Mitochondria and ALS: Implications from novel genes and pathways

Molecular and Cellular Neuroscience 55 (2013) 44–49

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Mitochondria and ALS: Implications from novel genes and pathways Mauro Cozzolino a, b, Alberto Ferri b, c, Cristiana Valle b, c, Maria Teresa Carrì b, d,⁎ a

Institute for Translational Pharmacology, CNR, Rome, Italy Fondazione Santa Lucia IRCCS, Rome, Italy c Institute for Cell Biology and Neurobiology, CNR, Rome, Italy d Department of Biology, University of Rome Tor Vergata, Rome, Italy b

a r t i c l e

i n f o

Article history: Received 2 April 2012 Accepted 6 June 2012 Available online 15 June 2012 Keywords: Amyotrophic lateral sclerosis Mitochondria Oxidative stress

a b s t r a c t Evidence from patients with sporadic and familiar amyotrophic lateral sclerosis (ALS) and from models based on the overexpression of mutant SOD1 found in a small subset of patients, clearly point to mitochondrial damage as a relevant facet of this neurodegenerative condition. In this mini-review we provide a brief update on the subject in the light of newly discovered genes (such as TDP-43 and FUS/TLS) associated to familial ALS and of a deeper knowledge of the mechanisms of derangement of mitochondria. This article is part of a Special Issue entitled 'Mitochondrial function and dysfunction in neurodegeneration'. © 2012 Elsevier Inc. All rights reserved.

Mitochondria, ALS and mutant SOD1 Since the first reports of mitochondrial abnormalities in tissues from patients with amyotrophic lateral sclerosis (ALS) (Afifi et al., 1966; Atsumi, 1981; Hart et al., 1977; Okamoto et al., 1990; Siklos et al., 1996), mitochondrial dysfunction has been steadily recognized as a central matter in the pathogenesis of this disease. All aspects of mitochondrial physiology have been analyzed in patients and models based on the expression of mutant SOD1s (mutSOD1) found in a subset of patients, and nearly all have been found to be variably compromised in this pathological condition. The notion that alterations in mitochondrial bioenergetics, clearance of dys-functional organelles, calcium buffering and induction of mitochondrial apoptosis are all important features of the disease has robust foundation deriving from different experimental evidence (Fig. 1) (Duffy et al., 2011). More recently, alterations in mitochondrial morphology, fusion/fission dynamics and biogenesis have attracted much attention, given the importance of such alterations in the overall homeostasis of mitochondrial function (Chen and Chan, 2009). That mitochondrial dynamics may play a relevant role in ALS was put forward by the observation of morphological alterations that are reminiscent of fragmentation in mitochondria of Abbreviations: ALS, Amyotrophic Lateral Sclerosis; CFP, Cyan Fluorescent Protein; ER, Endoplasmic Reticulum; FUS/TLS, Fused in Sarcoma/Translocated in Liposarcoma; GSH, Glutathione; HAT, Histone Acetyl Transferase; HDAC, Histone Deacetylase; ROS, Reactive Oxygen Species: SIRT, Sirtuin; SOD1, Cu, Zn Superoxide Dismutase; TDP-43, TAR DNA Binding Protein 43; UPR, Unfolded Protein Response; VAPB, VAMP Associated Protein. ⁎ Corresponding author at: Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica, 00133 Rome, Italy. Fax: + 39 06 50170 3323. E-mail address: [email protected] (M.T. Carrì). 1044-7431/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2012.06.001

