Respiratory Chain Enzyme Research Articles

Mitochondria and ALS

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10), a mitochondrial protein of unknown function, cause a disease spectrum with clinical features of motor neuron disease, dementia, myopathy and cardiomyopathy. To investigate the pathogenic mechanisms of CHCHD10, we generated mutant knock-in mice harboring the mouse-equivalent of a disease associated human S59L mutation, S55L in the endogenous mouse gene. CHCHD10S55Lmice develop progressive motor deficits, myopathy, cardiomyopathy and accelerated mortality. Critically, CHCHD10 accumulates in aggregates with its paralog CHCHD2 specifically in affected tissues of CHCHD10S55Lmice, leading to aberrant organelle morphology and function. Aggregates induce a potent mitochondrial integrated stress response (mtISR) through mTORC1 activation, with elevation of stress-induced transcription factors, secretion of myokines, upregulated serine and one-carbon metabolism, and downregulation of respiratory chain enzymes. Conversely, CHCHD10 ablation does not induce disease pathology or activate the mtISR, indicating that CHCHD10S55L-dependent disease pathology is not caused by loss-of-function. Overall, CHCHD10S55Lmice recapitulate crucial aspects of human disease and reveal a novel toxic gain-of-function mechanism through maladaptive mtISR and metabolic dysregulation.

P.109Congenital myopathy in patients with Kabuki and Au-Kline syndromes - Double trouble or expansion of the phenotypes?

Congenital myopathies (CM) are heterogeneous neuromuscular disorders with hypotonia and weakness, often from birth, classified on the morphological features on muscle biopsy. Cardiac muscle involvement, facial dysmorphisms and involvement of the heart and other organs is well recognized. Here we describe 5 unrelated patients with clinical and histopathological features of CM, assessed in two tertiary UK neuromuscular centers, later diagnosed with Kabuki (KS) or Au-Kline syndrome (AUS). KS and AUS are multisystem disorders, with overlapping features such as distinctive facial dysmorphisms, hypotonia, intellectual disability, growth and skeletal defects and congenital heart defects (CHD). Age of patients ranged from 2 to 13 years. Onset was at birth, with hypotonia, global developmental delay, dysmorphisms, CHD and severe feeding difficulties. On examination patients were hypotonic, but muscle strength was overall satisfactory. Three patients were hypermobile, one had hemivertebrae and one congenital hip dislocation. Microcephaly, Chiari I malformation and craniosynostosis was seen in one patient, each. Three patients had hearing loss. One patient had 3 rhabdomyolysis episodes triggered by viral infections. Respiratory chain enzyme analysis, performed in 3 patients, was normal. Analysis of genes for CM and syndromic conditions with learning disability and CHD identified pathogenic heterozygous variants in the KMT2D (3 patients with KS) or HNRNPK gene (1 patient with AUS), confirming their diagnoses (test ongoing in 5th patient). Muscle biopsies in 3 KMT2D patients showed mild non-specific changes and increased central nuclei with markedly abnormal myofibre architecture in the third case. Biopsy of the patient with variants in HNRNPK showed striking congenital fibre type disproportion. This is the first report of myopathology in KS and AUS patients and might suggest a link between the proteins disrupted in these conditions and muscle structure.

Mutations in NDUFS1 Cause Metabolic Reprogramming and Disruption of the Electron Transfer.

Complex I (CI) is the first enzyme of the mitochondrial respiratory chain and couples the electron transfer with proton pumping. Mutations in genes encoding CI subunits can frequently cause inborn metabolic errors. We applied proteome and metabolome profiling of patient-derived cells harboring pathogenic mutations in two distinct CI genes to elucidate underlying pathomechanisms on the molecular level. Our results indicated that the electron transfer within CI was interrupted in both patients by different mechanisms. We showed that the biallelic mutations in NDUFS1 led to a decreased stability of the entire N-module of CI and disrupted the electron transfer between two iron–sulfur clusters. Strikingly interesting and in contrast to the proteome, metabolome profiling illustrated that the pattern of dysregulated metabolites was almost identical in both patients, such as the inhibitory feedback on the TCA cycle and altered glutathione levels, indicative for reactive oxygen species (ROS) stress. Our findings deciphered pathological mechanisms of CI deficiency to better understand inborn metabolic errors.

Detection of novel mitochondrial mutations in cytochrome C oxidase subunit 1 (COX1) in patients with familial adenomatous polyposis (FAP).

