Publications from researchers at the Buck Institute for Research on Aging http://www.buckinstitute.org/melovLab © 2011 Buck Institute, All Rights Reserved Rapamycin has been shown to extend lifespan in numerous model organisms including mice, with the most dramatic longevity effects reported in females. However, little is known about the functional ramifications of this longevityenhancing paradigm in mammalian tissues. We treated 24month old female C57BL/6J mice with rapamycin for 3 months, and determined health outcomes via a variety of noninvasive measures of cardiovascular, skeletal, and metabolic health for individual mice. We determined that while rapamycin has mild transient metabolic effects, there are significant benefits to latelife cardiovascular function with a reversal or attenuation of agerelated changes in the heart. RNAseq analysis of cardiac tissue after treatment indicated inflammatory, metabolic, and antihypertrophic expression changes in cardiac tissue as potential mechanisms mediating the functional improvement. Rapamycin treatment also resulted in beneficial behavioral, skeletal, and motor changes in these mice compared to those fed a control diet. From these findings, we propose that latelife rapamycin therapy not only extends the lifespan of mammals, but also confers functional benefits to a number of tissues, and mechanistically implicates an improvement in contractile function and antihypertrophic signaling in the aged heart with a reduction in agerelated inflammation. This article is protected by copyright. All rights reserved. http://www.ncbi.nlm.nih.gov/pubmed/23734717 Mon, 31 Dec 2012 00:00:00 -0800 The brain is a highly metabolically active tissue that critically relies on oxidative phosphorylation as means for maintaining energy. One byproduct of this process is the production of potentially damaging radicals such as the superoxide anion (O2(.)). Superoxide has the capacity to damage components of the electron transport chain and other cellular constituents. Eukaryotic systems have evolved defenses against such damaging moieties, the chief member of which is superoxide dismutase (SOD2), an enzyme that efficiently converts superoxide to the less reactive hydrogen peroxide (H2O2), which can freely diffuse cross the mitochondrial membrane. Loss of SOD2 activity can result in numerous pathological phenotypes in metabolically active tissues particularly within the central nervous system. We will review SOD2's potential involvement in the progression of neurodegenerative disease such as stroke, Alzheimer's, Parkinson's, as well as its potential role in "normal" agerelated cognitive decline. We will also examine in vivo models of endogenous oxidative damage based upon loss of SOD2 and associated neurological phenotypes in relation to human neurodegenerative disorders. http://www.ncbi.nlm.nih.gov/pubmed/23727323 Mon, 31 Dec 2012 00:00:00 -0800 Massage therapy is commonly used during physical rehabilitation of skeletal muscle to ameliorate pain and promote recovery from injury. Although there is evidence that massage may relieve pain in injured muscle, how massage affects cellular function remains unknown. To assess the effects of massage, we administered either massage therapy or no treatment to separate quadriceps of 11 young male participants after exerciseinduced muscle damage. Muscle biopsies were acquired from the quadriceps (vastus lateralis) at baseline, immediately after 10 min of massage treatment, and after a 2.5hour period of recovery. We found that massage activated the mechanotransduction signaling pathways focal adhesion kinase (FAK) and extracellular signalregulated kinase 1/2 (ERK1/2), potentiated mitochondrial biogenesis signaling nuclear peroxisome proliferatoractivated receptor coactivator 1 (PGC1), and mitigated the rise in nuclear factor B (NFB) (p65) nuclear accumulation caused by exerciseinduced muscle trauma. Moreover, despite having no effect on muscle metabolites (glycogen, lactate), massage attenuated the production of the inflammatory cytokines tumor necrosis factor (TNF) and interleukin6 (IL6) and reduced heat shock protein 27 (HSP27) phosphorylation, thereby mitigating cellular stress resulting from myofiber injury. In summary, when administered to skeletal muscle that has been acutely damaged through exercise, massage therapy appears to be clinically beneficial by reducing inflammation and promoting mitochondrial biogenesis. http://www.ncbi.nlm.nih.gov/pubmed/22301554 Sat, 31 Dec 2011 00:00:00 -0800 ABSTRACT: BACKGROUND: Despite the success of highly active antiretroviral therapy (HAART), HIV infected individuals remain at increased risk for frailty and declines in physical function that are more often observed in older uninfected individuals. This may reflect premature or accelerated muscle aging. METHODS: Skeletal muscle gene expression profiles were evaluated in three uninfected independent microarray datasets including young (19 to 29 years old), middle aged (40 to 45 years old) and older (65 to 85 years old) subjects, and a muscle dataset from HIV infected subjects (36 to 51 years old). Using Bayesian analysis, a ten gene muscle aging signature was identified that distinguished young from old uninfected muscle and included the senescence and cell cycle arrest gene p21/Cip1 (CDKN1A). This ten gene signature was then evaluated in muscle specimens from a cohort of middle aged (30 to 55 years old) HIV infected individuals. Expression of p21/Cip1 and related pathways were validated and further analyzed in a rodent model for HIV infection. RESULTS: We identify and replicate the expression of a set of muscle aging genes that were prematurely expressed in HIV infected, but not uninfected, middle aged subjects. We validated select genes in a rodent model of chronic HIV infection. Because the signature included p21/Cip1, a cell cycle arrest gene previously associated with muscle aging and fibrosis, we explored pathways related to senescence and fibrosis. In addition to p21/Cip1, we observed HIV associated upregulation of the senescence factor p16INK4a (CDKN2A) and fibrosis associated TGFbeta1, CTGF, COL1A1 and COL1A2. Fibrosis in muscle tissue was quantified based on collagen deposition and confirmed to be elevated in association with infection status. Fiber type composition was also measured and displayed a significant increase in slow twitch fibers associated with infection. CONCLUSIONS: The expression of genes associated with a muscle aging signature is prematurely upregulated in HIV infection, with a prominent role for fibrotic pathways. Based on these data, therapeutic interventions that promote muscle function and attenuate profibrotic gene expression should be considered in future studies. http://www.ncbi.nlm.nih.gov/pubmed/22676806 Sat, 31 Dec 2011 00:00:00 -0800 In tissues with complex architectures such as bone, it is often difficult to purify and characterize specific cell types via molecular profiling. Single cell gene expression profiling is an emerging technology useful for characterizing transcriptional profiles of individual cells isolated from heterogeneous populations. In this study we describe a novel procedure for the isolation and characterization of gene expression profiles of single osteoblast lineage cells derived from cortical bone. Mixed populations of different cell types were isolated from adult long bones of C57BL/6J mice by enzymatic digestion, and subsequently subjected to FACS to purify and characterize osteoblast lineage cells via a selection strategy using antibodies against CD31, CD45, and alkaline phosphatase (AP), specific for mature osteoblasts. The purified individual osteoblast lineage cells were then profiled at the single cell level via nanofluidic PCR. This method permits robust gene expression profiling on single osteoblast lineage cells derived from mature bone, potentially from anatomically distinct sites. In conjunction with this technique, we have also shown that it is possible to carry out single cell profiling on cells purified from fixed and frozen bone samples without compromising the gene expression signal. The latter finding means the technique can be extended to biopsies of bone from diseased individuals. Our approach for single cell expression profiling provides a new dimension to the transcriptional profile of the primary osteoblast lineage population in vivo, and has the capacity to greatly expand our understanding of how these cells may function in vivo under normal and diseased states. http://www.ncbi.nlm.nih.gov/pubmed/23238121 Sat, 31 Dec 2011 00:00:00 -0800 Depressed cortical energy supply and impaired synaptic function are predominant associations of Alzheimer's disease (AD). To test the hypothesis that presynaptic bioenergetic deficits are associated with the progression of AD pathogenesis, we compared bioenergetic variables of cortical and hippocampal presynaptic nerve terminals (synaptosomes) from commonly used mouse models with ADlike phenotypes (J20 age 6 months, Tg2576 age 16 months, and APP/PS age 9 and 14 months) to agematched controls. No consistent bioenergetic deficiencies were detected in synaptosomes from the three models only APP/PS cortical synaptosomes from 14monthold mice showed an increase in respiration associated with proton leak. J20 mice were chosen for a highly stringent investigation of mitochondrial function and content. There were no significant differences in the quality of the synaptosomal preparations or the mitochondrial volume fraction. Furthermore, respiratory variables, calcium handling, and membrane potentials of synaptosomes from symptomatic J20 mice under calciumimposed stress were not consistently impaired. The recovery of marker proteins during synaptosome preparation was the same, ruling out the possibility that the lack of functional bioenergetic defects in synaptosomes from J20 mice was due to the selective loss of damaged synaptosomes during sample preparation. Our results support the conclusion that the intrinsic bioenergetic capacities of presynaptic nerve terminals are maintained in these symptomatic AD mouse models. http://www.ncbi.nlm.nih.gov/pubmed/23175831 Sat, 31 Dec 2011 00:00:00 -0800 This corrects the article on p. e28530 in vol. 6.. http://www.ncbi.nlm.nih.gov/pubmed/23071831 Sat, 31 Dec 2011 00:00:00 -0800 Alphasynuclein has been reported to be present in the nucleus and levels enhanced by oxidative stress. Herein, we sought to investigate the mechanistic role of nuclear alphasynuclein. We found that alphasynuclein nuclear localization coincided with enhanced chromatin binding both in an in vitro and a corresponding in vivo brain oxidative stress model previously characterized by our laboratory as well as in PD brain tissues. Genomewide chromatin immunoprecipitation (ChIP)onchip analysis of alphasynuclein:promoter binding in response to oxidative stress in vitro revealed that binding occurs at several promoters belonging to a range of functional categories including transcriptional regulation. Interestingly, given the important role of mitochondrial dysfunction in PD, this included binding to the promoter for the master mitochondrial transcription activator, PGC1alpha in vitro, in vivo, and in human brain tissue with age and PD. To test the possible mechanistic impact of alphasynuclein PGC1alpha promotor binding, we assessed PGC1alpha promoter activity, mRNA, and protein levels and expression of candidate PGC1alpha target genes in our in vitro model. All were found to be reduced in conjunction with increased levels of aberrant mitochondrial morphology and impaired mitochondrial function. Exogenous PGC1alpha expression was found to attenuate alphasynucleinmediated mitochondrial dysfunction and subsequent neurotoxicity in vitro. Our data suggest that nuclear alphasynuclein localization under conditions of oxidative stress may impact on mitochondrial function in part via the