cultured cells and mice expressing mutSOD1 (Cozzolino et al., 2009; Magrane et al., 2009; Vande Velde et al., 2011). Changes in the expression of proteins controlling mitochondrial fusion/fission have also been observed in a cellular model of the disease (Ferri et al., 2010). More recently, using live imaging microscopy, Magranè et al. were able to follow mitochondria reshaping occurring in mutSOD1 expressing motor neurons by virtue of a mitochondria targeted fluorescent probe, and from this analysis they were able to conclude that mitochondrial dynamics alterations and bioenergetic dysfunctions work together in triggering synaptic alterations in motor neurons expressing mutSOD1 (Magrane et al., 2012). Thus, impaired mitochondrial dynamics may have a primary role in the degeneration of motor neurons. Defective mitochondrial transportation along axons has also been repeatedly proposed to contribute to motor neuron degeneration in ALS models (Bilsland et al., 2010; De Vos et al., 2007; Morotz et al., 2012). However, this hypothesis has been questioned since increasing axonal mitochondrial mobility through genetic ablation of syntaphilin does not affect the onset of ALS-like symptoms in G93A-SOD1 mice (Zhu and Sheng, 2011) and in a recent paper Marinkovic et al. reported that axon degeneration is a much later event than defects in mitochondrial transport in G93A-SOD1 mice. Furthermore, transport deficits do not take place in the G85R-SOD1 model and are insufficient to cause motor neuron death in mice overexpressing wild type SOD1 (Marinkovic et al., 2012). In the case of familial ALS associated with mutSOD1, in the last years much effort has been put to discern whether association of mutant SOD1 to mitochondria is a leading mechanism driving mitochondrial dysfunction. Indeed, a small fraction of wild type SOD1 is normally localized in mitochondria, with an apparent role in preventing oxidative damage in the mitochondrial intermembrane space, a function that seems to be important for motor

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Fig. 1. Mitochondrial dysfunction in Amyotrophic Lateral Sclerosis: causes and consequences. Mutant SOD1 is imported into mitochondria and may interfere with different mitochondrial components. These include the mitochondrial apoptotic machinery, e.g. Bcl-2 (Pasinelli et al., 2004) and MPTP (Martin et al., 2009), triggering cytochrome c release in the cytosol and activation of the apoptotic cascade (Pasinelli et al., 2000); the protein import machinery (Li et al., 2010); the mitochondrial redox balance (GSH/GSSH ratio) (Ferri et al., 2006); cytosolic calcium buffering (Grosskreutz et al., 2010); mitochondrial axonal transport (De Vos et al., 2007); mitochondrial redox signalling by p66shc (Pesaresi et al., 2011); respiratory chain and oxidative phosphorylation (Mattiazzi et al., 2002); membrane potential (Carri et al., 1997; Wiedemann et al., 2002); mitochondrial dynamics (expression DRP1 and OPA1) (Ferri et al., 2010). Abnormal mitochondrial dynamics and fragmentation was observed also in some TDP-43 and FUS/TLS models (Tradewell et al., 2012; Xu et al., 2010). Mutant VAPB may alter maintenance of Ca2+ homeostasis and decreas anterograde axonal transport of mitochondria (De Vos et al., 2012). ATP, adenosine triphosphate; Bcl-2, B-cell lymphoma 2; CytoC, cytocrome c; DRP1, dynamin-related protein; FUS/TLS, fused in sarcoma; GSGG, glutathione disulfide; GSH, glutathione; MPTP, mitochondrial permeability transition pore; OPA1, optical atrophy 1; ROS, reactive oxygen species; SOD1, Cu–Zn Superoxide dismutase; TDP-43, TAR DNA-binding protein 43; TIM, transporter inner membrane; TOM, transporter outer membrane; VAPB, vesicle-associated membrane proteinassociated protein B; I, II, III, IV, respiratory chain complexes; Δψ, membrane potential.

axon maintenance (Fischer et al., 2011). Further, mutant SOD1s associate to mitochondria, probably to an increased amount compared to the wild type protein, and mostly on the outer membrane and intermembrane space (Bergemalm et al., 2006; Deng et al., 2006; Ferri et al., 2006; Higgins et al., 2002; Israelson et al., 2010; Jaarsma et al., 2001; Liu et al., 2004; Pasinelli et al., 2004; Vande Velde et al., 2008; Vijayvergiya et al., 2005). In this process, protein misfolding and aggregation, that is mediated by free Cysteine residues and prompted by mutations and/or by a pro-oxidant environment (Cozzolino et al., 2008; Ferri et al., 2006; Karch and Borchelt, 2010; Karch et al., 2009), play a central role, because it is likely that entrapment in mitochondria is the driving force that eventually leads to mitochondrial accumulation of the protein. This is particularly interesting, since the regulation of the mitochondrial localization of SOD1 is strictly controlled to respond to the physiological needs of mitochondria, that change as a consequence of variations of oxygen concentration, and mutant SOD1 might lose this regulation with significant consequences on mitochondrial function (Kawamata and Manfredi, 2008). Some specific targets of mitochondria-associated mutSOD1s have been already identified (Israelson et al., 2010; Kawamata et al., 2008; Pasinelli et al., 2004; Pedrini et al., 2010) and some of them were proposed to be relevant in the pathological ALS context. This

