafish model, we comprehensively validate the clinical relevance of TUFM mutations and identify probucol as a promising therapeutic approach for managing COXPD4. Our data offer valuable insights for understanding mitochondrial diseases and developing effective treatments.

A Rare Case of Neuronal Ceroid Lipofuscinosis-Type 1 (NCL-1) with Vitamin D-Dependent Rickets-Type 1 (VDDR-1), Complex 1 Mitochondrial Deficiency, and Mixed Variant-Checkerboard and Phylloid Type of Pigmentary Mosaicism.

Introduction Neuronal ceroid lipofuscinosis-type 1 (NCL-1) is a neurodegenerative lysosomal storage disorder. Vitamin D-dependent rickets type 1 (VDDR-1) is a rare cause of refractory rickets. Here, we report an unusual association of NCL-1 with VDDR-1. Case A 3-year-old boy presented with a history of seizures from 45 days of life, delayed development, and loss of attained milestones at 20 months of age, along with progressive vision impairment since 1 year. Examination showed a failure to thrive, microcephaly, rachitic rosary, checkerboard and phylloid type of pigmentary mosaicism, fundus showed disc pallor with generalized narrowing of arterioles, bilateral retinitis pigmentosa, spasticity and dystonia, brisk reflexes, extensor plantar, and left choreoathetoid movements. Investigations showed hypocalcemia (7.8 mg/dL), normal phosphorus (3.9 mg/dL), elevated alkaline phosphatase (508.8 U/L), elevated parathyroid hormone (513.35 pg/mL), low 1,25-dihydroxy-vitamin D (9.93 pg/mL), and normal renal function. The child had metabolic acidosis, elevated ammonia (403.9 micromol/L), lactate (95 mg/dL, normal range 4.5-19.8 mg/dL), and creatine phosphokinase (432 U/L) level, and normal tandem mass spectroscopy. X-ray wrist showed healing vitamin deficiency rickets. Abnormal electroencephalogram was suggestive of low voltage activity. Magnetic resonance imaging brain showed gross cerebral and cerebellar atrophy. A muscle biopsy showed scattered atrophic fibers and several ultrastructural granular osmiophilic deposits and some mitochondrial aggregates of varying size were observed. Mitochondrial respiratory chain enzyme assay exhibited complex-1 deficiency (activity < 30%). Genetic analysis showed two pathogenic mutations: homozygous nonsynonymous variation c.674T > C in exon 7 of the PPT1 gene and a homozygous frameshift variation c.1178_1179delAA in exon 7 of CYP27B1 confirming the diagnosis of NCL-1 with VDDR-1. The child was treated with a low protein diet, levetiracetam, clonazepam, trihexyphenidyl, haloperidol, calcium supplement, calcitriol, and sodium benzoate; some improvement in clinical and biochemical parameters was noted on follow-up. Conclusion This is a novel association of NCL-1 with VDDR-1 associated with complex-1 mitochondrial deficiency which has previously not been reported in the literature.

Alterations in oxidative phosphorylation complexes in arrhythmogenic cardiomyopathy using desmosomal KO cell model