protein's capacity to act as a transcriptional modulator of PGC1alpha. This represents a novel role for alphasynuclein as it relates to mitochondrial dysfunction in PD. http://www.ncbi.nlm.nih.gov/pubmed/22705949 Sat, 31 Dec 2011 00:00:00 -0800 Oxidative stress is frequently implicated in the pathology of neurodegenerative disease. The chief source of this stress is from mitochondrial respiration, via the passage of reducing equivalents through the respiratory chain resulting in a small but potentially pathological production of superoxide. The superoxide that is produced during normal respiration is primarily detoxified within the mitochondria by superoxide dismutase 2 (Sod2), a key protein for maintaining mitochondrial function. Mitochondria are distributed throughout the soma of neurons, as well as along neuronal processes and at the synaptic terminus. This distribution of potentially independent mitochondria throughout the neuron, at distinct subcellular locations, allows for the possibility of regional subcellular deficits in mitochondrial function. There has been increasing interest in the quantification and characterization of messages and proteins at the synapse, due to its importance in neurodegenerative disease, most notably Alzheimer's disease. Here, we report the transcriptomic and proteomic changes that occur in synaptosomes from frontal cortices of Sod2 null mice. Constitutively null Sod2 mice were differentially dosed with the synthetic catalytic antioxidant EUK189, which can extend the lifespan of these mice, as well as uncover or prevent neurodegeneration due to endogenous oxidative stress. This approach facilitated insight into quantification of trafficked messages and proteins to the synaptosome. We used two complementary methods to investigate the nature of the synaptosome under oxidative stress either whole genome gene expression microarrays or mass spectrometrybased proteomics using isobaric tagging for relative and absolute quantitation (iTRAQ) of proteins. We have characterized the relative enrichments of gene ontologies at both gene and protein expression that occur due to mitochondrial oxidative stress in the synaptosome, which may lead to new avenues of investigation in understanding the regulation of the synaptic function in normal and diseased states. As a result of using these approaches, we report for the first time an activation of the mTOR pathway in synaptosomes isolated from Sod2 null mice, confirmed by an upregulation of the phosphorylation of 4EBP1. http://www.ncbi.nlm.nih.gov/pubmed/22796328 Sat, 31 Dec 2011 00:00:00 -0800 Changes in fat content have been associated with dietary restriction (DR), but whether they playa causal role in mediating various responses to DR remains unknown. We demonstrate that upon DR, Drosophila melanogaster shift their metabolism toward increasing fattyacid synthesis and breakdown, which is required for various responses to DR. Inhibition of fattyacid synthesis or oxidation genes specifically in the muscle tissue inhibited lifespan extension upon DR. Furthermore, DR enhances spontaneous activity of flies, which was found to bedependent on the enhanced fattyacid metabolism. This increase in activity was found to be at leastpartially required for the lifespan extension upon DR. Overexpression of adipokinetic hormone (dAKH), the functional ortholog of glucagon, enhances fat metabolism, spontaneous activity, and life span. Together, these results suggest that enhanced fat metabolism in the muscle and physical activity play a key role in the protective effects of DR. http://www.ncbi.nlm.nih.gov/pubmed/22768842 Sat, 31 Dec 2011 00:00:00 -0800 Huntington's disease (HD) is caused by a CAG expansion in the huntingtin gene. Expansion of the polyglutamine tract in the huntingtin protein results in massive cell death in the striatum of HD patients. We report that human induced pluripotent stem cells (iPSCs) derived from HD patient fibroblasts can be corrected by the replacement of the expanded CAG repeat with a normal repeat using homologous recombination, and that the correction persists in iPSC differentiation into DARPP32positive neurons invitro and invivo. Further, correction of the HDiPSCs normalized pathogenic HD signaling pathways (cadherin, TGF, BDNF, and caspase activation) and reversed disease phenotypes such as susceptibility to cell death and altered mitochondrial bioenergetics in neural stem cells. The ability to make patientspecific, genetically corrected iPSCs from HD patients will provide relevant disease models in identical genetic backgrounds and is a critical step for the eventual use of these cells in cell replacement therapy. http://www.ncbi.nlm.nih.gov/pubmed/22748967 Sat, 31 Dec 2011 00:00:00 -0800 The C. elegans germline and somatic gonad are actively developing until the animal reaches adulthood, and then continue to undergo striking changes as the animal ages. Reported changes include a depletion of available sperm, a decrease in oocyte quality up till midlife, a reduction in germline nuclei, a decrease in fertility, and an accumulation of DNA in the midbody of aging C. elegans. Here, we have focused on the aging gonad in old animals, and show in detail that the aging gonad undergoes a massive uterine growth composed of endoreduplicating oocytes, yolk, and expanses of chromatin. We use a novel series of imaging techniques in combination with histological methodology for reconstructing aged worms in 3dimensions, and show in old animals growing masses swelling inside the uterus to occupy most of the diameter of the worm. We link this accelerated growth to the cep1/p53 tumor suppressor. Because cep1 is required for DNA damage induced apoptosis, and daf2 limits longevity, these results suggest a role for agerelated DNA damage in dysplastic uterine growths, which in some respects resemble premalignant changes that can occur in aging mammals. http://www.ncbi.nlm.nih.gov/pubmed/22562940 Sat, 31 Dec 2011 00:00:00 -0800 Medicinal benefits of Allium vegetables, such as garlic, have been noted throughout recorded history, including protection against cancer and cardiovascular disease. We now demonstrate that garlic constituent diallyl trisulfide (DATS) increases longevity of C. elegans by affecting the skn1 pathway. Treatment of worms with 510M DATS increased worm mean lifespan even when treatment is started during young adulthood. To explore the mechanisms involved in the DATSmediated increase in longevity, we treated daf2, daf16, and eat2 mutants and found that DATS increased the lifespan of daf2 and daf16 mutants, but not the eat2 mutants. Microarray experiments demonstrated that a number of genes regulated by oxidative stress and the skn1 transcription factor were also changed by DATS treatment. Consistently, DATS treatment leads to the induction of the skn1 target gene gst4, and this induction was dependent on skn1. We also found that the effects of DATS on worm lifespan depend on skn1 activity in both in the intestine and ASI neurons. Together our data suggest that DATS is able to increase worm lifespan by enhancing the function of the prolongevity transcription factor skn1. http://www.ncbi.nlm.nih.gov/pubmed/21296648 Mon, 31 Jan 2011 00:00:00 -0800 Presynaptic nerve terminals require high levels of ATP for the maintenance of synaptic function. Failure of synaptic mitochondria to generate adequate ATP has been implicated as a causative event preceding loss of synaptic networks in neurodegenerative disease. Endogenous oxidative stress has often been postulated as an etiological basis for this pathology, but has been difficult to test in vivo. Inactivation of the superoxide dismutase gene (Sod2) encoding the chief defense enzyme against mitochondrial superoxide radicals results in neonatal lethality. However, intervention with an SOD mimetic extends the lifespan of this model, and uncovers a neurodegenerative phenotype providing a unique model for the examination of in vivo oxidative stress. We present here studies on synaptic termini isolated from the frontal cortex of Sod2 null mice demonstrating impaired bioenergetic function as a result of mitochondrial oxidative stress. Cortical synaptosomes from Sod2 null mice demonstrate a severe decline in mitochondrial spare respiratory capacity to physiological demand induced by mitochondrial respiratory chain uncoupling with FCCP or plasma membrane depolarization induced by 4aminopyridine treatment. However, Sod2 null animals compensate for impaired oxidative metabolism in part by Pasteur effect allowing for normal neurotransmitter release at the synapse, setting up a potentially detrimental energetic paradigm. The results of this study demonstrate that high throughput respirometry is a facile method for analyzing specific regions of the brain in transgenic models, and can uncover bioenergetic deficits in subcellular regions due to endogenous oxidative stress. http://www.ncbi.nlm.nih.gov/pubmed/21215798 Fri, 31 Dec 2010 00:00:00 -0800 A causal role for mitochondrial DNA (mtDNA) mutagenesis in mammalian aging is supported by recent studies demonstrating that the mtDNA mutator mouse, harboring a defect in the proofreadingexonuclease activity of mitochondrial polymerase gamma, exhibits accelerated aging phenotypes characteristic of human aging, systemic mitochondrial dysfunction, multisystem pathology, and reduced lifespan. Epidemiologic studies in humans have demonstrated that endurance training reduces the risk of chronic diseases and extends life expectancy. Whether endurance exercise can attenuate the cumulative systemic decline observed in aging remains elusive. Here we show that 5 mo of endurance exercise induced systemic mitochondrial biogenesis, prevented mtDNA depletion and mutations, increased mitochondrial oxidative capacity and respiratory chain assembly, restored mitochondrial morphology, and blunted pathological levels of apoptosis in multiple tissues of mtDNA mutator mice. These adaptations conferred complete phenotypic protection, reduced multisystem pathology, and prevented premature mortality in these mice. The systemic mitochondrial rejuvenation through endurance exercise promises to be an effective therapeutic approach to mitigating mitochondrial dysfunction in aging and related comorbidities. http://www.ncbi.nlm.nih.gov/pubmed/21368114 Fri, 31 Dec 2010 00:00:00 -0800 The roundworm C. elegans is widely used as an aging model, with hundreds of genes identified that modulate aging(Kaeberlein et al. 2002). The development and bodyplan of the 959 cells comprising the adult have been well described and established for more than 25 years(Sulston Horvitz 1977 Sulston et al. 1983). However, morphological changes with age in this optically transparent animal are less well understood, with only a handful of studies investigating the pathobiology of aging. Age related changes in muscle(Herndon et al. 2002), neurons(Herndon et al. 2002), intestine and yolk granules(Garigan et al. 2002 Herndon et al. 2002), nuclear architecture(Haithcock et al. 2005), tail nuclei(Golden et al. 2007), and the germline(Golden et al. 2007) have been observed via a variety of traditional relatively lowthroughput methods. We report here a number of novel approaches to study the pathobiology of aging C. elegans. We combined histological staining of serialsectioned tissues, transmission electron microscopy, and confocal microscopy with 3D volumetric reconstructions, and characterized agerelated morphological changes of multiple wildtype individuals at different ages. This enabled us to identify several novel pathologies with age in the C. elegans intestine, including loss of critical nuclei, degradation of intestinal microvilli, changes in the size, shape, and cytoplasmic contents of the intestine, and altered morphologies due to ingested bacteria. The threedimensional models we have created of tissues and cellular components from multiple