is the case for VDAC1, a mitochondrial porin located on the outer mitochondrial membrane and is also a key player in mitochondriamediated apoptosis that was proposed to be required for the association of misfolded mutant SOD1 with mitochondria (Israelson et al., 2010) and thus mediate its toxicity. However, SOD1 remains associated with mitochondria despite genetic deletion of VDAC1 (Li et al., 2010). Since mating mutant SOD1 mice with VDAC1+/− mice accelerates disease onset and decreases survival (Israelson et al., 2010) it is likely that SOD1 and VDAC1 act independently from their association in generating mitochondrial damage. Mitochondrial mutSOD1s impinge on the overall mitochondrial protein composition and affects the pattern of mitochondrial protein import in spinal cord but not in other non-affected tissues (Li et al., 2010). This suggests that other, yet undiscovered, protein partners might be recruited by misfolded mutSOD1 in mitochondria that may be particularly relevant for motor neuron maintenance. Consequently, experimental approaches aimed at modifying the process of accumulation and aggregation of mutSOD1s in mitochondria may prove crucial to devise new therapeutic approaches for ALS, or at least for familial ALS linked to mutSOD1. We and others were able to provide circumstantial evidence that localization in mitochondria is both necessary and sufficient for mutSOD1s to drive mitochondrial

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damage and cell death (Cozzolino et al., 2009; Magrane et al., 2009) . A more direct support to this suggestion came recently from studies in transgenic mice where mutSOD1 is specifically overexpressed in the mitochondrial intermembrane space (Igoudjil et al., 2011). These mice show many, but not all, ALS-like characteristics found in transgenic mice overexpressing a normal mutSOD1, which is a robust confirmation of the hypothesis that mitochondrial mutSOD1s cause mitochondrial impairment. Why this is not sufficient to induce a complete ALS phenotype, including neuromuscular junction dismantling and denervation is unclear, but worth to be further investigated. Indeed these data suggest that a relevant fraction of disease phenotypes are associated to the toxic activities of mutSOD1 in other localizations, both mitochondrial (a fraction of mutSOD1 is associated to the outer face of the mitochondria outer membranes) and nonmitochondrial (e.g. nuclear or extracellular). Recent evidence from our lab further support this hypothesis. Indeed, the removal from G93A transgenic mice of p66Shc, a protein that acts as a regulator of mitochondrial redox signaling and that is known to mediate mitochondrial dysfunction and apoptosis, confers a significant protection from mutSOD1-induced mitochondrial damage and a considerable attenuation of disease phenotypes, but does not halt ALS progression entirely (Pesaresi et al., 2011). All these considerations point to mitochondrial dysfunction as a fundamental, albeit not unique, contributor to ALS. Thus it is not surprising that a big effort has been dedicated during the very last years to the development and testing of drugs able to target and prevent mitochondrial dysfunction. These include antioxidant agents, modulators of mitochondrial function, drugs activating the antioxidant response signalling program, and others (reviewed in (Duffy et al., 2011)). Some of them, like creatine and minocycline, proved to be effective in mouse models but did not give any significant result when translated into clinical trials (Benatar, 2007; Gordon et al., 2007). More recently, olesoxime (also called TRO19622), a drug targeting a specific mitochondrial function such as mitochondrial pore opening (Martin, 2010), and dexpramipexole, a drug with pleiotropic neuroprotective properties including inhibition of apoptotic enzymes and preservation of mitochondrial structure and activity (Cheah and Kiernan, 2010), have generated great expectations by virtue of significant effects in preclinical tests (Bordet et al., 2007). Unluckily, olesoxime did not prove effective in a clinical trial, as it does not prolong the survival of patients versus placebo (www.trophos.com); on the contrary, dexpramipexole is safe, well tolerated, and showed a positive effect in subjects with ALS (Cudkowicz et al., 2011). These quite unpredictable results might descend from the multi-factorial character of this disease, as well as from the nature of the specific mitochondrial target of these drugs. For these reasons, the development of new genetic models and the identification of new and specific pathways in ALS that could have a role relevant to mitochondrial physiology might be of great help.