Familial adenomatous polyposis (FAP) is an Autosomal dominant inherited disorder and a rare form‌ of colorectal cancer (CRC) that is characterized by the development of hundreds to thousands of adenomas in the rectum and colon. Mostly, cancers develop after the advent of the polyps. It appears in both sexes evenly, and the occurrence of the disease is in the second decade of life. Mitochondrial genome mutations have been reported with a variety of Tumors, but the precise role of these mutations in the pathogenicity and tumor progression is not exactly clear. Cytochrome c oxidase subunit I (COX1) is the terminal enzyme of the mitochondrial respiratory chain. The present study aims at assessing the occurrence of mtDNA mutations in COX1 gene in FAP patients and attempts to find out the cause and effect relationship between mitochondrial mutations and tumor progression. In this study, 56 FAP patients were investigated for the presence of the mutations in mitochondrial COX1 coding gene by PCR and sequencing analysis. All sequences that differed from the revised Cambridge Reference Sequence (rCRS) were classified as missense/ nonsense or silent mutations. Functional genomic studies using Bio-informatics tools were performed on the founded mutations to understand the downstream alterations in structure and function of protein. We identified 38 changes in the COX1 gene in patients with FAP symptoms. Most of them were heteroplasmic changes of missense type (25/38). Tree of the changes (G6145A, C6988A, and T7306G) were nonsense mutations and had not been reported in the literature before. Our results of bioinformatics predictions showed that the identified mutations can affect mitochondrial functions, especially if the conservative domain of the protein is concerned. Our findings indicate a high frequency of mtDNA mutations in all of the FAP cases compared to matched controls. These data significantly enhance our understanding of how such mutations contribute to cancer pathologies and develop the cancer treatment methods by new diagnostic biomarkers, and new drugs for gene therapy.

Antimicrobial activities of biologically synthesized metal nanoparticles: an insight into the mechanism of action

Increasing antimicrobial resistance is a clinical crisis worldwide. Recent progress in the field of green synthesis has fascinated scientists and researchers to explore its potentials against pathogenic microbes. Bioinspired-metal-based nanoparticles (silver, copper, gold, zinc, etc.) have been reported to be tested against both Gram-positive and Gram-negative bacteria such as B. subtilis, E. coli, Staphylococcus aureus, etc., as well as some pathogenic fungi including A. niger, F. oxysporum, A. fumigatus, etc., and are testified to exhibit inhibitory effects against pathogenic microbes. The possible modes of action of these metal nanoparticles include: (a) excess production of reactive oxygen species inside microbes; (b) disruption of vital enzymes in respiratory chain via damaging microbial plasma membranes; (c) accumulation of metal ions in microbial membranes; (d) electrostatic attraction between metal nanoparticles and microbial cells which disrupt metabolic activities; and (e) inhibition of microbial proteins/enzymes by increased production of H2O2. Although these pathways are interconnected, information on potential mechanism of most of these biogenic nanoparticles is still limited. Further exploration of these mechanisms could help in tackling the burning issue of antibiotics resistance.

Study of the effect of polyelectrolytes with antiviral effect on the activity of influenza virus neuraminidase and the process of oxidative phosphorylation in mitochondrias of cells host organism