individuals of different ages, represent a unique resource to demonstrate global heterogeneity of a multicellular organism. http://www.ncbi.nlm.nih.gov/pubmed/21501374 Fri, 31 Dec 2010 00:00:00 -0800 Reducing protein synthesis slows growth and development but can increase adult life span. We demonstrate that knockdown of eukaryotic translation initiation factor 4G (eIF4G), which is downregulated during starvation and dauer state, results in differential translation of genes important for growth and longevity in C.elegans. Genomewide mRNA translation state analysis showed that inhibition of IFG1, the C.elegans ortholog of eIF4G, results in a relative increase in ribosomal loading and translation of stress response genes. Some of these genes are required for life span extension when IFG1 is inhibited. Furthermore, enhanced ribosomal loading of certain mRNAs upon IFG1 inhibition was correlated with increased mRNA length. This association was supported by changes in the proteome assayed via quantitative mass spectrometry. Our results suggest that IFG1 mediates the antagonistic effects on growth and somatic maintenance by regulating mRNA translation of particular mRNAs based, in part, on transcript length. http://www.ncbi.nlm.nih.gov/pubmed/21723504 Fri, 31 Dec 2010 00:00:00 -0800 Cellular senescence is an established cellular stress response that acts primarily to prevent the proliferation of cells that experience potentially oncogenic stress. In recent years, it has become increasingly apparent that the senescence response is a complex phenotype, which has a variety of cell nonautonomous effects. The senescenceassociated secretory phenotype, or SASP, entails the secretion of numerous cytokines, growth factors and proteases. The SASP can have beneficial or detrimental effects, depending on the physiological context. One recently described beneficial effect is to aid tissue repair. Among the detrimental effects, the SASP can disrupt normal tissue structures and function, and, ironically, can promote malignant phenotypes in nearby cells. These detrimental effects in many ways recapitulate the degenerative and hyperplastic pathologies that develop during aging. Because the SASP is largely a response to genomic or epigenomic damage, we suggest it may be a model for a cellular damage response that can propagate damage signals both within and among tissues. We propose that both the degenerative and hyperplastic diseases of aging may be fueled by such damage signals. http://www.ncbi.nlm.nih.gov/pubmed/21925603 Fri, 31 Dec 2010 00:00:00 -0800 Understanding why and how senescence evolved is of great importance in investigating the multiple, complex mechanisms that influence the course of ageing in humans and other organisms. Compelling arguments eliminate the idea that death is generally programmed by genes for ageing, but there is still a widespread tendency to interpret data in terms of loosely defined 'age regulation', which does not usually make either evolutionary or mechanistic sense. This review critically addresses the role of natural selection in shaping ageing within the life history and examines the implications for research on genetic pathways that influence the life span. It is recognised that in exceptional circumstances the possibility exists for selection to favour limiting survival. In acknowledging that, at least in theory, ageing might occasionally be adaptive, however, the high barriers to validating actual instances of adaptive ageing are made clear. http://www.ncbi.nlm.nih.gov/pubmed/21959160 Fri, 31 Dec 2010 00:00:00 -0800 Two of the greatest challenges in regenerative medicine today remain (1) the ability to culture human embryonic stem cells (hESCs) at a scale sufficient to satisfy clinical demand and (2) the ability to eliminate teratomaforming cells from preparations of cells with clinically desirable phenotypes. Understanding the pathways governing apoptosis in hESCs may provide a means to address these issues. Limiting apoptosis could aid scaling efforts, whereas triggering selective apoptosis in hESCs could eliminate unwanted teratomaforming cells. We focus here on the BCL2 family of proteins, which regulate mitochondrialdependent apoptosis. We used quantitative PCR to compare the steadystate expression profile of all human BCL2 family members in hESCs with that of human primary cells from various origins and two cancer lines. Our findings indicate that hESCs express elevated levels of the proapoptotic BH3only BCL2 family members NOXA, BIK, BIM, BMF and PUMA when compared with differentiated cells and cancer cells. However, compensatory expression of prosurvival BCL2 family members in hESCs was not observed, suggesting a possible explanation for the elevated rates of apoptosis observed in proliferating hESC cultures, as well as a mechanism that could be exploited to limit hESCderived neoplasms. http://www.ncbi.nlm.nih.gov/pubmed/22174832 Fri, 31 Dec 2010 00:00:00 -0800 While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase specific target amplification (RTSTA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues. http://www.ncbi.nlm.nih.gov/pubmed/22231655 Fri, 31 Dec 2010 00:00:00 -0800 Cellular senescence arrests the proliferation of mammalian cells at risk for neoplastic transformation, and is also associated with aging. However, the factors that cause cellular senescence during aging are unclear. Excessive reactive oxygen species (ROS) have been shown to cause cellular senescence in culture, and accumulated molecular damage due to mitochondrial ROS has long been thought to drive aging phenotypesin vivo. Here, we test the hypothesis that mitochondrial oxidative stress can promote cellular senescence in vivo and contribute to aging phenotypes in vivo, specifically in the skin. We show that the number of senescent cells, as well as impaired mitochondrial (complex II) activity increase in naturally aged mouse skin. Using a mouse model of genetic Sod2 deficiency, we show that failure to express this important mitochondrial antioxidant enzyme also impairs mitochondrial complex II activity, causes nuclear DNA damage, and induces cellular senescence but not apoptosis in the epidermis. Sod2 deficiency also reduced the number of cells and thickness of the epidermis, while increasing terminal differentiation. Our results support the idea that mitochondrial oxidative stress and cellular senescence contribute to aging skin phenotypes in vivo. http://www.ncbi.nlm.nih.gov/pubmed/22278880 Fri, 31 Dec 2010 00:00:00 -0800 The insulinlike signaling (ILS) pathway regulates metabolism and is known to modulate adult life span in C. elegans. Altered stress responses and resistance to a wide range of stressors are also associated with changes in ILS and contribute to enhanced longevity. The transcription factors DAF16 and HSF1 are key effectors of the longevity phenotype. We demonstrate that increased intrinsic thermotolerance, due to lower ILS, is not dependent on stressinduced transcriptional responses but instead requires active protein translation. Translation profiling experiments reveal genes that are posttranscriptionally regulated in response to altered ILS during heat shock in a DAF16dependent manner. Furthermore, several novel proteins are specifically required for ILS effects on thermotolerance. We propose that lowered ILS results in metabolic and physiological changes. These DAF16induced changes precondition a translational response under acute stress to modulate survival. http://www.ncbi.nlm.nih.gov/pubmed/20816092 Tue, 31 Aug 2010 00:00:00 -0700 Unaccustomed eccentric exercise damages skeletal muscle tissue, activating mechanisms of recovery and remodeling that may be influenced by the female sex hormone 17betaestradiol (E2). Using high density oligonucleotide based microarrays, we screened for differences in mRNA expression caused by E2 and eccentric exercise. After random assignment to 8 days of either placebo (CON) or E2 (EXP), eighteen men performed 150 singleleg eccentric contractions. Muscle biopsies were collected at baseline (BL), following supplementation (PS), 3 hours (3H) and 48 hours (48H) after exercise. Serum E2 concentrations increased significantly with supplementation (P0.001) but did not affect microarray results. Exercise led to early transcriptional changes in striated muscle activator of Rho signaling (STARS), Rho family GTPase 3 (RND3), mitogen activated protein kinase (MAPK) regulation and the downstream transcription factor FOS. Targeted RTPCR analysis identified concurrent induction of negative regulators of calcineurin signaling RCAN (P0.001) and HMOX1 (P = 0.009). Protein contents were elevated for RND3 at 3H (P = 0.02) and FOS at 48H (P0.05). These findings indicate that early RhoA and NFAT signaling and regulation are altered following exercise for muscle remodeling and repair, but are not affected by E2. http://www.ncbi.nlm.nih.gov/pubmed/20502695 Fri, 30 Apr 2010 00:00:00 -0700 Advancements in animal models and cell culture techniques have been invaluable in the elucidation of the molecular mechanisms that regulate muscle atrophy. However, few studies have examined muscle atrophy in humans using modern experimental techniques. The purpose of this study was to examine changes in global gene transcription during immobilizationinduced muscle atrophy in humans and then explore the effects of the most prominent transcriptional alterations on protein expression and function. Healthy men and women (N = 24) were subjected to two weeks of unilateral limb immobilization, with muscle biopsies obtained before, after 48 hours (48 H) and 14 days (14 D) of immobilization. Muscle cross sectional area (approximately 5) and strength (1020) were significantly reduced in men and women (approximately 5 and 1020, respectively) after 14 D of immobilization. Microarray analyses of total RNA extracted from biopsy samples at 48 H and 14 D uncovered 575 and 3,128 probes, respectively, which were significantly altered during immobilization. As a group, genes involved in mitochondrial bioenergetics and carbohydrate metabolism were predominant features at both 48 H and 14 D, with genes involved in protein synthesis and degradation significantly downregulated and upregulated, respectively, at 14 D of muscle atrophy. There was also a significant decrease in the protein content of mitochondrial cytochrome c oxidase, and the enzyme activity of cytochrome c oxidase and citrate synthase after 14 D of immobilization. Furthermore, protein ubiquitination was significantly increased at 48 H but not 14 D of immobilization. These results suggest that transcriptional and posttranscriptional suppression of mitochondrial processes is sustained throughout 14 D of immobilization, while protein ubiquitination plays an early but transient role in muscle atrophy following shortterm immobilization in humans. http://www.ncbi.nlm.nih.gov/pubmed/19654872 Fri, 31 Jul 2009 00:00:00 -0700 We have mapped a protein interaction network of human homologs of proteins that modify longevity in invertebrate species. This network is derived from a proteomescale human protein interaction Core Network generated through unbiased highthroughput yeast twohybrid searches. The longevity network is composed of 175 human homologs of proteins known to confer increased longevity through loss of function in yeast, nematode, or fly, and 2,163 additional human proteins that interact with these homologs. Overall, the network consists of 3,271 binary interactions among 2,338 unique proteins. A comparison of the average node degree of the human longevity homologs with random sets of proteins in the Core Network indicates that human homologs of longevity proteins are highly connected hubs with a mean node degree of 18.8 partners. Shortest path length analysis shows that proteins in this network are significantly more connected than would be expected by chance. To