93 results on '"Giovanni Manfredi"'
Search Results
2. Mutant CHCHD10 causes an extensive metabolic rewiring that precedes OXPHOS dysfunction in a murine model of mitochondrial cardiomyopathy
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Nicole M. Sayles, Nneka Southwell, Kevin McAvoy, Kihwan Kim, Alba Pesini, Corey J. Anderson, Catarina Quinzii, Suzanne Cloonan, Hibiki Kawamata, and Giovanni Manfredi
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coiled-coil-helix-coiled-coil-helix domain containing 10 ,CHCHD10 ,mitochondria ,heart, cardiomyopathy ,integrated stress response ,metabolic rewiring ,Biology (General) ,QH301-705.5 - Abstract
Summary: Mitochondrial cardiomyopathies are fatal diseases, with no effective treatment. Alterations of heart mitochondrial function activate the mitochondrial integrated stress response (ISRmt), a transcriptional program affecting cell metabolism, mitochondrial biogenesis, and proteostasis. In humans, mutations in CHCHD10, a mitochondrial protein with unknown function, were recently associated with dominant multi-system mitochondrial diseases, whose pathogenic mechanisms remain to be elucidated. Here, in CHCHD10 knockin mutant mice, we identify an extensive cardiac metabolic rewiring triggered by proteotoxic ISRmt. The stress response arises early on, before the onset of bioenergetic impairments, triggering a switch from oxidative to glycolytic metabolism, enhancement of transsulfuration and one carbon (1C) metabolism, and widespread metabolic imbalance. In parallel, increased NADPH oxidases elicit antioxidant responses, leading to heme depletion. As the disease progresses, the adaptive metabolic stress response fails, resulting in fatal cardiomyopathy. Our findings suggest that early interventions to counteract metabolic imbalance could ameliorate mitochondrial cardiomyopathy associated with proteotoxic ISRmt.
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- 2022
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3. Fibroblast bioenergetics to classify amyotrophic lateral sclerosis patients
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Csaba Konrad, Hibiki Kawamata, Kirsten G. Bredvik, Andrea J. Arreguin, Steven A. Cajamarca, Jonathan C. Hupf, John M. Ravits, Timothy M. Miller, Nicholas J. Maragakis, Chadwick M. Hales, Jonathan D. Glass, Steven Gross, Hiroshi Mitsumoto, and Giovanni Manfredi
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Bioenergetics ,Mitochondria ,ALS ,Fibroblasts ,PLS ,Machine learning ,Neurology. Diseases of the nervous system ,RC346-429 ,Geriatrics ,RC952-954.6 - Abstract
Abstract Background The objective of this study was to investigate cellular bioenergetics in primary skin fibroblasts derived from patients with amyotrophic lateral sclerosis (ALS) and to determine if they can be used as classifiers for patient stratification. Methods We assembled a collection of unprecedented size of fibroblasts from patients with sporadic ALS (sALS, n = 171), primary lateral sclerosis (PLS, n = 34), ALS/PLS with C9orf72 mutations (n = 13), and healthy controls (n = 91). In search for novel ALS classifiers, we performed extensive studies of fibroblast bioenergetics, including mitochondrial membrane potential, respiration, glycolysis, and ATP content. Next, we developed a machine learning approach to determine whether fibroblast bioenergetic features could be used to stratify patients. Results Compared to controls, sALS and PLS fibroblasts had higher average mitochondrial membrane potential, respiration, and glycolysis, suggesting that they were in a hypermetabolic state. Only membrane potential was elevated in C9Orf72 lines. ATP steady state levels did not correlate with respiration and glycolysis in sALS and PLS lines. Based on bioenergetic profiles, a support vector machine (SVM) was trained to classify sALS and PLS with 99% specificity and 70% sensitivity. Conclusions sALS, PLS, and C9Orf72 fibroblasts share hypermetabolic features, while presenting differences of bioenergetics. The absence of correlation between energy metabolism activation and ATP levels in sALS and PLS fibroblasts suggests that in these cells hypermetabolism is a mechanism to adapt to energy dissipation. Results from SVM support the use of metabolic characteristics of ALS fibroblasts and multivariate analysis to develop classifiers for patient stratification.
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- 2017
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4. Mutant TDP-43 does not impair mitochondrial bioenergetics in vitro and in vivo
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Hibiki Kawamata, Pablo Peixoto, Csaba Konrad, Gloria Palomo, Kirsten Bredvik, Meri Gerges, Federica Valsecchi, Leonard Petrucelli, John M. Ravits, Anatoly Starkov, and Giovanni Manfredi
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TDP-43 ,TAR DNA-binding protein 43 ,Mitochondria ,Bioenergetics ,Calcium ,ALS ,Neurology. Diseases of the nervous system ,RC346-429 ,Geriatrics ,RC952-954.6 - Abstract
Abstract Background Mitochondrial dysfunction has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Functional studies of mitochondrial bioenergetics have focused mostly on superoxide dismutase 1 (SOD1) mutants, and showed that mutant human SOD1 impairs mitochondrial oxidative phosphorylation, calcium homeostasis, and dynamics. However, recent reports have indicated that alterations in transactivation response element DNA-binding protein 43 (TDP-43) can also lead to defects of mitochondrial morphology and dynamics. Furthermore, it was proposed that TDP-43 mutations cause oxidative phosphorylation impairment associated with respiratory chain defects and that these effects were caused by mitochondrial localization of the mutant protein. Here, we investigated the presence of bioenergetic defects in the brain of transgenic mice expressing human mutant TDP-43 (TDP-43A315T mice), patient derived fibroblasts, and human cells expressing mutant forms of TDP-43. Methods In the brain of TDP-43A315T mice, TDP-43 mutant fibroblasts, and cells expressing mutant TDP-43, we tested several bioenergetics parameters, including mitochondrial respiration, ATP synthesis, and calcium handling. Differences between mutant and control samples were evaluated by student t-test or by ANOVA, followed by Bonferroni correction, when more than two groups were compared. Mitochondrial localization of TDP-43 was investigated by immunocytochemistry in fibroblasts and by subcellular fractionation and western blot of mitochondrial fractions in mouse brain. Results We did not observe defects in any of the mitochondrial bioenergetic functions that were tested in TDP-43 mutants. We detected a small amount of TDP-43A315T peripherally associated with brain mitochondria. However, there was no correlation between TDP-43 associated with mitochondria and respiratory chain dysfunction. In addition, we observed increased calcium uptake in mitochondria from TDP-43A315T mouse brain and cells expressing A315T mutant TDP-43. Conclusions While alterations of mitochondrial morphology and dynamics in TDP-43 mutant neurons are well established, the present study did not demonstrate oxidative phosphorylation defects in TDP-43 mutants, in vitro and in vivo. On the other hand, the increase in mitochondrial calcium uptake in A315T TDP-43 mutants was an intriguing finding, which needs to be investigated further to understand its mechanisms and potential pathogenic implications.
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- 2017
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5. Energy deficit in parvalbumin neurons leads to circuit dysfunction, impaired sensory gating and social disability
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Melis Inan, Mingrui Zhao, Monica Manuszak, Cansu Karakaya, Anjali M. Rajadhyaksha, Virginia M. Pickel, Theodore H. Schwartz, Peter A. Goldstein, and Giovanni Manfredi
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COX ,Interneurons ,Mitochondria ,Oscillations ,Parvalbumin ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Parvalbumin-expressing, fast spiking interneurons have high-energy demands, which make them particularly susceptible to energy impairment. Recent evidence suggests a link between mitochondrial dysfunction in fast spiking cortical interneurons and neuropsychiatric disorders. However, the effect of mitochondrial dysfunction restricted to parvalbumin interneurons has not been directly addressed in vivo. To investigate the consequences of mitochondrial dysfunction in parvalbumin interneurons in vivo, we generated conditional knockout mice with a progressive decline in oxidative phosphorylation by deleting cox10 gene selectively in parvalbumin neurons (PV-Cox10 CKO). Cox10 ablation results in defective assembly of cytochrome oxidase, the terminal enzyme of the electron transfer chain, and leads to mitochondrial bioenergetic dysfunction. PV-Cox10 CKO mice showed a progressive loss of cytochrome oxidase in cortical parvalbumin interneurons. Cytochrome oxidase protein levels were significantly reduced starting at postnatal day 60, and this was not associated with a change in parvalbumin interneuron density. Analyses of intrinsic electrophysiological properties in layer 5 primary somatosensory cortex revealed that parvalbumin interneurons could not sustain their typical high frequency firing, and their overall excitability was enhanced. An increase in both excitatory and inhibitory input onto parvalbumin interneurons was observed in PV-Cox10 CKO mice, resulting in a disinhibited network with an imbalance of excitation/inhibition. Investigation of network oscillations in PV-Cox10 CKO mice, using local field potential recordings in anesthetized mice, revealed significantly increased gamma and theta frequency oscillation power in both medial prefrontal cortex and hippocampus. PV-Cox10 CKO mice did not exhibit muscle strength or gross motor activity deficits in the time frame of the experiments, but displayed impaired sensory gating and sociability. Taken together, these data reveal that mitochondrial dysfunction in parvalbumin interneurons can alter their intrinsic physiology and network connectivity, resulting in behavioral alterations similar to those observed in neuropsychiatric disorders, such as schizophrenia and autism.
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- 2016
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6. Mitochondria and endoplasmic reticulum crosstalk in amyotrophic lateral sclerosis
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Giovanni Manfredi and Hibiki Kawamata
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Endoplasmic reticulum ,Calcium ,MAMS ,Mitochondria ,Reactive oxygen species ,Protein misfolding ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Physical and functional interactions between mitochondria and the endoplasmic reticulum (ER) are crucial for cell life. These two organelles are intimately connected and collaborate to essential processes, such as calcium homeostasis and phospholipid biosynthesis. The connections between mitochondria and endoplasmic reticulum occur through structures named mitochondria associated membranes (MAMs), which contain lipid rafts and a large number of proteins, many of which serve multiple functions at different cellular sites. Growing evidence strongly suggests that alterations of ER–mitochondria interactions are involved in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), a devastating and rapidly fatal motor neuron disease. Mutations in proteins that participate in ER–mitochondria interactions and MAM functions are increasingly being associated with genetic forms of ALS and other neurodegenerative diseases. This evidence strongly suggests that, rather than considering the two organelles separately, a better understanding of the disease process can derive from studying the alterations in their crosstalk. In this review we discuss normal and pathological ER–mitochondria interactions and the evidence that link them to ALS.
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- 2016
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7. The Mitochondrial Unfolded Protein Response as a Non-Oncogene Addiction to Support Adaptation to Stress during Transformation in Cancer and Beyond
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Timothy C. Kenny, Giovanni Manfredi, and Doris Germain
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mitochondria ,mitochondrial unfolded protein response ,cancer ,ALS ,sirtuin deacetylase ,estrogen receptor ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Upon accumulation of misfolded proteins in the mitochondria, the mitochondrial unfolded protein response (UPRmt) is activated. This review focuses on the role of this response in cancer. We discuss evidence that during transformation, the UPRmt may play an essential role in the maintenance of the integrity of the mitochondria in the face of increased oxidative stress. However, the role of the UPRmt in other diseases is also emerging and is therefore also briefly discussed.
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- 2017
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8. Mitochondrial Transport and Turnover in the Pathogenesis of Amyotrophic Lateral Sclerosis
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Veronica Granatiero and Giovanni Manfredi
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mitochondria ,ALS ,axonal transport ,mitophagy ,SOD1 ,Miro1 ,PINK1 ,Parkin ,Biology (General) ,QH301-705.5 - Abstract
Neurons are high-energy consuming cells, heavily dependent on mitochondria for ATP generation and calcium buffering. These mitochondrial functions are particularly critical at specific cellular sites, where ionic currents impose a large energetic burden, such as at synapses. The highly polarized nature of neurons, with extremely large axoplasm relative to the cell body, requires mitochondria to be efficiently transported along microtubules to reach distant sites. Furthermore, neurons are post-mitotic cells that need to maintain pools of healthy mitochondria throughout their lifespan. Hence, mitochondrial transport and turnover are essential processes for neuronal survival and function. In neurodegenerative diseases, the maintenance of a healthy mitochondrial network is often compromised. Numerous lines of evidence indicate that mitochondrial impairment contributes to neuronal demise in a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), where degeneration of motor neurons causes a fatal muscle paralysis. Dysfunctional mitochondria accumulate in motor neurons affected by genetic or sporadic forms of ALS, strongly suggesting that the inability to maintain a healthy pool of mitochondria plays a pathophysiological role in the disease. This article critically reviews current hypotheses on mitochondrial involvement in the pathogenesis of ALS, focusing on the alterations of mitochondrial axonal transport and turnover in motor neurons.
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- 2019
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9. Tau interactome maps synaptic and mitochondrial processes associated with neurodegeneration
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Tara E. Tracy, Jesus Madero-Pérez, Danielle L. Swaney, Timothy S. Chang, Michelle Moritz, Csaba Konrad, Michael E. Ward, Erica Stevenson, Ruth Hüttenhain, Grant Kauwe, Maria Mercedes, Lauren Sweetland-Martin, Xu Chen, Sue-Ann Mok, Man Ying Wong, Maria Telpoukhovskaia, Sang-Won Min, Chao Wang, Peter Dongmin Sohn, Jordie Martin, Yungui Zhou, Wenjie Luo, John Q. Trojanowski, Virginia M.Y. Lee, Shiaoching Gong, Giovanni Manfredi, Giovanni Coppola, Nevan J. Krogan, Daniel H. Geschwind, and Li Gan
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Proteomics ,Aging ,interactome ,Neurodegenerative ,Alzheimer's Disease ,Severity of Illness Index ,Medical and Health Sciences ,protein-protein interaction ,synapse ,2.1 Biological and endogenous factors ,Protein Interaction Maps ,Amino Acids ,APEX ,Aetiology ,Alzheimer's Disease Related Dementias (ADRD) ,Neurons ,Stem Cell Research - Induced Pluripotent Stem Cell - Human ,neurodegeneration ,Brain ,Biological Sciences ,Mitochondria ,Frontotemporal Dementia (FTD) ,Tauopathies ,Frontotemporal Dementia ,Neurological ,Disease Progression ,Tau secretion ,Subcellular Fractions ,Protein Binding ,Induced Pluripotent Stem Cells ,tau Proteins ,General Biochemistry, Genetics and Molecular Biology ,Protein Domains ,Alzheimer Disease ,Acquired Cognitive Impairment ,Humans ,Biotinylation ,Cell Nucleus ,Stem Cell Research - Induced Pluripotent Stem Cell ,affinity purification mass spectrometry ,Neurosciences ,Alzheimer's Disease including Alzheimer's Disease Related Dementias (AD/ADRD) ,Stem Cell Research ,Brain Disorders ,Synapses ,Nerve Degeneration ,Mutation ,Mutant Proteins ,Dementia ,Tau ,Energy Metabolism ,Developmental Biology - Abstract
Tau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established interactions of Tau with presynaptic vesicle proteins during activity-dependent Tau secretion and mapped the Tau-binding sites to the cytosolic domains of integral synaptic vesicle proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau's interaction with mitochondria proteins, which were downregulated inAD brains of multiple cohorts and correlated with disease severity. These multimodal and dynamic Tau interactomes with exquisite spatial resolution shed light on Tau's role in neuronal function and disease and highlight potential therapeutic targets to block Tau-mediated pathogenesis.