Aim. Study of the inhibitory effect of polyelectrolytes with antiviral effect against influenza neuraminidase. Determination of the type and inhibition constant. The study of the effect of polyelectrolytes (PE) on the process of oxidative phosphorylation in the mitochondria of cells host organism. Materials and methods. Purified influenza virus strains were used: A/VPCH/Weybridge (H7N7), A/Mallard Pennsylvania/10218/84 (H5N2), A/NIBRG-14 (H5N1) with an initial infectious titer of 4,5 lgTCD50/ml. PE solutions of polystyrenesulfonate with a degree of polymerization of 8 (PSS-8) in concentrations of 0,5—4,0 mM and polyallylamine (6 kDa) PAA (6 kDa) in concentrations of 0,5—4,0 μM. To determine the activity of influenza neuraminidase, influenza virus strains were used after the removal of low molecular weight inhibitors of neuraminidase by dialysis against water. The neuraminidase substrate was fetuin at final concentrations of 0,052 to 1,2 μM for PAA (6 kDa) and from 0,052 to 1,2 mM for PSS-8. As quantitative characteristics of respiration and phosphorylation of mitochondria, respiratory coefficients according to Lardi-Velman and Chans-Williams, as well as the ratio of ADP/O were used. Mitochondria were isolated from skeletal muscle. The determination of respiratory coefficients and the ratio of ADP/O) was determined by the polarographic method. Results. A noncompetitive type of inhibition of these PEs was detected in relation to the neuraminidase activity of influenza viruses with inhibition constants KI = 1,6 ± 0.08 μM for PAA (6 kDa) and KI = 1,7 ± 0.085 mM for PSS-8. Respiratory coefficients and the ratio of ADP / 0 were determined in the absence and after addition of PSS-8 and PAA (6 kDa) to mitochondria at concentrations of 20 mM and 10 μM, respectively. A decrease in respiratory coefficients and an ADP/O ratio was observed, indicating an inhibition of the enzymes of the electron-transport chain of mitochondria. At concentrations of less than 20 mM and 10 μM for PSS-8 and PAA (6 kDa), all indicators did not significantly change. Conclusion. The non-competitive mechanism of inhibition of the neuraminidase activity of the influenza PE viruses is explained by the conformational changes in the molecules of the enzyme and/or enzyme-substrate complex and, accordingly, the structural and functional changes in its secondary structure. When going beyond the range of non-toxic concentrations of20 mM for PSS-8 and 10 μM for PAA (6 kDa), inhibition of the mitochondrial respiratory chain enzymes was observed while maintaining the antiviral effect.

Protective mechanisms of melatonin on caprine spleen injury induced by cadmium (Cd): an in vitro study

Current study explores
 the potential mechanisms of melatonin on cadmium-induced spleen tissue injury
 of goat. Spleen tissues were incubated with different concentrations (50, 100,
 200, 400 and 600 µM) of cadmium acetate (Cd), respectively and the lipid
 peroxidation of the tissue was measured. It was found that Cd at the level of
 400 µM induced maximum spleen damage among other concentrations. Thus, Cd 400
 µM was selected to examine whether melatonin treatment can protect against this
 damage. The results showed that Cd increased the oxidative stress in the spleen
 tissue either by elevating pro-oxidant enzymes, or, by suppressing the variety
 of antioxidant enzymes and thus, to increase the intracellular reactive oxygen
 species (ROS). Melatonin treatment at the concentrations of 0.25, 0.5 and 1 mM
 significantly reduced all these alterations, respectively. At the level of
 cellular organelles, Cd caused mitochondrial morphological and functional
 injuries. These include mitochondrial surface distortion and inhibitions of glycolytic,
 Krebs cycle, and respiratory chain enzymes. Melatonin at a concentration of 0.5
 mM almost completely preserved Cd induced mitochondrial pathological
 alterations. Cd
 pollution is a cause of serious health hazard world wide, particularly in the
 developing areas and currently, there is no specific remedy for Cd toxicities.
 The results suggest that melatonin is a promising therapeutic agent to
 combat Cd-induced oxidative stress and it deserves further investigation
 clinically.

Arabidopsis SCO Proteins Oppositely Influence Cytochrome c Oxidase Levels and Gene Expression during Salinity Stress.

SCO (synthesis of cytochrome c oxidase) proteins are involved in the insertion of copper during the assembly of cytochrome c oxidase (COX), the final enzyme of the mitochondrial respiratory chain. Two SCO proteins, namely, homolog of copper chaperone 1 and 2 (HCC1 and HCC2) are present in seed plants, but HCC2 lacks the residues involved in copper binding, leading to uncertainties about its function. In this study, we performed a transcriptomic and phenotypic analysis of Arabidopsis thaliana plants with reduced expression of HCC1 or HCC2. We observed that a deficiency in HCC1 causes a decrease in the expression of several stress-responsive genes, both under basal growth conditions and after applying a short-term high salinity treatment. In addition, HCC1 deficient plants show a faster decrease in chlorophyll content, photosystem II quantum efficiency and COX levels after salinity stress, as well as a faster increase in alternative oxidase capacity. Notably, HCC2 deficiency causes opposite changes in most of these parameters. Bimolecular fluorescence complementation analysis indicated that both proteins are able to interact. We postulate that HCC1 is a limiting factor for COX assembly during high salinity conditions and that HCC2 probably acts as a negative modulator of HCC1 activity through protein-protein interactions. In addition, a direct or indirect role of HCC1 and HCC2 in the gene expression response to stress is proposed.