Mitochondria, ALS and novel genes The landscape of current knowledge on ALS has considerably changed over the very last years, when new genes associated to the familial form of the disease have been identified (Andersen and AlChalabi, 2011). Among these, mutations in genes coding for proteins involved in RNA processing (TDP-43 and FUS), or genetic alterations that might give rise to toxic RNA species (C9ORF72) led scientists to try and reconcile previous knowledge indicating that protein misfolding, oxidative damage, defective axonal transport and excitotoxicity play a central role in the disease with the knowledge that defects in RNA metabolism generate the same clinical phenotype. Do we also need to reconsider the concept that mitochondria play a central role in ALS, as indicated by previous studies on SOD1-linked forms of the disease? Although an answer to this question is still premature, different lines of evidence

suggest that mitochondrial dysfunction might be a common determinant of ALS. Mutations in the gene encoding vesicle-associated membrane protein-associated protein B (VAPB) have been identified to be causative of some cases of ALS type-8 (Chen et al., 2010; Nishimura et al., 2004). VAPB function has not been fully characterized, but roles in unfolded protein response and ER stress, cytoskeleton organization and ephrin receptors signaling have been proposed (Morotz et al., 2012). A yeast-two-hybrid screening has recently disclosed that VAPB interacts with PTPIP51, a protein anchored through its N-terminal domain to the outer mitochondrial membrane (De Vos et al., 2012). Interaction between VAPB and PTPIP51 is enriched in mitochondria-associated membranes, i.e. those regions of ER that are in close proximity with mitochondria and that have a central position in the exchange of Ca2+ between ER and mitochondria and thus in Ca2+ homeostasis. Interestingly, the mutant P56S-VAPB shows an increased interaction with PTPIP51, induces clustering of mitochondria in perinuclear regions and disrupts mitochondrial Ca2+ handling. In turn, this might underlie elevation in cytosolic Ca 2+ concentrations, release of Miro-1 and associated kinesin-1 from microtubules and the resulting disruption of anterograde axonal transport of mitochondria. The latter feature characterizes neurons overexpressing the P56S mutant, and was proposed to have a significant role in SOD1-linked models of familial ALS (De Vos et al., 2008, 2012). More recently, mutations in the TAR DNA binding protein (TDP-43) have been described in a subset of ALS patients. TDP-43 is DNA and RNA binding protein with a major nuclear localization and a role in regulating different aspects of RNA metabolism, with pre-mRNA splicing among the most prominent (Polymenidou et al., 2011; Tollervey et al., 2011)). ALS linked mutations impact on the subcellular localization of TDP-43, leading to the accumulation of the protein in the cytosol, often in the form of aggregates (Guo et al., 2011; Johnson et al., 2009; Nonaka et al., 2009). Whether mutations in TDP-43 affect the expression and/or splicing of genes that control mitochondrial function, or confer to the mutated/aggregated protein a new property that is toxic for mitochondria is not definitively proven. Yet, in a yeast model, TDP-43 aggregates around mitochondria and there is an inverse correlation between respiratory activity and toxicity of the mutant protein, which suggests that mitochondria and oxidative stress are relevant to TDP-43-triggered cell death (Braun et al., 2011). More interestingly, clear mitochondrial phenotypes have been described in mouse models that mimic some, but not all, aspects of human ALS through the overexpression of wild type or mutant TDP-43. Mice overexpressing human TDP-43 under the control of the Thy-1.2 promoter, that drives expression in neurons including motor neurons, display various motor defects including tremors, abnormal reflex of the hindlimbs, gait abnormalities, paralysis and premature death (Shan et al., 2010). At the histological level, ubiquitin-positive, TDP-43-negative cytoplasmic inclusions are present in spinal motor neurons. These inclusions are strikingly labeled by antibodies recognizing mitochondrial markers such as HSP60 and VDAC, and appear to be composed of abnormally aggregated mitochondria when analyzed in electron microscopy. A similar result is obtained when TDP-43 mice are crossbred with mice where mitochondria are fluorescently labeled with neuron specific, mitochondria-targeted CFP. Consistent with the hypothesis that this feature is a consequence of abnormal mitochondria trafficking, TDP-43 transgenic mice show a significant depletion of mitochondria at neuromuscular junctions, and cytoplasmic inclusions in these mice contain proteins associated to kinesin-based motors that control axonal transport, such as Kif3a and KAP3 (Shan et al., 2010). Similar findings have been reported in mice with mouse prion promoter (mPrP) driving the expression of human TDP-43 (Xu et al., 2010). These animals develop motor deficits and reactive gliosis, but no motor neuron loss, and show signs of impaired mitochondrial function, as suggested by abnormal clustering of mitochondria within

