GCN2 inhibition reduces mutant SOD1 clustering and toxicity and delays disease progression in an Amyotrophic Lateral Sclerosis mouse model

GCN2 inhibition reduces mutant SOD1 clustering and toxicity and delays disease progression in an Amyotrophic Lateral Sclerosis mouse model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article GCN2 inhibition reduces mutant SOD1 clustering and toxicity and delays disease progression in an Amyotrophic Lateral Sclerosis mouse model Didio Alberto Ortiz, Nuria Peregrín, Miguel Valencia, Rodrigo Vinueza-Gavilanes, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4544133/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The disruption of protein folding homeostasis in motoneurons (MNs), and the ensuing accumulation of protein aggregates, is one of the main molecular hallmarks of amyotrophic lateral sclerosis (ALS) pathology, and has been recapitulated in cellular and animal disease models. The loss of proteostasis and other stresses in the MN trigger the activation of a general stress mechanism, the integrated stress response (ISR). The ISR is initiated by either of four stress-sensing kinases (GCN2, HRI, PERK and PKR) which, upon activation by distinct insults, promote a dramatic remodeling of gene expression to combat stress and promote survival. Paradoxically, in pathologies where stress is chronic or overwhelming, the ISR can also promote neuronal death. In ALS experimental models, extensive evidence demonstrates a key role of this mechanism in the progression of disease, and has inspired many attempts to develop ALS therapies based on ISR modulation. In our group, we recently discovered that the downstream ISR inhibitor ISRIB increases survival of a neuronal ALS model based on the expression of the neurotoxic ALS allele, SOD1 G93A. In the current study, we found that ISR inhibition is sufficient to prevent the concentration of mutant SOD1 into cytosolic foci, suggesting that ISR is required for SOD1 protein aggregation. Through a systematic CRISPR Cas9 approach and pharmacological inhibition, we demonstrate that, unexpectedly, the ISR kinase GCN2 is required for SOD1 clustering in cell lines and primary neuronal cultures. Moreover, genetic or pharmacological GCN2 inhibition strongly enhances survival of neurons overexpressing mutant SOD1. Finally, GCN2 pharmacological inhibition in fALS SOD1G93A transgenic mice delayed muscle denervation, strength loss, weight loss, and the appearance of ALS symptoms. Based on these findings, we propose GCN2 as a new potential therapeutic target for ALS. Amyotrophic Lateral Sclerosis Integrated Stress Response GCN2 SOD1 clustering neuronal models SOD1G93A transgenic mouse electromyographic analysis Figures Figure 1 Full Text The disruption of protein folding homeostasis in motoneurons (MNs) (and the ensuing accumulation of protein aggregates) is one of the main molecular hallmarks of amyotrophic lateral sclerosis (ALS). Both in sporadic ALS patients, in patients with inheritable forms of the disease (familial ALS, or fALS), or in neuronal and animal transgenic disease models, protein aggregation correlates and – in many experimental models – directly participates in neurodegeneration. In turn, the loss of motoneuron homeostasis triggers a coping mechanism, the integrated stress response (ISR) [1]. The ISR can be initiated by four independent stress-sensing kinases (PKR, PERK, GCN2, and HRI), each of which is activated by distinct stresses: GCN2 by aminoacid and glucose starvation or bribosome collision, HRI by heme deprivation, mitochondrial stress and proteasome deficiency, PERK by protein misfolding at the endoplasmic reticulum (ER) and PKR by double-strand RNA. Once activated, either of them phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2a), leading to 1) the general reduction in translation of most mRNAs and 2) the enhanced translation of transcripts encoding stress response factors, such as the activating transcription factor 4 (ATF4). While these changes in gene expression are in principle neuroprotective, under chronic stress the ISR can also drive apoptosis [1]. In most ALS studies, the ISR kinase PERK was proposed to be the ISR trigger. Importantly, in ALS animal