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Thomas Langer - One of the best experts on this subject based on the ideXlab platform.
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Astrocyte-specific deletion of the mitochondrial m-AAA protease reveals glial contribution to neurodegeneration.
Glia, 2019Co-Authors: Sara Murru, Esther Barth, Thomas Langer, Simon Hess, Eva R. Almajan, Désirée Schatton, Steffen Hermans, Susanne Brodesser, Peter Kloppenburg, Elena I. RugarliAbstract:Mitochondrial dysfunction causes neurodegeneration but whether impairment of mitochondrial homeostasis in astrocytes contributes to this pathological process remains largely unknown. The m-AAA protease exerts quality control and regulatory functions crucial for mitochondrial homeostasis. AFG3L2, which encodes one of the subunits of the m-AAA protease, is mutated in spinocerebellar ataxia SCA28 and in infantile syndromes characterized by spastic-ataxia, epilepsy and premature death. Here, we investigate the role of AFG3L2 and its redundant homologue Afg3l1 in the Bergmann glia (BG), radial astrocytes of the cerebellum that have functional connections with Purkinje cells (PC) and regulate glutamate homeostasis. We show that astrocyte-specific deletion of AFG3L2 in the mouse leads to late-onset motor impairment and to degeneration of BG, which display aberrant morphology, altered expression of the glutamate transporter EAAT2, and a reactive inflammatory signature. The neurological and glial phenotypes are drastically exacerbated when astrocytes lack both Afg31l and AFG3L2, and therefore, are totally depleted of the m-AAA protease. Moreover, mitochondrial stress responses and necroptotic markers are induced in the cerebellum. In both mouse models, targeted BG show a fragmented mitochondrial network and loss of mitochondrial cristae, but no signs of respiratory dysfunction. Importantly, astrocyte-specific deficiency of Afg3l1 and AFG3L2 triggers secondary morphological degeneration and electrophysiological changes in PCs, thus demonstrating a non-cell-autonomous role of glia in neurodegeneration. We propose that astrocyte dysfunction amplifies both neuroinflammation and glutamate excitotoxicity in patients carrying mutations in AFG3L2, leading to a vicious circle that contributes to neuronal death.
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m-AAA proteases, mitochondrial calcium homeostasis and neurodegeneration
Cell Research, 2018Co-Authors: Maria Patron, Hans-georg Sprenger, Thomas LangerAbstract:The function of mitochondria depends on ubiquitously expressed and evolutionary conserved m -AAA proteases in the inner membrane. These ATP-dependent peptidases form hexameric complexes built up of homologous subunits. AFG3L2 subunits assemble either into homo-oligomeric isoenzymes or with SPG7 (paraplegin) subunits into hetero-oligomeric proteolytic complexes. Mutations in AFG3L2 are associated with dominant spinocerebellar ataxia (SCA28) characterized by the loss of Purkinje cells, whereas mutations in SPG7 cause a recessive form of hereditary spastic paraplegia (HSP7) with motor neurons of the cortico-spinal tract being predominantly affected. Pleiotropic functions have been assigned to m -AAA proteases, which act as quality control and regulatory enzymes in mitochondria. Loss of m -AAA proteases affects mitochondrial protein synthesis and respiration and leads to mitochondrial fragmentation and deficiencies in the axonal transport of mitochondria. Moreover m -AAA proteases regulate the assembly of the m itochondrial c alcium u niporter (MCU) complex. Impaired degradation of the MCU subunit EMRE in AFG3L2-deficient mitochondria results in the formation of deregulated MCU complexes, increased mitochondrial calcium uptake and increased vulnerability of neurons for calcium-induced cell death. A reduction of calcium influx into the cytosol of Purkinje cells rescues ataxia in an AFG3L2-deficient mouse model. In this review, we discuss the relationship between the m -AAA protease and mitochondrial calcium homeostasis and its relevance for neurodegeneration and describe a novel mouse model lacking MCU specifically in Purkinje cells. Our results pledge for a novel view on m -AAA proteases that integrates their pleiotropic functions in mitochondria to explain the pathogenesis of associated neurodegenerative disorders.
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The Mitochondrial m-AAA Protease Prevents Demyelination and Hair Greying
PLoS Genetics, 2016Co-Authors: Shuaiyu Wang, Julie Jacquemyn, Sara Murru, Esther Barth, Paola Martinelli, Carien M. Niessen, Thomas Langer, Elena I. RugarliAbstract:The m-AAA protease preserves proteostasis of the inner mitochondrial membrane. It ensures a functional respiratory chain, by controlling the turnover of respiratory complex subunits and allowing mitochondrial translation, but other functions in mitochondria are conceivable. Mutations in genes encoding subunits of the m-AAA protease have been linked to various neurodegenerative diseases in humans, such as hereditary spastic paraplegia and spinocerebellar ataxia. While essential functions of the m-AAA protease for neuronal survival have been established, its role in adult glial cells remains enigmatic. Here, we show that deletion of the highly expressed subunit AFG3L2 in mature mouse oligodendrocytes provokes early-on mitochondrial fragmentation and swelling, as previously shown in neurons, but causes only late-onset motor defects and myelin abnormalities. In contrast, total ablation of the m-AAA protease, by deleting both AFG3L2 and its paralogue Afg3l1, triggers progressive motor dysfunction and demyelination, owing to rapid oligodendrocyte cell death. Surprisingly, the mice showed premature hair greying, caused by progressive loss of melanoblasts that share a common developmental origin with Schwann cells and are targeted in our experiments. Thus, while both neurons and glial cells are dependant on the m-AAA protease for survival in vivo, complete ablation of the complex is necessary to trigger death of oligodendrocytes, hinting to cell-autonomous thresholds of vulnerability to m-AAA protease deficiency.