examine the relationship of this network to human aging phenotypes, we compared the genes encoding longevity network proteins to genes known to be changed transcriptionally during aging in human muscle. In the case of both the longevity protein homologs and their interactors, we observed enrichments for differentially expressed genes in the network. To determine whether homologs of human longevity interacting proteins can modulate life span in invertebrates, homologs of 18 human FRAP1 interacting proteins showing significant changes in human aging muscle were tested for effects on nematode life span using RNAi. Of 18 genes tested, 33 extended life span when knockeddown in Caenorhabditis elegans. These observations indicate that a broad class of longevity genes identified in invertebrate models of aging have relevance to human aging. They also indicate that the longevity protein interaction network presented here is enriched for novel conserved longevity proteins. http://www.ncbi.nlm.nih.gov/pubmed/19293945 Sat, 28 Feb 2009 00:00:00 -0800 Lymphomas adapt to their environment by undergoing a complex series of biochemical changes that are currently not well understood. To better define these changes, we examined the gene expression and gene ontology profiles of thymic lymphomas from a commonly used model of carcinogenesis, the p53(/) mouse. These tumors show a highly significant upregulation of mitochondrial biogenesis, mitochondrial protein translation, mtDNA copy number, reactive oxygen species, antioxidant defenses, proton transport, ATP synthesis, hypoxia response, and glycolysis, indicating a fundamental change in the bioenergetic profile of the transformed T cell. Our results suggest that T cell tumorigenesis involves a simultaneous upregulation of mitochondrial biogenesis, mitochondrial respiration, and glycolytic activity. These processes would allow cells to adapt to the stressful tumor environment by facilitating energy production and thereby promote tumor growth. Understanding these adaptations is likely to result in improved therapeutic strategies for this tumor type. http://www.ncbi.nlm.nih.gov/pubmed/19038329 Wed, 31 Dec 2008 00:00:00 -0800 There has been a great deal of interest in identifying potential biomarkers of aging. Biomarkers of aging would be useful to predict potential vulnerabilities in an individual that may arise well before they are chronologically expected, due to idiosyncratic aging rates that occur between individuals. Prior attempts to identify biomarkers of aging have often relied on the comparisons of longlived animals to a wildtype control. However, the effect of interventions in model systems that prolong lifespan (such as single gene mutations or caloric restriction) can sometimes be difficult to interpret due to the manipulation itself having multiple unforeseen consequences on physiology, unrelated to aging itself. The search for predictive biomarkers of aging therefore is problematic, and the identification of metrics that can be used to predict either physiological or chronological age would be of great value. One methodology that has been used to identify biomarkers for numerous pathologies is gene expression profiling. Here, we report wholegenome expression profiles of individual wildtype Caenorhabditis elegans covering the entire wildtype nematode lifespan. Individual nematodes were scored for either agerelated behavioral phenotypes, or survival, and then subsequently associated with their respective gene expression profiles. This facilitated the identification of transcriptional profiles that were highly associated with either physiological or chronological age. Overall, our approach serves as a paradigm for identifying potential biomarkers of aging in higher organisms that can be repeatedly sampled throughout their lifespan. http://www.ncbi.nlm.nih.gov/pubmed/18778409 Sun, 30 Nov 2008 00:00:00 -0800 We used cDNA microarrays to screen for differentially expressed genes during recovery from exerciseinduced muscle damage in humans. Male subjects (n = 4) performed 300 maximal eccentric contractions, and skeletal muscle biopsy samples were analyzed at 3 h and 48 h after exercise. In total, 113 genes increased 3 h postexercise, and 34 decreased. At 48 h postexercise, 59 genes increased and 29 decreased. On the basis of these data, we chose 19 gene changes and conducted secondary analyses using realtime RTPCR from muscle biopsy samples taken from 11 additional subjects who performed an identical bout of exercise. Realtime RTPCR analyses confirmed that exerciseinduced muscle damage led to a rapid (3 h) increase in sterol response element binding protein 2 (SREBP2), followed by a delayed (48 h) increase in the SREBP2 gene targets Acyl CoA:cholesterol acyltransferase (ACAT)2 and insulininduced gene 1 (insig1). The expression of the IL1 receptor, a known regulator of SREBP2, was also elevated after exercise. Taken together, these expression changes suggest a transcriptional program for increasing cholesterol and lipid synthesis and/or modification. Additionally, damaging exercise induced the expression of protein kinase H11, capping protein Z alpha (capZalpha), and modulatory calcineurininteracting protein 1 (MCIP1), as well as cardiac ankryin repeat protein 1 (CARP1), DNAJB2, cmyc, and junD, each of which are likely involved in skeletal muscle growth, remodeling, and stress management. In summary, using DNA microarrays and RTPCR, we have identified novel genes that respond to skeletal muscle damage, which, given the known biological functions, are likely involved in recovery from and/or adaptation to damaging exercise. http://www.ncbi.nlm.nih.gov/pubmed/18321953 Sat, 31 May 2008 00:00:00 -0700 Creatine monohydrate (CrM) supplementation has been shown to increase fatfree mass and muscle power output possibly via cell swelling. Little is known about the cellular response to CrM. We investigated the effect of shortterm CrM supplementation on global and targeted mRNA expression and protein content in human skeletal muscle. In a randomized, placebocontrolled, crossover, doubleblind design, 12 young, healthy, nonobese men were supplemented with either a placebo (PL) or CrM (loading phase, 20 g/day x 3 days maintenance phase, 5 g/day x 7 days) for 10 days. Following a 28day washout period, subjects were put on the alternate supplementation for 10 days. Muscle biopsies of the vastus lateralis were obtained and were assessed for mRNA expression (cDNA microarrays realtime PCR) and protein content (Kinetworks KPKS 1.0 Protein Kinase screen). CrM supplementation significantly increased fatfree mass, total body water, and body weight of the participants (P 0.05). Also, CrM supplementation significantly upregulated (1.3 to 5.0fold) the mRNA content of genes and protein content of kinases involved in osmosensing and signal transduction, cytoskeleton remodeling, protein and glycogen synthesis regulation, satellite cell proliferation and differentiation, DNA replication and repair, RNA transcription control, and cell survival. We are the first to report this largescale gene expression in the skeletal muscle with shortterm CrM supplementation, a response that suggests changes in cellular osmolarity. http://www.ncbi.nlm.nih.gov/pubmed/17957000 Mon, 31 Dec 2007 00:00:00 -0800 Lithium (Li()) has been used to treat mood affect disorders, including bipolar, for decades. This drug is neuroprotective and has several identified molecular targets. However, it has a narrow therapeutic range and the one or more underlying mechanisms of its therapeutic action are not understood. Here we describe a pharmacogenetic study of Li() in the nematode Caenorhabditis elegans. Exposure to Li() at clinically relevant concentrations throughout adulthood increases survival during normal aging (up to 46 median increase). Longevity is extended via a novel mechanism with altered expression of genes encoding nucleosomeassociated functions. Li() treatment results in reduced expression of the worm ortholog of LSD1 (T08D10.2), a histone demethylase knockdown by RNA interference of T08D10.2 is sufficient to extend longevity ( approximately 25 median increase), suggesting Li() regulates survival by modulating histone methylation and chromatin structure. http://www.ncbi.nlm.nih.gov/pubmed/17959600 Fri, 30 Nov 2007 00:00:00 -0800 Great inroads into the understanding of aging have been made using C. elegans as a model system. Several genes have been identified that, when mutated, can extend lifespan. Yet, much about aging remains a mystery, and new technologies that allow the simultaneous assay of expression levels of thousands of genes have been applied to the question of how and why aging might occur. With correct experimental design and statistical analysis, differential gene expression between two or more populations can be obtained with high confidence. The ability to survey the entire genome in an unbiased way is a great asset for the study of complex biological phenomena such as aging. Aging undoubtedly involves changes in multiple genes involved in multiple processes, some of which may not yet be known. Gene expression profiling of wild type aging, and of strains with increased life spans, has provided some insight into potential mechanisms, and more can be expected in the future. http://www.ncbi.nlm.nih.gov/pubmed/18050504 Wed, 31 Oct 2007 00:00:00 -0700 Agerelated neurodegenerative disease has been mechanistically linked with mitochondrial dysfunction via damage from reactive oxygen species produced within the cell. We determined whether increased mitochondrial oxidative stress could modulate or regulate two of the key neurochemical hallmarks of Alzheimer's disease (AD): tau phosphorylation, and betaamyloid deposition. Mice lacking superoxide dismutase 2 (SOD2) die within the first week of life, and develop a complex heterogeneous phenotype arising from mitochondrial dysfunction and oxidative stress. Treatment of these mice with catalytic antioxidants increases their lifespan and rescues the peripheral phenotypes, while uncovering central nervous system pathology. We examined sod2 null mice differentially treated with high and low doses of a catalytic antioxidant and observed striking elevations in the levels of tau phosphorylation (at Ser396 and other phosphoepitopes of tau) in the lowdose antioxidant treated mice at ADassociated residues. This hyperphosphorylation of tau was prevented with an increased dose of the antioxidant, previously reported to be sufficient to prevent neuropathology. We then genetically combined a wellcharacterized mouse model of AD (Tg2576) with heterozygous sod2 knockout mice to study the interactions between mitochondrial oxidative stress and cerebral Ass load. We found that mitochondrial SOD2 deficiency exacerbates amyloid burden and significantly reduces metal levels in the brain, while increasing levels of Ser396 phosphorylated tau. These findings mechanistically link mitochondrial oxidative stress with the pathological features of AD. http://www.ncbi.nlm.nih.gov/pubmed/17579710 Thu, 31 May 2007 00:00:00 -0700 Human aging is associated with skeletal muscle atrophy and functional impairment (sarcopenia). Multiple lines of evidence suggest that mitochondrial dysfunction is a major contributor to sarcopenia. We evaluated whether healthy aging was associated with a transcriptional profile reflecting mitochondrial impairment and whether resistance exercise could reverse this signature to that approximating a younger physiological age. Skeletal muscle biopsies from healthy older (N = 25) and younger (N = 26) adult men and women were compared using gene expression profiling, and a subset of these were related to measurements of muscle strength. 