models ISR activation is most prominent in fast-fatigable MNs, the most vulnerable MN subpopulation [2]. Driven by this compelling evidence, many groups have developed pharmacological ISR activators or suppressors to treat ALS, some of which have reached clinical trials [3]; yet, it is still unclear what stress/es drives ISR activation in ALS and which is the most robust therapeutic strategy. Mutations in the SOD1 gene cause fALS and SOD1 protein aggregation in patient postmortem samples and transgenic mouse models. We previously developed a neuronal ALS model based on the expression of the mutant allele SOD1 G93A, where we can quantitatively score the risk of neuronal death. Importantly, this experimental setting recapitulates both ISR activation and neuronal death. In this model, the ISR downstream inhibitor (ISRIB, [4]) tuned neuronal ISR and enhanced neuronal survival [5]. Intriguingly, ISRIB also mitigated a distinct proteostatic mechanism, the unfolded protein (UPR), suggesting that ISR dampening could somehow reduce neurotoxicity [5]. The neuroprotective effect of ISR inhibition could not be recapitulated by the inhibition of PERK, suggesting that ISR stress sensing kinase/s other than PERK trigger ISR activation in ALS. To investigate if ISR modulation affects the localization and neurotoxicity of mutant SOD1 protein we explored the effect of ISRIB in mutant SOD1 clustering when overexpressed in HEK293 cells. Consistent with previous studies, SOD1 overexpression led to the formation of protein clusters in approximately 50% of cells (FigS1B-C). In this setting, ISRIB prevented mutant SOD1 clustering (Fig1A, FigS3A). This effect was not associated to changes in SOD1 steady-state levels (FigS3B). By relieving the translational inhibition imposed by eIF2a phosphorylation, ISRIB prevents the formation of stress granules (SGs), dynamic assemblies of untranslated mRNPs formed upon abrupt translational shutdown [4]. Since SGs may contribute to the aggregation of ALS neurotoxic proteins such as FUS and TDP43 [6], we asked whether SOD1 foci were related to SGs. Mutant SOD1 failed to promote the clustering of the SG marker G3BP2 in HEK293 cells. Moreover, when formation of SGs was pharmacologically induced by arsenite treatment (ARS), SG foci did not colocalize with SOD1 clusters, confirming that SOD1 foci are not SG-related (FigS3C-D). We concluded that basal or stress induced ISR is necessary for SOD1 clustering. To identify the ISR kinase/s required for mutant SOD1 clustering, we generated CRISPR-Cas9 gene editing constructs that prevent the expression of human ISR kinases (FigS4C), and analyzed the clustering of WT and mutant SOD1 whenever either of these kinases was knocked down. This approach provides the effective shutdown of each of the ISR kinases and enables to address the role of ISR kinases in neuronal survival (see below). Surprisingly, the clustering ability of WT and mutant SOD1 was affected by distinct ISR kinases. In the case of WT SOD1, gene editing of PERK increased the fraction of SOD1-expressing cells containing foci. On the other hand, mutant SOD1 clustering ability was strongly reduced by GCN2 gene editing (Fig1B). We confirmed that these effects were indeed due to the loss of their kinase activity by using two well-established pharmacological inhibitors of GCN2 or PERK kinase activity: PERKib (GSK2606414), and GCN2ib (GCN2iB). As anticipated, PERKib was able to prevent PERK autophosphorylation/activation and ATF4 translation in cells treated with the ER stress pharmacological inducer thapsigargin (Thap) (Fig1B). Similarly, upon treatment with the histidine analog histidinol (HisOH), GCN2ib prevented the autophosphorylation of GCN2 and the ensuing translation of ATF4 protein. These effects were specific since neither of the inhibitors prevented the activation of the non-cognate ISR kinase (FigS4B). In line with the CRISPR/Cas9 observations, PERKib enhanced the appearance of WT SOD1 foci and displayed a tendency to increase the number of cells with SOD1G93A foci (Fig1C), while GCN2ib strongly reduced mutant SOD1 clustering (Fig1D). Next, we tested if PERK and GCN2 inhibition would affect SOD1 distribution in primary neurons. In neurons overexpressing