Chronic melatonin treatment prevents age-dependent cardiac mitochondrial dysfunction in senescence-accelerated mice

Heart mitochondria from female senescence-accelerated (SAMP8) and senescence-resistant (SAMR1) mice of 5 or 10 months of age, were studied. Mitochondrial oxidative stress was determined by measuring the levels of lipid peroxidation, glutathione and glutathione disulfide and glutathione peroxidase and reductase activities. Mitochondrial function was assessed by measuring the activity of the respiratory chain complexes and ATP content. The results show that the age-dependent mitochondrial oxidative damage in the heart of SAMP8 mice was accompanied by a reduction in the electron transport chain complex activities and in ATP levels. Chronic melatonin administration between 1 and 10 months of age normalized the redox and the bioenergetic status of the mitochondria and increased ATP levels. The results support the presence of significant mitochondrial oxidative stress in SAM mice at 10 months of age, and they suggest a beneficial effect of chronic pharmacological intervention with melatonin, which reduces the deteriorative and functional oxidative changes in cardiac mitochondria with age.     

Curcumin prevents mitochondrial dysfunction in the brain of the senescence-accelerated mouse-prone 8

The aging brain suffers mitochondrial dysfunction and a reduced availability of energy in the form of ATP, which in turn may cause or promote the decline in cognitive, sensory, and motor function observed with advancing age. There is a need for animal models that display some of the pathological features of human brain aging in order to study their prevention by e.g. dietary factors. We thus investigated the suitability of the fast-aging senescence-accelerated mouse-prone 8 (SAMP8) strain and its normally aging control senescence-accelerated mouse-resistant 1 (SAMR1) as a model for the age-dependent changes in mitochondrial function in the brain. To this end, 2-months old male SAMR1 (n = 10) and SAMP8 mice (n = 7) were fed a Western type diet (control groups) for 5 months and one group of SAMP8 mice (n = 6) was fed an identical diet fortified with 500 mg curcumin per kg. Dissociated brain cells and brain tissue homogenates were analyzed for malondialdehyde, heme oxygenase-1 mRNA, mitochondrial membrane potential (MMP), ATP concentrations, protein levels of mitochondrial marker proteins for mitochondrial membranes (TIMM, TOMM), the mitochondrial permeability transition pore (ANT1, VDAC1, TSPO), respiration complexes, and fission and fusion (Fis, Opa1, Mfn1, Drp1). Dissociated brain cells isolated from SAMP8 mice showed significantly reduced MMP and ATP levels, probably due to significantly diminished complex V protein expression, and increased expression of TSPO. Fission and fusion marker proteins indicate enhanced mitochondrial fission in brains of SAMP8 mice. Treatment of SAMP8 mice with curcumin improved MMP and ATP and restored mitochondrial fusion, probably by up-regulating nuclear factor PGC1α protein expression. In conclusion, SAMP8 compared to SAMR1 mice are a suitable model to study age-dependent changes in mitochondrial function and curcumin emerges as a promising nutraceutical for the prevention of neurodegenerative diseases that are accompanied or caused by mitochondrial dysfunction.     

Diastolic dysfunction is associated with cardiac fibrosis in the senescence-accelerated mouse

Diastolic heart failure is a major cause of mortality in the elderly population. It is often preceded by diastolic dysfunction, which is characterized by impaired active relaxation and increased stiffness. We tested the hypothesis that senescence-prone (SAMP8) mice would develop diastolic dysfunction compared with senescence-resistant controls (SAMR1). Pulsed-wave Doppler imaging of the ratio of blood flow velocity through the mitral valve during early (E) vs. late (A) diastole was reduced from 1.3 ± 0.03 in SAMR1 mice to 1.2 ± 0.03 in SAMP8 mice (P < 0.05). Tissue Doppler imaging of the early (E') and late (A') diastolic mitral annulus velocities found E' reduced from 25.7 ± 0.9 mm/s in SAMR1 to 21.1 ± 0.8 mm/s in SAMP8 mice and E'/A' similarly reduced from 1.1 ± 0.02 to 0.8 ± 0.03 in SAMR1 vs. SAMP8 mice, respectively (P < 0.05). Invasive hemodynamics revealed an increased slope of the end-diastolic pressure-volume relationship (0.5 ± 0.05 vs. 0.8 ± 0.14; P < 0.05), indicating increased left ventricular chamber stiffness. There were no differences in systolic function or mean arterial pressure; however, diastolic dysfunction was accompanied by increased fibrosis in the hearts of SAMP8 mice. In SAMR1 vs. SAMP8 mice, interstitial collagen area increased from 0.3 ± 0.04 to 0.8 ± 0.09% and perivascular collagen area increased from 1.0 ± 0.11 to 1.6 ± 0.14%. Transforming growth factor-β and connective tissue growth factor gene expression were increased in the hearts of SAMP8 mice (P < 0.05 for all data). In summary, SAMP8 mice show increased fibrosis and diastolic dysfunction similar to those seen in humans with aging and may represent a suitable model for future mechanistic studies.     