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III (Fondo Investigación Sanitaria FIS) Introduction Arrhythmogenic cardiomyopathy (ACM) is an inherited disease that predominantly affects the right ventricle characterized by progressive cardiomyocyte loss and the replacement of the myocardium by fibro-fatty tissue, which can lead to sudden cardiac death (1). The main genetic causes of ACM are variants in desmosomal genes (PKP2, DSP, DSG2, DSC2 and JUP), representing up to 50% of patients (2). We lack sufficient understanding at molecular level of the pathophysiological mechanisms that may trigger ACM and determine its progression. Recent studies have proposed a deficit in transcripts encoding for proteins from the electron transport chain, which may be a potential ACM molecular signature in samples from patients with ACM carrying mutations in PKP2 (3). Purpose Our study aims to deepen into the knowledge of this proposed mechanism in PKP2 deficiency using edited cell model and elucidating whether these mechanisms are also triggered in DSG2 and DSC2 deficiency, being a potential common ACM pathomechanism, or they are gene specific. Methods HL-1 edited cell lines for desmosomal genes previously generated by CRISPR/Cas9 (PKP2-KO, DSC2-KO and DSG2-KO) in our research group were used to perform our analysis (4). Expression profiling was performed by RNA-seq analysis. To experimentally validate the RNA-seq analysis results, we performed western blot assays using total protein lysate to test protein levels of the five oxidative phosphorylation (OXPHOS) complexes for the PKP2-KO, DSC2-KO and DSG2-KO groups. To test if these differences have an impact at functional level, XFp cell Mito Stress Test (Agilent Technologies) was performed. Results RNA-seq analysis results showed numerous downregulated pathways related to aerobic respiration, the electron transport chain, oxidative phosphorylation and ATP synthesis in all three desmosomal KO lines. Our experimental results showed a significant reduction of the OXPHOS complexes of the mitochondrial respiration chain at protein level in all PKP2-KO, DSC2-KO and DSG2-KO (Fig. 1). At functional level, our preliminary results of Mito Stress Test suggest differences in basal and maximal respiration, ATP production, non-mitochondrial oxygen consumption and spare respiratory capacity between experimental groups. Conclusions Our results support previously published studies describing oxidative phosphorylation as a pathomechanism for PKP2 deficiency and also suggest that these mechanisms may be a common feature for desmosomal mutations in ACM.Total expression of OXPHOS complexes

Mitochondrial GRIM19 Loss Induces Liver Fibrosis through NLRP3/IL33 Activation via Reactive Oxygen Species/NF-кB Signaling.

Hepatic fibrosis (HF) is a critical step in the progression of hepatocellular carcinoma (HCC). Gene associated with retinoid-IFN-induced mortality 19 (GRIM19), an essential component of mitochondrial respiratory chain complex I, is frequently attenuated in various human cancers, including HCC. Here, we aimed to investigate the potential relationship and underlying mechanism between GRIM19 loss and HF pathogenesis. GRIM19 expression was evaluated in normal liver tissues, hepatitis, hepatic cirrhosis, and HCC using human liver disease spectrum tissue microarrays. We studied hepatocyte-specific GRIM19 knockout mice and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) lentivirus-mediated GRIM19 gene-editing in murine hepatocyte AML12 cells in vitro and in vivo. We performed flow cytometry, immunofluorescence, immunohistochemistry, western blotting, and pharmacological intervention to uncover the potential mechanisms underlying GRIM19 loss-induced HF. Mitochondrial GRIM19 was progressively downregulated in chronic liver disease tissues, including hepatitis, cirrhosis, and HCC tissues. Hepatocyte-specific GRIM19 heterozygous deletion induced spontaneous hepatitis and subsequent liver fibrogenesis in mice. In addition, GRIM19 loss caused chronic liver injury through reactive oxygen species (ROS)-mediated oxidative stress, resulting in aberrant NF-кB activation via an IKK/IкB partner in hepatocytes. Furthermore, GRIM19 loss activated NLRP3-mediated IL33 signaling via the ROS/NF-кB pathway in hepatocytes. Intraperitoneal administration of the NLRP3 inhibitor MCC950 dramatically alleviated GRIM19 loss-driven HF in vivo. The mitochondrial GRIM19 loss facilitates liver fibrosis through NLRP3/IL33 activation via ROS/NF-кB signaling, providing potential therapeutic approaches for earlier HF prevention.