Prospects for the Use of Thermal Extracts of Brucella abortusI-206 in S-and L-Forms in the Diagnosis and Prevention of Brucellosis

Currently, one of the topical areas of research is the development of new antigen preparations for the specific diagnosis and prevention of brucellosis, since indication of the pathogen and prevention of the disease is complicated by the ability of brucella to dissociate, and live vaccines used for specific prevention of brucellosis have residual virulence. Thermal extracts (TE) obtained from Brucella abortus I-206 in the L- and S-form can be used as such promising antigens. It is known that TE in the L- and S-forms have immunogenic properties, as well as a modulating effect on the proliferation of immunocompetent cells, morphological changes in the immunocompetent organs of experimental animals.The aimof the work is to study the effect of Brucella abortus thermal extracts in L- and S-forms on the functional state of the cells of experimental animals.Materials and methods. The study was performed on 100 certified white mice. As objects of study, we used the B. abortus I-206 TE in L- and S-forms. Evaluation of the effect of antigenic drugs on the functional state of phagocytes of laboratory animals in vitro was performed on peritoneal macrophages. The total activity of the respiratory chain enzymes in the NBT-test and superoxide dismutase was determined. Cells of intact animals served as controls. As a positive control, a commercial antigenic LPS preparation Escherichia coli was used. The content of cyclic nucleotides in homogenates of immunocompetent organs was determined using ELISA.Results.This study presents materials on the study of the effect of TE on the bactericidal activity of phagocytes and the level of cyclic nucleotides in immunocompetent organs. It has been established that TEs activate oxygen-dependent bactericidal systems of phagocytes. When studying the effect of TE on the content of cyclic nucleotides in immunocompetent organs of white mice, an increase in their concentration was revealed, indicating an increase in the functional activity of the cells.Conclusion.The obtained data make it possible to substantiate the need for a further detailed study of the immunogenic properties of B. abortus TE in the L- or S-form on the organism of experimental animals.

Biallelic variants in COX4I1 associated with a novel phenotype resembling Leigh syndrome with developmental regression, intellectual disability, and seizures

Autosomal recessive COX4I1 deficiency has been previously reported in a single individual with a homozygous pathogenic variant in COX4I1, who presented with short stature, poor weight gain, dysmorphic features, and features of Fanconi anemia. COX4I1 encodes subunit 4, isoform 1 of cytochrome c oxidase. Cytochrome c oxidase is a respiratory chain enzyme that plays an important role in mitochondrial electron transport and reduces molecular oxygen to water leading to the formation of ATP. Defective production of cytochrome c oxidase leads to a variable phenotypic spectrum ranging from isolated myopathy to Leigh syndrome. Here, we describe two siblings, born to consanguineous parents, who presented with encephalopathy, developmental regression, hypotonia, pathognomonic brain imaging findings resembling Leigh-syndrome, and a novel homozygous variant on COX4I1, expanding the known clinical phenotype associated with pathogenic variants in COX4I1.

Time-resolved ATP measurements during vesicle respiration

In vitro synthesis of ATP catalyzed by the ATP-synthase requires membrane vesicles, in which the ATP-synthase is present within the bilayer membrane. Inverted vesicle prepared from Gram negative cells (e.g., Escherichia coli or Pseudomonas putida) can be readily obtained and used for in vitro ATP-synthesis. Up to now, quantification of ATP synthesized by membrane vesicles has been mostly analyzed via bioluminescence-based assays. Alternatively, vesicle respiration and the associated ATP level can be determined using biosensors, which not only provide high selectivity, but allow ATP measurements without the sample being illuminated. Here, we present a microbiosensor for ATP in combination with scanning electrochemical microscopy (SECM) using an innovative two-compartment electrochemical cell for the determination of ATP levels at E.coli or P. putida inverted vesicles. For a protein concentration of 22 mg/ml, a total amount of 0.29 ± 0.03 μM/μl ATP per vesicle was determined in case of E.coli; in turn, P. putida derived vesicles yielded 0.48 ± 0.02 μM/μl ATP per vesicle at a total protein concentration of 25.2 mg/ml. Inhibition experiments with Venturicidin A clearly revealed that the respiratory chain enzyme complex responsible for ATP generation is effectively involved.

Synthesis and Biological Assessment of Pyrrolobenzoxazine Scaffold as a Potent Antioxidant.