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Transcriptional activation of LON Gene by a new form of mitochondrial stress: A role for the nuclear respiratory factor 2 in StAR overload response (SOR)
Molecular and cellular endocrinology, 2015Co-Authors: Assaf Bahat, Thomas Langer, Shira Perlberg, Naomi Melamed-book, Ines Lauria, Sara Isaac, Amir Eden, Joseph OrlyAbstract:High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peri-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases.
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loss of the m aaa protease subunit AFG3L2 causes mitochondrial transport defects and tau hyperphosphorylation
The EMBO Journal, 2014Co-Authors: Arun Kumar Kondadi, Paola Martinelli, Shuaiyu Wang, Sara Montagner, Nikolay Kladt, Anne Korwitz, David Herholz, Michael J. Baker, Astrid C. Schauss, Thomas LangerAbstract:The m-AAA protease subunit AFG3L2 is involved in degradation and processing of substrates in the inner mitochondrial membrane. Mutations in AFG3L2 are associated with spinocerebellar ataxia SCA28 in humans and impair axonal development and neuronal survival in mice. The loss of AFG3L2 causes fragmentation of the mitochondrial network. However, the pathogenic mechanism of neurodegeneration in the absence of AFG3L2 is still unclear. Here, we show that depletion of AFG3L2 leads to a specific defect of anterograde transport of mitochondria in murine cortical neurons. We observe similar transport deficiencies upon loss of AFG3L2 in OMA1-deficient neurons, indicating that they are not caused by OMA1-mediated degradation of the dynamin-like GTPase OPA1 and inhibition of mitochondrial fusion. Treatment of neurons with antioxidants, such as N-acetylcysteine or vitamin E, or decreasing tau levels in axons restored mitochondrial transport in AFG3L2-depleted neurons. Consistently, tau hyperphosphorylation and activation of ERK kinases are detected in mouse neurons postnatally deleted for AFG3L2. We propose that reactive oxygen species signaling leads to cytoskeletal modifications that impair mitochondrial transport in neurons lacking AFG3L2.
Giorgio Casari - One of the best experts on this subject based on the ideXlab platform.
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Upregulation of Peroxiredoxin 3 Protects AFG3L2-KO Cortical Neurons In Vitro from Oxidative Stress: A Paradigm for Neuronal Cell Survival under Neurodegenerative Conditions.
Oxidative medicine and cellular longevity, 2019Co-Authors: Barbara Bettegazzi, Francesca Maltecca, Giorgio Casari, Daniele Zacchetti, Fabio Grohovaz, Ilaria Pelizzoni, Floramarida Salerno Scarzella, Lisa Michelle Restelli, Franca CodazziAbstract:Several neurodegenerative disorders exhibit selective vulnerability, with subsets of neurons more affected than others, possibly because of the high expression of an altered gene or the presence of particular features that make them more susceptible to insults. On the other hand, resilient neurons may display the ability to develop antioxidant defenses, particularly in diseases of mitochondrial origin, where oxidative stress might contribute to the neurodegenerative process. In this work, we investigated the oxidative stress response of embryonic fibroblasts and cortical neurons obtained from AFG3L2-KO mice. AFG3L2 encodes a subunit of a protease complex that is expressed in mitochondria and acts as both quality control and regulatory enzyme affecting respiration and mitochondrial dynamics. When cells were subjected to an acute oxidative stress protocol, the survival of AFG3L2-KO MEFs was not significantly influenced and was comparable to that of WT; however, the basal level of the antioxidant molecule glutathione was higher. Indeed, glutathione depletion strongly affected the viability of KO, but not of WT MEF, thereby indicating that oxidative stress is more elevated in KO MEF even though well controlled by glutathione. On the other hand, when cortical KO neurons were put in culture, they immediately appeared more vulnerable than WT to the acute oxidative stress condition, but after few days in vitro, the situation was reversed with KO neurons being more resistant than WT to acute stress. This compensatory, protective competence was not due to the upregulation of glutathione, rather of two mitochondrial antioxidant proteins: superoxide dismutase 2 and, at an even higher level, peroxiredoxin 3. This body of evidence sheds light on the capability of neurons to activate neuroprotective pathways and points the attention to peroxiredoxin 3, an antioxidant enzyme that might be critical for neuronal survival also in other disorders affecting mitochondria.
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m-AAA and i-AAA complexes coordinate to regulate OMA1, the stress-activated supervisor of mitochondrial dynamics.
Journal of cell science, 2018Co-Authors: Francesco Consolato, Francesca Maltecca, Susanna Tulli, Irene Sambri, Giorgio CasariAbstract:The proteolytic processing of dynamin like GTPase OPA1, mediated by the activity of both YME1L1 ( i- AAA protease complex) and OMA1, is a crucial step in the regulation of mitochondrial dynamics. OMA1 is a zinc metallopeptidase of the inner mitochondrial membrane that undergoes pre-activating proteolytic and auto-proteolytic cleavage after mitochondrial import. Here, we identify AFG3L2 ( m- AAA complex) as the major protease mediating this event by maturing the pre-pro-OMA1 of 60 kDa to the pro-OMA1 form of 40 kDa by severing the amino-terminal part without recognizing specific consensus sequence. Therefore, m- AAA and i- AAA complexes coordinately regulate OMA1 processing and turnover, and consequently OPA1 isoforms, thus adding new information in the comprehension of the molecular mechanisms in mitochondrial dynamics and of neurodegenerative diseases affected by these phenomena.