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- 2022
10. Doxycycline promotes proteasome fitness in the central nervous system
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Matthew J. O'Connell, Doris Germain, Giovanni Manfredi, and Edmund C. Jenkins
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Central Nervous System ,Male ,Proteasome Endopeptidase Complex ,Transcription, Genetic ,Mitochondrial translation ,Science ,Central nervous system ,Saccharomyces cerevisiae ,Biology ,Article ,Mice ,Mitochondrial unfolded protein response ,medicine ,Cyclic AMP ,Animals ,Doxycycline ,Multidisciplinary ,Estrogen Receptor alpha ,Yeast ,Cell biology ,Mitochondria ,Cytosol ,Proteostasis ,medicine.anatomical_structure ,Proteasome ,Gene Expression Regulation ,Proteolysis ,Unfolded Protein Response ,Medicine ,Female ,medicine.drug - Abstract
Several studies reported that mitochondrial stress induces cytosolic proteostasis in yeast and C. elegans. Notably, inhibition of mitochondrial translation with doxcycyline decreases the toxicity of β-amyloid aggregates, in a C. elegans. However, how mitochondrial stress activates cytosolic proteostasis remains unclear. Further whether doxycycline has this effect in mammals and in disease relevant tissues also remains unclear. We show here that doxycycline treatment in mice drastically reduces the accumulation of proteins destined for degradation by the proteasome in a CNS region-specific manner. This effect is associated with the activation of the ERα axis of the mitochondrial unfolded protein response (UPRmt), in both males and females. However, sexually dimorphic mechanisms of proteasome activation were observed. Doxycycline also activates the proteasome in fission yeast, where ERα is not expressed. Rather, the ancient ERα-coactivator Mms19 regulates this response in yeast. Our results suggest that the UPRmt initiates a conserved mitochondria-to-cytosol stress signal, resulting in proteasome activation, and that this signal has adapted during evolution, in a sex and tissue specific-manner. Therefore, while our results support the use of doxycycline in the prevention of proteopathic diseases, they also indicate that sex is an important variable to consider in the design of future clinical trials using doxycycline.
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- 2021
11. Distal denervation in the SOD1 knockout mouse correlates with loss of mitochondria at the motor nerve terminal
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Giovanni Manfredi, Seneshaw Asress, Hibiki Kawamata, Anna Stepanova, Yingjie Li, Lindsey R. Hayes, Jonathan D. Glass, and Alexander Galkin
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rho GTP-Binding Proteins ,0301 basic medicine ,Mitochondrial intermembrane space ,SOD1 ,Neuromuscular Junction ,Motor nerve ,Mice, Transgenic ,Mitochondrion ,Article ,Mitochondrial Proteins ,Mice ,03 medical and health sciences ,Superoxide Dismutase-1 ,0302 clinical medicine ,Developmental Neuroscience ,medicine ,Animals ,Humans ,Axon ,Muscle, Skeletal ,Mitochondrial transport ,Mice, Knockout ,Denervation ,Chemistry ,Neurodegeneration ,nutritional and metabolic diseases ,medicine.disease ,Mitochondria ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,nervous system ,Neurology ,030217 neurology & neurosurgery - Abstract
Impairment of mitochondrial transport has long been implicated in the pathogenesis of neuropathy and neurodegeneration. However, the role of mitochondria in stabilizing motor nerve terminals at neuromuscular junction (NMJ) remains unclear. We previously demonstrated that mice lacking the antioxidant enzyme, superoxide dismutase-1 (Sod1−/−), develop progressive NMJ denervation. This was rescued by expression of SOD1 exclusively in the mitochondrial intermembrane space (MitoSOD1/Sod1−/−), suggesting that oxidative stress within mitochondria drives denervation in these animals. However, we also observed reduced mitochondrial density in Sod1−/− motor axons in vitro. To investigate the relationship between mitochondrial density and NMJ innervation in vivo, we crossed Sod1−/− mice with the fluorescent reporter strains Thy1-YFP and Thy1-mitoCFP. We identified an age-dependent loss of mitochondria at motor nerve terminals in Sod1−/− mice, that closely correlated with NMJ denervation, and was rescued by MitoSOD1 expression. To test whether augmenting mitochondrial transport rescues Sod1−/− axons, we generated transgenic mice overexpressing the mitochondrial cargo adaptor, Miro1. This led to a partial rescue of mitochondrial density at motor nerve terminals by 12 months of age, but was insufficient to prevent denervation. These findings suggest that loss of mitochondria in the distal motor axon may contribute to denervation in Sod1−/− mice, perhaps via loss of key mitochondrial functions such as calcium buffering and/or energy production.
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- 2019
12. Sex Differences in Ischemia/Reperfusion Injury: The Role of Mitochondrial Permeability Transition
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Giovanni Manfredi and Jasmine A. Fels
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0301 basic medicine ,Programmed cell death ,medicine.medical_specialty ,medicine.drug_class ,Ischemia ,Estrogen receptor ,Biochemistry ,Article ,Mitochondrial Transmembrane Permeability-Driven Necrosis ,Permeability ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Internal medicine ,Animals ,Humans ,Medicine ,Receptor ,Sex Characteristics ,business.industry ,General Medicine ,medicine.disease ,Mitochondria ,030104 developmental biology ,Endocrinology ,Mitochondrial permeability transition pore ,Estrogen ,Reperfusion Injury ,Calcium ,business ,Reperfusion injury ,030217 neurology & neurosurgery ,Intracellular - Abstract
Brain and heart ischemia are among the leading causes of death and disability in both men and women, but there are significant sex differences in the incidence and severity of these diseases. Ca(2+) dysregulation in response to ischemia/reperfusion injury (I/RI) is a well-recognized pathogenic mechanism leading to the death of affected cells. Excess intracellular Ca(2+) causes mitochondrial matrix Ca(2+) overload that can result in mitochondrial permeability transition (MPT), which can have severe consequences for mitochondrial function and trigger cell death. Recent findings indicate that estrogens and their related receptors are involved in the regulation of MPT, suggesting that sex differences in I/RI could be linked to estrogen-dependent modulation of mitochondrial Ca(2+). Here, we review the evidence supporting sex differences in I/RI and the role of estrogen and estrogen receptors (ERs) in producing these differences, the involvement of mitochondrial Ca(2+) overload in disease pathogenesis, and the estrogen-dependent modulation of MPT that may contribute to sex differences.
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- 2019
13. Prohibitin is a positive modulator of mitochondrial function in PC12 cells under oxidative stress
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Anatoly A. Starkov, Liping Qian, Corey Anderson, Anja Kahl, Ping Zhou, Anna Stepanova, Giovanni Manfredi, and Costantino Iadecola
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0301 basic medicine ,Time Factors ,Cardiolipins ,Cell Survival ,Respiratory chain ,macromolecular substances ,Mitochondrion ,Transfection ,medicine.disease_cause ,PC12 Cells ,Biochemistry ,Neuroprotection ,Article ,Mice ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Oxygen Consumption ,0302 clinical medicine ,Prohibitins ,Cardiolipin ,medicine ,Animals ,Humans ,Respiratory function ,Viability assay ,Enzyme Inhibitors ,RNA, Small Interfering ,Prohibitin ,Cells, Cultured ,Neurons ,Dose-Response Relationship, Drug ,technology, industry, and agriculture ,Hydrogen Peroxide ,Embryo, Mammalian ,Oxidants ,Mitochondria ,Rats ,Cell biology ,Mice, Inbred C57BL ,Repressor Proteins ,Oxidative Stress ,030104 developmental biology ,Electron Transport Chain Complex Proteins ,chemistry ,Oligomycins ,lipids (amino acids, peptides, and proteins) ,030217 neurology & neurosurgery ,Oxidative stress - Abstract
Prohibitin (PHB) is a ubiquitously expressed and evolutionarily conserved mitochondrial protein with multiple functions. We have recently shown that PHB up-regulation offers robust protection against neuronal injury in models of cerebral ischemia in vitro and in vivo, but the mechanism by which PHB affords neuroprotection remains to be elucidated. Here, we manipulated PHB expression in PC12 neural cells to investigate its impact on mitochondrial function and the mechanisms whereby it protects cells exposed to oxidative stress. PHB over-expression promoted cell survival, whereas PHB down-regulation diminished cell viability. Functionally, manipulation of PHB levels did not affect basal mitochondrial respiration, but it increased spare respiratory capacity. Moreover, PHB over-expression preserved mitochondrial respiratory function of cells exposed to oxidative stress. Preserved respiratory capacity in differentiated PHB over-expressing cells exposed to oxidative stress was associated with an elongated mitochondrial morphology, whereas PHB down-regulation enhanced fragmentation. Mitochondrial complex I oxidative degradation was attenuated by PHB over-expression and increased in PHB knockdown cells. Changes in complex I degradation were associated with alterations of respiratory chain supercomplexes. Furthermore, we showed that PHB directly interacts with cardiolipin and that down-regulation of PHB results in loss of cardiolipin in mitochondria, which may contribute to destabilizing respiratory chain supercomplexes. Taken together, these data demonstrate that PHB modulates mitochondrial integrity and bioenergetics under oxidative stress, and suggest that the protective effect of PHB is mediated by stabilization of the mitochondrial respiratory machinery and its functional capacity, by the regulation of cardiolipin content. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.
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- 2018
14. Proteinopathies and OXPHOS dysfunction in neurodegenerative diseases
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Giovanni Manfredi and Hibiki Kawamata
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0301 basic medicine ,Reviews ,Review ,Oxidative phosphorylation ,Gene mutation ,Biology ,Mitochondrion ,Oxidative Phosphorylation ,Mitochondrial Proteins ,03 medical and health sciences ,Protein Interaction Mapping ,medicine ,Animals ,Humans ,Proteostasis Deficiencies ,Regulation of gene expression ,Amyloid beta-Peptides ,Neurodegeneration ,Correction ,Neurodegenerative Diseases ,Cell Biology ,medicine.disease ,Mitochondria ,3. Good health ,Cell biology ,030104 developmental biology ,Gene Expression Regulation ,Apoptosis ,alpha-Synuclein ,Protein folding ,Homeostasis - Abstract
Kawamata and Manfredi review proposed mechanisms of how the accumulation of misfolded proteins in neurodegenerative diseases causes mitochondrial dysfunction., Mitochondria participate in essential processes in the nervous system such as energy and intermediate metabolism, calcium homeostasis, and apoptosis. Major neurodegenerative diseases are characterized pathologically by accumulation of misfolded proteins as a result of gene mutations or abnormal protein homeostasis. Misfolded proteins associate with mitochondria, forming oligomeric and fibrillary aggregates. As mitochondrial dysfunction, particularly of the oxidative phosphorylation system (OXPHOS), occurs in neurodegeneration, it is postulated that such defects are caused by the accumulation of misfolded proteins. However, this hypothesis and the pathological role of proteinopathies in mitochondria remain elusive. In this study, we critically review the proposed mechanisms whereby exemplary misfolded proteins associate with mitochondria and their consequences on OXPHOS.
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- 2017
15. Selected mitochondrial DNA landscapes activate the SIRT3 axis of the UPRmt to promote metastasis
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Sneha Grandhi, Jerry E. Chipuk, Thomas LaFramboise, M D'Aurello, Janine H. Santos, Doris Germain, Luena Papa, Marcelo G. Bonini, Abdul Kader Sagar, Peter C. Hart, Giovanni Manfredi, M Sersinghe, Timothy C. Kenny, M Ragazzi, Kevin W. Eliceiri, and Amanjot Kaur Riar
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0301 basic medicine ,Cancer Research ,Mitochondrial DNA ,SIRT3 ,SOD2 ,Repressor ,Breast Neoplasms ,Biology ,DNA, Mitochondrial ,03 medical and health sciences ,Cell Line, Tumor ,Sirtuin 3 ,Mitochondrial unfolded protein response ,Genetics ,Humans ,Neoplasm Metastasis ,Enhancer ,Molecular Biology ,Superoxide Dismutase ,Forkhead Box Protein O3 ,Mitochondria ,030104 developmental biology ,Proteostasis ,Cancer cell ,Unfolded Protein Response ,Cancer research ,Original Article ,Female - Abstract
By causing mitochondrial DNA (mtDNA) mutations and oxidation of mitochondrial proteins, reactive oxygen species (ROS) leads to perturbations in mitochondrial proteostasis. Several studies have linked mtDNA mutations to metastasis of cancer cells but the nature of the mtDNA species involved remains unclear. Our data suggests that no common mtDNA mutation identifies metastatic cells; rather the metastatic potential of several ROS-generating mutations is largely determined by their mtDNA genomic landscapes, which can act either as an enhancer or repressor of metastasis. However, mtDNA landscapes of all metastatic cells are characterized by activation of the SIRT/FOXO/SOD2 axis of the mitochondrial unfolded protein response (UPRmt). The UPRmt promotes a complex transcription program ultimately increasing mitochondrial integrity and fitness in response to oxidative proteotoxic stress. Using SOD2 as a surrogate marker of the UPRmt, we found that in primary breast cancers, SOD2 is significantly increased in metastatic lesions. We propose that the ability of selected mtDNA species to activate the UPRmt is a process that is exploited by cancer cells to maintain mitochondrial fitness and facilitate metastasis.