14 of the older adults had muscle samples taken before and after a sixmonth resistance exercisetraining program. Before exercise training, older adults were 59 weaker than younger, but after six months of training in older adults, strength improved significantly (P0.001) such that they were only 38 lower than young adults. As a consequence of age, we found 596 genes differentially expressed using a false discovery rate cutoff of 5. Prior to the exercise training, the transcriptome profile showed a dramatic enrichment of genes associated with mitochondrial function with age. However, following exercise training the transcriptional signature of aging was markedly reversed back to that of younger levels for most genes that were affected by both age and exercise. We conclude that healthy older adults show evidence of mitochondrial impairment and muscle weakness, but that this can be partially reversed at the phenotypic level, and substantially reversed at the transcriptome level, following six months of resistance exercise training. http://www.ncbi.nlm.nih.gov/pubmed/17520024 Mon, 30 Apr 2007 00:00:00 -0700 The nematode Caenorhabditis elegans has become one of the most widely used model systems for the study of aging, yet very little is known about how C. elegans age. The development of the worm, from egg to young adult has been completely mapped at the cellular level, but such detailed studies have not been extended throughout the adult lifespan. Numerous single gene mutations, drug treatments and environmental manipulations have been found to extend worm lifespan. To interpret the mechanism of action of such aging interventions, studies to characterize normal worm aging, similar to those used to study worm development are necessary. We have used 4',6'diamidino2phenylindole hydrochloride staining and quantitative polymerase chain reaction to investigate the integrity of nuclei and quantify the nuclear genome copy number of C. elegans with age. We report both systematic loss of nuclei or nuclear DNA, as well as dramatic agerelated changes in nuclear genome copy number. These changes are delayed or attenuated in longlived daf2 mutants. We propose that these changes are important pathobiological characteristics of aging nematodes. http://www.ncbi.nlm.nih.gov/pubmed/17286610 Wed, 28 Feb 2007 00:00:00 -0800 Aging is generally defined and studied as a population phenomenon. However, there is great interest, especially when discussing human aging, in the identification of factors that influence the life span of an individual organism. The nematode Caenorhabditis elegans provides an excellent model system for the study of aging at the level of the individual, since young nematodes are essentially clonal yet experience a large range of individual life spans. We are conducting gene expression profiling of individual nematodes, with the aim of discovering genes that vary stochastically in expression between individuals of the same age. Such genes are candidates to modulate the ultimate life span achieved by each individual. We here present statistical analysis of gene expression profiles of individual nematodes from two different microarray platforms, examining the issue of technical vs. biological variance as it pertains to uncovering genes of interest in this paradigm of individual aging. http://www.ncbi.nlm.nih.gov/pubmed/16876364 Thu, 30 Nov 2006 00:00:00 -0800 The murine mutant weaver (gene symbol, wv) mouse, which carries a mutation in the gene encoding the Gprotein inwardly rectifying potassium channel Girk2, exhibits a diverse range of defects as a result of postnatal cell death in several different brain neuron subtypes. Loss of dopaminergic nigrostriatal neurons in the weaver, unlike cerebellar granule neuronal loss, is via a noncaspasemediated mechanism. Here, we present data demonstrating that degeneration of midbrain dopaminergic neurons in weaver is mediated via neuroinflammation. Furthermore, in vivo administration of the antiinflammatory agent minocycline attenuates nigrostriatal dopaminergic neurodegeneration. This has novel implications for the use of the weaver mouse as a model for Parkinson's disease, which has been associated with increased neuroinflammation. http://www.ncbi.nlm.nih.gov/pubmed/17093086 Tue, 31 Oct 2006 00:00:00 -0800 RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)expressed short hairpin RNA (shRNA) has been used to make shRNAexpressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of wellestablished Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species. http://www.ncbi.nlm.nih.gov/pubmed/16450009 Tue, 31 Jan 2006 00:00:00 -0800 Mice that lack the mitochondrial form of superoxide dismutase (SOD2) incur severe pathologies and mitochondrial deficiencies, including major depletion of complex II, as a consequence of buildup of endogenous reactive oxygen species (Melov, S., Coskun, P., Patel, M., Tuinstra, R., Cottrell, B., Jun, A. S., Zastawny, T. H., Dizdaroglu, M., Goodman, S. I., Huang, T. T., Miziorko, H., Epstein, C. J., and Wallace, D. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 846851 and Li, Y., Huang, T. T., Carlson, E. J., Melov, S., Ursell, P. C., Olson, J. L., Noble, L. J., Yoshimura, M. P., Berger, C., Chan, P. H., Wallace, D. C., and Epstein, C. J. (1995) Nat. Genet. 11, 376381). These problems can be greatly attenuated or rescued by synthetic antioxidant treatment, such as with the catalytic antioxidant EUK189 (Hinerfeld, D., Traini, M. D., Weinberger, R. P., Cochran, B., Doctrow, S. R., Harry, J., and Melov, S. (2004) J. Neurochem. 88, 657667). We have used heart mitochondria from sod2 null mice to better understand mitochondrial reactive oxygen species production both in the absence of SOD2 and following in vivo antioxidant treatment. Isolated heart mitochondria from 5dayold sod2 null animals respiring on the complex II substrate succinate exhibited statistically significant higher levels of mitochondrial O2 (157, p 0.01) but significantly less H2O2 (33, p 0.001) than wild type littermates. Treatment of sod2 nullizygous mice with EUK189 proportionately increased the levels of complex II and H2O2. Increased production of O2 resulting from complex II normalization had no effect on steady state levels due to the rapid conversion to H2O2, a process presumably aided by the presence of the EUK189, an SOD mimetic. http://www.ncbi.nlm.nih.gov/pubmed/16326710 Tue, 31 Jan 2006 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/16264422 Mon, 31 Oct 2005 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/16235574 Fri, 30 Sep 2005 00:00:00 -0700 The abnormal accumulation of amyloid betapeptide (Abeta) in the form of senile (or amyloid) plaques is one of the main characteristics of Alzheimer disease (AD). Both cholesterol and Cu2 have been implicated in AD pathogenesis and plaque formation. Abeta binds Cu2 with very high affinity, forming a redoxactive complex that catalyzes H2O2 production from O2 and cholesterol. Here we show that Abeta:Cu2 complexes oxidize cholesterol selectively at the C3 hydroxyl group, catalytically producing 4cholesten3one and therefore mimicking the activity of cholesterol oxidase, which is implicated in cardiovascular disease. Abeta toxicity in neuronal cultures correlated with this activity, which was inhibited by Cu2 chelators including clioquinol. Cell death induced by staurosporine or H2O2 did not elevate 4cholesten3one levels. Brain tissue from AD subjects had 98 more 4cholesten3one than tissue from agematched control subjects. We observed a similar increase in the brains of Tg2576 transgenic mice compared with nontransgenic littermates the increase was inhibited by in vivo treatment with clioquinol, which suggests that brain Abeta accumulation elevates 4cholesten3one levels in AD. Cu2mediated oxidation of cholesterol may be a pathogenic mechanism common to atherosclerosis and AD. http://www.ncbi.nlm.nih.gov/pubmed/16127459 Wed, 31 Aug 2005 00:00:00 -0700 Mitochondrial diseases are a heterogeneous array of disorders with a complex etiology. Use of microarrays as a tool to investigate complex human disease is increasingly common, however, a principle drawback of microarrays is their limited dynamic range, due to the poor quantification of weak signals. Although it is generally understood that lowintensity microarray 'spots' may be unreliable, there exists little documentation of their accuracy. Quantitative PCR (QPCR) is frequently used to validate microarray data, yet few QPCR validation studies have focused on the accuracy of lowintensity microarray signals. Hence, we have used QPCR to systematically assess microarray accuracy as a function of signal strength in a mouse model of mitochondrial disease, the superoxide dismutase 2 (SOD2) nullizygous mouse. We have focused on a unique category of dataspots with only one weak signal in a twodye comparative hybridizationand show that such 'highlow' signal intensities are common for differentially expressed genes. This category of differential expression may be more important in mitochondrial disease in which there are often mosaic expression patterns due to the idiosyncratic distribution of mutant mtDNA in heteroplasmic individuals. Using RNA from the SOD2 mouse, we found that when spotted cDNA microarray data are filtered for quality (low variance between many technical replicates) and spot intensity (above a negative control threshold in both channels), there is an excellent quantitative concordance with QPCR (R2 = 0.94). The accuracy of gene expression ratios from lowintensity spots (R2 = 0.27) and 'highlow' spots (R2 = 0.32) is considerably lower. Our results should serve as guidelines for microarray interpretation and the selection of genes for validation in mitochondrial disorders. http://www.ncbi.nlm.nih.gov/pubmed/16120406 Sun, 31 Jul 2005 00:00:00 -0700 To investigate the dysregulating effect of excess oxidative stress on mammary gland development, mammary anlage from newborn female mice with normal (/) or absent (null, /) manganese superoxide dismutase (SOD2) were excised and implanted under the renal capsule of normal host female nude mice with/without concurrent estrogen supplementation. After 30 days the transplanted glands were excised for wholemount, microscopic and immunohistochemical evaluation. In contrast to the normal growth and maturation of transplanted SOD2/ glands, SOD2/ glands showed arrested development, reduced ductal outgrowth and branching, and absent lumen. These hypomorphic SOD2/ ducts contained hyperplastic epithelium with increased Ki67 labelling, loss of Ecadherin expression, and disorganized p63 and cytokeratin (K)14 expressing basal and myoepithelial components. Estrogen treatment failed to upregulate progesterone receptor or normalize development. These findings suggest that excess oxidative stress from loss of SOD2 function can arrest mammary gland maturation and induce hyperplastic epithelium with early premalignant features. http://www.ncbi.nlm.nih.gov/pubmed/16085231 Sun, 31 Jul 2005 00:00:00 -0700 To search for novel transcriptional pathways that are activated in skeletal muscle after endurance exercise, we used cDNA microarrays to measure global mRNA expression after an exhaustive bout of highintensity cycling (approximately 75 min). Healthy, young, sedentary males performed the cycling bout, and skeletal muscle biopsies were taken from the vastus lateralis before, and at 3 and 48 h after exercise. We examined mRNA expression in individual muscle samples from four subjects using cDNA microarrays, used repeatedmeasures significance analysis of microarray (SAM) to determine statistically significant expression changes, and confirmed selected results using realtime RTPCR. In total, the expression of 118 genes significantly increased 3 h postcycling and 8 decreased. At 48 h, the expression of 29 genes significantly increased and 5 decreased. Many of these are potentially important novel genes involved in exercise recovery and adaptation, including several involved in 1) metabolism and mitochondrial biogenesis (FOXO1, PPARdelta, PPARgamma, nuclear