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ROLE OF MITOCHONDRIA IN CORTICAL NEURONS FROM SPINOCEREBELLAR ATAXIA 28 (SCA28) MOUSE MODEL.
Journal of World Mitochondria Society, 2015Co-Authors: Franca Codazzi, Francesca Maltecca, Giorgio Casari, Floramarida Salerno, Federico Zucca, Barbara Bettegazzi, Daniele Zacchetti, Fabio GrohovazAbstract:In this study we investigated the role of mitochondria in cortical neurons obtained from a mouse model of spinocerebellar ataxia 28 (SCA28) characterized by dark degeneration of Purkinje cells. Primary cortical neurons from mice, WT and KO for AFG3L2 (the mitochondrial gene mutated in SCA28), were analyzed by single-cell videomicroscopy after iron overload, a condition able to to promote oxidative stress. Different fluorescent probes were employed to monitor iron oxidative status, ROS formation, labile iron pool, glutathione content and cell death. Detoxifying proteins were quantified by Western blot. Upon iron overload, AFG3L2-KO cortical neurons showed a significantly higher percentage of cell death than the WT counterpart. Surprisingly, with culture time, AFG3L2-KO neurons acquired a protective phenotype that made them more resistant than WT neurons to oxidative conditions. Further investigation revealed that the main antioxidant cellular molecule, glutathione, was not involved in this acquired resistance. On the other hand, during their ageing in culture, KO neurons showed an increase in the expression of SOD2 and peroxiredoxin III, two mitochondrial detoxifying proteins that appear to be involved in this compensatory and protective mechanism.
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Respiratory dysfunction by AFG3L2 deficiency causes decreased mitochondrial calcium uptake via organellar network fragmentation
Human molecular genetics, 2012Co-Authors: Francesca Maltecca, Laura Cassina, Diego De Stefani, Francesco Consolato, Michal Wasilewski, Luca Scorrano, Rosario Rizzuto, Giorgio CasariAbstract:The mitochondrial protein AFG3L2 forms homo-oligomeric and hetero-oligomeric complexes with paraplegin in the inner mitochondrial membrane, named m-AAA proteases. These complexes are in charge of quality control of misfolded proteins and participate in the regulation of OPA1 proteolytic cleavage, required for mitochondrial fusion. Mutations in AFG3L2 cause spinocerebellar ataxia type 28 and a complex neurodegenerative syndrome of childhood. In this study, we demonstrated that the loss of AFG3L2 in mouse embryonic fibroblasts (MEFs) reduces mitochondrial Ca(2+) uptake capacity. This defect is neither a consequence of global alteration in cellular Ca(2+) homeostasis nor of the reduced driving force for Ca(2+) internalization within mitochondria, since cytosolic Ca(2+) transients and mitochondrial membrane potential remain unaffected. Moreover, experiments in permeabilized cells revealed unaltered mitochondrial Ca(2+) uptake speed in AFG3L2(-/-) cells, indicating the presence of functional Ca(2+) uptake machinery. Our results show that the defective Ca(2+) handling in AFG3L2(-/-) cells is caused by fragmentation of the mitochondrial network, secondary to respiratory dysfunction and the consequent processing of OPA1. This leaves a number of mitochondria devoid of connections to the ER and thus without Ca(2+) elevations, hampering the proper Ca(2+) diffusion along the mitochondrial network. The recovery of mitochondrial fragmentation in AFG3L2(-/-) MEFs by overexpression of OPA1 rescues the impaired mitochondrial Ca(2+) buffering, but fails to restore respiration. By linking mitochondrial morphology and Ca(2+) homeostasis, these findings shed new light in the molecular mechanisms underlining neurodegeneration caused by AFG3L2 mutations.
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In vivo detection of oxidized proteins: a practical approach to tissue-derived mitochondria.
Methods in molecular biology (Clifton N.J.), 2010Co-Authors: Francesca Maltecca, Giorgio CasariAbstract:Mitochondria are the major producers of free radical oxygen species (ROS) as well as the major target of oxidative damage. Defects in the mitochondrial respiratory chain complexes can increase ROS production and reduce ROS removal, leading to oxidative modification of proteins, lipids, and DNA. AAA proteases of the inner mitochondrial membrane, paraplegin and AFG3L2, participate in the biogenesis and maintenance of respiratory chain complexes. These proteins form hetero-oligomeric paraplegin/ AFG3L2 and homo-oligomeric AFG3L2 complexes named m-AAA proteases. Inactivation of m-AAA proteases causes respiratory defects and altered mitochondrial morphology both in yeast and in mammals. In fact, mouse models defective for AFG3L2 display a very severe neurological syndrome and die within two weeks after birth. They display widespread morphological alterations of mitochondria in the central and peripheral nervous system and deficiencies in respiratory chain complex I and in complex III, which are major producers of ROS in physiological and especially in pathological conditions. Therefore, an efficient and reliable methodology to monitor the effect of increased ROS production is useful for accurately phenotyping cellular and animal models mutants in m-AAA. By measuring carbonyl formation as marker of protein oxidation, we have shown that respiratory defects cause oxidative damage in AFG3L2 mutants, indicating that oxidative stress is crucial in the pathogenesis of m-AAA deficiency.