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- 2017
16. Sex specific activation of the ERα axis of the mitochondrial UPR (UPRmt) in the G93A-SOD1 mouse model of familial ALS
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Gloria M. Palomo, Suzanne R. Burstein, Amanjot Kaur Riar, Doris Germain, Andrea J. Arreguin, and Giovanni Manfredi
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Male ,0301 basic medicine ,Proteasome Endopeptidase Complex ,Mitochondrial intermembrane space ,animal diseases ,SOD1 ,Biology ,Mitochondrion ,Mitochondrial Proteins ,Mice ,03 medical and health sciences ,0302 clinical medicine ,ATP-Dependent Proteases ,Heat shock protein ,Mitochondrial unfolded protein response ,Genetics ,Animals ,Humans ,Molecular Biology ,Heat-Shock Proteins ,Genetics (clinical) ,Sex Characteristics ,Superoxide Dismutase ,Amyotrophic Lateral Sclerosis ,Serine Endopeptidases ,Estrogen Receptor alpha ,nutritional and metabolic diseases ,Articles ,General Medicine ,High-Temperature Requirement A Serine Peptidase 2 ,Molecular biology ,Mitochondria ,nervous system diseases ,Disease Models, Animal ,030104 developmental biology ,Proteostasis ,Proteasome ,Unfolded Protein Response ,Female ,Estrogen receptor alpha ,030217 neurology & neurosurgery - Abstract
The mitochondrial unfolded protein response (UPRmt) is a transcriptional program aimed at restoring proteostasis in mitochondria. Upregulation of mitochondrial matrix proteases and heat shock proteins was initially described. Soon thereafter, a distinct UPRmt induced by misfolded proteins in the mitochondrial intermembrane space (IMS) and mediated by the estrogen receptor alpha (ERα), was found to upregulate the proteasome and the IMS protease OMI. However, the IMS-UPRmt was never studied in a neurodegenerative disease in vivo. Thus, we investigated the IMS-UPRmt in the G93A-SOD1 mouse model of familial ALS, since mutant SOD1 is known to accumulate in the IMS of neural tissue and cause mitochondrial dysfunction. As the ERα is most active in females, we postulated that a differential involvement of the IMS-UPRmt could be linked to the longer lifespan of females in the G93A-SOD1 mouse. We found a significant sex difference in the IMS-UPRmt, because the spinal cords of female, but not male, G93A-SOD1 mice showed elevation of OMI and proteasome activity. Then, using a mouse in which G93A-SOD1 was selectively targeted to the IMS, we demonstrated that the IMS-UPRmt could be specifically initiated by mutant SOD1 localized in the IMS. Furthermore, we showed that, in the absence of ERα, G93A-SOD1 failed to activate OMI and the proteasome, confirming the ERα dependence of the response. Taken together, these results demonstrate the IMS-UPRmt activation in SOD1 familial ALS, and suggest that sex differences in the disease phenotype could be linked to differential activation of the ERα axis of the IMS-UPRmt.
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- 2017
17. Redox-Dependent Loss of Flavin by Mitochondrial Complex I in Brain Ischemia/Reperfusion Injury
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Zoya V. Niatsetskaya, Csaba Konrad, Alexander Galkin, Anna Stepanova, Anatoly A. Starkov, Sergey A. Sosunov, Giovanni Manfredi, Ilka Wittig, and Vadim S. Ten
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0301 basic medicine ,animal structures ,Physiology ,Flavin Mononucleotide ,Clinical Biochemistry ,Flavin mononucleotide ,Flavin group ,Biochemistry ,Redox ,Brain ischemia ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,Structure-Activity Relationship ,Flavins ,medicine ,Animals ,Molecular Biology ,General Environmental Science ,Forum Original Research Communications ,Electron Transport Complex I ,030102 biochemistry & molecular biology ,Cell Biology ,Hydrogen Peroxide ,medicine.disease ,Cell biology ,Mitochondria ,Oxygen ,Oxidative Stress ,030104 developmental biology ,chemistry ,Animals, Newborn ,Reperfusion Injury ,Hypoxia-Ischemia, Brain ,General Earth and Planetary Sciences ,Reactive Oxygen Species ,Reperfusion injury ,Oxidation-Reduction ,Function (biology) ,Mitochondrial Complex I - Abstract
Aims: Brain ischemia/reperfusion (I/R) is associated with impairment of mitochondrial function. However, the mechanisms of mitochondrial failure are not fully understood. This work was undertaken to determine the mechanisms and time course of mitochondrial energy dysfunction after reperfusion following neonatal brain hypoxia-ischemia (HI) in mice. Results: HI/reperfusion decreased the activity of mitochondrial complex I, which was recovered after 30 min of reperfusion and then declined again after 1 h. Decreased complex I activity occurred in parallel with a loss in the content of noncovalently bound membrane flavin mononucleotide (FMN). FMN dissociation from the enzyme is caused by succinate-supported reverse electron transfer. Administration of FMN precursor riboflavin before HI/reperfusion was associated with decreased infarct volume, attenuation of neurological deficit, and preserved complex I activity compared with vehicle-treated mice. In vitro, the rate of FMN release during oxidation of succinate was not affected by the oxygen level and amount of endogenously produced reactive oxygen species. Innovation: Our data suggest that dissociation of FMN from mitochondrial complex I may represent a novel mechanism of enzyme inhibition defining respiratory chain failure in I/R. Strategies preventing FMN release during HI and reperfusion may limit the extent of energy failure and cerebral HI injury. The proposed mechanism of acute I/R-induced complex I impairment is distinct from the generally accepted mechanism of oxidative stress-mediated I/R injury. Conclusion: Our study is the first to highlight a critical role of mitochondrial complex I-FMN dissociation in the development of HI-reperfusion injury of the neonatal brain. Antioxid. Redox Signal. 31, 608–622.
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- 2019
18. ALS/FTD mutant CHCHD10 mice reveal a tissue-specific toxic gain-of-function and mitochondrial stress response
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Samantha Meadows, Crystal Davis, Suzanne R. Burstein, Corey Anderson, Aamir Zuberi, Kirsten Bredvik, Teresa A. Milner, Jalia Dash, Alessandra Piersigilli, Giovanni Manfredi, Cathleen M. Lutz, Hibiki Kawamata, and Laure Case
- Subjects
0301 basic medicine ,Respiratory chain ,Mice, Transgenic ,Biology ,medicine.disease_cause ,Article ,Pathology and Forensic Medicine ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Downregulation and upregulation ,Mitochondrial myopathy ,Gene knockin ,medicine ,Integrated stress response ,Animals ,Myopathy ,Genetic Association Studies ,Mutation ,Neurodegeneration ,Parkinson Disease ,medicine.disease ,Cell biology ,Mitochondria ,030104 developmental biology ,Frontotemporal Dementia ,Gain of Function Mutation ,Mitochondrial Membranes ,Neurology (clinical) ,medicine.symptom ,030217 neurology & neurosurgery - Abstract
Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10), a mitochondrial protein of unknown function, cause a disease spectrum with clinical features of motor neuron disease, dementia, myopathy and cardiomyopathy. To investigate the pathogenic mechanisms of CHCHD10, we generated mutant knock-in mice harboring the mouse-equivalent of a disease associated human S59L mutation, S55L in the endogenous mouse gene. CHCHD10(S55L) mice develop progressive motor deficits, myopathy, cardiomyopathy and accelerated mortality. Critically, CHCHD10 accumulates in aggregates with its paralog CHCHD2 specifically in affected tissues of CHCHD10(S55L) mice, leading to aberrant organelle morphology and function. Aggregates induce a potent mitochondrial integrated stress response (mtISR) through mTORC1 activation, with elevation of stress-induced transcription factors, secretion of myokines, upregulated serine and one-carbon metabolism, and downregulation of respiratory chain enzymes. Conversely, CHCHD10 ablation does not induce disease pathology or activate the mtISR, indicating that CHCHD10(S55L)-dependent disease pathology is not caused by loss-of-function. Overall, CHCHD10(S55L) mice recapitulate crucial aspects of human disease and reveal a novel toxic gain-of-function mechanism through maladaptive mtISR and metabolic dysregulation.
- Published
- 2019
19. The dependence of brain mitochondria reactive oxygen species production on oxygen level is linear, except when inhibited by antimycin A
- Author
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Roger Springett, Anna Stepanova, Csaba Konrad, Giovanni Manfredi, Vadim S. Ten, and Alexander Galkin
- Subjects
0301 basic medicine ,Electron-Transferring Flavoproteins ,Cell Respiration ,Antimycin A ,Mitochondrion ,Biochemistry ,Article ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Oxygen Consumption ,medicine ,Animals ,chemistry.chemical_classification ,Reactive oxygen species ,Brain ,Metabolism ,medicine.disease ,Cell Hypoxia ,Mitochondria ,Oxygen ,030104 developmental biology ,Mitochondrial respiratory chain ,chemistry ,Coenzyme Q – cytochrome c reductase ,Biophysics ,Limiting oxygen concentration ,Energy Metabolism ,Reactive Oxygen Species ,Reperfusion injury ,030217 neurology & neurosurgery - Abstract
Reactive oxygen species (ROS) are by-products of physiological mitochondrial metabolism that are involved in several cellular signaling pathways as well as tissue injury and pathophysiological processes, including brain ischemia/reperfusion injury. The mitochondrial respiratory chain is considered a major source of ROS; however, there is little agreement on how ROS release depends on oxygen concentration. The rate of H2 O2 release by intact brain mitochondria was measured with an Amplex UltraRed assay using a high-resolution respirometer (Oroboros) equipped with a fluorescent optical module and a system of controlled gas flow for varying the oxygen concentration. Three types of substrates were used: malate and pyruvate, succinate and glutamate, succinate alone or glycerol 3-phosphate. For the first time we determined that, with any substrate used in the absence of inhibitors, H2 O2 release by respiring brain mitochondria is linearly dependent on the oxygen concentration. We found that the highest rate of H2 O2 release occurs in conditions of reverse electron transfer when mitochondria oxidize succinate or glycerol 3-phosphate. H2 O2 production by complex III is significant only in the presence of antimycin A and, in this case, the oxygen dependence manifested mixed (linear and hyperbolic) kinetics. We also demonstrated that complex II in brain mitochondria could contribute to ROS generation even in the absence of its substrate succinate when the quinone pool is reduced by glycerol 3-phosphate. Our results underscore the critical importance of reverse electron transfer in the brain, where a significant amount of succinate can be accumulated during ischemia providing a backflow of electrons to complex I at the early stages of reperfusion. Our study also demonstrates that ROS generation in brain mitochondria is lower under hypoxic conditions than in normoxia. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
- Published
- 2019
20. Deactivation of mitochondrial complex I after hypoxia-ischemia in the immature brain
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Csaba Konrad, Susan J. Vannucci, Sergio Guerrero-Castillo, Susanne Arnold, Giovanni Manfredi, Anna Stepanova, and Alexander Galkin
- Subjects
Male ,medicine.medical_specialty ,Neurological disability ,Ischemia ,Hypoxia ischemia ,Electron Transport ,03 medical and health sciences ,0302 clinical medicine ,All institutes and research themes of the Radboud University Medical Center ,Internal medicine ,Medicine ,Animals ,Humans ,Rats, Wistar ,Cells, Cultured ,chemistry.chemical_classification ,Reactive oxygen species ,Electron Transport Complex I ,business.industry ,Brain ,Metabolic Disorders Radboud Institute for Molecular Life Sciences [Radboudumc 6] ,Original Articles ,Hydrogen Peroxide ,medicine.disease ,Mitochondria ,Endocrinology ,Neurology ,chemistry ,Animals, Newborn ,Hypoxia-Ischemia, Brain ,Immature brain ,Female ,Neurology (clinical) ,Cardiology and Cardiovascular Medicine ,business ,Reactive Oxygen Species ,030217 neurology & neurosurgery ,Mitochondrial Complex I - Abstract
Mortality from perinatal hypoxic–ischemic (HI) brain injury reached 1.15 million worldwide in 2010 and is also a major factor for neurological disability in infants. HI directly influences the oxidative phosphorylation enzyme complexes in mitochondria, but the exact mechanism of HI-reoxygenation response in brain remains largely unresolved. After induction of HI-reoxygenation in postnatal day 10 rats, activities of mitochondrial respiratory chain enzymes were analysed and complexome profiling was performed. The effect of conformational state (active/deactive (A/D) transition) of mitochondrial complex I on H2O2release was measured simultaneously with mitochondrial oxygen consumption. In contrast to cytochrome c oxidase and succinate dehydrogenase, HI-reoxygenation resulted in inhibition of mitochondrial complex I at 4 h after reoxygenation. Immediately after HI, we observed a robust increase in the content of deactive (D) form of complex I. The D-form is less active in reactive oxygen species (ROS) production via reversed electron transfer, indicating the key role of the deactivation of complex I in ischemia/reoxygenation. We describe a novel mechanism of mitochondrial response to ischemia in the immature brain. HI induced a deactivation of complex I in order to reduce ROS production following reoxygenation. Delayed activation of complex I represents a novel mitochondrial target for pathological-activated therapy.
- Published
- 2019
21. Mitochondrial Transport and Turnover in the Pathogenesis of Amyotrophic Lateral Sclerosis
- Author
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Giovanni Manfredi and Veronica Granatiero
- Subjects
0301 basic medicine ,Calcium buffering ,SOD1 ,PINK1 ,Review ,Mitochondrion ,Biology ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,0302 clinical medicine ,Mitophagy ,medicine ,Amyotrophic lateral sclerosis ,lcsh:QH301-705.5 ,Mitochondrial transport ,Parkin ,General Immunology and Microbiology ,medicine.disease ,Cell biology ,mitochondria ,030104 developmental biology ,mitophagy ,lcsh:Biology (General) ,nervous system ,Miro1 ,Axoplasmic transport ,ALS ,axonal transport ,General Agricultural and Biological Sciences ,030217 neurology & neurosurgery - Abstract
Neurons are high-energy consuming cells, heavily dependent on mitochondria for ATP generation and calcium buffering. These mitochondrial functions are particularly critical at specific cellular sites, where ionic currents impose a large energetic burden, such as at synapses. The highly polarized nature of neurons, with extremely large axoplasm relative to the cell body, requires mitochondria to be efficiently transported along microtubules to reach distant sites. Furthermore, neurons are post-mitotic cells that need to maintain pools of healthy mitochondria throughout their lifespan. Hence, mitochondrial transport and turnover are essential processes for neuronal survival and function. In neurodegenerative diseases, the maintenance of a healthy mitochondrial network is often compromised. Numerous lines of evidence indicate that mitochondrial impairment contributes to neuronal demise in a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), where degeneration of motor neurons causes a fatal muscle paralysis. Dysfunctional mitochondria accumulate in motor neurons affected by genetic or sporadic forms of ALS, strongly suggesting that the inability to maintain a healthy pool of mitochondria plays a pathophysiological role in the disease. This article critically reviews current hypotheses on mitochondrial involvement in the pathogenesis of ALS, focusing on the alterations of mitochondrial axonal transport and turnover in motor neurons.