WT SOD1, the protein is evenly distributed through soma and processes; quite differently, mutant SOD1 protein is discontinuously distributed through soma, dendrites, and axons. Treatment of WT SOD1-expressing neurons with PERKib subverted the protein distribution into a discontinuous pattern (FigS5), while blocking the ISR with ISRIB or GCN2ib restored the homogeneous distribution of mutant SOD1 (Fig1E). These results put forward an unanticipated role of GCN2 in mutant SOD1 behaviour. We determined the effect of GCN2 in mutant SOD1-induced neurodegeneration by using longitudinal survival analysis (Fig1F) [5]. We generated and validated constructs expressing the endonuclease Cas9 with three different guide RNAs (gRNAs) targeting the coding sequence of rat GCN2 (FigS4E). Then, we co-transfected into primary neuronal cultures these constructs (or a control plasmid expressing a non-targeting gRNA) with plasmids expressing a recombinant version of SOD1G93A bearing a C-terminal mCherry tag (G93ACh) [5]. To assess the effect of GCN2 gene editing in neuronal survival in a non-ALS context, CRISPR-Cas9 constructs were co-transfected with a plasmid expressing mCherry (Ch). As shown in Fig1G, GCN2 knock-down with two different gRNAs significantly enhanced the survival of neurons overexpressing G93ACh, while it did not affect the survival of Ch-expressing neurons (FigS6A). In line with these results, pharmacological inhibition of GCN2 also reduced the risk of death of G93ACh-expressing neurons (Fig1H). Therefore, GCN2 plays an important role in mutant SOD1 neuronal distribution and toxicity. Finally, we evaluated the therapeutic potential of GCN2 inhibition by treating the SOD1 G93A (G93A) transgenic ALS mouse model with a pharmacological GCN2 inhibitor. The small molecule GCN2ib was intraperitoneally delivered to WT and G93A mice at 10mg/kg (twice a day) from 6 to 16 weeks of age. To track the denervation process, we made electromyographic (EMG) recordings, where the detection of spontaneous activation potentials (SAPs) serves as a non-invasive readout of muscle denervation. EMG analysis serves to document the higher vulnerability of fast-fatigable MNs in the tibialis anterior (TA, a muscle mainly innervated by fast-fatigable MNs), when compared to slow-resistant MNs (more represented in the soleus, SOL). Starting at 10 weeks of age, a significant number of SAPs was detected in G93A TA muscles compared to WT mice. GCN2ib treatment of G93A mice delayed the appearance of SAP in TA muscles, only becoming significant at 14 weeks. A similar trend was observed in SOL recordings from G93A mice. No effects in EMG recordings whatsoever were found in GCN2ib-treated WT mice (Fig1I-J) (Supplementary Material). Accordingly, GCN2ib treatment delayed the clinical score and the motor phenotype of G93A mice until 14 weeks old (Fig1K-L). Moreover, GCN2ib treatment increased the weight gain of both treated WT and G93A mice (Fig1M). To directly test if GCN2 enhanced survival of spinal cord MNs, independent WT and G93A mice cohorts were treated and sacrificed at 12 weeks of age (FigS7) and surviving anti-choline acetyltransferase (ChAT+) neurons (MNs) were counted after immunohistochemistry. Importantly, in GCN2ib-treated ALS mice, the number of MNs tended to be higher than in vehicle-treated G93A mice (Figure S7C). Altogether, these results indicate that GCN2 pharmacological inhibition delays disease progression in ALS mice. Our findings support the notion that stress-induced ISR contributes to the neurotoxicity of fALS neurotoxic proteins. Indeed, ISR activation has been proposed to facilitate ALS neurotoxicity in other fALS experimental models: In the case of the pathological (GGGGCC) expansions in C9ORF72 intron, RAN translation of dipeptide repeats (DpR) is enhanced by ISR activation [7]; in turn, DpR peptides enhance ISR activation [8]. In the case of TDP43 or FUS fALS, ISR-induced assembly of SGs may act as “seeds” for protein aggregation, promoting cytosolic toxicity and/or nuclear loss-of-function neurotoxicity [6]. Together with these works, our study demonstrates that ISR inhibition can change the behavior/toxicity of fALS neurotoxic proteins, and thereby dampen the initial trigger