receptor binding protein 2, IL6 receptor, ribosomal protein L2, aminolevulinate deltasynthase 2) 2) the oxidant stress response (metalothioneins 1B, 1F, 1G, 1H, 1L, 2A, 3, interferon regulatory factor 1) and 3) electrolyte transport across membranes NaKATPase (beta3), SERCA3, chloride channel 4. Others include genes involved in cell stress, proteolysis, apoptosis, growth, differentiation, and transcriptional activation, as well as all three nuclear receptor subfamily 4A family members (Nur77, Nurr1, and Nor1). This study is the first to characterize global mRNA expression during recovery from endurance exercise, and the results provide potential insight into 1) the transcriptional contributions to homeostatic recovery in human skeletal muscle after endurance exercise, and 2) the transcriptional contributions from a single bout of endurance exercise to the adaptive processes that occur after a period of endurance exercise training. http://www.ncbi.nlm.nih.gov/pubmed/15985525 Sun, 31 Jul 2005 00:00:00 -0700 The majority of cellular superoxide is generated in the mitochondria as a byproduct of normal oxidative metabolism. In the mitochondria, superoxide is detoxified by manganese superoxide dismutase (SOD2). Mice lacking SOD2 demonstrate a multifaceted neonatal lethal phenotype, including a spongiform encephalopathy that is preventable through antioxidant treatment. The molecular events behind the observed pathology in the cortex of these mice are unknown. We hypothesized that the lack of SOD2 would result in significant changes in cortical gene expression and that therapeutically beneficial antioxidant treatment would normalize the expression of some genes, providing insight into the mechanism by which mitochondrial oxidative stress results in neurodegeneration. We report the identification of gene expression profiles associated with this paradigm, which characterize the degree of response to the pharmacologic intervention. We have identified specific pathways targeted by endogenous oxidative stress, including glutathione metabolism, iron metabolism, and cellsurvival pathways centering on the kinase AKT. The normalization of expression of some of these pathways by antioxidant treatment suggests approaches to treating disease in which endogenous oxidative stress plays a role. http://www.ncbi.nlm.nih.gov/pubmed/15964507 Tue, 31 May 2005 00:00:00 -0700 Alzheimer disease is characterized by cerebral Abeta deposition, which we have recently discovered occurs also in the lens as cataracts in Alzheimer disease patients. Here we report the presence of significantly increased cataracts in the lenses of an Abetatransgenic mouse model for Alzheimer disease and their amelioration upon treatment with EUK189, a synthetic SOD/catalase mimetic. These data support an oxidative etiology for ADassociated lens cataracts and their potential to be treated preventatively with antioxidants. http://www.ncbi.nlm.nih.gov/pubmed/15607908 Tue, 30 Nov 2004 00:00:00 -0800 The use of microarrays as a tool to investigate fundamental biological questions has become ubiquitous over the past several years. Microarrays are becoming as common as the polymerase chain reaction or any of the other tools in the molecular biologist's armory. Unlike experiments involving other tools, however, the design and analysis of microarray experiments present some unique problems to molecular biologists, problems with which statisticians have long been familiar. In this overview of microarrays and agingrelated research, we will review selected highlights of microarray studies that have been carried out to study aging to date, as well as discuss some of the potential problems that routinely arise during these types of experiments, especially in the context of aging. http://www.ncbi.nlm.nih.gov/pubmed/15498758 Thu, 30 Sep 2004 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/15482810 Thu, 30 Sep 2004 00:00:00 -0700 Mitochondria and aging are fast becoming two of the most paired terms in biology. As a crucial nexus for the cell, mitochondria are involved in numerous essential aspects of cell function, from energy production via the respiratory chain to steroid biosynthesis, heme assembly, pyrimidine biosynthesis, the tricarboxylic acid cycle, and apoptosis. Mitochondria are also the main producers of reactive oxygen species within the cell. Theories about aging have revolved around mitochondria for decades, but it is only in the past few years that animal models have started to give significant insights into mitochondriamediated pathophysiology that might be intimately associated with aging. This review will highlight several new animal models of mitochondrial dysfunction in the context of aging. http://www.ncbi.nlm.nih.gov/pubmed/15374671 Tue, 31 Aug 2004 00:00:00 -0700 Fractionation enhances the resolution of proteins with similar characteristics by reducing the number of proteins that comigrate in gels, thus facilitating the detection of lowerabundance proteins and the accurate determination of quantitative and qualitative differences in disease and normal samples. An efficient, reproducible microscale fractionation protocol for complex protein mixtures using novel ionexchange membrane chromatographic substrates (PerkinElmer, Boston, MA, USA Vivascience, Carlsbad, CA, USA) is described. The fractionation techniques were used in combination with twodimensional (2D) gels and orthogonal matrixassisted laser desorption/ionizationtime of flight (MALDITOF) mass spectrometry to identify differentially expressed proteins in brain samples from persons with and without Alzheimer's disease. http://www.ncbi.nlm.nih.gov/pubmed/15300776 Sat, 31 Jul 2004 00:00:00 -0700 We compare the aging of wildtype and longlived C. elegans by gene expression profiling of individual nematodes. Using a custom cDNA array, we have characterized the gene expression of 45 individuals at 4 distinct ages throughout the adult lifespan of wildtype N2 nematodes, and at the same ages for individuals of the longlived strain daf2(e1370). Using statistical tools developed for microarray data analysis, we identify genes that differentiate aging N2 from aging daf2, as well as classes of genes that change with age in a similar way in both genotypes. Our novel approach of studying individual nematodes provides practical advantages, since it obviates the use of mutants or drugs to block reproduction, as well as the use of stressful massculturing procedures, that have been required for previous microarray studies of C. elegans. In addition, this approach has the potential to uncover the molecular variability between individuals of a population, variation that is missed when studying pools of thousands of individuals. http://www.ncbi.nlm.nih.gov/pubmed/15153179 Fri, 30 Apr 2004 00:00:00 -0700 Oxidative stress and mitochondrial dysfunction have been linked to neurodegenerative disorders such as Parkinson's and Alzheimer's disease. However, it is not yet understood how endogenous mitochondrial oxidative stress may result in mitochondrial dysfunction. Most prior studies have tested oxidative stress paradigms in mitochondria through either chemical inhibition of specific components of the respiratory chain, or adding an exogenous insult such as hydrogen peroxide or paraquat to directly damage mitochondria. In contrast, mice that lack mitochondrial superoxide dismutase (SOD2 null mice) represent a model of endogenous oxidative stress. SOD2 null mice develop a severe neurological phenotype that includes behavioral defects, a severe spongiform encephalopathy, and a decrease in mitochondrial aconitase activity. We tested the hypothesis that specific components of the respiratory chain in the brain were differentially sensitive to mitochondrial oxidative stress, and whether such sensitivity would lead to neuronal cell death. We carried out proteomic differential display and examined the activities of respiratory chain complexes I, II, III, IV, V, and the tricarboxylic acid cycle enzymes alphaketoglutarate dehydrogenase and citrate synthase in SOD2 null mice in conjunction with efficacious antioxidant treatment and observed differential sensitivities of mitochondrial proteins to oxidative stress. In addition, we observed a striking pattern of neuronal cell death as a result of mitochondrial oxidative stress, and were able to significantly reduce the loss of neurons via antioxidant treatment. http://www.ncbi.nlm.nih.gov/pubmed/14720215 Wed, 31 Dec 2003 00:00:00 -0800 An article in the current Advance Online Publication section of Nature Genetics reports the results of a largescale RNA interference (RNAi) screen for genes that, when downregulated, produce enhanced longevity in the nematode Caenorhabditis elegans. The downregulation of a large number of genes related to energy metabolism and mitochondrial function yielded animals with increased lifespans, albeit with some additional deleterious phenotypes in some cases. In this Perspective, I discuss the implications of these results and make a plea for a more integrated approach in assessing the role of mitochondrial function, single gene mutations, and longevity. http://www.ncbi.nlm.nih.gov/pubmed/14603016 Fri, 31 Oct 2003 00:00:00 -0800 Reactive oxygen species are an inevitable byproduct of mitochondrial respiration. It has been estimated that between 0.4 and 4 of molecular oxygen is converted to the radical superoxide (O2) and this level is significantly influenced by the functional status of the mitochondria. It is well established that exogenous oxidative stress and high doses of mitochondrial poisons such as paraquat and carbonyl cyanide 4 (trifluoromethoxy) phenylhydrazone (FCCP) can lead to genomic instability. In this report we show for the first time that endogenous mitochondrial oxidative stress in standard cell culture conditions results in nuclear genomic instability in primary mouse embryonic fibroblasts (MEFs). We show that lack of mitochondrial superoxide dismutase in MEFs leads to a severe increase of double strand breaks, endtoend fusions, chromosomal translocations, and loss of cell viability and proliferative capacity. Our results predict that endogenous mitochondrial oxidative stress can induce genomic instability, and therefore may have a profound effect in cancer and aging. http://www.ncbi.nlm.nih.gov/pubmed/14570235 Tue, 30 Sep 2003 00:00:00 -0700 Most mammalian cells do not divide indefinitely, owing to a process termed replicative senescence. In human cells, replicative senescence is caused by telomere shortening, but murine cells senesce despite having long stable telomeres. Here, we show that the phenotypes of senescent human fibroblasts and mouse embryonic fibroblasts (MEFs) differ under standard culture conditions, which include 20 oxygen. MEFs did not senesce in physiological (3) oxygen levels, but underwent a spontaneous event that allowed indefinite proliferation in 20 oxygen. The proliferation and cytogenetic profiles of DNA repairdeficient MEFs suggested that DNA damage limits MEF proliferation in 20 oxygen. Indeed, MEFs accumulated more DNA damage in 20 oxygen than 3 oxygen, and more damage than human fibroblasts in 20 oxygen. Our results identify oxygen sensitivity as a critical difference between mouse and human cells, explaining their proliferative differences in culture, and possibly their different rates of cancer and ageing. http://www.ncbi.nlm.nih.gov/pubmed/12855956 Thu, 31 Jul 2003 00:00:00 -0700 The oxidative stress theory of aging has become increasingly accepted as playing a role in the aging process, based primarily on a substantial accumulation of circumstantial evidence. In recent years, the hypothesis that mitochondrially generated reactive oxygen species play a role in organismal aging has been directly tested in both invertebrate and mammalian model systems. Initial results imply that oxidative damage, specifically the level of superoxide, does