- Published
- 2018
22. Energy deficit in parvalbumin neurons leads to circuit dysfunction, impaired sensory gating and social disability
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Anjali M. Rajadhyaksha, Mingrui Zhao, Peter A. Goldstein, Giovanni Manfredi, Cansu Karakaya, Melis Inan, Virginia M. Pickel, Theodore H. Schwartz, and M. Manuszak
- Subjects
0301 basic medicine ,Oscillations ,Interneuron ,Prefrontal Cortex ,Hippocampus ,Mice, Transgenic ,Somatosensory system ,Inhibitory postsynaptic potential ,lcsh:RC321-571 ,Social Skills ,03 medical and health sciences ,0302 clinical medicine ,Interneurons ,medicine ,Animals ,Prefrontal cortex ,lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry ,Parvalbumin ,Neurons ,Sensory gating ,biology ,musculoskeletal, neural, and ocular physiology ,COX ,Somatosensory Cortex ,Sensory Gating ,Mitochondria ,Electrophysiology ,Parvalbumins ,030104 developmental biology ,medicine.anatomical_structure ,Neurology ,nervous system ,biology.protein ,Neuroscience ,030217 neurology & neurosurgery - Abstract
Parvalbumin-expressing, fast spiking interneurons have high-energy demands, which make them particularly susceptible to energy impairment. Recent evidence suggests a link between mitochondrial dysfunction in fast spiking cortical interneurons and neuropsychiatric disorders. However, the effect of mitochondrial dysfunction restricted to parvalbumin interneurons has not been directly addressed in vivo. To investigate the consequences of mitochondrial dysfunction in parvalbumin interneurons in vivo, we generated conditional knockout mice with a progressive decline in oxidative phosphorylation by deleting cox10 gene selectively in parvalbumin neurons (PV-Cox10 CKO). Cox10 ablation results in defective assembly of cytochrome oxidase, the terminal enzyme of the electron transfer chain, and leads to mitochondrial bioenergetic dysfunction. PV-Cox10 CKO mice showed a progressive loss of cytochrome oxidase in cortical parvalbumin interneurons. Cytochrome oxidase protein levels were significantly reduced starting at postnatal day 60, and this was not associated with a change in parvalbumin interneuron density. Analyses of intrinsic electrophysiological properties in layer 5 primary somatosensory cortex revealed that parvalbumin interneurons could not sustain their typical high frequency firing, and their overall excitability was enhanced. An increase in both excitatory and inhibitory input onto parvalbumin interneurons was observed in PV-Cox10 CKO mice, resulting in a disinhibited network with an imbalance of excitation/inhibition. Investigation of network oscillations in PV-Cox10 CKO mice, using local field potential recordings in anesthetized mice, revealed significantly increased gamma and theta frequency oscillation power in both medial prefrontal cortex and hippocampus. PV-Cox10 CKO mice did not exhibit muscle strength or gross motor activity deficits in the time frame of the experiments, but displayed impaired sensory gating and sociability. Taken together, these data reveal that mitochondrial dysfunction in parvalbumin interneurons can alter their intrinsic physiology and network connectivity, resulting in behavioral alterations similar to those observed in neuropsychiatric disorders, such as schizophrenia and autism.
- Published
- 2016
23. Estrogen Receptor Beta Modulates Permeability Transition in Brain Mitochondria
- Author
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Anatoly A. Starkov, Suzanne R. Burstein, Jasmine A. Fels, Ping Zhou, Liping Qian, Costantino Iadecola, Hyun Jeong Kim, Giovanni Manfredi, and Sheng Zhang
- Subjects
0301 basic medicine ,Male ,Estrogen receptor ,Mitochondrion ,Biochemistry ,Hippocampus ,Mitochondrial Membrane Transport Proteins ,Tissue Culture Techniques ,Cyclophilins ,Mice ,0302 clinical medicine ,Piperidines ,Chlorocebus aethiops ,Adenosine Triphosphatases ,Membrane Potential, Mitochondrial ,Mice, Knockout ,biology ,Chemistry ,Neurodegeneration ,Glutamate receptor ,Mitochondrial Proton-Translocating ATPases ,Cell biology ,Mitochondria ,COS Cells ,Cyclosporine ,Female ,Cyclophilin D ,Protein Binding ,Biophysics ,Article ,03 medical and health sciences ,Mitochondrial membrane transport protein ,Prosencephalon ,Sex Factors ,medicine ,Animals ,Estrogen Receptor beta ,Estrogen receptor beta ,Mitochondrial Permeability Transition Pore ,Membrane Proteins ,Cell Biology ,Microtomy ,medicine.disease ,Mice, Inbred C57BL ,030104 developmental biology ,Mitochondrial permeability transition pore ,Forebrain ,biology.protein ,Pyrazoles ,Calcium ,Carrier Proteins ,030217 neurology & neurosurgery - Abstract
Recent evidence highlights a role for sex and hormonal status in regulating cellular responses to ischemic brain injury and neurodegeneration. A key pathological event in ischemic brain injury is the opening of a mitochondrial permeability transition pore (MPT) induced by excitotoxic calcium levels, which can trigger irreversible damage to mitochondria accompanied by the release of pro-apoptotic factors. However, sex differences in brain MPT modulation have not yet been explored. Here, we show that mitochondria isolated from female mouse forebrain have a lower calcium threshold for MPT than male mitochondria, and that this sex difference depends on the MPT regulator cyclophilin D (CypD). We also demonstrate that an estrogen receptor beta (ERβ) antagonist inhibits MPT and knockout of ERβ decreases the sensitivity of mitochondria to the CypD inhibitor, cyclosporine A. These results suggest a functional relationship between ERβ and CypD in modulating brain MPT. Moreover, co-immunoprecipitation studies identify several ERβ binding partners in mitochondria. Among these, we investigate the mitochondrial ATPase as a putative site of MPT regulation by ERβ. We find that previously described interaction between the oligomycin sensitivity-conferring subunit of ATPase (OSCP) and CypD is decreased by ERβ knockout, suggesting that ERβ modulates MPT by regulating CypD interaction with OSCP. Functionally, in primary neurons and hippocampal slice cultures, modulation of ERβ has protective effects against glutamate toxicity and oxygen glucose deprivation, respectively. Taken together, these results reveal a novel pathway of brain MPT regulation by ERβ that could contribute to sex differences in ischemic brain injury and neurodegeneration.
- Published
- 2018
24. In vitro and in vivo studies of the ALS-FTLD protein CHCHD10 reveal novel mitochondrial topology and protein interactions
- Author
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Teresa A. Milner, Myriam Bourens, Suzanne R. Burstein, Aamir Zuberi, Hibiki Kawamata, Suzanne M. Cloonan, R Zeng, Giovanni Manfredi, Federica Valsecchi, Antoni Barrientos, and Cathleen M. Lutz
- Subjects
0301 basic medicine ,Models, Molecular ,Mitochondrial intermembrane space ,Substantia nigra ,Biology ,Topology ,medicine.disease_cause ,Mitochondrial Proteins ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Protein Interaction Mapping ,Genetics ,medicine ,Animals ,Humans ,Protein Interaction Domains and Motifs ,Myopathy ,Molecular Biology ,Genetics (clinical) ,Loss function ,Genetic Association Studies ,Mice, Knockout ,Mutation ,Amyotrophic Lateral Sclerosis ,Skeletal muscle ,General Medicine ,Articles ,Motor neuron ,Molecular biology ,Cell biology ,Mitochondria ,DNA-Binding Proteins ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,HEK293 Cells ,Frontotemporal Dementia ,Knockout mouse ,Protein Structural Elements ,medicine.symptom ,Carrier Proteins ,030217 neurology & neurosurgery ,HeLa Cells ,Transcription Factors - Abstract
Mutations in coiled-coil-helix-coiled-coil-helix-domain containing 10 (CHCHD10), a mitochondrial twin CX9C protein whose function is still unknown, cause myopathy, motor neuron disease, frontotemporal dementia, and Parkinson's disease. Here, we investigate CHCHD10 topology and its protein interactome, as well as the effects of CHCHD10 depletion or expression of disease-associated mutations in wild-type cells. We find that CHCHD10 associates with membranes in the mitochondrial intermembrane space, where it interacts with a closely related protein, CHCHD2. Furthermore, both CHCHD10 and CHCHD2 interact with p32/GC1QR, a protein with various intra and extra-mitochondrial functions. CHCHD10 and CHCHD2 have short half-lives, suggesting regulatory rather than structural functions. Cell lines with CHCHD10 knockdown do not display bioenergetic defects, but, unexpectedly, accumulate excessive intramitochondrial iron. In mice, CHCHD10 is expressed in many tissues, most abundantly in heart, skeletal muscle, liver, and in specific CNS regions, notably the dopaminergic neurons of the substantia nigra and spinal cord neurons, which is consistent with the pathology associated with CHCHD10 mutations. Homozygote CHCHD10 knockout mice are viable, have no gross phenotypes, no bioenergetic defects or ultrastructural mitochondrial abnormalities in brain, heart or skeletal muscle, indicating that functional redundancy or compensatory mechanisms for CHCHD10 loss occur in vivo. Instead, cells expressing S59L or R15L mutant versions of CHCHD10, but not WT, have impaired mitochondrial energy metabolism. Taken together, the evidence obtained from our in vitro and in vivo studies suggest that CHCHD10 mutants cause disease through a gain of toxic function mechanism, rather than a loss of function.
- Published
- 2017
25. Exploring new pathways of neurodegeneration in ALS: The role of mitochondria quality control
- Author
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Giovanni Manfredi and Gloria M. Palomo
- Subjects
General Neuroscience ,Amyotrophic Lateral Sclerosis ,SOD1 ,Neurodegeneration ,Mitophagy ,Mitochondrion ,Biology ,medicine.disease ,medicine.disease_cause ,Article ,Parkin ,Mitochondria ,Proteostasis ,mitochondrial fusion ,Nerve Degeneration ,medicine ,Animals ,Humans ,Neurology (clinical) ,Molecular Biology ,Neuroscience ,Oxidative stress ,Developmental Biology - Abstract
Neuronal cells are highly dependent on mitochondria, and mitochondrial dysfunction is associated with neurodegenerative diseases. As perturbed mitochondrial function renders neurons extremely sensitive to a wide variety of insults, such as oxidative stress and bioenergetic defects, mitochondrial defects can profoundly affect neuronal fate. Several studies have linked ALS with mitochondrial dysfunction, stemming from observations of mitochondrial abnormalities, both in patients and in cellular and mouse models of familial forms of ALS. Mitochondrial changes have been thoroughly investigated in mutants of superoxide dismutase 1 (SOD1), one of the most common causes of familial ALS, for which excellent cellular and animal models are available, but recently evidence is emerging also in other forms of ALS, both familial and sporadic. Mitochondrial defects in ALS involve many critical physiopathological processes, from defective bioenergetics to abnormal calcium homeostasis, altered morphology and impaired trafficking. In this review, we summarize established evidence of mitochondrial dysfunction in ALS, especially in SOD1 mutant models of familial ALS. The main focus of the review is on defective mitochondrial quality control (MQC) in ALS. MQC operates at multiple levels to clear damaged proteins through proteostasis and to eliminate irreparably damaged organelles through mitophagy. However, since ALS motor neurons progressively accumulate damaged mitochondria, it is plausible that the MQC is ineffective or overwhelmed by excessive workload imposed by the chronic and extensive mitochondrial damage. This article is part of a Special Issue entitled ALS complex pathogenesis .
- Published
- 2015
26. Role of soluble adenylyl cyclase in mitochondria
- Author
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Giovanni Manfredi, Federica Valsecchi, and Csaba Konrad
- Subjects
sAC ,Oxidative phosphorylation ,Mitochondrion ,Biology ,Oxidative Phosphorylation ,Article ,ADCY10 ,Protein phosphorylation ,cAMP ,Humans ,PKA ,Protein kinase A ,education ,Molecular Biology ,education.field_of_study ,ADCY9 ,Soluble adenylyl cyclase ,Cyclic AMP-Dependent Protein Kinases ,Mitochondria ,Cell biology ,Enzyme Activation ,Biochemistry ,Molecular Medicine ,Phosphorylation ,Adenylyl Cyclases - Abstract
The soluble adenylyl cyclase (sAC) catalyzes the conversion of ATP into cyclic AMP (cAMP). Recent studies have shed new light on the role of sAC localized in mitochondria and its product cAMP, which drives mitochondrial protein phosphorylation and regulation of the oxidative phosphorylation system and other metabolic enzymes, presumably through the activation of intra-mitochondrial PKA. In this review article, we summarize recent findings on mitochondrial sAC activation by bicarbonate (HCO3−) and calcium (Ca2+) and the effects on mitochondrial metabolism. We also discuss putative mechanisms whereby sAC-mediated mitochondrial protein phosphorylation regulates mitochondrial metabolism. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.
- Published
- 2014
27. Fibroblast bioenergetics to classify amyotrophic lateral sclerosis patients
- Author
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John Ravits, Steven S. Gross, Kirsten Bredvik, Andrea J. Arreguin, Nicholas J. Maragakis, Csaba Konrad, Jonathan Hupf, Steven A. Cajamarca, Jonathan D. Glass, Hibiki Kawamata, Hiroshi Mitsumoto, Timothy M. Miller, Giovanni Manfredi, and Chadwick M. Hales
- Subjects
Adult ,Male ,0301 basic medicine ,Pathology ,medicine.medical_specialty ,Bioenergetics ,PLS ,lcsh:Geriatrics ,Mitochondrion ,Biology ,lcsh:RC346-429 ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,C9orf72 ,Machine learning ,medicine ,Humans ,Glycolysis ,Amyotrophic lateral sclerosis ,Fibroblast ,Molecular Biology ,lcsh:Neurology. Diseases of the nervous system ,Aged ,Skin ,Primary Lateral Sclerosis ,Aged, 80 and over ,Amyotrophic Lateral Sclerosis ,Middle Aged ,Fibroblasts ,medicine.disease ,Mitochondria ,lcsh:RC952-954.6 ,030104 developmental biology ,medicine.anatomical_structure ,Hypermetabolism ,Female ,Neurology (clinical) ,ALS ,Energy Metabolism ,030217 neurology & neurosurgery ,Research Article - Abstract
Background The objective of this study was to investigate cellular bioenergetics in primary skin fibroblasts derived from patients with amyotrophic lateral sclerosis (ALS) and to determine if they can be used as classifiers for patient stratification. Methods We assembled a collection of unprecedented size of fibroblasts from patients with sporadic ALS (sALS, n = 171), primary lateral sclerosis (PLS, n = 34), ALS/PLS with C9orf72 mutations (n = 13), and healthy controls (n = 91). In search for novel ALS classifiers, we performed extensive studies of fibroblast bioenergetics, including mitochondrial membrane potential, respiration, glycolysis, and ATP content. Next, we developed a machine learning approach to determine whether fibroblast bioenergetic features could be used to stratify patients. Results Compared to controls, sALS and PLS fibroblasts had higher average mitochondrial membrane potential, respiration, and glycolysis, suggesting that they were in a hypermetabolic state. Only membrane potential was elevated in C9Orf72 lines. ATP steady state levels did not correlate with respiration and glycolysis in sALS and PLS lines. Based on bioenergetic profiles, a support vector machine (SVM) was trained to classify sALS and PLS with 99% specificity and 70% sensitivity. Conclusions sALS, PLS, and C9Orf72 fibroblasts share hypermetabolic features, while presenting differences of bioenergetics. The absence of correlation between energy metabolism activation and ATP levels in sALS and PLS fibroblasts suggests that in these cells hypermetabolism is a mechanism to adapt to energy dissipation. Results from SVM support the use of metabolic characteristics of ALS fibroblasts and multivariate analysis to develop classifiers for patient stratification. Electronic supplementary material The online version of this article (10.1186/s13024-017-0217-5) contains supplementary material, which is available to authorized users.