Heat stress upregulates arachidonic acid to trigger autophagy in sertoli cells via dysfunctional mitochondrial respiratory chain function

As a key factor in determining testis size and sperm number, sertoli cells (SCs) play a crucial role in male infertility. Heat stress (HS) reduces SCs counts, negatively impacting nutrient transport and supply to germ cells, and leading to spermatogenesis failure in humans and animals. However, how HS affects the number of SCs remains unclear. We hypothesized that changes in SC metabolism contribute to the adverse effects of HS. In this study, we first observed an upregulation of arachidonic acid (AA), an unsaturated fatty acid after HS exposure by LC-MS/MS metabolome detection. By increasing ROS levels, expression of KEAP1 and NRF2 proteins as well as LC3 and LAMP2, 100 µM AA induced autophagy in SCs by activating oxidative stress (OS). We observed adverse effects of AA on mitochondria under HS with a decrease of mitochondrial number and an increase of mitochondrial membrane potential (MMP). We also found that AA alternated the oxygen transport and absorption function of mitochondria by increasing glycolysis flux and decreasing oxygen consumption rate as well as the expression of mitochondrial electron transport chain (ETC) proteins Complex I, II, V. However, pretreatment with 5 mM NAC (ROS inhibitor) and 2 µM Rotenone (mitochondrial ETC inhibitor) reversed the autophagy induced by AA. In summary, AA modulates autophagy in SCs during HS by disrupting mitochondrial ETC function, inferring that the release of AA is a switch-like response, and providing insight into the underlying mechanism of high temperatures causing male infertility.

Fmo induction as a tool to screen for pro-longevity drugs.

Dietary restriction (DR) and hypoxia (low oxygen) extend lifespan in Caenorhabditis elegans through the induction of a convergent downstream longevity gene, fmo-2. Flavin-containing monooxygenases (FMOs) are highly conserved xenobiotic-metabolizing enzymes with a clear role in promoting longevity in nematodes and a plausible similar role in mammals. This makes them an attractive potential target of small molecule drugs to stimulate the health-promoting effects of longevity pathways. Here, we utilize an fmo-2 fluorescent transcriptional reporter in C. elegans to screen a set of 80 compounds previously shown to improve stress resistance in mouse fibroblasts. Our data show that 19 compounds significantly induce fmo-2, and 10 of the compounds induce fmo-2 more than twofold. Interestingly, 9 of the 10 high fmo-2 inducers also extend lifespan in C. elegans. Two of these drugs, mitochondrial respiration chain complex inhibitors, interact with the hypoxia pathway to induce fmo-2, whereas two dopamine receptor type 2 (DRD2) antagonists interact with the DR pathway to induce fmo-2, indicating that dopamine signaling is involved in DR-mediated fmo-2 induction. Together, our data identify nine drugs that each (1) increase stress resistance in mouse fibroblasts, (2) induce fmo-2 in C. elegans, and (3) extend nematode lifespan, some through known longevity pathways. These results define fmo-2 induction as a viable approach to identifying and understanding mechanisms of putative longevity compounds.

#1546 Mitochondrial oxidative phosphorylation in human proximal tubular epithelial cells is impaired by iron deficiency