Unique mutations in mitochondrial DNA of senescence-accelerated mouse (SAM) strains

Mitochondrial DNA (mtDNA) is exclusively inherited maternally and hence could offer a good method for tracing the lineage of mouse strains. We examined the mtDNA sequence of senescence-accelerated mouse (SAM) strains as well as other laboratory strains of inbred mice to deduce the ancestral strain of SAM. Four unique mutations were identified at bases 2256, 10,847, 11,181, and 13,053 in SAM strains. The mutations were not found in other mouse strains including AKR/J, one of the parental strains of SAM. Comparison of the mtDNA sequences also led to the consensus mtDNA sequence of laboratory strains of inbred mice. The seven laboratory strains of common inbred mice showed polymorphisms at base 9348, thymine repeat from base 9818, and adenine repeat from base 9821, and could be classified into five types by combination of the differences. Although we could not identify mouse strains with the same type of mtDNA as SAM in this study, the polymorphisms would provide a promising clue to ascertain the ancestral strain(s) of SAM. The polymorphism in mtDNA could be used to ascertain the genealogy of other mouse strains as well.     

Comprehensive proteomics analysis of autophagy-deficient mouse liver

Autophagy is a bulk protein degradation system for the entire organelles and cytoplasmic proteins. Previously, we have shown the liver dysfunction by autophagy deficiency. To examine the pathological effect of autophagy deficiency, we examined protein composition and their levels in autophagy-deficient liver by the proteomic analysis. While impaired autophagy led to an increase in total protein mass, the protein composition was largely unchanged, consistent with non-selective proteins/organelles degradation of autophagy. However, a series of oxidative stress-inducible proteins, including glutathione S-transferase families, protein disulfide isomerase and glucose-regulated proteins were specifically increased in autophagy-deficient liver, probably due to enhanced gene expression, which is induced by accumulation of Nrf2 in the nuclei of mutant hepatocytes. Our results suggest that autophagy deficiency causes oxidative stress, and such stress might be the main cause of liver injury in autophagy-deficient liver.     

Superoxide dismutase in senescence-accelerated mouse retina

To examine the relationship between retinal ageing and superoxide dismutase, we studied the dismutase, with immunohistochemistry and immunoquantitative analysis, in the retina of senescence-accelerated mice P8/Ta (SAMP8/Ta) 3 and 12 months after birth. Accelerated senescence-resistant mice R1TA (SAMR1TA), which show no acceleration of senescence, were used as controls. In SAMP8/Ta, copper-zinc superoxide dismutase and manganese superoxide dismutase immunoreactivity in the photoreceptor inner segments, the outer nuclear layer and the inner nuclear layer increased earlier than in the controls. The increase in both superoxide dismutases with age occurred not only in SAMP8/Ta retinas but also in the controls. In conclusion, we propose the possibility that SAMP8/Ta undergo deterioration not only of learning and memory but also acceleration of senescence in the retina. The dismutases also appear to increase with normal ageing in the retina.     

Placental mitochondrial dysfunction with metabolic diseases: Therapeutic approaches

Both obesity and gestational diabetes mellitus (GDM) lead to poor maternal and fetal outcomes, including pregnancy complications, fetal growth issues, stillbirth, and developmental programming of adult-onset disease in the offspring. Increased placental oxidative/nitrative stress and reduced placental (trophoblast) mitochondrial respiration occur in association with the altered maternal metabolic milieu of obesity and GDM. The effect is particularly evident when the fetus is male, suggesting a sexually dimorphic influence on the placenta. In addition, obesity and GDM are associated with inflexibility in trophoblast, limiting the ability to switch between usage of glucose, fatty acids, and glutamine as substrates for oxidative phosphorylation, again in a sexually dimorphic manner. Here we review mechanisms underlying placental mitochondrial dysfunction: its relationship to maternal and fetal outcomes and the influence of fetal sex. Prevention of placental oxidative stress and mitochondrial dysfunction may improve pregnancy outcomes. We outline pathways to ameliorate deficient mitochondrial respiration, particularly the benefits and pitfalls of mitochondria-targeted antioxidants.     