Reduction of mitochondrial oxidative stress-mediated diseases is an important pharmaceutical objective in recent biomedical research. In this context, a series of novel pyrrolobenzoxazines (PyBs) framework with enormous diversity (compounds 5a-w) was synthesized by employing a low-temperature greener pathway, and antioxidant property of the synthesized compounds was successfully demonstrated on preclinical model goat heart mitochondria, in vitro. Copper-ascorbate (Cu-As) was utilized as an oxidative stress generator. Out of screened PyBs, the compound possessing -OH and -OMe groups on benzene nucleus along with pyrrolobenzoxazine core moiety (compound 5w) displayed magnificent antioxidant property with a minimum effective dose of 66 μM during the biochemical assessment. The ameliorative effect of synthesized pyrrolobenzoxazine moiety on levels of biomarkers of oxidative stress, antioxidant enzyme, activities of Krebs cycle and respiratory chain enzymes, mitochondrial morphology, and Ca2+ permeability of mitochondrial membrane was investigated in the presence of Cu-As. Furthermore, the binding mode of Cu-As by compound 5w was explored successfully using isothermal titration calorimetry (ITC) analysis.

Exogenous H2S reduces the acetylation levels of mitochondrial respiratory enzymes via regulating the NAD+-SIRT3 pathway in cardiac tissues of db/db mice.

Hydrogen sulfide (H2S), a gaseous molecule, is involved in modulating multiple physiological functions, such as antioxidant, antihypertension, and the production of polysulfide cysteine. H2S may inhibit reactive oxygen species generation and ATP production through modulating respiratory chain enzyme activities; however, the mechanism of this effect remains unclear. In this study, db/db mice, neonatal rat cardiomyocytes, and H9c2 cells treated with high glucose, oleate, and palmitate were used as animal and cellular models of type 2 diabetes. The mitochondrial respiratory rate, respiratory chain complex activities, and ATP production were decreased in db/db mice compared with those in db/db mice treated with exogenous H2S. Liquid chromatography with tandem mass spectrometry analysis showed that the acetylation level of proteins involved in the mitochondrial respiratory chain were increased in the db/db mice hearts compared with those with sodium hydrosulfide (NaHS) treatment. Exogenous H2S restored the ratio of NAD+/NADH, enhanced the expression and activity of sirtuin 3 (SIRT3) and decreased mitochondrial acetylation level in cardiomyocytes under hyperglycemia and hyperlipidemia. As a result of SIRT3 activation, acetylation of the respiratory complexe enzymes NADH dehydrogenase 1 (ND1), ubiquinol cytochrome c reductase core protein 1, and ATP synthase mitochondrial F1 complex assembly factor 1 was reduced, which enhanced the activities of the mitochondrial respiratory chain activity and ATP production. We conclude that exogenous H2S plays a critical role in improving cardiac mitochondrial function in diabetes by upregulating SIRT3.

Ursolic acid modulates MMPs, collagen-I, α-SMA, and TGF-β expression in isoproterenol-induced myocardial infarction in rats.

In the present study, the modulatory effect of ursolic acid (UA) on cardiac fibrosis and mitochondrial and lysosomal enzymes activity in isoproterenol-induced myocardial infarction (MI) in rats were examined. Isoproterenol hydrochloride (ISO; 85 mg/kg body weight) was administered subcutaneously for first two consecutive days. ISO-induced MI in rats significantly decreased the activities of mitochondrial tricarboxylic acid cycle enzymes and respiratory chain enzymes while increased the activities of lysosomal glycohydrolases and cathepsins. The expression of matrix metalloproteinase 2 (MMP-2), MMP-9, collagen type I, α-smooth muscle actin (α-SMA), and transforming growth factor-β (TGF-β) were upregulated in ISO-induced MI in rats. UA administration to rats showed increased activities of mitochondrial tricarboxylic acid cycle enzymes and respiratory chain enzymes and decreased activities of lysosomal glycohydrolases and cathepsins in ISO-induced rats. Furthermore, expression of MMP-2, MMP-9, collagen type I, α-SMA, and TGF-β downregulated in UA-administered rats. Thus, our results demonstrate that UA has an anti-fibrotic effect and attenuates the mitochondrial and lysosomal dysfunction in ISO-induced MI in rats.

Primary Coenzyme Q deficiency Due to Novel ADCK3 Variants, Studies in Fibroblasts and Review of Literature.