Francesca Maltecca - One of the best experts on this subject based on the ideXlab platform.
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A novel AFG3L2 mutation close to AAA domain leads to aberrant OMA1 and OPA1 processing in a family with optic atrophy.
Acta neuropathologica communications, 2020Co-Authors: Valentina Baderna, Joshua Schultz, Lisa S. Kearns, Michael C Fahey, Bryony A. Thompson, Jonathan B Ruddle, Aamira Huq, Francesca MalteccaAbstract:Autosomal dominant optic atrophy (ADOA) is a neuro-ophthalmic condition characterized by bilateral degeneration of the optic nerves. Although heterozygous mutations in OPA1 represent the most common genetic cause of ADOA, a significant number of cases remain undiagnosed. Here, we describe a family with a strong ADOA history with most family members spanning three generation having childhood onset of visual symptoms. The proband, in addition to optic atrophy, had neurological symptoms consistent with relapsing remitting multiple sclerosis. Clinical exome analysis detected a novel mutation in the AFG3L2 gene (NM_006796.2:c.1010G > A; p.G337E), which segregated with optic atrophy in family members. AFG3L2 is a metalloprotease of the AAA subfamily which exerts quality control in the inner mitochondrial membrane. Interestingly, the identified mutation localizes close to the AAA domain of AFG3L2, while those localized in the proteolytic domain cause dominant spinocerebellar ataxia type 28 (SCA28) or recessive spastic ataxia with epilepsy (SPAX5). Functional studies in patient fibroblasts demonstrate that the p.G337E AFG3L2 mutation strongly destabilizes the long isoforms of OPA1 via OMA hyper-activation and leads to mitochondrial fragmentation, thus explaining the family phenotype. This study widens the clinical spectrum of neurodegenerative diseases caused by AFG3L2 mutations, which shall be considered as genetic cause of ADOA.
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Upregulation of Peroxiredoxin 3 Protects AFG3L2-KO Cortical Neurons In Vitro from Oxidative Stress: A Paradigm for Neuronal Cell Survival under Neurodegenerative Conditions.
Oxidative medicine and cellular longevity, 2019Co-Authors: Barbara Bettegazzi, Francesca Maltecca, Giorgio Casari, Daniele Zacchetti, Fabio Grohovaz, Ilaria Pelizzoni, Floramarida Salerno Scarzella, Lisa Michelle Restelli, Franca CodazziAbstract:Several neurodegenerative disorders exhibit selective vulnerability, with subsets of neurons more affected than others, possibly because of the high expression of an altered gene or the presence of particular features that make them more susceptible to insults. On the other hand, resilient neurons may display the ability to develop antioxidant defenses, particularly in diseases of mitochondrial origin, where oxidative stress might contribute to the neurodegenerative process. In this work, we investigated the oxidative stress response of embryonic fibroblasts and cortical neurons obtained from AFG3L2-KO mice. AFG3L2 encodes a subunit of a protease complex that is expressed in mitochondria and acts as both quality control and regulatory enzyme affecting respiration and mitochondrial dynamics. When cells were subjected to an acute oxidative stress protocol, the survival of AFG3L2-KO MEFs was not significantly influenced and was comparable to that of WT; however, the basal level of the antioxidant molecule glutathione was higher. Indeed, glutathione depletion strongly affected the viability of KO, but not of WT MEF, thereby indicating that oxidative stress is more elevated in KO MEF even though well controlled by glutathione. On the other hand, when cortical KO neurons were put in culture, they immediately appeared more vulnerable than WT to the acute oxidative stress condition, but after few days in vitro, the situation was reversed with KO neurons being more resistant than WT to acute stress. This compensatory, protective competence was not due to the upregulation of glutathione, rather of two mitochondrial antioxidant proteins: superoxide dismutase 2 and, at an even higher level, peroxiredoxin 3. This body of evidence sheds light on the capability of neurons to activate neuroprotective pathways and points the attention to peroxiredoxin 3, an antioxidant enzyme that might be critical for neuronal survival also in other disorders affecting mitochondria.
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m-AAA and i-AAA complexes coordinate to regulate OMA1, the stress-activated supervisor of mitochondrial dynamics.
Journal of cell science, 2018Co-Authors: Francesco Consolato, Francesca Maltecca, Susanna Tulli, Irene Sambri, Giorgio CasariAbstract:The proteolytic processing of dynamin like GTPase OPA1, mediated by the activity of both YME1L1 ( i- AAA protease complex) and OMA1, is a crucial step in the regulation of mitochondrial dynamics. OMA1 is a zinc metallopeptidase of the inner mitochondrial membrane that undergoes pre-activating proteolytic and auto-proteolytic cleavage after mitochondrial import. Here, we identify AFG3L2 ( m- AAA complex) as the major protease mediating this event by maturing the pre-pro-OMA1 of 60 kDa to the pro-OMA1 form of 40 kDa by severing the amino-terminal part without recognizing specific consensus sequence. Therefore, m- AAA and i- AAA complexes coordinately regulate OMA1 processing and turnover, and consequently OPA1 isoforms, thus adding new information in the comprehension of the molecular mechanisms in mitochondrial dynamics and of neurodegenerative diseases affected by these phenomena.