Standards requirements for systems biology approaches to health care: mitochondrial proteomics

A review of the standards needs of the mitochondrial proteomics communities is presented based on the presentations and discussions at National Institute of Standards and Technology (NIST) workshop, Systems Biology Approaches to Health Care: Mitochondrial Proteomics, held on September 17-18, 2002. The mitochondrial proteomics areas addressed for standards needs are model systems, methods and data. This review outlines the challenges in the field, proposes standards efforts that the community would like to see pursued to meet those challenges, and is followed by a summary and NIST's planned efforts to address these standards requirements.     

Comparative studies on mitochondrial development in yeasts

Summary High molecular weight mitochondrial RNA from Saccharomyces cerevisiae can be isolated rapidly and in relatively high yield from mitochondria prepared from cells prefixed with glutaraldehyde and disrupted mechanically. The RNA has lower electrophoretic mobilities than corresponding species from cytoplasmic ribosomes, and can also be distinguished from cytoplasmic RNA on the basis of the sensitivity of the mobility to temperature. RNA from cytoplasmic ribosomes and mitochondria of Candida parapsilosis shows a similar differential response to temperature.Mitochondrial ribosomes in Saccharomyces cerevisiae do not appear to be distinguishable from the cytoplasmic particles on the basis of sedimentation velocity. They can be identified, however, by pulse-labelling cells in the presence of cycloheximide. Cytoplasmic ribosomes under these conditions do not label. The labelling of mitochondrial ribosomes is sensitive to chloramphenicol, and is dispersed over the polysomal or ribosomal aggregate region of density gradients.     

Mitochondrial dysfunction in platelets and hippocampi of senescence-accelerated mice

Senescence-accelerated mice (SAM) strains are useful models to understand the mechanisms of age-dependent degeneration. In this study, measurements of the mitochondrial membrane potential (Δψ_m) of platelets and the Adenosine 5^′-triphosphate (ATP) content of hippocampi and platelets were made, and platelet mitochondria were observed in SAMP8 (faster aging mice) and SAMR1 (aging resistant control mice) at 2, 6 and 9 months of age. In addition, an Aβ-induced (Amyloid beta-protein) damage model of platelets was established. After the addition of Aβ, the Δψ_m of platelets of SAMP8 at 1and 6 months of age were measured. We found that platelet Δψ_m, and hippocampal and platelet ATP content of SAMP8 mice decreased at a relatively early age compared with SAMR1. The platelets of 6 month-old SAMP8 showed a tolerance to Aβ-induced damages. These results suggest that mitochondrial dysfunction might be one of the mechanisms leading to age-associated degeneration in SAMP mice at an early age and the platelets could serve as a biomarker for detection of mitochondrial function and age related disease.     

Peptide fractionation in proteomics approaches

Peptide fractionation is extremely important in proteomics approaches. Full proteome characterization is desired from complex organisms, and with growing interest in post-translational modifications an extended protein sequence coverage is required. Peptide fractionation techniques have the great challenge of feeding current mass spectrometers in a way in which these issues are met. Peptide fractionation can be divided into three simple components: the column characteristics; the mobile phase; and peptide properties (charge, polarity, hydrophobicity and size). The current challenges are in the combination of these three components to allow comprehensive proteomics studies to be improved.     

Bioinformatics approaches in clinical proteomics

Protein expression profiling is increasingly being used to discover, validate and characterize biomarkers that can potentially be used for diagnostic purposes and to aid in pharmaceutical development. Correct analysis of data obtained from these experiments requires an understanding of the underlying analytic procedures used to obtain the data, statistical principles underlying high-dimensional data and clinical statistical tools used to determine the utility of the interpreted data. This review summarizes each of these steps, with the goal of providing the nonstatistician proteomics researcher with a working understanding of the various approaches that may be used by statisticians. Emphasis is placed on the process of mining high-dimensional data to identify a specific set of biomarkers that may be used in a diagnostic or other assay setting.     