Primary deficiency of coenzyme Q10 (CoQ10 ubiquinone), is classified as a mitochondrial respiratory chain disorder with phenotypic variability. The clinical manifestation may involve one or multiple tissue with variable severity and presentation may range from infancy to late onset. ADCK3 gene mutations are responsible for the most frequent form of hereditary CoQ10 deficiency (Q10 deficiency-4 OMIM #612016) which is mainly associated with autosomal recessive spinocerebellar ataxia (ARCA2, SCAR9). Here we provide the clinical, biochemical and genetic investigation for unrelated three nuclear families presenting an autosomal form of Spino-Cerebellar Ataxia due to novel mutations in the ADCK3 gene. Using next generation sequence technology we identified a homozygous Gln343Ter mutation in one family with severe, early onset of the disease and compound heterozygous mutations of Gln343Ter and Ser608Phe in two other families with variable manifestations. Biochemical investigation in fibroblasts showed decreased activity of the CoQ dependent mitochondrial respiratory chain enzyme succinate cytochrome c reductase (complex II + III). Exogenous CoQ slightly improved enzymatic activity, ATP production and decreased oxygen free radicals in some of the patient's cells. Our results are presented in comparison to previously reported mutations and expanding the clinical, molecular and biochemical spectrum of ADCK3 related CoQ10 deficiencies.

Novel compound mutations in the mitochondrial translation elongation factor (TSFM) gene cause severe cardiomyopathy with myocardial fibro-adipose replacement

Primary mitochondrial dysfunction is an under-appreciated cause of cardiomyopathy, especially when cardiac symptoms are the unique or prevalent manifestation of disease. Here, we report an unusual presentation of mitochondrial cardiomyopathy, with dilated phenotype and pathologic evidence of biventricular fibro-adipose replacement, in a 33-year old woman who underwent cardiac transplant. Whole exome sequencing revealed two novel compound heterozygous variants in the TSFM gene, coding for the mitochondrial translation elongation factor EF-Ts. This protein participates in the elongation step of mitochondrial translation by binding and stabilizing the translation elongation factor Tu (EF-Tu). Bioinformatics analysis predicted a destabilization of the EF-Ts variants complex with EF-Tu, in agreement with the dramatic steady-state level reduction of both proteins in the clinically affected myocardium, which demonstrated a combined respiratory chain enzyme deficiency. In patient fibroblasts, the decrease of EF-Ts was paralleled by up-regulation of EF-Tu and induction of genes involved in mitochondrial biogenesis, along with increased expression of respiratory chain subunits and normal oxygen consumption rate. Our report extends the current picture of morphologic phenotypes associated with mitochondrial cardiomyopathies and confirms the heart as a main target of TSFM dysfunction. The compensatory response detected in patient fibroblasts might explain the tissue-specific expression of TSFM-associated disease.

Photobiomodulation enhancement of cell proliferation at 660 nm does not require cytochrome c oxidase

Photobiomodulation (PBM) therapy is based on the use of specific light parameters to promote tissue repair. Although demonstrated in different cell models and tissues, the mechanism by which photobiomodulation operates is not well understood. Previous studies suggested that the cell proliferation enhancement triggered by red and near-infrared PBM involves the activation of the mitochondrial respiratory chain enzyme cytochrome c oxidase (CCO). It was suggested that light in this range would displace inhibitory nitric oxide bound to CCO. To test this mechanism, we took advantage of cell lines lacking CCO, including a mouse line knockout for Cox10 (a gene required for the synthesis of heme a, the prosthetic group of CCO) and a human cell line with an mtDNA mutation in the tRNA Lysine gene, leading to mitochondrial protein synthesis impairment and the lack of three critical CCO subunits. In both models we showed the complete absence of assembled CCO. PBM (660 nm) was applied to these proliferating cells using various parameters. In most of the conditions tested, increased cell proliferation was associated with PBM in both control and CCO negative cells, demonstrating that CCO is not required for PBM enhancement of cellular proliferation. Additional experiments showed that PBM increased both ATP levels and citrate synthase activity and levels. These results showed that although metabolic changes are associated with PBM, CCO is not required for its cell proliferation enhancing effect.