of motoneuron death. Remarkably, PERK inhibition promotes the clustering of WT SOD1 but does not affect mutant SOD1 aggregation. Tampering with basal or ALS-induced PERK activity may alter ER homeostasis, affecting the redox regulation of WT SOD1 and promoting its aggregation as described previously [9]. That said, the fact that PERK inhibition [5] cannot improve the survival of mutant SOD1-expressing neurons suggests that PERK is not the main/sole driver of ISR in ALS [9]. In this regard, our results agree with a recent work where PERK inhibition did not improve disease progression in transgenic mutant SOD1 mice [10]. GCN2 can be activated by either uncharged tRNAs or by ribosome stalling/collision [11]. The low amino acid levels documented in the serum and cerebrospinal fluid of ALS patients may be a plausible GCN2 trigger in ALS. A recent report describes in a C9ORF72 model that glucose hypometabolism induces GCN2 activation, and plays a neurotoxic role [12]. GCN2 was also identified as a modulator of DpR toxicity [8]. Based on these evidences we postulate that GCN2 inhibition could serve to treat (at least) C9orf72 and SOD1 fALS patients, that account for 50% of all fALS cases. The discovery of GCN2 as a key determinant of mutant SOD1 behavior and neurotoxicity opens up a new perspective, where understanding how ISR determines proteostasis, and exploring the neuroprotective effect of GCN2 in other ALS models will serve to establish its therapeutic potential. Abbreviations FUS: Fused in sarcoma GCN2: general control nonderepressible 2 HRI: heme-regulated inhibitor iPSC: Induced pluripotent stem cells PERK: Protein kinase RNA- like endoplasmic reticulum kinase PKR: protein kinase R RAN: Repeat Associated Non-AUG SOD1: superoxide dismutase type 1 TDP43: TAR DNA-binding protein 43 Declarations Funding This work was supported by PID2020-120497RB-I00 MCIU/AEI/10.13039/501100011033, BFU2017-90043-P MCINN/AEI/10.13039/501100011033/ and by FEDER “Una manera de hacer Europa” (MA and TA), Proyecto Intramural IdisNa 2022 (MA) and Fundación para la Investigación Médica Aplicada (FIMA) Proyectos I+D, 2017 (TA). DO was supported by República de Panamá, Programa de Becas IFARHU-SENACYT (reference number 270-2018-922), and NP by AC FIMA pre-doctoral fellowship. Author´s contributions Conception and design: MA, TA. Acquisition of data: DO, NP, EMO, RVG, RF, MJN. Analysis of data: DO, NP, MV, EMO, RVG, RF, MA, TA. Interpretation of data: DO, NP, MV, EMO, RVG, RF, MA, TA. Drafting of the manuscript: DO, NP, MA, TA. Substantial revision of the manuscript: DO, NP, MV, MA, TA. Critical revision of the data: GGA. Each author has approved the submitted version of this manuscript as well as subsequent modifications. Acknowledgments We would like to acknowledge Dr. Julio Artieda (Neurophysiology Unit, Clinica Universidad de Navarra) and Dr. Ivone Jericó (Motoneuron Disease Unit, Hospital Universitario de Navarra) for their continuous support and advice in this project. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Animal handling was carried out in accordance with the European Community Council Directive 2010/63/EU and Spanish legislation (Real Decreto 53/2013). All protocols were approved by the Ethics Committee of the University of Navarra (CEEA): rat primary neuronal cultures (CEEA 038-18, CEEA 018-23), breeding SOD1 G93A transgenic mouse colony (CEEA104c-17, CEEA 111c-22) and procedures in SOD1 G93A transgenic mouse (CEEA 005-18, CEEA E49-19(005-18E1), CEEA E30-23 (019-23E1)). Consent for publication Not applicable Competing interests The authors declare that they have no competing interests References Costa-Mattioli M, Walter P. The integrated stress response: From mechanism to disease. Science. 2020;368. Saxena S, et al. A role for motoneuron subtype-selective ER stress in disease manifestations of FALS mice. Nat Neurosci. 