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neurons and altered expression of proteins controlling mitochondrial dynamics. In particular, TDP-43PrP mice possess enhanced levels of components of the mitochondrial fission machinery (Fis1 and phosphorylated DLP1) and a reduction in mitofusin 1 expression, which is involved in mitochondrial fusion. Mitochondrial dynamics is apparently unaffected in mice overexpressing the ALS-linked mutant M337V-TDP-43 under the same mPrP promoter, although these mice otherwise show phenotypes similar to those overexpressing the wild type protein (and partially overlapping the human phenotype), including abnormal aggregates of vacuolated and degenerated mitochondria (Xu et al., 2011). The lack of modification in mitochondrial dynamics by the M337V substitution in TDP-43 may be ascribed to the fact that this mutation gives rise to a truncated protein that may lack the ability to interfere with the fusion/fission machinery. Interestingly, neuronal loss due to apoptosis was not detected in TDP43M337V mice, which suggests that alteration of mitochondrial dynamics are a crucial event in the induction of neuronal death. It is not completely clear whether wild type or mutant TDP-43 cytosolic aggregates co-localize with mitochondrial clusters, although data from the works mentioned above seem to exclude this possibility. However, that TDP-43 accumulation in mitochondria might directly impact on mitochondrial function and/or trafficking is an option that deserves further investigation in the light of evidence from sporadic ALS patients, where TDP-43 immunogold-labeled deposits occasionally appear in different sub-compartments of mitochondria in the anterior horns of spinal cord (Mori et al., 2008; Sasaki et al., 2010). As for TDP-43, also FUS is found mutated in ALS patients and is involved in different aspects of RNA metabolism, and thus RNA dysmetabolism may account for the pathogenic effects that FUS mutations exert in humans. Compared to TDP-43, not many animal models, and in particular rodents, have been produced to study the pathological consequences of overexpressing or silencing wild type or mutant FUS, and thus evidence that substantiate or dismiss mitochondrial dysfunction in FUS-linked ALS are still poor. Nonetheless, it has been recently shown that microinjection of mutant FUS in mouse motor neurons causes a significant shortening of mitochondria, a feature that is reminiscent of other ALS models (Tradewell et al., 2012) This effect is enhanced by the concurrent downregulation of PRMT1, an arginine methyltransferase whose knockdown augments cytosolic accumulation of mutant FUS. These results therefore suggest that redistribution from the nucleus to the cytoplasm, a prominent feature of mutant FUS, is correlated with accumulation of mitochondrial abnormalities in motor neurons. Furthermore, transgenic rats expressing mutant FUS show ubiquitinated aggregates that are positive for the mitochondrial marker COXIV, suggesting that mitochondria are damaged also in this model and may be ubiquitinated for degradation (Huang et al., 2011). Finally, mitochondrial abnormalities, and in particular basophilic inclusions containing both FUS proteins and disorganized mitochondria, have been also revealed in a neuropathological characterization of two patients with rapidly progressive ALS (Huang et al., 2010). Overall, these results link mitochondrial dysfunction to toxicity from mutation in other (non-SOD1) ALS genes, and suggest a (dys)functional convergence between the products of these genes and mutant SOD1 in the pathogenesis of ALS, but indeed further analysis is needed to corroborate these observations and understand the mechanisms linking these proteins to mitochondrial dysfunction. Mitochondria, ALS and novel targets Beside new ALS-linked mutant proteins, other players may have a previously mis-recognized role in mitochondrial damage in ALS. As for other neurodegenerative conditions, evidence for the involvement of altered epigenetic control in ALS due to an unbalance between histone acetyl transferases (HATs) and histone deacetylases (HDACs) activities is accumulating (Janssen et al., 2010; Schmalbach and Petri,