Abstract Background and Aims We recently reported that iron deficiency may precede decline in renal function in patients with diabetes who had normal renal function at baseline but had a subsequent annual rate of eGFR decline of 3.3% or more (J Diabetes Investig. 2023;14:874-882.). Mitochondria contain many mitochondrial respiratory chain (MRC) enzymes that require iron as an active center, and consequently iron deficiency is known to impair mitochondrial function. Since the proximal tubular epithelial cells (PTECs) of the kidney are the cells with the greatest number of mitochondria in the body, we investigated whether iron deficiency impairs the mitochondrial capacity of the PTECs. Method Human cultured PTECs were analyzed in ‘control group’, ‘iron-deficient group ‘(co-cultured with deferiprone (DFP), an iron chelator), and ‘rescue group’ (co-cultured with DFP and FeCl3 which saturates the chelating ability of DFP), each with n ≥ 4 (MRC activities n = 4-5, others n ≥ 5). All analyses were performed after 24 hours of incubation. Values are shown as averages ± SEM. Results A Seahorse Extracellular Flux Analyzer showed that the basal oxygen consumption rates (OCRs) of control, iron-deficiency and rescue groups were 30.5 ± 1.2 pmol/min, 11.5 ± 0.7 pmol/min, and 31.1 ± 1.2 pmol/min, respectively, which revealed that basal OCRs of PTECs significantly decreased in the iron-deficient group. In addition, the analyzer also revealed that the activities of MRC for complexes I, II, and III, which have iron-sulfur clusters as their active centers, were significantly decreased in citrate synthase ratio to 77.6 ± 6.2%, 75.1 ± 6.4%, and 72.8 ± 9.3% of the control group, respectively, while complex IV was not significantly different at 84.9% of the control group. These reductions in MRC activities were not observed in the rescue group. Western blotting results also revealed that complex I, II, and III subunit proteins, NDUF9, SDHA, and UQCR2, were significantly reduced in the iron-deficient group (reduced to 38 ± 5.8%, 87 ± 8.8%, and 55 ± 5.7% of the control group, respectively). On the other hand, lactate concentrations in the culture medium of control group, iron-deficient group and rescue group were 1.73 ± 0.13 mmol, 3.01 ± 0.15 mmol and 1.61 ± 0.12 mmol, which revealed that glycolytic system was increased in the iron-deficient group, presumably to compensate for decreased oxidative phosphorylation. In response to intracellular iron deficiency, the iron regulatory protein 2 (IRP2) was shown in Western blotting to be unchanged in the rescue group (123 ± 4.0% of the control group), but increased in the iron-deficient group (170 ± 20% of the control group). The qRT-PCR analyses showed that mRNA expression of iron-sulfur cluster assembly enzyme (ISCU) was significantly decreased in the iron-deficiency group compared with the control group (41.8%), but not in the rescue group (95.1%), which is presumed that IRP2 suppressed ISCU expression, as has been proved previously. These results should indicate that iron deficiency suppressed iron utilization in the mitochondria of PTECs. The number of viable PTECs in the iron-deficient group after 24 hours was 1.99 ± 0.09 × 10^5 cells/well, which was significantly lower than that of the control (3.15 ± 0.15 × 10^5 cells/well) and rescue (2.68 ± 0.18 × 10^5 cells/well) groups. On the other hand, the number of dead PTECs was 0.042 ± 0.019 × 10^5 cells in the iron-deficient group, which was not significantly different from the control (0.023 ± 0.005 × 10^5 cells) and the rescue (0.020 ± 0.011 × 10^5 cells) groups. These data suggest that cell growth should be suppressed by iron deficiency. Conclusion Mitochondrial oxidative phosphorylation through the MRC was impaired by iron deficiency in PTECs. This impairment of mitochondrial capacity in PTECs may lead to PTECs dysfunction, potentially inducing renal damage such as cell death and interstitial injury. Further analysis of the effects of this mitochondrial dysfunction on ATP production capacity and reactive oxygen species production is needed.

Upregulation of cholesterol synthesis by lysosomal defects requires a functional mitochondrial respiratory chain

Mitochondria and lysosomes are two organelles that carry out both signaling and metabolic roles in cells. Recent evidence has shown that mitochondria and lysosomes are dependent on one another, as primary defects in one cause secondary defects in the other. Although there are functional impairments in both cases, the signaling consequences of primary mitochondrial dysfunction and lysosomal defects are dissimilar. Here, we used RNA sequencing to obtain transcriptomes from cells with primary mitochondrial or lysosomal defects to identify the global cellular consequences associated with mitochondrial or lysosomal dysfunction. We used these data to determine the pathways affected by defects in both organelles, which revealed a prominent role for the cholesterol synthesis pathway. We observed a transcriptional upregulation of this pathway in cellular and murine models of lysosomal defects, while it is transcriptionally downregulated in cellular and murine models of mitochondrial defects. We identified a role for the posttranscriptional regulation of transcription factor SREBF1, a master regulator of cholesterol and lipid biosynthesis, in models of mitochondrial respiratory chain deficiency. Furthermore, we found that retention of Ca2+ in lysosomes of cells with mitochondrial respiratory chain defects contributes to the differential regulation of the cholesterol synthesis pathway in the mitochondrial and lysosomal defects tested. Finally, we verified invivo, using a model of mitochondria-associated disease in Caenorhabditis elegans that normalization of lysosomal Ca2+ levels results in partial rescue of the developmental delay induced by the respiratory chain deficiency.