play a role in limiting the lifespans of invertebrates such as Drosophila melanogaster and Caenorhabditis elegans. In mammalian model systems, the effect of oxidative stress on lifespan is less clear, but there is evidence that antioxidant treatment protects against agerelated dysfunction, including cognitive decline. http://www.ncbi.nlm.nih.gov/pubmed/12882341 Mon, 30 Jun 2003 00:00:00 -0700 The advent of microarrays in studying gene expression in aging has created tremendous excitement due to its potential for uncovering molecular mechanisms of aging and agerelated disease. However, the appropriate implementation of this technology in the science of aging requires serious attention to methodological detail and statistical rigor. This report highlights discussions from the microarray workshop on aging held at the First Conference on Functional Genomics of Aging in Seville, Spain. The topics discussed by the participants included technical issues relating to the printing of arrays, RNA isolation, cDNA labeling and hybridization, optimal design of microarray experiments, and statistical analysis of these data. Microarray analysis of complex tissues through the use of laser capture microdissection was also discussed. http://www.ncbi.nlm.nih.gov/pubmed/12618016 Fri, 28 Feb 2003 00:00:00 -0800 Oxidative stress is a ubiquitous phenomena in all cell types, and it is primarily produced in mitochondria which are essential for multicellular life. Oxidative stress targets can be wide ranging and include nucleic acids and a variety of macromolecules. This review discusses the role of oxidative stress in the context of animal models, focusing in particular on animal models of aging, as well as the development of a new class of therapeutic small molecular weight antioxidants that have proven effective in extending the lifespan of a simple invertebrate nematode. http://www.ncbi.nlm.nih.gov/pubmed/12200034 Wed, 31 Jul 2002 00:00:00 -0700 During the course of normal metabolism, reactive oxygen species (ROS) are produced from within the respiratory chain of the mitochondria. These ROS have the capacity to oxidize and damage a variety of cellular constituents including lipids, DNA, and proteins. We have taken a genetic and pharmacological approach in delineating the range of molecular targets that can be oxidatively damaged by mitochondrial ROS. Specifically, we use mice that are lacking the mitochondrial form of superoxide dismutase (sod 2(/) mice) to better understand the possible phenotypes that can arise from mitochondrial oxidative stress. sod 2(/) mice can be used to test the efficacy of antioxidants, and more generally the efficacy of antioxidants against mitochondrial oxidative stress. We have evaluated superoxide dismutase/catalase mimetics in this mammalian model of mitochondrial oxidative stress, and have shown a high degree of efficacy in protecting against ROS produced within the mitochondria. Similarly, we have employed the nematode Caenorhabditis elegans to test the hypothesis that effective antioxidant therapy can prolong the life span of an invertebrate. http://www.ncbi.nlm.nih.gov/pubmed/11976207 Sun, 31 Mar 2002 00:00:00 -0800 The identification of a majority of the polypeptides in mitochondria would be invaluable because they play crucial and diverse roles in many cellular processes and diseases. The endogenous production of reactive oxygen species (ROS) is a major limiter of life as illustrated by studies in which the transgenic overexpression in invertebrates of catalytic antioxidant enzymes results in increased lifespans. Mitochondria have received considerable attention as a principal sourceand targetof ROS. Mitochondrial oxidative stress has been implicated in heart disease including myocardial preconditioning, ischemia/reperfusion, and other pathologies. In addition, oxidative stress in the mitochondria is associated with the pathogenesis of Alzheimer's disease, Parkinson's disease, prion diseases, and amyotrophic lateral sclerosis (ALS) as well as aging itself. The rapidly emerging field of proteomics can provide powerful strategies for the characterization of mitochondrial proteins. Current approaches to mitochondrial proteomics include the creation of detailed catalogues of the protein components in a single sample or the identification of differentially expressed proteins in diseased or physiologically altered samples versus a reference control. It is clear that for any proteomics approach prefractionation of complex protein mixtures is essential to facilitate the identification of lowabundance proteins because the dynamic range of protein abundance within cells has been estimated to be as high as 10(7). The opportunities for identification of proteins directly involved in diseases associated with or caused by mitochondrial dysfunction are compelling. Future efforts will focus on linking genomic array information to actual protein levels in mitochondria. http://www.ncbi.nlm.nih.gov/pubmed/11884366 Thu, 28 Feb 2002 00:00:00 -0800 Alzheimer's disease (AD) is a devastating agerelated neurodegenerative disorder that has been intensively studied over the last several years. In vitro and in vivo studies have led to an understanding of some of the physicochemical properties of amyloid, a wellcharacterized hallmark of AD. Clioquinol is a drug that acts on amyloid by perturbing amyloid's metallochemistry, and Clioquinol treatment has been shown to be beneficial in a mouse model of AD. This short review examines the recent studies relating to Clioquinol and AD, and anticipates the imminent results of a Phase II trial of Clioquinol in AD, due in March 2002. http://www.ncbi.nlm.nih.gov/pubmed/11852134 Thu, 31 Jan 2002 00:00:00 -0800 Superoxide is produced as a result of normal energy metabolism within the mitochondria and is scavenged by the mitochondrial form of superoxide dismutase (sod2). Mice with inactivated SOD2 (sod2 nullizygous mice) die prematurely, exhibiting several metabolic and mitochondrial defects and severe tissue pathologies, including a lethal spongiform neurodegenerative disorder (Li et al., 1995 Melov et al., 1998, 1999). We show that treatment of sod2 nullizygous mice with synthetic superoxide dismutase (SOD)catalase mimetics extends their lifespan by threefold, rescues the spongiform encephalopathy, and attenuates mitochondrial defects. This class of antioxidant compounds has been shown previously to extend lifespan in the nematode Caenorhabditis elegans (Melov et al., 2000). These new findings in mice suggest novel therapeutic approaches to neurodegenerative diseases associated with oxidative stress such as Friedreich ataxia, spongiform encephalopathies, and Alzheimer's and Parkinson's diseases, in which chronic oxidative damage to the brain has been implicated. http://www.ncbi.nlm.nih.gov/pubmed/11606622 Sun, 30 Sep 2001 00:00:00 -0700 Advances in understanding of mitochondrial physiology and genetics in relation to pathology have exploded in the last decade. Paralleling this increase has been an active debate about the role of mitochondrial oxidative stress with regard to mitochondrial DNA mutations, aging, and disease. We discuss in a historical context the rapid progress in our understanding of the role of mitochondrial DNA mutations in disease, mitochondrial oxidative stress in aging, and the potential interplay between these two phenomena. http://www.ncbi.nlm.nih.gov/pubmed/11511398 Tue, 31 Jul 2001 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/11226736 Wed, 28 Feb 2001 00:00:00 -0800 We tested the theory that reactive oxygen species cause aging. We augmented the natural antioxidant systems of Caenorhabditis elegans with small synthetic superoxide dismutase/catalase mimetics. Treatment of wildtype worms increased their mean lifespan by a mean of 44 percent, and treatment of prematurely aging worms resulted in normalization of their lifespan (a 67 percent increase). It appears that oxidative stress is a major determinant of lifespan and that it can be counteracted by pharmacological intervention. http://www.ncbi.nlm.nih.gov/pubmed/10968795 Thu, 31 Aug 2000 00:00:00 -0700 During the last 10 years, the theory known as the "free radical theory of aging" has achieved prominence as one of the most compelling explanations for many of the degenerative changes associated with aging. Although its appeal derives from a longstanding body of supporting correlative data, the theory was only recently more rigorously tested. Ongoing researches in the study of free radical biochemistry and the genetics of aging have been at the forefront of this work. First, transgenic approaches in invertebrate models with candidate genes such as superoxide dismutase (SOD) involved in the detoxification of reactive oxygen species (ROS) have shown that the endogenous production of ROS due to normal physiologic processes is a major limiter of life span. Genes involved in ROS detoxification are highly conserved among eukaryotes hence, the physiologic processes that limit life span in invertebrates are likely to be similar in higher eukaryotes. Secondly, transgenic mice deficient in the antioxidant enzyme mitochondrial superoxide dismutase (SOD2) die within their first week of life, demonstrating the importance of limiting endogenous mitochondrial free radicals in mammals. Together, data from studies using transgenic invertebrates and those using sod2 mutant mice demonstrate that modulation of metabolic ROS can have a profound effect on life span. We show here that the effects of mitochondrial ROS can be modulated through appropriate catalytic antioxidant intervention. These catalytic antioxidants are discussed in the context of mitochondrial oxidative stress and their potential role in intervening in mitochondrial oxidative stress and aging. http://www.ncbi.nlm.nih.gov/pubmed/10911961 Mon, 31 Jul 2000 00:00:00 -0700 Deletions of the mitochondrial DNA (mtDNA) have been shown to accumulate with age in a variety of species regardless of mean or maximal life span. This implies that such mutations are either a molecular biomarker of senescence or that they are more causally linked to senescence itself. One assay that can be used to detect these mtDNA mutations is the longextension polymerase chain reaction assay. This assay amplifies approximately 16 kb of the mtDNA in mammalian mitochondria and preferentially amplifies mtDNAs that are either deleted or duplicated. We have applied this assay to the aging human brain and found a heterogeneous array of rearranged mtDNAs. In addition, we have developed in situ polymerase chain reaction to detect mtDNA within individual cells of both the mouse and the human brain as a first step in identifying and enumerating cells containing mutant mtDNAs in situ. http://www.ncbi.nlm.nih.gov/pubmed/10638530 Mon, 31 Jan 2000 00:00:00 -0800 During the course of normal respiration, reactive oxygen species are produced which are particularly detrimental to mitochondrial function. This is shown by recent studies with a mouse that lacks the mitochondrial form of superoxide dismutase (Sod2). Tissues that are heavily dependent on mitochondrial function such as the brain and heart are most severely affected in the Sod2 mutant mouse. Recent work with a mouse mutant for the heart/muscle specific isoform of the mitochondrial adenine nuclear translocator (Ant1) demonstrates a potential link between mitochondrial oxidative stress and mitochondrial DNA mutations. These mutations can be detected by Longextension PCR, a method for detecting a wide variety of mutations of the mitochondrial genome. Such mutations have also been observed in the mitochondrial genome with senescence regardless of the mean or maximal lifespan of the organism being studied. Mutations have been detected with age in Caenorhabditis elegans, mice, chimpanzees, and humans. This implies that a causal relationship may exist between mitochondrial reactive