- Published
- 2017
28. The Mitochondrial Unfolded Protein Response as a Non-Oncogene Addiction to Support Adaptation to Stress during Transformation in Cancer and Beyond
- Author
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Giovanni Manfredi, Timothy C. Kenny, and Doris Germain
- Subjects
0301 basic medicine ,Cancer Research ,SIRT3 ,Mini Review ,Mitochondrion ,Biology ,medicine.disease_cause ,lcsh:RC254-282 ,03 medical and health sciences ,Mitochondrial unfolded protein response ,sirtuin deacetylase ,medicine ,cancer ,Cancer ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Oncogene Addiction ,medicine.disease ,Cell biology ,mitochondria ,030104 developmental biology ,Oncology ,mitochondrial unfolded protein response ,Protein folding ,Adaptation ,ALS ,Oxidative stress ,estrogen receptor - Abstract
Upon accumulation of misfolded proteins in the mitochondria, the mitochondrial unfolded protein response (UPRmt) is activated. This review focuses on the role of this response in cancer. We discuss evidence that during transformation, the UPRmt may play an essential role in the maintenance of the integrity of the mitochondria in the face of increased oxidative stress. However, the role of the UPRmt in other diseases is also emerging and is therefore also briefly discussed.
- Published
- 2017
29. Distinct intracellular sAC-cAMP domains regulate ER Ca
- Author
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Federica, Valsecchi, Csaba, Konrad, Marilena, D'Aurelio, Lavoisier S, Ramos-Espiritu, Anna, Stepanova, Suzanne R, Burstein, Alexander, Galkin, Jordi, Magranè, Anatoly, Starkov, Jochen, Buck, Lonny R, Levin, and Giovanni, Manfredi
- Subjects
Fibroblasts ,Cell Fractionation ,Endoplasmic Reticulum ,Oxidative Phosphorylation ,Cell Line ,Mitochondria ,Gene Knockout Techniques ,Mice ,Oxygen Consumption ,Gene Expression Regulation ,Cyclic AMP ,Animals ,Inositol 1,4,5-Trisphosphate Receptors ,Calcium ,Calcium Signaling ,Adenylyl Cyclases ,Research Article - Abstract
cAMP regulates a wide variety of physiological functions in mammals. This single second messenger can regulate multiple, seemingly disparate functions within independently regulated cell compartments. We have previously identified one such compartment inside the matrix of the mitochondria, where soluble adenylyl cyclase (sAC) regulates oxidative phosphorylation (OXPHOS). We now show that sAC knockout fibroblasts have a defect in OXPHOS activity and attempt to compensate for this defect by increasing OXPHOS proteins. Importantly, sAC knockout cells also exhibit decreased probability of endoplasmic reticulum (ER) Ca2+ release associated with diminished phosphorylation of the inositol 3-phosphate receptor. Restoring sAC expression exclusively in the mitochondrial matrix rescues OXPHOS activity and reduces mitochondrial biogenesis, indicating that these phenotypes are regulated by intramitochondrial sAC. In contrast, Ca2+ release from the ER is only rescued when sAC expression is restored throughout the cell. Thus, we show that functionally distinct, sAC-defined, intracellular cAMP signaling domains regulate metabolism and Ca2+ signaling.
- Published
- 2017
30. Mutant TDP-43 does not impair mitochondrial bioenergetics in vitro and in vivo
- Author
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Giovanni Manfredi, Federica Valsecchi, Meri Gerges, Csaba Konrad, Gloria M. Palomo, Pablo M. Peixoto, Kirsten Bredvik, John Ravits, Leonard Petrucelli, Anatoly A. Starkov, and Hibiki Kawamata
- Subjects
0301 basic medicine ,TAR DNA-binding protein 43 ,TDP-43 ,SOD1 ,Mutant ,Respiratory chain ,Mice, Transgenic ,Oxidative phosphorylation ,Bioenergetics ,lcsh:Geriatrics ,Mitochondrion ,Oxidative Phosphorylation ,lcsh:RC346-429 ,Cell Line ,Mice ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Mutant protein ,mental disorders ,Animals ,Humans ,Mitochondrial calcium uptake ,Molecular Biology ,lcsh:Neurology. Diseases of the nervous system ,ATP synthase ,biology ,Brain ,nutritional and metabolic diseases ,Molecular biology ,Mitochondria ,nervous system diseases ,DNA-Binding Proteins ,lcsh:RC952-954.6 ,030104 developmental biology ,Mutation ,biology.protein ,Calcium ,Neurology (clinical) ,ALS ,Energy Metabolism ,030217 neurology & neurosurgery ,Research Article - Abstract
Background Mitochondrial dysfunction has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Functional studies of mitochondrial bioenergetics have focused mostly on superoxide dismutase 1 (SOD1) mutants, and showed that mutant human SOD1 impairs mitochondrial oxidative phosphorylation, calcium homeostasis, and dynamics. However, recent reports have indicated that alterations in transactivation response element DNA-binding protein 43 (TDP-43) can also lead to defects of mitochondrial morphology and dynamics. Furthermore, it was proposed that TDP-43 mutations cause oxidative phosphorylation impairment associated with respiratory chain defects and that these effects were caused by mitochondrial localization of the mutant protein. Here, we investigated the presence of bioenergetic defects in the brain of transgenic mice expressing human mutant TDP-43 (TDP-43A315T mice), patient derived fibroblasts, and human cells expressing mutant forms of TDP-43. Methods In the brain of TDP-43A315T mice, TDP-43 mutant fibroblasts, and cells expressing mutant TDP-43, we tested several bioenergetics parameters, including mitochondrial respiration, ATP synthesis, and calcium handling. Differences between mutant and control samples were evaluated by student t-test or by ANOVA, followed by Bonferroni correction, when more than two groups were compared. Mitochondrial localization of TDP-43 was investigated by immunocytochemistry in fibroblasts and by subcellular fractionation and western blot of mitochondrial fractions in mouse brain. Results We did not observe defects in any of the mitochondrial bioenergetic functions that were tested in TDP-43 mutants. We detected a small amount of TDP-43A315T peripherally associated with brain mitochondria. However, there was no correlation between TDP-43 associated with mitochondria and respiratory chain dysfunction. In addition, we observed increased calcium uptake in mitochondria from TDP-43A315T mouse brain and cells expressing A315T mutant TDP-43. Conclusions While alterations of mitochondrial morphology and dynamics in TDP-43 mutant neurons are well established, the present study did not demonstrate oxidative phosphorylation defects in TDP-43 mutants, in vitro and in vivo. On the other hand, the increase in mitochondrial calcium uptake in A315T TDP-43 mutants was an intriguing finding, which needs to be investigated further to understand its mechanisms and potential pathogenic implications.
- Published
- 2017
31. IRE1α-XBP1 controls T cell function in ovarian cancer by regulating mitochondrial activity
- Author
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Ievgen Motorykin, Sheng Zhang, Tito A. Sandoval, Sahil Chopra, Csaba Konrad, Melanie R. Rutkowski, Gabriel A. Rabinovich, Mahesh Raundhal, Minkyung Song, Kevin Holcomb, Paulo C. Rodriguez, Chang-Suk Chae, Jose R. Conejo-Garcia, Dmitriy Zamarin, Laurie H. Glimcher, Michael J. Crowley, Andrew V. Kossenkov, Sarah E. Bettigole, Hee Rae Shin, Giovanni Manfredi, Juan R. Cubillos-Ruiz, Kyle K. Payne, Chen Tan, Ricardo A. Chaurio, and Juan P. Cerliani
- Subjects
0301 basic medicine ,X-Box Binding Protein 1 ,Glycosylation ,Glutamine ,T-Lymphocytes ,Mitochondrion ,Glutamine transport ,purl.org/becyt/ford/1 [https] ,Mice ,Neoplasm Metastasis ,Ovarian Neoplasms ,Multidisciplinary ,Otras Medicina Básica ,Ascites ,purl.org/becyt/ford/3.1 [https] ,Endoplasmic Reticulum Stress ,OVARIAN CANCER ,3. Good health ,XBP1 ,Mitochondria ,Gene Expression Regulation, Neoplastic ,Survival Rate ,Medicina Básica ,medicine.anatomical_structure ,Disease Progression ,purl.org/becyt/ford/3 [https] ,Female ,CIENCIAS NATURALES Y EXACTAS ,Signal Transduction ,CIENCIAS MÉDICAS Y DE LA SALUD ,T cell ,Otras Ciencias Biológicas ,Cell Respiration ,Inmunología ,IRE1 ,Biology ,Protein Serine-Threonine Kinases ,Ciencias Biológicas ,03 medical and health sciences ,Interferon-gamma ,Downregulation and upregulation ,Endoribonucleases ,medicine ,Animals ,Humans ,purl.org/becyt/ford/1.6 [https] ,Endoplasmic reticulum ,T CELL ,medicine.disease ,030104 developmental biology ,Glucose ,Unfolded protein response ,Cancer research ,Unfolded Protein Response ,Amino Acid Transport Systems, Basic ,Tumor Escape ,Ovarian cancer ,Neoplasm Transplantation - Abstract
Tumours evade immune control by creating hostile microenvironments that perturb T cell metabolism and effector function 1?4 . However, it remains unclear how intra-tumoral T cells integrate and interpret metabolic stress signals. Here we report that ovarian cancer?an aggressive malignancy that is refractory to standard treatments and current immunotherapies 5?8 ?induces endoplasmic reticulum stress and activates the IRE1α?XBP1 arm of the unfolded protein response 9,10 in T cells to control their mitochondrial respiration and anti-tumour function. In T cells isolated from specimens collected from patients with ovarian cancer, upregulation of XBP1 was associated with decreased infiltration of T cells into tumours and with reduced IFNG mRNA expression. Malignant ascites fluid obtained from patients with ovarian cancer inhibited glucose uptake and caused N-linked protein glycosylation defects in T cells, which triggered IRE1α?XBP1 activation that suppressed mitochondrial activity and IFNγ production. Mechanistically, induction of XBP1 regulated the abundance of glutamine carriers and thus limited the influx of glutamine that is necessary to sustain mitochondrial respiration in T cells under glucose-deprived conditions. Restoring N-linked protein glycosylation, abrogating IRE1α?XBP1 activation or enforcing expression of glutamine transporters enhanced mitochondrial respiration in human T cells exposed to ovarian cancer ascites. XBP1-deficient T cells in the metastatic ovarian cancer milieu exhibited global transcriptional reprogramming and improved effector capacity. Accordingly, mice that bear ovarian cancer and lack XBP1 selectively in T cells demonstrate superior anti-tumour immunity, delayed malignant progression and increased overall survival. Controlling endoplasmic reticulum stress or targeting IRE1α?XBP1 signalling may help to restore the metabolic fitness and anti-tumour capacity of T cells in cancer hosts. Fil: Song, Minkyung. Weill Cornell Medicine; Estados Unidos Fil: Sandoval, Tito A.. Weill Cornell Medicine; Estados Unidos Fil: Chae, Chang-Suk. Weill Cornell Medicine; Estados Unidos Fil: Chopra, Sahil. Weill Cornell Medicine; Estados Unidos Fil: Tan, Chen. Weill Cornell Medicine; Estados Unidos Fil: Rutkowski, Melanie R.. University of Virginia; Estados Unidos Fil: Raundhal, Mahesh. Dana Farber Cancer Institute; Estados Unidos. Harvard Medical School; Estados Unidos Fil: Chaurio, Ricardo A.. H. Lee Moffitt Cancer Center & Research Institute; Estados Unidos Fil: Payne, Kyle K.. H. Lee Moffitt Cancer Center & Research Institute; Estados Unidos Fil: Konrad, Csaba. Weill Cornell Medicine; Estados Unidos Fil: Bettigole, Sarah E.. Quentis Therapeutics Inc.; Estados Unidos Fil: Shin, Hee Rae. Quentis Therapeutics Inc.; Estados Unidos Fil: Crowley, Michael J. P.. Weill Cornell Graduate School of Medical Sciences; Estados Unidos Fil: Cerliani, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Kossenkov, Andrew V.. The Wistar Institute; Estados Unidos Fil: Motorykin, Ievgen. Weill Cornell Medicine,; Estados Unidos Fil: Zhang, Sheng. Weill Cornell Medicine,; Estados Unidos Fil: Manfredi, Giovanni. Weill Cornell Medicine,; Estados Unidos Fil: Zamarin, Dmitriy. Memorial Sloan Kettering Cancer Center; Estados Unidos Fil: Holcomb, Kevin. Weill Cornell Medicine,; Estados Unidos Fil: Rodriguez, Paulo C.. H. Lee Moffitt Cancer Center & Research Institute; Estados Unidos Fil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Conejo Garcia, Jose R.. H. Lee Moffitt Cancer Center & Research Institute; Estados Unidos Fil: Glimcher, Laurie H.. Dana Farber Cancer Institute; Estados Unidos. Harvard Medical School; Estados Unidos Fil: Cubillos-Ruiz, Juan R.. Weill Graduate School Of Medical Sciences; Estados Unidos. Weill Graduate School Of Medical Sciences; Estados Unidos
- Published
- 2017
32. Dichotomous Effects of Chronic Intermittent Hypoxia on Focal Cerebral Ischemic Injury
- Author
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Giovanni Manfredi, Pablo M. Peixoto, Christal G. Coleman, Giuseppe Faraco, Costantino Iadecola, Henning U. Voss, Katherine A Jackman, Ping Zhou, and Virginia M. Pickel
- Subjects
Male ,Ischemia ,Mitochondrion ,Article ,Mice ,Laser-Doppler Flowmetry ,Animals ,Medicine ,Hypoxia ,Stroke ,Advanced and Specialized Nursing ,chemistry.chemical_classification ,Reactive oxygen species ,business.industry ,Sleep apnea ,Infarction, Middle Cerebral Artery ,Depolarization ,Isolated brain ,medicine.disease ,Mitochondria ,Mice, Inbred C57BL ,Disease Models, Animal ,Cerebral blood flow ,chemistry ,Cerebrovascular Circulation ,Anesthesia ,Chronic Disease ,Neurology (clinical) ,Reactive Oxygen Species ,Cardiology and Cardiovascular Medicine ,business - Abstract
Background and Purpose— Obstructive sleep apnea, a condition associated with chronic intermittent hypoxia (CIH), carries an increased risk of stroke. However, CIH has been reported to either increase or decrease brain injury in models of focal cerebral ischemia. The factors determining the differential effects of CIH on ischemic injury and their mechanisms remain unclear. Here, we tested the hypothesis that the intensity of the hypoxic challenge determines the protective or destructive nature of CIH by modulating mitochondrial resistance to injury. Methods— Male C57Bl/6J mice were exposed to CIH with 10% or 6% O 2 for ≤35 days and subjected to transient middle cerebral artery occlusion. Motor deficits and infarct volume were assessed 3 days later. Intraischemic cerebral blood flow was measured by laser-Doppler flowmetry and resting cerebral blood flow by arterial spin labeling MRI. Ca 2+ -induced mitochondrial depolarization and reactive oxygen species production were evaluated in isolated brain mitochondria. Results— We found that 10% CIH is neuroprotective, whereas 6% CIH exacerbates tissue damage. No differences in resting or intraischemic cerebral blood flow were observed between 6% and 10% CIH. However, 10% CIH reduced, whereas 6% CIH increased, mitochondrial reactive oxygen species production and susceptibility to Ca 2+ -induced depolarizations. Conclusions— The influence of CIH on the ischemic brain is dichotomous and can be attributed, in part, to changes in the mitochondrial susceptibility to injury. The findings highlight a previously unappreciated complexity in the effect of CIH on the brain, which needs to be considered in evaluating the neurological effect of conditions associated with cyclic hypoxia.