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ROLE OF MITOCHONDRIA IN CORTICAL NEURONS FROM SPINOCEREBELLAR ATAXIA 28 (SCA28) MOUSE MODEL.
Journal of World Mitochondria Society, 2015Co-Authors: Franca Codazzi, Francesca Maltecca, Giorgio Casari, Floramarida Salerno, Federico Zucca, Barbara Bettegazzi, Daniele Zacchetti, Fabio GrohovazAbstract:In this study we investigated the role of mitochondria in cortical neurons obtained from a mouse model of spinocerebellar ataxia 28 (SCA28) characterized by dark degeneration of Purkinje cells. Primary cortical neurons from mice, WT and KO for AFG3L2 (the mitochondrial gene mutated in SCA28), were analyzed by single-cell videomicroscopy after iron overload, a condition able to to promote oxidative stress. Different fluorescent probes were employed to monitor iron oxidative status, ROS formation, labile iron pool, glutathione content and cell death. Detoxifying proteins were quantified by Western blot. Upon iron overload, AFG3L2-KO cortical neurons showed a significantly higher percentage of cell death than the WT counterpart. Surprisingly, with culture time, AFG3L2-KO neurons acquired a protective phenotype that made them more resistant than WT neurons to oxidative conditions. Further investigation revealed that the main antioxidant cellular molecule, glutathione, was not involved in this acquired resistance. On the other hand, during their ageing in culture, KO neurons showed an increase in the expression of SOD2 and peroxiredoxin III, two mitochondrial detoxifying proteins that appear to be involved in this compensatory and protective mechanism.
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Purkinje neuron Ca2+ influx reduction rescues ataxia in SCA28 model.
The Journal of clinical investigation, 2014Co-Authors: Francesca Maltecca, Francesco Consolato, Davide Mazza, Elisa Baseggio, Paola Podini, Samuel M. Young, Ilaria Drago, Ben A. Bahr, Aldamaria Puliti, Franca CodazziAbstract:Spinocerebellar ataxia type 28 (SCA28) is a neurodegenerative disease caused by mutations of the mitochondrial protease AFG3L2. The SCA28 mouse model, which is haploinsufficient for AFG3L2, exhibits a progressive decline in motor function and displays dark degeneration of Purkinje cells (PC-DCD) of mitochondrial origin. Here, we determined that mitochondria in cultured AFG3L2-deficient PCs ineffectively buffer evoked Ca²⁺ peaks, resulting in enhanced cytoplasmic Ca²⁺ concentrations, which subsequently triggers PC-DCD. This Ca²⁺-handling defect is the result of negative synergism between mitochondrial depolarization and altered organelle trafficking to PC dendrites in AFG3L2-mutant cells. In SCA28 mice, partial genetic silencing of the metabotropic glutamate receptor mGluR1 decreased Ca²⁺ influx in PCs and reversed the ataxic phenotype. Moreover, administration of the β-lactam antibiotic ceftriaxone, which promotes synaptic glutamate clearance, thereby reducing Ca²⁺ influx, improved ataxia-associated phenotypes in SCA28 mice when given either prior to or after symptom onset. Together, the results of this study indicate that ineffective mitochondrial Ca²⁺ handling in PCs underlies SCA28 pathogenesis and suggest that strategies that lower glutamate stimulation of PCs should be further explored as a potential treatment for SCA28 patients.
Elena I. Rugarli - One of the best experts on this subject based on the ideXlab platform.
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Astrocyte-specific deletion of the mitochondrial m-AAA protease reveals glial contribution to neurodegeneration.
Glia, 2019Co-Authors: Sara Murru, Esther Barth, Thomas Langer, Simon Hess, Eva R. Almajan, Désirée Schatton, Steffen Hermans, Susanne Brodesser, Peter Kloppenburg, Elena I. RugarliAbstract:Mitochondrial dysfunction causes neurodegeneration but whether impairment of mitochondrial homeostasis in astrocytes contributes to this pathological process remains largely unknown. The m-AAA protease exerts quality control and regulatory functions crucial for mitochondrial homeostasis. AFG3L2, which encodes one of the subunits of the m-AAA protease, is mutated in spinocerebellar ataxia SCA28 and in infantile syndromes characterized by spastic-ataxia, epilepsy and premature death. Here, we investigate the role of AFG3L2 and its redundant homologue Afg3l1 in the Bergmann glia (BG), radial astrocytes of the cerebellum that have functional connections with Purkinje cells (PC) and regulate glutamate homeostasis. We show that astrocyte-specific deletion of AFG3L2 in the mouse leads to late-onset motor impairment and to degeneration of BG, which display aberrant morphology, altered expression of the glutamate transporter EAAT2, and a reactive inflammatory signature. The neurological and glial phenotypes are drastically exacerbated when astrocytes lack both Afg31l and AFG3L2, and therefore, are totally depleted of the m-AAA protease. Moreover, mitochondrial stress responses and necroptotic markers are induced in the cerebellum. In both mouse models, targeted BG show a fragmented mitochondrial network and loss of mitochondrial cristae, but no signs of respiratory dysfunction. Importantly, astrocyte-specific deficiency of Afg3l1 and AFG3L2 triggers secondary morphological degeneration and electrophysiological changes in PCs, thus demonstrating a non-cell-autonomous role of glia in neurodegeneration. We propose that astrocyte dysfunction amplifies both neuroinflammation and glutamate excitotoxicity in patients carrying mutations in AFG3L2, leading to a vicious circle that contributes to neuronal death.