oxygen species production, and the senescence specific occurrence of mitochondrial DNA mutations. http://www.ncbi.nlm.nih.gov/pubmed/10486594 Tue, 31 Aug 1999 00:00:00 -0700 It has been hypothesized that a major factor in the progression of mitochondrial disease resulting from defects in oxidative phosphorylation (OXPHOS) is the stimulation of the mitochondrial production of reactive oxygen species (ROS) and the resulting damage to the mtDNA. To test this hypothesis, we examined the mitochondria from mice lacking the heart/muscle isoform of the adenine nucleotide translocator (Ant1), designated Ant1(tm2Mgr) (/) mice. The absence of Ant1 blocks the exchange of ADP and ATP across the mitochondrial inner membrane, thus inhibiting OXPHOS. Consistent with Ant1 expression, mitochondria isolated from skeletal muscle, heart, and brain of the Ant1deficient mice produced markedly increased amounts of the ROS hydrogen peroxide, whereas liver mitochondria, which express a different Ant isoform, produced normally low levels of hydrogen peroxide. The increased production of ROS by the skeletal muscle and heart was associated with a dramatic increase in the ROS detoxification enzyme manganese superoxide dismutase (Sod2, also known as MnSod) in muscle tissue and muscle mitochondria, a modest increase in Sod2 in heart tissue, and no increase in heart mitochondria. The level of glutathione peroxidase1 (Gpx1), a second ROS detoxifying enzyme, was increased moderately in the mitochondria of both tissues. Consistent with the lower antioxidant defenses in heart, the heart mtDNAs of the Ant1deficient mice showed a striking increase in the accumulation of mtDNA rearrangements, whereas skeletal muscle, with higher antioxidant defenses, had fewer mtDNA rearrangements. Hence, inhibition of OXPHOS does increase mitochondrial ROS production, eliciting antioxidant defenses. If the antioxidant defenses are insufficient to detoxify the ROS, then an increased mtDNA mutation rate can result. http://www.ncbi.nlm.nih.gov/pubmed/10220377 Mon, 31 May 1999 00:00:00 -0700 Oxidative stress has been implicated in many diseases. The chief source of reactive oxygen species within the cell is the mitochondrion. We have characterized a variety of the biochemical and metabolic effects of inactivation of the mouse gene for the mitochondrial superoxide dismutase (CD1Sod2(tm1Cje)). The Sod2 mutant mice exhibit a tissuespecific inhibition of the respiratory chain enzymes NADHdehydrogenase (complex I) and succinate dehydrogenase (complex II), inactivation of the tricarboxylic acid cycle enzyme aconitase, development of a urine organic aciduria in conjunction with a partial defect in 3hydroxy3methylglutarylCoA lyase, and accumulation of oxidative DNA damage. These results indicate that the increase in mitochondrial reactive oxygen species can result in biochemical aberrations with features reminiscent of mitochondrial myopathy, Friedreich ataxia, and 3hydroxy3methylglutarylCoA lyase deficiency. http://www.ncbi.nlm.nih.gov/pubmed/9927656 Sun, 28 Feb 1999 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/9620757 Tue, 30 Jun 1998 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/9568243 Sun, 31 May 1998 00:00:00 -0700 Reactive oxygen species (ROS) have been implicated in a wide range of degenerative processes including amyotrophic lateral sclerosis, ischemic heart disease, Alzheimer disease, Parkinson disease and aging. ROS are generated by mitochondria as the toxic byproducts of oxidative phosphorylation, their energy generating pathway. Genetic inactivation of the mitochondrial form of superoxide dismutase in mice results in dilated cardiomyopathy, hepatic lipid accumulation and early neonatal death. We report that treatment with the superoxide dismutase (SOD) mimetic Manganese 5, 10, 15, 20tetrakis (4benzoic acid) porphyrin (MnTBAP) rescues these Sod2tm1Cje(/) mutant mice from this systemic pathology and dramatically prolongs their survival. The animals instead develop a pronounced movement disorder progressing to total debilitation by three weeks of age. Neuropathologic evaluation reveals a striking spongiform degeneration of the cortex and specific brain stem nuclei associated with gliosis and intramyelinic vacuolization similar to that observed in cytotoxic edema and disorders associated with mitochondrial abnormalities such as Leighs disease and Canavans disease. We believe that due to the failure of MnTBAP to cross the blood brain barrier progressive neuropathology is caused by excessive mitochondrial production of ROS. Consequently, MnTBAPtreated Sod2tm1Cje(/) mice may provide an excellent model for examining the relationship between free radicals and neurodegenerative diseases and for screening new drugs to treat these disorders. http://www.ncbi.nlm.nih.gov/pubmed/9462746 Sat, 31 Jan 1998 00:00:00 -0800 Mitochondrial DNA (mtDNA) rearrangements have been shown to accumulate with age in the postmitotic tissues of a variety of animals and have been hypothesized to result in the agerelated decline of mitochondrial bioenergetics leading to tissue and organ failure. Caloric restriction in rodents has been shown to extend life span supporting an association between bioenergetics and senescence. In the present study, we use full length mtDNA amplification by longextension polymerase chain reaction (LXPCR) to demonstrate that mice accumulate a wide variety of mtDNA rearrangements with age in post mitotic tissues. Similarly, using an alternative PCR strategy, we have found that 24 kb minicircles containing the origin of heavystrand replication accumulate with age in heart but not brain. Analysis of mtDNA structure and conformation by Southern blots of unrestricted DNA resolved by field inversion gel electrophoresis have revealed that the brain mtDNAs of young animals contain the traditional linear, nicked, and supercoiled mtDNAs while old animals accumulate substantial levels of a slower migrating species we designate agespecific mtDNAs. In old caloric restricted animals, a wide variety of rearranged mtDNAs can be detected by LXPCR in post mitotic tissues, but Southern blots of unrestricted DNA reveals a marked reduction in the levels of the age specific mtDNA species. These observations confirm that mtDNA mutations accumulate with age in mice and suggest that caloric restriction impedes this progress. http://www.ncbi.nlm.nih.gov/pubmed/9023106 Mon, 31 Mar 1997 00:00:00 -0800 The Sod2 gene for Mnsuperoxide dismutase (MnSOD), an intramitochondrial free radical scavenging enzyme that is the first line of defense against superoxide produced as a byproduct of oxidative phosphorylation, was inactivated by homologous recombination. Homozygous mutant mice die within the first 10 days of life with a dilated cardiomyopathy, accumulation of lipid in liver and skeletal muscle, and metabolic acidosis. Cytochemical analysis revealed a severe reduction in succinate dehydrogenase (complex II) and aconitase (a TCA cycle enzyme) activities in the heart and, to a lesser extent, in other organs. These findings indicate that MnSOD is required for normal biological function of tissues by maintaining the integrity of mitochondrial enzymes susceptible to direct inactivation by superoxide. http://www.ncbi.nlm.nih.gov/pubmed/7493016 Sun, 31 Dec 1995 00:00:00 -0800 Several reports have shown that individual mitochondrial DNA (mtDNA) deletions accumulate with age. However, the overall extent of somatic mtDNA damage with age remains unclear. We have utilized fulllength PCR to concurrently screen for multiple mtDNA rearrangements in total DNA extracted from skeletal muscle derived from physiologically normal individuals (n = 35). This revealed that both the number and variety of mtDNA rearrangements increases dramatically between young and old individuals (P 0.0001). We further examined the mtDNA from both the younger and older subjects by Southern blot analysis and observed an agerelated increase in mtDNA(s) comparable in size to mtDNA products unique to patients with known mtDNA deletions. These data imply that a wide spectrum of mtDNA rearrangements accumulate in old individuals, which correlates with the marked age related decrease in OXPHOS capacity observed in postmitotic tissues. http://www.ncbi.nlm.nih.gov/pubmed/7479075 Thu, 30 Nov 1995 00:00:00 -0800 We have discovered that three longevity mutants of the nematode Caenorhabditis elegans also exhibit increased intrinsic thermotolerance (Itt) as young adults. Mutation of the age1 gene causes not only 65 longer life expectancy but also Itt. The Itt phenotype cosegregates with age1. Longlived spe26 and daf2 mutants also exhibit Itt. We investigated the relationship between increased thermotolerance and increased lifespan by developing conditions for environmental induction of thermotolerance. Such pretreatments at sublethal temperatures induce significant increases in thermotolerance and small but statistically highly significant increases in life expectancy, consistent with a causal connection between these two traits. Thus, when an animal's resistance to stress is increased, by either genetic or environmental manipulation, we also observe an increase in life expectancy. These results suggest that ability to respond to stress limits the life expectancy of C. elegans and might do so in other metazoa as well. http://www.ncbi.nlm.nih.gov/pubmed/7638227 Thu, 31 Aug 1995 00:00:00 -0700 We have developed a longextensionPCR strategy which amplifies approximately half of the mitochondrial genome (6.3 kb) of Caenorhabditis elegans using an individual worm as target. We analyzed three strains over their life span to assess the number of detectable deletions in the mitochondrial genome. Two of these strains are wildtype for life span while the third is mutant in the age1 gene, approximately doubling its maximum life span. At the mean life span in wildtype strains, there was a significant difference between the frequency of deletions detected in the mitochondrial genome compared with the mean number of deletions in young animals. In addition, deletions in the mitochondrial genome occur at a significantly lower rate in age1 mutants as compared with wild type. We cloned and identified the breakpoints of two deletions and found that one of the deletions had a direct repeat of 8 bp at the breakpoint. This is the largest single study (over 900 individual animals) characterizing the frequency of deletions in the mitochondrial genome as a function of age yet carried out. http://www.ncbi.nlm.nih.gov/pubmed/7753635 Wed, 31 May 1995 00:00:00 -0700 We have examined an aging population of Caenorhabditis elegans via a PCR assay to determine if deletions in the mitochondrial genome occur in the nematode. We detected eight such deletions, identified the breakpoints of four of these, and discovered direct repeats of 48 base pairs at the site of all four deletions. Six of the eight repeats involved in the deletions are located in or immediately adjacent to tRNAs. Without a biochemical bias, the probability of direct repeats being present at all four breakpoints was 4 x 10(6). http://www.ncbi.nlm.nih.gov/pubmed/8152911 Sat, 30 Apr 1994 00:00:00 -0700 We have cloned and sequenced the gene for the 180 kDa subunit of the a polymerase from Drosophila melanogaster. The protein shows high similarity to the 180 kDa subunits from other species. Comparative expression analysis for the transcript, protein and enzymic activity suggests that control occurs mainly at the level of transcription. In situ analyses of the RNA suggest that high levels of the transcript are synthesised in the ovaries and deposited uniformly in the egg. Immunolocalisation of the 180 kDa polypeptide in whole embryos shows that its location is mainly nuclear however, dispersal of the protein can be seen to occur during mitotic phases of the cell cycle. http://www.ncbi.nlm.nih.gov/pubmed/1429896 Mon, 30 Nov 1992 00:00:00 -0800