- Published
- 2014
33. Abnormal mitochondrial transport and morphology are common pathological denominators in SOD1 and TDP43 ALS mouse models
- Author
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Wen-Biao Gan, Czrina Cortez, Jordi Magrané, and Giovanni Manfredi
- Subjects
Male ,Genetically modified mouse ,Transgene ,SOD1 ,Mutant ,Mice, Transgenic ,Biology ,Mitochondrion ,Mice ,Superoxide Dismutase-1 ,Genetics ,medicine ,Animals ,Humans ,Amyotrophic lateral sclerosis ,Molecular Biology ,Genetics (clinical) ,Mitochondrial transport ,Motor Neurons ,Neurons ,Superoxide Dismutase ,Amyotrophic Lateral Sclerosis ,nutritional and metabolic diseases ,Articles ,General Medicine ,Anatomy ,medicine.disease ,Sciatic Nerve ,Mitochondria ,Cell biology ,DNA-Binding Proteins ,Disease Models, Animal ,nervous system ,Disease Progression ,Axoplasmic transport - Abstract
Neuronal mitochondrial morphology abnormalities occur in models of familial amyotrophic lateral sclerosis (ALS) associated with SOD1 and TDP43 mutations. These abnormalities have been linked to mitochondrial axonal transport defects, but the temporal and spatial relationship between mitochondrial morphology and transport alterations in these two distinct genetic forms of ALS has not been investigated in vivo. To address this question, we crossed SOD1 (wild-type SOD1(WT) and mutant SOD1(G93A)) or TDP43 (mutant TDP43(A315T)) transgenic mice with mice expressing the fluorescent protein Dendra targeted to mitochondria in neurons (mitoDendra). At different time points during the disease course, we studied mitochondrial transport in the intact sciatic nerve of living mice and analyzed axonal mitochondrial morphology at multiple sites, spanning from the spinal cord to the motor terminals. Defects of retrograde mitochondrial transport were detected at 45 days of age, before the onset of symptoms, in SOD1(G93A) and TDP43(A315T) mice, but not in SOD1(WT). At later disease stages, also anterograde mitochondrial transport was affected in both mutant mouse lines. In SOD1(G93A) mice, mitochondrial morphological abnormalities were apparent at 15 days of age, thus preceding transport abnormalities. Conversely, in TDP43(A315T) mice, morphological abnormalities appeared after the onset of transport defects. Taken together, these findings demonstrate that neuronal mitochondrial transport and morphology abnormalities occur in vivo and that they are common denominators of different genetic forms of the ALS. At the same time, differences in the temporal and spatial manifestation of mitochondrial abnormalities between the two mouse models of familial ALS imply that different molecular mechanisms may be involved.
- Published
- 2013
34. Barth syndrome: Cellular compensation of mitochondrial dysfunction and apoptosis inhibition due to changes in cardiolipin remodeling linked to tafazzin (TAZ) gene mutation
- Author
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Frédéric M. Vaz, Thomas Landes, Patrice X. Petit, Guillaume Vial, Pascale Bellenguer, Nellie Taleux, Christian Slomianny, Jean-Philippe Puech, Francois Gonzalvez, Ian M. Møller, Marilena D'Aurelio, Marie Boutant, Giovanni Manfredi, Aoula Moustapha, Eyal Gottlieb, Ronald J.A. Wanders, Laeticia Arnauné-Pelloquin, Riekelt H. Houtkooper, AGEM - Amsterdam Gastroenterology Endocrinology Metabolism, and Laboratory Genetic Metabolic Diseases
- Subjects
Cardiolipins ,Tafazzin ,Respiratory chain ,Apoptosis ,Citrate (si)-Synthase ,Biology ,Mitochondrion ,Cell Line ,chemistry.chemical_compound ,Adenosine Triphosphate ,Superoxides ,medicine ,Cardiolipin ,Humans ,Citrate synthase ,Respiratory complex ,Lymphocytes ,Molecular Biology ,Caspase 8 ,Cell Death ,Monolysocardiolipin ,Barth syndrome ,medicine.disease ,Mitochondria ,Cell biology ,Electron Transport Chain Complex Proteins ,chemistry ,Barth Syndrome ,Mutation ,Respirasome ,biology.protein ,Molecular Medicine ,Lysophospholipids ,Reactive oxygen species ,Acyltransferases ,Signal Transduction ,Transcription Factors - Abstract
Cardiolipin is a mitochondrion-specific phospholipid that stabilizes the assembly of respiratory chain complexes, favoring full-yield operation. It also mediates key steps in apoptosis. In Barth syndrome, an X chromosome-linked cardiomyopathy caused by tafazzin mutations, cardiolipins display acyl chain modifications and are present at abnormally low concentrations, whereas monolysocardiolipin accumulates. Using immortalized lymphoblasts from Barth syndrome patients, we showed that the production of abnormal cardiolipin led to mitochondrial alterations. Indeed, the lack of normal cardiolipin led to changes in electron transport chain stability, resulting in cellular defects. We found a destabilization of the supercomplex (respirasome) I+III2+IVn but also decreased amounts of individual complexes I and IV and supercomplexes I+III and III+IV. No changes were observed in the amounts of individual complex III and complex II. We also found decreased levels of complex V. This complex is not part of the supercomplex suggesting that cardiolipin is required not only for the association/stabilization of the complexes into supercomplexes but also for the modulation of the amount of individual respiratory chain complexes. However, these alterations were compensated by an increase in mitochondrial mass, as demonstrated by electron microscopy and measurements of citrate synthase activity. We suggest that this compensatory increase in mitochondrial content prevents a decrease in mitochondrial respiration and ATP synthesis in the cells. We also show, by extensive flow cytometry analysis, that the type II apoptosis pathway was blocked at the mitochondrial level and that the mitochondria of patients with Barth syndrome cannot bind active caspase-8. Signal transduction is thus blocked before any mitochondrial event can occur. Remarkably, basal levels of superoxide anion production were slightly higher in patients' cells than in control cells as previously evidenced via an increased protein carbonylation in the taz1Δ mutant in the yeast. This may be deleterious to cells in the long term. The consequences of mitochondrial dysfunction and alterations to apoptosis signal transduction are considered in light of the potential for the development of future treatments.
- Published
- 2013
35. Differential susceptibility of mitochondrial complex II to inhibition by oxaloacetate in brain and heart
- Author
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Anna, Stepanova, Yevgeniya, Shurubor, Federica, Valsecchi, Giovanni, Manfredi, and Alexander, Galkin
- Subjects
Oxaloacetic Acid ,Q1, 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone ,I/R, ischemia/reperfusion ,Article ,A/D, active/de-active transition ,Oxaloacetate ,Mice ,Ischemia ,Animals ,DDM, n-Dodecyl β-d-maltoside ,GOT1, glutamic/oxaloacetate transaminase, Type 1 ,HAR, hexaammineruthenium ,SMP, submitochondrial particles ,Electron Transport Complex II ,Myocardium ,Brain ,FAD, flavin adenine dinucleotide ,Mitochondria ,Mitochondrial complex II ,Succinate dehydrogenase ,SET medium, sucrose/EDTA/Tris medium ,RET, reverse electron transfer ,SDH, succinate dehydrogenase ,OAA, oxaloacetate ,Krebs cycle ,TCA, tricarboxylic acid cycle - Abstract
Mitochondrial Complex II is a key mitochondrial enzyme connecting the tricarboxylic acid (TCA) cycle and the electron transport chain. Studies of complex II are clinically important since new roles for this enzyme have recently emerged in cell signalling, cancer biology, immune response and neurodegeneration. Oxaloacetate (OAA) is an intermediate of the TCA cycle and at the same time is an inhibitor of complex II with high affinity (Kd ~ 10− 8 M). Whether or not OAA inhibition of complex II is a physiologically relevant process is a significant, but still controversial topic. We found that complex II from mouse heart and brain tissue has similar affinity to OAA and that only a fraction of the enzyme in isolated mitochondrial membranes (30.2 ± 6.0% and 56.4 ± 5.6% in the heart and brain, respectively) is in the free, active form. Since OAA could bind to complex II during isolation, we established a novel approach to deplete OAA in the homogenates at the early stages of isolation. In heart, this treatment significantly increased the fraction of free enzyme, indicating that OAA binds to complex II during isolation. In brain the OAA-depleting system did not significantly change the amount of free enzyme, indicating that a large fraction of complex II is already in the OAA-bound inactive form. Furthermore, short-term ischemia resulted in a dramatic decline of OAA in tissues, but it did not change the amount of free complex II. Our data show that in brain OAA is an endogenous effector of complex II, potentially capable of modulating the activity of the enzyme., Highlights • Complex II in mitochondrial membranes is inhibited by tightly-bound oxaloacetate. • Oxaloacetate binds to the heart enzyme during isolation. • In brain a large fraction of Complex II is present in oxaloacetate-bound form. • Short-time tissue ischemia does not affect the content of the free Complex II in brain.
- Published
- 2016
36. Mitochondrial Dynamics and Bioenergetic Dysfunction Is Associated with Synaptic Alterations in Mutant SOD1 Motor Neurons
- Author
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Mary Anne Sahawneh, Alvaro G. Estévez, Jordi Magrané, Giovanni Manfredi, and Serge Przedborski
- Subjects
Mitochondrial Diseases ,Neurite ,Primary Cell Culture ,SOD1 ,Mitochondrion ,Biology ,Mitochondrial Size ,Article ,Rats, Sprague-Dawley ,Superoxide Dismutase-1 ,Pregnancy ,medicine ,Animals ,Humans ,Amyotrophic lateral sclerosis ,Motor Neurons ,Superoxide Dismutase ,General Neuroscience ,Amyotrophic Lateral Sclerosis ,Motor neuron ,medicine.disease ,Mitochondria ,Rats ,Disease Models, Animal ,medicine.anatomical_structure ,nervous system ,mitochondrial fusion ,Synapses ,Axoplasmic transport ,Female ,Rats, Transgenic ,Energy Metabolism ,Neuroscience - Abstract
Mutations in Cu,Zn superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis (FALS), a rapidly fatal motor neuron disease. Mutant SOD1 has pleiotropic toxic effects on motor neurons, among which mitochondrial dysfunction has been proposed as one of the contributing factors in motor neuron demise. Mitochondria are highly dynamic in neurons; they are constantly reshaped by fusion and move along neurites to localize at sites of high-energy utilization, such as synapses. The finding of abnormal mitochondria accumulation in neuromuscular junctions, where the SOD1-FALS degenerative process is though to initiate, suggests that impaired mitochondrial dynamics in motor neurons may be involved in pathogenesis. We addressed this hypothesis by live imaging microscopy of photo-switchable fluorescent mitoDendra in transgenic rat motor neurons expressing mutant or wild-type human SOD1. We demonstrate that mutant SOD1 motor neurons have impaired mitochondrial fusion in axons and cell bodies. Mitochondria also display selective impairment of retrograde axonal transport, with reduced frequency and velocity of movements. Fusion and transport defects are associated with smaller mitochondrial size, decreased mitochondrial density, and defective mitochondrial membrane potential. Furthermore, mislocalization of mitochondria at synapses among motor neurons,in vitro, correlates with abnormal synaptic number, structure, and function. Dynamics abnormalities are specific to mutant SOD1 motor neuron mitochondria, since they are absent in wild-type SOD1 motor neurons, they do not involve other organelles, and they are not found in cortical neurons. Together, these results suggest that impaired mitochondrial dynamics may contribute to the selective degeneration of motor neurons in SOD1-FALS.
- Published
- 2012
37. Hiding in plain sight: Uncovering a new function of vitamin A in redox signaling
- Author
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Ulrich Hämmerling, Giovanni Manfredi, Rebeca Acín-Pérez, Beatrice Hoyos, and Donald A. Fischman
- Subjects
Cytochrome ,Citric Acid Cycle ,Respiratory chain ,Pyruvate Dehydrogenase Complex ,Oxidative phosphorylation ,Oxidative Phosphorylation ,Electron Transport ,Acetyl Coenzyme A ,Animals ,Humans ,Vitamin A ,Protein kinase A ,Molecular Biology ,biology ,Cytochrome c ,Cytochromes c ,Cell Biology ,Pyruvate dehydrogenase complex ,Mitochondria ,Cell biology ,Citric acid cycle ,Protein Kinase C-delta ,biology.protein ,Intermembrane space ,Oxidation-Reduction ,Signal Transduction - Abstract
The protein kinase Cδ signalosome modulates the generation of acetyl-Coenzyme A from glycolytic sources. This module is composed of four interlinked components: PKCδ, the signal adapter p66Shc, cytochrome c, and vitamin A. It resides in the intermembrane space of mitochondria, and is at the center of a feedback loop that senses upstream the redox balance between oxidized and reduced cytochrome c as a measure of the workload of the respiratory chain, and transmits a forward signal to the pyruvate dehydrogenase complex to adjust the flux of fuel entering the tricarboxylic acid cycle. The novel role of vitamin A as co-activator and potential electron carrier, required for redox activation of PKCδ, is discussed. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.
- Published
- 2012
38. In VivoPathogenic Role of Mutant SOD1 Localized in the Mitochondrial Intermembrane Space
- Author
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Jordi Magrané, Isabel Hervias, Magali Dumont, Czrina Cortez, Hyun Jeong Kim, Jonathan D. Glass, Anissa Igoudjil, Giovanni Manfredi, Anatoly A. Starkov, and Lindsey R. Fischer
- Subjects
Male ,Mitochondrial intermembrane space ,Mutant ,SOD1 ,Mice, Transgenic ,Nerve Tissue Proteins ,Kaplan-Meier Estimate ,Mitochondrion ,Biology ,Article ,Mice ,Superoxide Dismutase-1 ,Microscopy, Electron, Transmission ,medicine ,Animals ,Humans ,Amyotrophic lateral sclerosis ,Muscle, Skeletal ,Analysis of Variance ,Superoxide Dismutase ,Myocardium ,General Neuroscience ,Amyotrophic Lateral Sclerosis ,Body Weight ,Neurodegeneration ,Brain ,nutritional and metabolic diseases ,Heart ,Motor neuron ,medicine.disease ,Mitochondria ,nervous system diseases ,Cell biology ,Disease Models, Animal ,medicine.anatomical_structure ,Spinal Cord ,nervous system ,Biochemistry ,Mutation ,Calcium ,Energy Metabolism ,Intermembrane space - Abstract
Mutations in Cu,Zn superoxide dismutase (SOD1) are associated with familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 causes a complex array of pathological events, through toxic gain of function mechanisms, leading to selective motor neuron degeneration. Mitochondrial dysfunction is among the well established toxic effects of mutant SOD1, but its mechanisms are just starting to be elucidated. A portion of mutant SOD1 is localized in mitochondria, where it accumulates mostly on the outer membrane and inside the intermembrane space (IMS). Evidence in cultured cells suggests that mutant SOD1 in the IMS causes mitochondrial dysfunction and compromises cell viability. Therefore, to test its pathogenic rolein vivowe generated transgenic mice expressing G93A mutant or wild-type (WT) human SOD1 targeted selectively to the mitochondrial IMS (mito-SOD1). We show that mito-SOD1 is correctly localized in the IMS, where it oligomerizes and acquires enzymatic activity. Mito-G93ASOD1 mice, but not mito-WTSOD1 mice, develop a progressive disease characterized by body weight loss, muscle weakness, brain atrophy, and motor impairment, which is more severe in females. These symptoms are associated with reduced spinal motor neuron counts and impaired mitochondrial bioenergetics, characterized by decreased cytochrome oxidase activity and defective calcium handling. However, there is no evidence of muscle denervation, a cardinal pathological feature of ALS. Together, our findings indicate that mutant SOD1 in the mitochondrial IMS causes mitochondrial dysfunction and neurodegeneration, butper seit is not sufficient to cause a full-fledged ALS phenotype, which requires the participation of mutant SOD1 localized in other cellular compartments.