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The Mitochondrial m-AAA Protease Prevents Demyelination and Hair Greying
PLoS Genetics, 2016Co-Authors: Shuaiyu Wang, Julie Jacquemyn, Sara Murru, Esther Barth, Paola Martinelli, Carien M. Niessen, Thomas Langer, Elena I. RugarliAbstract:The m-AAA protease preserves proteostasis of the inner mitochondrial membrane. It ensures a functional respiratory chain, by controlling the turnover of respiratory complex subunits and allowing mitochondrial translation, but other functions in mitochondria are conceivable. Mutations in genes encoding subunits of the m-AAA protease have been linked to various neurodegenerative diseases in humans, such as hereditary spastic paraplegia and spinocerebellar ataxia. While essential functions of the m-AAA protease for neuronal survival have been established, its role in adult glial cells remains enigmatic. Here, we show that deletion of the highly expressed subunit AFG3L2 in mature mouse oligodendrocytes provokes early-on mitochondrial fragmentation and swelling, as previously shown in neurons, but causes only late-onset motor defects and myelin abnormalities. In contrast, total ablation of the m-AAA protease, by deleting both AFG3L2 and its paralogue Afg3l1, triggers progressive motor dysfunction and demyelination, owing to rapid oligodendrocyte cell death. Surprisingly, the mice showed premature hair greying, caused by progressive loss of melanoblasts that share a common developmental origin with Schwann cells and are targeted in our experiments. Thus, while both neurons and glial cells are dependant on the m-AAA protease for survival in vivo, complete ablation of the complex is necessary to trigger death of oligodendrocytes, hinting to cell-autonomous thresholds of vulnerability to m-AAA protease deficiency.
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AFG3L2 supports mitochondrial protein synthesis and Purkinje cell survival
The Journal of clinical investigation, 2012Co-Authors: Eva-ruxandra Almajan, Esther Barth, Paola Martinelli, Thomas Langer, Peter Kloppenburg, Ricarda Richter, Lars Paeger, Thorsten Decker, Nils-göran Larsson, Elena I. RugarliAbstract:Mutations in the AFG3L2 gene have been linked to spinocerebellar ataxia type 28 and spastic ataxia-neuropathy syndrome in humans; however, the pathogenic mechanism is still unclear. AFG3L2 encodes a subunit of the mitochondrial m-AAA protease, previously implicated in quality control of misfolded inner mitochondrial membrane proteins and in regulatory functions via processing of specific substrates. Here, we used a conditional AFG3L2 mouse model that allows restricted deletion of the gene in Purkinje cells (PCs) to shed light on the pathogenic cascade in the neurons mainly affected in the human diseases. We demonstrate a cell-autonomous requirement of AFG3L2 for survival of PCs. Examination of PCs prior to neurodegeneration revealed fragmentation and altered distribution of mitochondria in the dendritic tree, indicating that abnormal mitochondrial dynamics is an early event in the pathogenic process. Moreover, PCs displayed features pointing to defects in mitochondrially encoded respiratory chain subunits at early stages. To unravel the underlying mechanism, we examined a constitutive knockout of AFG3L2, which revealed a decreased rate of mitochondrial protein synthesis associated with impaired mitochondrial ribosome assembly. We therefore propose that defective mitochondrial protein synthesis, leading to early-onset fragmentation of the mitochondrial network, is a central causative factor in AFG3L2-related neurodegeneration.
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Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1
The Journal of cell biology, 2009Co-Authors: Sarah Ehses, Elena I. Rugarli, Andrea Bernacchia, Ines Raschke, Giuseppe Mancuso, Stefan Geimer, Daniel Tondera, Jean-claude Martinou, Benedikt Westermann, Thomas LangerAbstract:Mitochondrial fusion depends on the dynamin-like guanosine triphosphatase OPA1, whose activity is controlled by proteolytic cleavage. Dysfunction of mitochondria induces OPA1 processing and results in mitochondrial fragmentation, allowing the selective removal of damaged mitochondria. In this study, we demonstrate that two classes of metallopeptidases regulate OPA1 cleavage in the mitochondrial inner membrane: isoenzymes of the adenosine triphosphate (ATP)-dependent matrix AAA (ATPase associated with diverse cellular activities [m-AAA]) protease, variable assemblies of the conserved subunits paraplegin, AFG3L1 and -2, and the ATP-independent peptidase OMA1. Functionally redundant isoenzymes of the m-AAA protease ensure the balanced accumulation of long and short isoforms of OPA1 required for mitochondrial fusion. The loss of AFG3L2 in mouse tissues, down-regulation of AFG3L1 and -2 in mouse embryonic fibroblasts, or the expression of a dominant-negative AFG3L2 variant in human cells decreases the stability of long OPA1 isoforms and induces OPA1 processing by OMA1. Moreover, cleavage by OMA1 causes the accumulation of short OPA1 variants if mitochondrial DNA is depleted or mitochondrial activities are impaired. Our findings link distinct peptidases to constitutive and induced OPA1 processing and shed new light on the pathogenesis of neurodegenerative disorders associated with mutations in m-AAA protease subunits.