Bioinformatics approaches in clinical proteomics

Protein expression profiling is increasingly being used to discover, validate and characterize biomarkers that can potentially be used for diagnostic purposes and to aid in pharmaceutical development. Correct analysis of data obtained from these experiments requires an understanding of the underlying analytic procedures used to obtain the data, statistical principles underlying high-dimensional data and clinical statistical tools used to determine the utility of the interpreted data. This review summarizes each of these steps, with the goal of providing the nonstatistician proteomics researcher with a working understanding of the various approaches that may be used by statisticians. Emphasis is placed on the process of mining high-dimensional data to identify a specific set of biomarkers that may be used in a diagnostic or other assay setting.     

Peptide fractionation in proteomics approaches

Peptide fractionation is extremely important in proteomics approaches. Full proteome characterization is desired from complex organisms, and with growing interest in post-translational modifications an extended protein sequence coverage is required. Peptide fractionation techniques have the great challenge of feeding current mass spectrometers in a way in which these issues are met. Peptide fractionation can be divided into three simple components: the column characteristics; the mobile phase; and peptide properties (charge, polarity, hydrophobicity and size). The current challenges are in the combination of these three components to allow comprehensive proteomics studies to be improved.     

Comparative analyses of cell disruption methods for mitochondrial isolation in high-throughput proteomics study

One of the most crucial steps in mitochondrial isolation is disruption of intact cells to denude intracellular organelles, but the yield and purity of different disruption protocols have not been well addressed. In the present study, MDCK cells were disrupted by mechanical (sonication and homogenization), physical (repeated freeze/thaw cycles and hypoosmotic burst), and chemical (using Triton X-100, NP-40, or CHAPS) methods. Efficacy of cell disruption was evaluated by trypan blue staining and mitochondria were subsequently isolated by standardized differential centrifugation. The yield of isolation was also determined by measuring protein concentrations, whereas the purity was examined by Janus green B staining, Western blot analyses of markers for mitochondria (COX-4) and other subcellular organelles/locales (i.e., nucleus, cytoplasm, endoplasmic reticulum, and lysosome), transmission electron microscopy, two-dimensional electrophoresis, and Q-TOF MS and/or MS/MS analyses. Our data demonstrated that sonication is the method of choice for disruption of cells prior to mitochondrial isolation for proteome analysis.     

神经退行性疾病的早期信号:线粒体功能障碍

线粒体是广泛存在于各种真核细胞中,可以进行独立复制的特殊的细胞器,它既能提供细胞内各种生命活动所需要的能源,也参与多种其他极为重要的生理活动。线粒体呼吸功能的障碍是许多神经退行性疾病发病早期共识的病理现象,探索线粒体在疾病发生过程中的变化,不仅对研究AD等神经退行性疾病的发病机理,对设计和开发创新药物也具有重要的指导意义。本文就线粒体的结构功能及其在神经退行性疾病发病过程中出现功能障碍的证据、诱因和可能的治疗方案作一简要综述。     

Recovery of adriamycin induced mitochondrial dysfunction in liver by selenium

Adriamycin (ADR) is a chemotherapeutic drug. Its toxicities may associate with mitochondriopathy. Selenium (Se) is a trace element for essential intracellular antioxidant enzymes. However, there is lack of data related to the effect of selenium on the liver tissue of ADR-induced mitochondrial dysfunction. The study was to investigate whether Se could restore mitochondrial dysfunction of liver-exposed ADR. Rats were divided into four groups as a control, ADR, Se, co-treated ADR with Se groups. The biochemical measurements of the liver were made in mitochondrial and cytosol. ATP level and mitochondria membrane potential (MMP) were measured. Total oxidant (TOS), total antioxidant (TAS) status were determined and oxidative stress index (OSI) was calculated by using TOS and TAS. ADR increased TOS in mitochondria and also oxidative stress in mitochondria. ADR sligtly decreased MMP, and ATP level. Partial recovery of MMP by Se was able to elevate the ATP production in cotreatment of ADR with Se. TOS in mitochondria and cytosol was diminished, as well as OSI. We concluded that selenium could potentially be used against oxidative stress induced by ADR in liver, resulting from the restoration of MMP and ATP production and prevention of mitochondrial damage in vivo.     

线粒体蛋白质组学

线粒体作为真核细胞内一种重要的细胞器,在许多生理病理过程中发挥重要作用。蛋白质组学研究技术的不断发展推动了线粒体蛋白质组的研究。本文对近年来线粒体蛋白质组学的研究现状、存在的影响因素及发展前景进行了综述。