- Published
- 2011
39. A Phosphodiesterase 2A Isoform Localized to Mitochondria Regulates Respiration
- Author
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Jochen Buck, Michael Russwurm, Clemens Steegborn, Rebeca Acín-Pérez, Lonny R. Levin, Kathrin Günnewig, Melanie Gertz, Georg Zoidl, Joachim Rassow, Lavoisier S. Ramos, and Giovanni Manfredi
- Subjects
Gene isoform ,Cell Respiration ,Green Fluorescent Proteins ,Respiratory chain ,Mitochondrion ,Biology ,Biochemistry ,Animals ,Humans ,Protein Isoforms ,education ,Cyclic GMP ,Molecular Biology ,education.field_of_study ,Microscopy, Confocal ,Brain ,Phosphodiesterase ,Cell Biology ,Soluble adenylyl cyclase ,Cyclic Nucleotide Phosphodiesterases, Type 2 ,Mitochondria ,Protein Structure, Tertiary ,Rats ,Cell biology ,Liver ,3',5'-Cyclic-AMP Phosphodiesterases ,Mitochondrial matrix ,Second messenger system ,Endopeptidase K ,Signal transduction ,Signal Transduction - Abstract
Mitochondria are central organelles in cellular energy metabolism, apoptosis, and aging processes. A signaling network regulating these functions was recently shown to include soluble adenylyl cyclase as a local source of the second messenger cAMP in the mitochondrial matrix. However, a mitochondrial cAMP-degrading phosphodiesterase (PDE) necessary for switching off this cAMP signal has not yet been identified. Here, we describe the identification and characterization of a PDE2A isoform in mitochondria from rodent liver and brain. We find that mitochondrial PDE2A is located in the matrix and that the unique N terminus of PDE2A isoform 2 specifically leads to mitochondrial localization of this isoform. Functional assays show that mitochondrial PDE2A forms a local signaling system with soluble adenylyl cyclase in the matrix, which regulates the activity of the respiratory chain. Our findings complete a cAMP signaling cascade in mitochondria and have implications for understanding the regulation of mitochondrial processes and for their pharmacological modulation.
- Published
- 2011
40. Pink1 regulates the oxidative phosphorylation machinery via mitochondrial fission
- Author
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Giovanni Manfredi, Wencheng Liu, Chenjian Li, Kindiya Geghman, Bingwei Lu, and Rebeca Acín-Pérez
- Subjects
Blotting, Western ,PINK1 ,Oxidative phosphorylation ,Protein Serine-Threonine Kinases ,Biology ,Mitochondrion ,Oxidative Phosphorylation ,Animals, Genetically Modified ,Electron Transport ,Electron Transport Complex IV ,Gene Knockout Techniques ,Adenosine Triphosphate ,Oxygen Consumption ,GTP-Binding Proteins ,Animals ,Drosophila Proteins ,Humans ,Electron Transport Complex I ,Multidisciplinary ,ATP synthase ,fungi ,Parkinson Disease ,Biological Sciences ,Mitochondria ,Cell biology ,Cytoskeletal Proteins ,Disease Models, Animal ,Drosophila melanogaster ,mitochondrial fusion ,Biochemistry ,Mutation ,DNAJA3 ,biology.protein ,Mitochondrial fission - Abstract
Mutations in PTEN-induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson's disease (PD), PARK6. To investigate the mechanism of PINK1 pathogenesis, we used the Drosophila Pink1 knockout (KO) model. In mitochondria isolated from Pink1-KO flies, mitochondrial respiration driven by the electron transport chain (ETC) is significantly reduced. This reduction is the result of a decrease in ETC complex I and IV enzymatic activity. As a consequence, Pink1-KO flies also display a reduced mitochondrial ATP synthesis. Because mitochondrial dynamics is important for mitochondrial function and Pink1-KO flies have defects in mitochondrial fission, we explored whether fission machinery deficits underlie the bioenergetic defect in Pink1-KO flies. We found that the bioenergetic defects in the Pink1-KO can be ameliorated by expression of Drp1 , a key molecule in mitochondrial fission. Further investigation of the ETC complex integrity in wild type, Pink1-KO, PInk1-KO/Drp1 transgenic, or Drp1 transgenic flies indicates that the reduced ETC complex activity is likely derived from a defect in the ETC complex assembly, which can be partially rescued by increasing mitochondrial fission. Taken together, these results suggest a unique pathogenic mechanism of PINK1 PD: The loss of PINK1 impairs mitochondrial fission, which causes defective assembly of the ETC complexes, leading to abnormal bioenergetics.
- Published
- 2011
41. A kinetic assay of mitochondrial ADP–ATP exchange rate in permeabilized cells
- Author
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Christos Chinopoulos, Hibiki Kawamata, Giovanni Manfredi, and Anatoly A. Starkov
- Subjects
Biophysics ,Citrate (si)-Synthase ,Mitochondrion ,Biochemistry ,Article ,Permeability ,Cell Line ,Myoblasts ,Fluorides ,Mice ,chemistry.chemical_compound ,Adenosine Triphosphate ,Adenine nucleotide ,Animals ,Citrate synthase ,Magnesium ,Molecular Biology ,Membrane Potential, Mitochondrial ,biology ,Adenine nucleotide translocator ,Intracellular Membranes ,Cell Biology ,Mitochondria ,Adenosine Diphosphate ,Kinetics ,Proton-Translocating ATPases ,Adenosine diphosphate ,Digitonin ,chemistry ,biology.protein ,Beryllium ,ATP–ADP translocase ,Vanadates ,Mitochondrial ADP, ATP Translocases ,Adenosine triphosphate - Abstract
We previously described a method to measure ADP-ATP exchange rates in isolated mitochondria by recording the changes in free extramitochondrial [Mg(2+)] reported by an Mg(2+)-sensitive fluorescent indicator, exploiting the differential affinity of ADP and ATP to Mg(2+). In the current article, we describe a modification of this method suited for following ADP-ATP exchange rates in environments with competing reactions that interconvert adenine nucleotides such as in permeabilized cells that harbor phosphorylases and kinases, ion pumps exhibiting substantial ATPase activity, and myosin ATPase activity. Here we report that the addition of BeF(3)(-) and sodium orthovanadate (Na(3)VO(4)) to medium containing digitonin-permeabilized cells inhibits all ADP-ATP-using reactions except the adenine nucleotide translocase (ANT)-mediated mitochondrial ADP-ATP exchange. An advantage of this assay is that mitochondria that may have been also permeabilized by digitonin do not contribute to ATP consumption by the exposed F(1)F(o)-ATPase due to its sensitivity to BeF(3)(-) and Na(3)VO(4). With this assay, ADP-ATP exchange rate mediated by the ANT in permeabilized cells is measured for the entire range of mitochondrial membrane potential titrated by stepwise additions of an uncoupler and expressed as a function of citrate synthase activity per total amount of protein.
- Published
- 2010
42. SOD1 targeted to the mitochondrial intermembrane space prevents motor neuropathy in the Sod1 knockout mouse
- Author
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Jonathan D. Glass, Yingjie Li, Anissa Igoudjil, Jason M. Hansen, Giovanni Manfredi, Lindsey R. Fischer, and Jordi Magrané
- Subjects
Mitochondrial intermembrane space ,animal diseases ,Blotting, Western ,SOD1 ,Mice, Transgenic ,Mitochondrion ,Superoxide dismutase ,Mice ,chemistry.chemical_compound ,Superoxide Dismutase-1 ,medicine ,Animals ,Axon ,Cells, Cultured ,Motor Neurons ,Analysis of Variance ,biology ,Superoxide Dismutase ,Superoxide ,nutritional and metabolic diseases ,Original Articles ,Intracellular Membranes ,Motor neuron ,Mitochondria ,nervous system diseases ,medicine.anatomical_structure ,nervous system ,chemistry ,biology.protein ,Neurology (clinical) ,Intermembrane space ,Neuroscience - Abstract
Motor axon degeneration is a critical but poorly understood event leading to weakness and muscle atrophy in motor neuron diseases. Here, we investigated oxidative stress-mediated axonal degeneration in mice lacking the antioxidant enzyme, Cu,Zn superoxide dismutase (SOD1). We demonstrate a progressive motor axonopathy in these mice and show that Sod1(-/-) primary motor neurons extend short axons in vitro with reduced mitochondrial density. Sod1(-/-) neurons also show oxidation of mitochondrial--but not cytosolic--thioredoxin, suggesting that loss of SOD1 causes preferential oxidative stress in mitochondria, a primary source of superoxide in cells. SOD1 is widely regarded as the cytosolic isoform of superoxide dismutase, but is also found in the mitochondrial intermembrane space. The functional significance of SOD1 in the intermembrane space is unknown. We used a transgenic approach to express SOD1 exclusively in the intermembrane space and found that mitochondrial SOD1 is sufficient to prevent biochemical and morphological defects in the Sod1(-/-) model, and to rescue the motor phenotype of these mice when followed to 12 months of age. These results suggest that SOD1 in the mitochondrial intermembrane space is fundamental for motor axon maintenance, and implicate oxidative damage initiated at mitochondrial sites in the pathogenesis of motor axon degeneration.
- Published
- 2010
43. Parkin overexpression selects against a deleterious mtDNA mutation in heteroplasmic cybrid cells
- Author
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Der-Fen Suen, Derek P. Narendra, Richard J. Youle, Atsushi Tanaka, and Giovanni Manfredi
- Subjects
Mitochondrial DNA ,Mitochondrial Diseases ,Recombinant Fusion Proteins ,Ubiquitin-Protein Ligases ,Gene Expression ,Hybrid Cells ,Mitochondrion ,Biology ,medicine.disease_cause ,DNA, Mitochondrial ,Membrane Fusion ,Models, Biological ,Human mitochondrial genetics ,Parkin ,Cell Line ,Electron Transport Complex IV ,Mitochondrial myopathy ,medicine ,Humans ,Genetics ,Mutation ,Multidisciplinary ,Biological Sciences ,Mitochondrial Proton-Translocating ATPases ,medicine.disease ,Heteroplasmy ,Mitochondria ,nervous system diseases ,Protein Transport ,mitochondrial fusion - Abstract
Mitochondrial genomes with deleterious mutations can replicate in cells along with wild-type genomes in a state of heteroplasmy, and are a cause of severe inherited syndromes, such as mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke (MELAS), neuropathy, ataxia, retinitis pigmentosa-maternally inherited Leigh syndrome (NARP-MILS), and Leber's hereditary optic neuropathy (LHON). The cytosolic E3 ligase, Parkin, commonly mutated in recessive familial parkinsonism, translocates to depolarized mitochondria and induces their autophagic elimination, suggesting that Parkin may signal the selective removal of defective mitochondria within the cell. We report that long-term overexpression of Parkin can eliminate mitochondria with deleterious COXI mutations in heteroplasmic cybrid cells, thereby enriching cells for wild-type mtDNA and restoring cytochrome c oxidase activity. After relieving cybrid cells of Parkin overexpression, a more favorable wild-type to mutant mitochondrial genome ratio is stably maintained. These data support the model that Parkin functions in a mitochondrial quality control pathway. Additionally, they suggest that transiently increasing levels of Parkin expression might ameliorate certain mitochondrial diseases.
- Published
- 2010
44. Rewiring of Glutamine Metabolism Is a Bioenergetic Adaptation of Human Cells with Mitochondrial DNA Mutations
- Author
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Steven S. Gross, Valerio Carelli, Ifrah Shahi, Yevgeniya I. Shurubor, Andrea J. Arreguin, Elizabeth L. Calder, Dazhi Zhao, M. Flint Beal, Qiuying Chen, Guido Primiano, Kathryne Kirk, Marilena D'Aurelio, Travis T. Denton, Giovanni Manfredi, Federica Valsecchi, Chen, Qiuying, Kirk, Kathryne, Shurubor, Yevgeniya I., Zhao, Dazhi, Arreguin, Andrea J., Shahi, Ifrah, Valsecchi, Federica, Primiano, Guido, Calder, Elizabeth L., Carelli, Valerio, Denton, Travis T., Beal, M. Flint, Gross, Steven S., Manfredi, Giovanni, and D'Aurelio, Marilena
- Subjects
Male ,0301 basic medicine ,Mitochondrial DNA ,Physiology ,Glutamine ,glutamate ,Oxidative phosphorylation ,Mitochondrion ,DNA, Mitochondrial ,Oxidative Phosphorylation ,Mice ,03 medical and health sciences ,Mitochondrial myopathy ,medicine ,Animals ,Humans ,skeletal muscle ,Molecular Biology ,anaplerosi ,Alanine ,Catabolism ,Chemistry ,Mitochondrial Myopathies ,Cell Biology ,Metabolism ,medicine.disease ,OXPHOS dysfunction ,Adaptation, Physiological ,Mitochondria ,Cell biology ,mitochondrial disease ,Disease Models, Animal ,α-ketoglutarate ,030104 developmental biology ,Mutation ,Ketoglutaric Acids ,Energy Metabolism ,metabolism ,Flux (metabolism) ,myopathy ,HeLa Cells - Abstract
Using molecular, biochemical, and untargeted stable isotope tracing approaches, we identify a previously unappreciated glutamine-derived α-ketoglutarate (αKG) energy-generating anaplerotic flux to be critical in mitochondrial DNA (mtDNA) mutant cells that harbor human disease-associated oxidative phosphorylation defects. Stimulating this flux with αKG supplementation enables the survival of diverse mtDNA mutant cells under otherwise lethal obligatory oxidative conditions. Strikingly, we demonstrate that when residual mitochondrial respiration in mtDNA mutant cells exceeds 45% of control levels, αKG oxidative flux prevails over reductive carboxylation. Furthermore, in a mouse model of mitochondrial myopathy, we show that increased oxidative αKG flux in muscle arises from enhanced alanine synthesis and release into blood, concomitant with accelerated amino acid catabolism from protein breakdown. Importantly, in this mouse model of mitochondriopathy, muscle amino acid imbalance is normalized by αKG supplementation. Taken together, our findings provide a rationale for αKG supplementation as a therapeutic strategy for mitochondrial myopathies. Chen et al. show that patient cells with mtDNA mutations and a mouse model of mitochondrial myopathy have compensatory glutamine-derived anaplerotic flux that provides αKG to the TCA cycle to enable mutant cell survival. The metabolic fate of αKG (oxidative versus reductive) depends on the severity of OXPHOS impairment.