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Genetic interaction between the m -AAA protease isoenzymes reveals novel roles in cerebellar degeneration
Human Molecular Genetics, 2009Co-Authors: Paola Martinelli, Raffaealla Magnoni, Federica Cerri, Veronica La Mattina, Angelo Quattrini, Andrea Bernacchia, Giorgio Casari, Elena I. RugarliAbstract:: The mitochondrial m-AAA protease has a crucial role in axonal development and maintenance. Human mitochondria possess two m-AAA protease isoenzymes: a hetero-oligomeric complex, composed of paraplegin and AFG3L2 (Afg3 like 2), and a homo-oligomeric AFG3L2 complex. Loss of function of paraplegin (encoded by the SPG7 gene) causes hereditary spastic paraplegia, a disease characterized by retrograde degeneration of cortical motor axons. Spg7(-/-) mice show a late-onset degeneration of long spinal and peripheral axons with accumulation of abnormal mitochondria. In contrast, AFG3L2(Emv66/Emv66) mutant mice, lacking the AFG3L2 protein, are affected by a severe neuromuscular phenotype, due to defects in motor axon development. The role of the homo-oligomeric m-AAA protease and the extent of cooperation and redundancy between the two isoenzymes in adult neurons are still unclear. Here we report an early-onset severe neurological phenotype in Spg7(-/-) AFG3L2(Emv66/+) mice, characterized by loss of balance, tremor and ataxia. Spg7(-/-) AFG3L2(Emv66/+) mice display acceleration and worsening of the axonopathy observed in paraplegin-deficient mice. In addition, they show prominent cerebellar degeneration with loss of Purkinje cells and parallel fibers, and reactive astrogliosis. Mitochondria from affected tissues are prone to lose mt-DNA and have unstable respiratory complexes. At late stages, neurons contain structural abnormal mitochondria defective in COX-SDH reaction. Our data demonstrate genetic interaction between the m-AAA isoenzymes and suggest that different neuronal populations have variable thresholds of susceptibility to reduced levels of the m-AAA protease. Moreover, they implicate impaired mitochondrial proteolysis as a novel pathway in cerebellar degeneration.
Paola Martinelli - One of the best experts on this subject based on the ideXlab platform.
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The Mitochondrial m-AAA Protease Prevents Demyelination and Hair Greying
PLoS Genetics, 2016Co-Authors: Shuaiyu Wang, Julie Jacquemyn, Sara Murru, Esther Barth, Paola Martinelli, Carien M. Niessen, Thomas Langer, Elena I. RugarliAbstract:The m-AAA protease preserves proteostasis of the inner mitochondrial membrane. It ensures a functional respiratory chain, by controlling the turnover of respiratory complex subunits and allowing mitochondrial translation, but other functions in mitochondria are conceivable. Mutations in genes encoding subunits of the m-AAA protease have been linked to various neurodegenerative diseases in humans, such as hereditary spastic paraplegia and spinocerebellar ataxia. While essential functions of the m-AAA protease for neuronal survival have been established, its role in adult glial cells remains enigmatic. Here, we show that deletion of the highly expressed subunit AFG3L2 in mature mouse oligodendrocytes provokes early-on mitochondrial fragmentation and swelling, as previously shown in neurons, but causes only late-onset motor defects and myelin abnormalities. In contrast, total ablation of the m-AAA protease, by deleting both AFG3L2 and its paralogue Afg3l1, triggers progressive motor dysfunction and demyelination, owing to rapid oligodendrocyte cell death. Surprisingly, the mice showed premature hair greying, caused by progressive loss of melanoblasts that share a common developmental origin with Schwann cells and are targeted in our experiments. Thus, while both neurons and glial cells are dependant on the m-AAA protease for survival in vivo, complete ablation of the complex is necessary to trigger death of oligodendrocytes, hinting to cell-autonomous thresholds of vulnerability to m-AAA protease deficiency.
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loss of the m aaa protease subunit AFG3L2 causes mitochondrial transport defects and tau hyperphosphorylation
The EMBO Journal, 2014Co-Authors: Arun Kumar Kondadi, Paola Martinelli, Shuaiyu Wang, Sara Montagner, Nikolay Kladt, Anne Korwitz, David Herholz, Michael J. Baker, Astrid C. Schauss, Thomas LangerAbstract:The m-AAA protease subunit AFG3L2 is involved in degradation and processing of substrates in the inner mitochondrial membrane. Mutations in AFG3L2 are associated with spinocerebellar ataxia SCA28 in humans and impair axonal development and neuronal survival in mice. The loss of AFG3L2 causes fragmentation of the mitochondrial network. However, the pathogenic mechanism of neurodegeneration in the absence of AFG3L2 is still unclear. Here, we show that depletion of AFG3L2 leads to a specific defect of anterograde transport of mitochondria in murine cortical neurons. We observe similar transport deficiencies upon loss of AFG3L2 in OMA1-deficient neurons, indicating that they are not caused by OMA1-mediated degradation of the dynamin-like GTPase OPA1 and inhibition of mitochondrial fusion. Treatment of neurons with antioxidants, such as N-acetylcysteine or vitamin E, or decreasing tau levels in axons restored mitochondrial transport in AFG3L2-depleted neurons. Consistently, tau hyperphosphorylation and activation of ERK kinases are detected in mouse neurons postnatally deleted for AFG3L2. We propose that reactive oxygen species signaling leads to cytoskeletal modifications that impair mitochondrial transport in neurons lacking AFG3L2.