Age-dependent expression of the vitamin D receptor and the protective effect of vitamin D receptor activation on H2O2-induced apoptosis in rat intervertebral disc cells

Accumulating evidence shows that genetic polymorphism of the vitamin D receptor (VDR) gene is associated with intervertebral disc degeneration (IDD), implying that VDR may be involved in the pathogenesis of IDD. However, the exact relationship between VDR and IDD remains unknown. The aim of this study was to investigate the age-dependent expression of VDR in rat intervertebral discs and to determine the effect of VDR on oxidative stress-induced cell apoptosis of the annulus fibrosus (AF) and the underlying mechanism. Sprague-Dawley rats were subjected to magnetic resonance imaging (MRI) and CT scans at young (2–3 months), adult (6–7 months), and old (14–15 months) ages. The images revealed age-related degeneration of the lumbar intervertebral discs and endplates. Immunohistochemistry demonstrated positive expression of VDR in the AF. The expression level of VDR in aged rats was significantly reduced compared with that in the young and adult animals and exhibited a negative correlation to IDD severity. Western blot analysis further demonstrated that the amount of VDR protein was significantly decreased in severe degenerative discs. AF cells were also isolated from young rat lumbar discs and subjected to different concentrations of hydrogen peroxide (H2O2) for various amounts of time. The results revealed that H2O2 inhibited the viability of AF cells and induced mitochondrial pathway apoptosis. However, pretreatment of AF cells with 10−7 and 10-8 M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] effectively increased cell viability, increased mitochondrial membrane potential, decreased the level of reactive oxygen species, increased mitochondrial ATP content, reserved the activity of key enzymes in the oxidative respiratory chain, and thus protected the mitochondria from H2O2-induced damage. Whereas, siRNA knock-down of VDR abolished the protective effects of 1,25(OH)2D3. Moreover, 1,25(OH)2D3 inhibited H2O2-induced autophagy of AF cells through inhibition of the mTOR/p70S6K signal pathway. Our study demonstrated that decreased expression of VDR may play a role in age-related intervertebral disc degeneration in rats and that activation of VDR ameliorates oxidative stress-induced apoptosis in AF cells by preserving mitochondrial functions.

ALS/FTD mutant CHCHD10 mice reveal a tissue-specific toxic gain-of-function and mitochondrial stress response.

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10), a mitochondrial protein of unknown function, cause a disease spectrum with clinical features of motor neuron disease, dementia, myopathy and cardiomyopathy. To investigate the pathogenic mechanisms of CHCHD10, we generated mutant knock-in mice harboring the mouse-equivalent of a disease-associated human S59L mutation, S55L in the endogenous mouse gene. CHCHD10S55L mice develop progressive motor deficits, myopathy, cardiomyopathy and accelerated mortality. Critically, CHCHD10 accumulates in aggregates with its paralog CHCHD2 specifically in affected tissues of CHCHD10S55L mice, leading to aberrant organelle morphology and function. Aggregates induce a potent mitochondrial integrated stress response (mtISR) through mTORC1 activation, with elevation of stress-induced transcription factors, secretion of myokines, upregulated serine and one-carbon metabolism, and downregulation of respiratory chain enzymes. Conversely, CHCHD10 ablation does not induce disease pathology or activate the mtISR, indicating that CHCHD10S55L-dependent disease pathology is not caused by loss-of-function. Overall, CHCHD10S55L mice recapitulate crucial aspects of human disease and reveal a novel toxic gain-of-function mechanism through maladaptive mtISR and metabolic dysregulation.

Heart failure and iron deficiency

Heart failure with reduced ejection fraction (HFrEF) is a clinical reality with an incidence of >10% in the population over 65 years of age, expected to increase in the coming years. Alongside the well-known pharmacological options (e.g. angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, neprilysin inhibitors, beta-blockers, aldosterone antagonists), which have significantly improved the clinical and prognostic outcomes of HFrEF patients, we now have a new therapeutic target: iron deficiency, defined as a ferritin concentration <100 μg/l or between 100-300 μg/l in the presence of a transferrin saturation <20%. Iron plays a major role in the transport of oxygen as a component of hemoglobin, as an oxygen reservoir, and as a component of myoglobin, as well as in the formation of energy, as a constituent of respiratory chain enzymes. Iron deficiency can therefore lead to anemia, changes in cognitive performance, behavior, emotions, reduced exercise capacity and myocardial structural and functional changes. Several clinical studies have shown that intravenous iron carboxymaltose supplementation can improve anemia, NYHA class, quality of life and exercise capacity of HFrEF patients. There are no randomized studies of adequate sample size that positively correlate iron supplementation with the higher endpoints of mortality and morbidity both in chronic and acute heart failure. Several studies are ongoing to answer these questions in the next few years.