- Published
- 2018
45. Novel Role of ATPase Subunit C Targeting Peptides Beyond Mitochondrial Protein Import
- Author
-
Jordi Magrané, Antoni L. Andreu, Cristofol Vives-Bauza, and Giovanni Manfredi
- Subjects
Gene isoform ,Protein subunit ,Respiratory chain ,Down-Regulation ,Mitochondrion ,Protein Sorting Signals ,Gene Expression Regulation, Enzymologic ,Oxidative Phosphorylation ,Electron Transport Complex IV ,Mice ,Adenosine Triphosphate ,RNA interference ,Cytochrome c oxidase ,Animals ,Humans ,Gene Silencing ,RNA, Messenger ,Molecular Biology ,ATP synthase ,biology ,Genetic Complementation Test ,Cell Biology ,Articles ,Mitochondrial Proton-Translocating ATPases ,Recombinant Proteins ,Mitochondria ,Isoenzymes ,Protein Subunits ,Protein Transport ,Mitochondrial respiratory chain ,Biochemistry ,biology.protein ,Peptides ,HeLa Cells - Abstract
Mammals have three isoforms of F1F0-ATP synthase subunit c, only differing by their mitochondrial targeting peptides. Here, we show that these isoforms are non-redundant, because of different functions conferred by the targeting peptides, which in addition to mediating protein import, play a yet undiscovered role in respiratory chain maintenance., In mammals, subunit c of the F1F0-ATP synthase has three isoforms (P1, P2, and P3). These isoforms differ by their cleavable mitochondrial targeting peptides, whereas the mature peptides are identical. To investigate this apparent genetic redundancy, we knocked down each of the three subunit c isoform by RNA interference in HeLa cells. Silencing any of the subunit c isoforms individually resulted in an ATP synthesis defect, indicating that these isoforms are not functionally redundant. We found that subunit c knockdown impaired the structure and function of the mitochondrial respiratory chain. In particular, P2 silencing caused defective cytochrome oxidase assembly and function. Because the expression of exogenous P1 or P2 was able to rescue the respective silencing phenotypes, but the two isoforms were unable to cross-complement, we hypothesized that their functional specificity resided in their targeting peptides. In fact, the expression of P1 and P2 targeting peptides fused to GFP variants rescued the ATP synthesis and respiratory chain defects in the silenced cells. Our results demonstrate that the subunit c isoforms are nonredundant, because they differ functionally by their targeting peptides, which, in addition to mediating mitochondrial protein import, play a yet undiscovered role in respiratory chain maintenance.
- Published
- 2010
46. Analysis of mouse models of cytochrome c oxidase deficiency owing to mutations in Sco2
- Author
-
Ira J. Goldberg, Hua Yang, Joseph H. Graziano, Raffay S. Khan, Sonja Brosel, Rebeca Acín-Pérez, Vesna Slavkovich, Ichizo Nishino, Eric A. Schon, and Giovanni Manfredi
- Subjects
Blotting, Western ,Respiratory chain ,Cytochrome-c Oxidase Deficiency ,Mitochondrion ,medicine.disease_cause ,Compound heterozygosity ,Electron Transport Complex IV ,Mice ,Genetics ,medicine ,Animals ,Cytochrome c oxidase ,Allele ,Molecular Biology ,Genetics (clinical) ,Enzyme Assays ,Mice, Knockout ,Mutation ,biology ,Muscles ,Articles ,General Medicine ,Embryo, Mammalian ,Immunohistochemistry ,Molecular biology ,Mitochondria ,Disease Models, Animal ,Mitochondrial respiratory chain ,Organ Specificity ,biology.protein ,Copper ,Molecular Chaperones - Abstract
Mutations in SCO2, a protein required for the proper assembly and functioning of cytochrome c oxidase (COX; complex IV of the mitochondrial respiratory chain), cause a fatal infantile cardioencephalomyopathy with COX deficiency. We have generated mice harboring a Sco2 knock-out (KO) allele and a Sco2 knock-in (KI) allele expressing an E-->K mutation at position 129 (E129K), corresponding to the E140K mutation found in almost all human SCO2-mutated patients. Whereas homozygous KO mice were embryonic lethals, homozygous KI and compound heterozygous KI/KO mice were viable, but had muscle weakness; biochemically, they had respiratory chain deficiencies as well as complex IV assembly defects in multiple tissues. There was a concomitant reduction in mitochondrial copper content, but the total amount of copper in examined tissues was not reduced. These mouse models should be of use in further studies of Sco2 function, as well as in testing therapeutic approaches to treat the human disorder.
- Published
- 2009
47. Control of oxidative phosphorylation by vitamin A illuminates a fundamental role in mitochondrial energy homoeostasis
- Author
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William S. Blaner, Donald A. Fischman, Robert A. Harris, Michael Leitges, Valerie Vinogradov, Ulrich Hämmerling, Nuttaporn Wongsiriroj, Giovanni Manfredi, Beatrice Hoyos, Rebeca Acín-Pérez, and Feng Zhao
- Subjects
Male ,Vitamin ,medicine.drug_class ,Retinoic acid ,Pyruvate Dehydrogenase Complex ,Biology ,Biochemistry ,Oxidative Phosphorylation ,Research Communications ,Jurkat Cells ,Mice ,Retinoids ,chemistry.chemical_compound ,Oxygen Consumption ,Genetics ,medicine ,Animals ,Homeostasis ,Humans ,Retinoid ,Vitamin A ,Molecular Biology ,Vitamin A Deficiency ,Retinol ,Mitochondrial Proton-Translocating ATPases ,Pyruvate dehydrogenase complex ,Mitochondria ,Protein Kinase C-delta ,Retinoic acid receptor ,chemistry ,Retinaldehyde ,Energy Metabolism ,Retinol binding ,Signal Transduction ,Biotechnology - Abstract
The physiology of two metabolites of vitamin A is understood in substantial detail: retinaldehyde functions as the universal chromophore in the vertebrate and invertebrate eye; retinoic acid regulates a set of vertebrate transcription factors, the retinoic acid receptor superfamily. The third member of this retinoid triumvirate is retinol. While functioning as the precursor of retinaldehyde and retinoic acid, a growing body of evidence suggests a far more fundamental role for retinol in signal transduction. Here we show that retinol is essential for the metabolic fitness of mitochondria. When cells were deprived of retinol, respiration and ATP synthesis defaulted to basal levels. They recovered to significantly higher energy output as soon as retinol was restored to physiological concentration, without the need for metabolic conversion to other retinoids. Retinol emerged as an essential cofactor of protein kinase Cδ (PKCδ), without which this enzyme failed to be activated in mitochondria. Furthermore, retinol needed to physically bind PKCδ, because mutation of the retinol binding site rendered PKCδ unresponsive to Rol, while retaining responsiveness to phorbol ester. The PKCδ/retinol complex signaled the pyruvate dehydrogenase complex for enhanced flux of pyruvate into the Krebs cycle. The baseline response was reduced in vitamin A-deficient lecithin:retinol acyl transferase-knockout mice, but this was corrected within 3 h by intraperitoneal injection of vitamin A; this suggests that vitamin A is physiologically important. These results illuminate a hitherto unsuspected role of vitamin A in mitochondrial bioenergetics of mammals, acting as a nutritional sensor. As such, retinol is of fundamental importance for energy homeostasis. The data provide a mechanistic explanation to the nearly 100-yr-old question of why vitamin A deficiency causes so many pathologies that are independent of retinoic acid action.—Acin-Perez, T., Hoyos, B., Zhao, F., Vinogradov, V., Fischman, D. A., Harris, R. A., Leitges, M., Wongsiriroj, N., Blaner, W. S., Manfredi, G., Hammerling, U. Control of oxidative phosphorylation by vitamin A illuminates a fundamental role in mitochondrial energy homoeostasis.
- Published
- 2009
48. Mitochondrial Function, Morphology, and Axonal Transport in Amyotrophic Lateral Sclerosis
- Author
-
Giovanni Manfredi and Jordi Magrané
- Subjects
Pathology ,medicine.medical_specialty ,Physiology ,Angiogenesis ,DNA repair ,Clinical Biochemistry ,Mitochondrion ,Biology ,Biochemistry ,medicine ,Animals ,Humans ,Amyotrophic lateral sclerosis ,Molecular Biology ,Mitochondrial transport ,General Environmental Science ,Neurons ,Rna processing ,Amyotrophic Lateral Sclerosis ,Cell Biology ,Forum Review Articles ,medicine.disease ,Axons ,Mitochondria ,Cell biology ,Vesicular transport protein ,Axoplasmic transport ,General Earth and Planetary Sciences - Abstract
Perturbation of organellar axonal transport is increasingly recognized as an important contributor in a number of neurodegenerative diseases. Although the specificity of this impairment remains to be elucidated, growing evidence suggests that in certain disease conditions, mitochondria are affected primarily by transport defects. Many hypotheses have been formulated to explain the pathogenic mechanisms involved in amyotrophic lateral sclerosis (ALS). The mutations described so far in genetic forms of ALS (familial ALS, fALS) affect proteins involved in a wide variety of cellular mechanisms, including free radical scavenging, energy metabolism, axonal transport, RNA processing, DNA repair, vesicular transport, and angiogenesis. Here we review the current knowledge on mitochondrial transport and its role in ALS. Antioxid. Redox Signal. 11, 1615–1626.
- Published
- 2009
49. Different regulation of wild-type and mutant Cu,Zn superoxide dismutase localization in mammalian mitochondria
- Author
-
Hibiki Kawamata and Giovanni Manfredi
- Subjects
Models, Molecular ,Protein Folding ,Molecular Sequence Data ,SOD1 ,Mitochondrion ,Mitochondrial Membrane Transport Proteins ,Gene Expression Regulation, Enzymologic ,Superoxide dismutase ,Mice ,Mitochondrial membrane transport protein ,Superoxide Dismutase-1 ,Cell Line, Tumor ,Chlorocebus aethiops ,Genetics ,Animals ,Humans ,Amino Acid Sequence ,Cysteine ,Frameshift Mutation ,Molecular Biology ,Cytochrome Reductases ,Genetics (clinical) ,Mammals ,biology ,Superoxide Dismutase ,Amyotrophic Lateral Sclerosis ,fungi ,Wild type ,nutritional and metabolic diseases ,Articles ,General Medicine ,Mitochondria ,Protein Structure, Tertiary ,nervous system diseases ,Cell biology ,Protein Transport ,Cytosol ,Biochemistry ,Chaperone (protein) ,COS Cells ,Mutation ,biology.protein ,Intermembrane space ,Molecular Chaperones - Abstract
The antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) is predominantly localized in the cytosol, but it is also found in mitochondria. Studies in yeast suggest that apoSOD1 is imported into mitochondria and trapped inside by folding and maturation, which is facilitated by its copper chaperone for SOD1 (CCS). Here, we show that in mammalian cells, SOD1 mitochondrial localization is dictated by its folding state, which is modulated by several interconnected factors. First, the intracellular distribution of CCS determines SOD1 partitioning in cytosol and mitochondria: CCS localization in the cytosol prevents SOD1 mitochondrial import, whereas CCS in mitochondria increases it. Second, the Mia40/Erv1 pathway for import of small intermembrane space proteins participates in CCS mitochondrial import in a respiratory chain-dependent manner. Third, CCS mitochondrial import is regulated by oxygen concentration: high (20%) oxygen prevents import, whereas physiological (6%) oxygen promotes it. Therefore, SOD1 localization responds to changes in environmental conditions following redistribution of CCS, which operates as an oxygen sensor. Fourth, all of the cysteine residues in human SOD1 are critical for its retention in mitochondria due to their involvement in intramolecular disulfide bonds and in the interaction with CCS. Mutations in SOD1 are associated with autosomal dominant familial amyotrophic lateral sclerosis. Like the wild-type protein, mutant SOD1 localizes to mitochondria, where it induces bioenergetic defects. We find that the physiological regulation of mitochondrial localization is either inefficient or absent in SOD1 pathogenic mutants. We propose misfolding and aggregation of these mutants that trap them inside mitochondria.
- Published
- 2008
50. The mitochondrial respiratory chain is a modulator of apoptosis
- Author
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Matthew S. Henning, Giovanni Manfredi, Anatoly A. Starkov, and Jennifer Q. Kwong
- Subjects
Respiratory chain ,Apoptosis ,Mitochondrion ,Biology ,Endoplasmic Reticulum ,DNA, Mitochondrial ,Mitochondrial apoptosis-induced channel ,Article ,Cell Line ,Electron Transport ,Electron Transport Complex IV ,Mitochondrial Proteins ,Electron Transport Complex III ,03 medical and health sciences ,Adenosine Triphosphate ,0302 clinical medicine ,Genetic model ,medicine ,Humans ,Enzyme Inhibitors ,Research Articles ,030304 developmental biology ,0303 health sciences ,Electron Transport Complex I ,Electron Transport Complex II ,Neurodegeneration ,Cell Biology ,Staurosporine ,medicine.disease ,Molecular biology ,Mitochondria ,Cell biology ,Mitochondrial respiratory chain ,030220 oncology & carcinogenesis ,Mutation ,Unfolded protein response ,Calcium ,Reactive Oxygen Species - Abstract
Mitochondrial dysfunction and dysregulation of apoptosis are implicated in many diseases such as cancer and neurodegeneration. We investigate here the role of respiratory chain (RC) dysfunction in apoptosis, using mitochondrial DNA mutations as genetic models. Although some mutations eliminate the entire RC, others target specific complexes, resulting in either decreased or complete loss of electron flux, which leads to impaired respiration and adenosine triphosphate (ATP) synthesis. Despite these similarities, significant differences in responses to apoptotic stimuli emerge. Cells lacking RC are protected against both mitochondrial- and endoplasmic reticulum (ER) stress–induced apoptosis. Cells with RC, but unable to generate electron flux, are protected against mitochondrial apoptosis, although they have increased sensitivity to ER stress. Finally, cells with a partial reduction in electron flux have increased apoptosis under both conditions. Our results show that the RC modulates apoptosis in a context-dependent manner independent of ATP production and that apoptotic responses are the result of the interplay between mitochondrial functional state and environmental cues.
- Published
- 2007
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