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Loss of the m‐AAA protease subunit AFG3L2 causes mitochondrial transport defects and tau hyperphosphorylation
The EMBO journal, 2014Co-Authors: Arun Kumar Kondadi, Paola Martinelli, Shuaiyu Wang, Sara Montagner, Nikolay Kladt, Anne Korwitz, David Herholz, Michael J. Baker, Astrid C. Schauss, Thomas LangerAbstract:The m-AAA protease subunit AFG3L2 is involved in degradation and processing of substrates in the inner mitochondrial membrane. Mutations in AFG3L2 are associated with spinocerebellar ataxia SCA28 in humans and impair axonal development and neuronal survival in mice. The loss of AFG3L2 causes fragmentation of the mitochondrial network. However, the pathogenic mechanism of neurodegeneration in the absence of AFG3L2 is still unclear. Here, we show that depletion of AFG3L2 leads to a specific defect of anterograde transport of mitochondria in murine cortical neurons. We observe similar transport deficiencies upon loss of AFG3L2 in OMA1-deficient neurons, indicating that they are not caused by OMA1-mediated degradation of the dynamin-like GTPase OPA1 and inhibition of mitochondrial fusion. Treatment of neurons with antioxidants, such as N-acetylcysteine or vitamin E, or decreasing tau levels in axons restored mitochondrial transport in AFG3L2-depleted neurons. Consistently, tau hyperphosphorylation and activation of ERK kinases are detected in mouse neurons postnatally deleted for AFG3L2. We propose that reactive oxygen species signaling leads to cytoskeletal modifications that impair mitochondrial transport in neurons lacking AFG3L2.
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AFG3L2 supports mitochondrial protein synthesis and Purkinje cell survival
The Journal of clinical investigation, 2012Co-Authors: Eva-ruxandra Almajan, Esther Barth, Paola Martinelli, Thomas Langer, Peter Kloppenburg, Ricarda Richter, Lars Paeger, Thorsten Decker, Nils-göran Larsson, Elena I. RugarliAbstract:Mutations in the AFG3L2 gene have been linked to spinocerebellar ataxia type 28 and spastic ataxia-neuropathy syndrome in humans; however, the pathogenic mechanism is still unclear. AFG3L2 encodes a subunit of the mitochondrial m-AAA protease, previously implicated in quality control of misfolded inner mitochondrial membrane proteins and in regulatory functions via processing of specific substrates. Here, we used a conditional AFG3L2 mouse model that allows restricted deletion of the gene in Purkinje cells (PCs) to shed light on the pathogenic cascade in the neurons mainly affected in the human diseases. We demonstrate a cell-autonomous requirement of AFG3L2 for survival of PCs. Examination of PCs prior to neurodegeneration revealed fragmentation and altered distribution of mitochondria in the dendritic tree, indicating that abnormal mitochondrial dynamics is an early event in the pathogenic process. Moreover, PCs displayed features pointing to defects in mitochondrially encoded respiratory chain subunits at early stages. To unravel the underlying mechanism, we examined a constitutive knockout of AFG3L2, which revealed a decreased rate of mitochondrial protein synthesis associated with impaired mitochondrial ribosome assembly. We therefore propose that defective mitochondrial protein synthesis, leading to early-onset fragmentation of the mitochondrial network, is a central causative factor in AFG3L2-related neurodegeneration.
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whole exome sequencing identifies homozygous AFG3L2 mutations in a spastic ataxia neuropathy syndrome linked to mitochondrial m aaa proteases
PLOS Genetics, 2011Co-Authors: Tyler Mark Pierson, Paola Martinelli, Nancy F Hansen, Praveen F. Cherukuri, Robert W Blakesley, Florian Bonn, Jamie K Teer, Pedro Cruz, David H. Adams, Gretchen GolasAbstract:We report an early onset spastic ataxia-neuropathy syndrome in two brothers of a consanguineous family characterized clinically by lower extremity spasticity, peripheral neuropathy, ptosis, oculomotor apraxia, dystonia, cerebellar atrophy, and progressive myoclonic epilepsy. Whole-exome sequencing identified a homozygous missense mutation (c.1847G>A; p.Y616C) in AFG3L2, encoding a subunit of an m-AAA protease. m-AAA proteases reside in the mitochondrial inner membrane and are responsible for removal of damaged or misfolded proteins and proteolytic activation of essential mitochondrial proteins. AFG3L2 forms either a homo-oligomeric isoenzyme or a hetero-oligomeric complex with paraplegin, a homologous protein mutated in hereditary spastic paraplegia type 7 (SPG7). Heterozygous loss-of-function mutations in AFG3L2 cause autosomal-dominant spinocerebellar ataxia type 28 (SCA28), a disorder whose phenotype is strikingly different from that of our patients. As defined in yeast complementation assays, the AFG3L2Y616C gene product is a hypomorphic variant that exhibited oligomerization defects in yeast as well as in patient fibroblasts. Specifically, the formation of AFG3L2Y616C complexes was impaired, both with itself and to a greater extent with paraplegin. This produced an early-onset clinical syndrome that combines the severe phenotypes of SPG7 and SCA28, in additional to other “mitochondrial” features such as oculomotor apraxia, extrapyramidal dysfunction, and myoclonic epilepsy. These findings expand the phenotype associated with AFG3L2 mutations and suggest that AFG3L2-related disease should be considered in the differential diagnosis of spastic ataxias.