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2010). Alteration in these activities would cause disturbances in genes accessibility and transcription through modification of the pattern of acetylation of histones and transcriptions factors, and may directly follow expression of mutant SOD1 (Petri et al., 2006) or TDP-43 and FUS (Kim et al., 2010). Some class I HDACs are modulated by oxidative stress, and the carbonylation of reactive Cysteine residues causes the reduction of their activity and change in the transcription of genes controlled by these HDACs (Doyle and Fitzpatrick, 2010). Thus, one can speculate that mitochondria-dependent oxidative stress may be a modulator of gene expression through the epigenetic modulation of DNA accessibility. Noticeably, oxidative stress is also a well-known modulator of several transcription factors including NF-κB, that is known be activated in ALS patients (Migheli et al., 1997) and in models linked to both SOD1 (Casciati et al., 2002) and TDP-43 (Swarup et al., 2011). Thus, mitochondria-dependent generation of ROS and oxidative stressdependent alteration of HDACs and transcription factors may concur in the generation of a pathological phenotype through the same mechanism, i.e. altered accessibility of a set of genes crucial for neuronal survival. That HDACs are modulated by oxidative stress is even more interesting in the light of a recent report that the activity of this class of enzymes influences also splice site selection (Hnilicova et al., 2011). This may explain (at least in part) our observation that mitochondrial damage is a cause of modification in the abundance of selected splicing variants (Maracchioni et al., 2007) and that defective RNA metabolism seems to descend directly from mitochondrial/oxidative stress in models for SOD1-linked ALS (Lenzken et al., 2011). HDAC inhibitors are broadly neuroprotective in vivo, and pan-HDAC inhibition is effective in promoting neuronal survival also in conditions of oxidative stress caused by glutathione depletion (Langley et al., 2008). For these reasons, unspecific HDAC inhibitors such as valproic acid, sodium phenylbutyrate and trichostatin A have been tested for the treatment of ALS with some negative outcome (Piepers et al., 2009) and several positive results (Cudkowicz et al., 2009; Feng et al., 2008; Petri et al., 2006; Sugai et al., 2004; Yoo and Ko, 2011) that may also be related to their multiple neuroprotective functions (including antioxidative, antiapoptotic and anti-glutamate toxicity properties, as in the case of valproic acid (Piepers et al., 2009)). However, a word of caution must be spent on the concept that modulation of HDACs may represent a promising approach for the treatment of ALS in the light of conflicting results even in SOD1 mice (Rouaux et al., 2007), which suggest that a better approach would be targeting specific HDACs. In this light, it is interesting that many NAD +-dependent class III HDACs (sirtuins, Sirt) are localized in mitochondria and are involved in the regulation of a number of genes related to energy metabolism and oxidative stress. Sirtuins seem to have a beneficial effect on life-span in several models, a function that may be linked to their ability to regulate systems that control the redox environment (Webster et al., 2012). This is particularly well known for Sirt3, that deacetylates a substantial subset of mitochondrial proteins (Lombard et al., 2007) including enzymes involved in crucial energetic pathways such as β-oxidation, oxidative phosphorylation and the TCA cycle ((Bell and Guarente, 2011) and refs. therein). Sirt3, which is induced by oxidative stress, seems to control the levels of ROS by multiple mechanisms, including the de-acetylation and post-translational activation of mitochondrial SOD2 (Chen et al., 2011; Tao et al., 2010) and isocitrate dehydrogenase 2, which is a major source of NADPH and protects (non-neuronal) cells from oxidative stress through increased levels of reduced glutathione (Yu et al., 2012). In this light, sirtuins modulation may represent an interesting strategy to overcome or bypass mitochondrial damage in ALS. However, at present there is no direct evidence of the involvement of this class of proteins in ALS, except for Sirt1 that is mostly cytosolic but resides also in mitochondria (Aquilano et al., 2010). Sirt1 up-

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