2009;12:627–36. Marlin E et al. The Role and Therapeutic Potential of the Integrated Stress Response in Amyotrophic Lateral Sclerosis. Int J Mol Sci. 2022;23. Sidrauski C et al. The small molecule ISRIB reverses the effects of eIF2alpha phosphorylation on translation and stress granule assembly. Elife 2015;4. Bugallo R, et al. Fine tuning of the unfolded protein response by ISRIB improves neuronal survival in a model of amyotrophic lateral sclerosis. Cell Death Dis. 2020;11:397. Song J. Molecular mechanisms of phase separation and amyloidosis of ALS/FTD-linked FUS and TDP-43. Aging Dis. 2023;0. Goodman LD, Bonini NM. Repeat-associated non-AUG (RAN) translation mechanisms are running into focus for GGGGCC-repeat associated ALS/FTD. Prog Neurobiol. 2019;183:101697. Kramer NJ, et al. CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity. Nat Genet. 2018;50:603–12. Medinas DB, et al. Endoplasmic reticulum stress leads to accumulation of wild-type SOD1 aggregates associated with sporadic amyotrophic lateral sclerosis. PNAS. 2018;115:8209–14. Dzhashiashvili Y, et al. The UPR-PERK pathway is not a promising therapeutic target for mutant SOD1-induced ALS. Neurobiol Dis. 2019;127:527–44. Wu CCC, et al. Ribosome Collisions Trigger General Stress Responses to Regulate Cell Fate. Cell. 2020;182:404–e41614. Nelson AT, et al. Glucose hypometabolism prompts RAN translation and exacerbates C9orf72-related ALS/FTD phenotypes. EMBO Rep. 2024;25:2479–510. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4544133","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":314233067,"identity":"4fea51ff-fbe0-48a8-a477-7c2571873ec1","order_by":0,"name":"Didio Alberto Ortiz","email":"","orcid":"","institution":"Center for Applied Medical Research, University of Navarra","correspondingAuthor":false,"prefix":"","firstName":"Didio","middleName":"Alberto","lastName":"Ortiz","suffix":""},{"id":314233068,"identity":"0de39001-bf1f-4ea8-a7b6-bd2463f7c756","order_by":1,"name":"Nuria Peregrín","email":"","orcid":"","institution":"Center for Applied Medical Research, University of Navarra","correspondingAuthor":false,"prefix":"","firstName":"Nuria","middleName":"","lastName":"Peregrín","suffix":""},{"id":314233069,"identity":"65c68ecb-f57d-420f-becc-8ef86bf2edae","order_by":2,"name":"Miguel Valencia","email":"","orcid":"","institution":"Center For Applied Medical Research, University of Navarra","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"","lastName":"Valencia","suffix":""},{"id":314233070,"identity":"d6c8410d-3fcd-458a-ad23-abd8154d7a3e","order_by":3,"name":"Rodrigo Vinueza-Gavilanes","email":"","orcid":"","institution":"Center For Applied Medical Research, University of Navarra","correspondingAuthor":false,"prefix":"","firstName":"Rodrigo","middleName":"","lastName":"Vinueza-Gavilanes","suffix":""},{"id":314233071,"identity":"3be2bca9-6840-4212-b8ec-172bafe8a896","order_by":4,"name":"Elisa Marín-Ordovas","email":"","orcid":"","institution":"Vall d'Hebron Research Institute: Vall d'Hebron Institut de Recerca","correspondingAuthor":false,"prefix":"","firstName":"Elisa","middleName":"","lastName":"Marín-Ordovas","suffix":""},{"id":314233072,"identity":"1013b8ea-8ba4-479f-8da0-9afa8c88d130","order_by":5,"name":"Roberto Ferrero","email":"","orcid":"","institution":"Center for Applied Medical Research, University of Navarra","correspondingAuthor":false,"prefix":"","firstName":"Roberto","middleName":"","lastName":"Ferrero","suffix":""},{"id":314233073,"identity":"aeeafb7c-99e6-4367-ab00-d88555206123","order_by":6,"name":"María Jesús Nicolás","email":"","orcid":"","institution":"Center for Applied Medical Research, University of Navarra","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Jesús","lastName":"Nicolás","suffix":""},{"id":314233074,"identity":"24360f0d-40d4-43e3-85f0-7eba9d477a7d","order_by":7,"name":"Gloria González-Aseguinolaza","email":"","orcid":"","institution":"Center for Applied Medical Research, University of Navarra","correspondingAuthor":false,"prefix":"","firstName":"Gloria","middleName":"","lastName":"González-Aseguinolaza","suffix":""},{"id":314233075,"identity":"debd55f2-cba4-4641-8ad2-40f2a30bfe47","order_by":8,"name":"Montserrat Arrasate","email":"","orcid":"","institution":"Center for Applied Medical Research, University of Navarra","correspondingAuthor":false,"prefix":"","firstName":"Montserrat","middleName":"","lastName":"Arrasate","suffix":""},{"id":314233076,"identity":"d113cea6-e156-4399-ab3d-278a263619c3","order_by":9,"name":"Tomás Aragón","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYBADOeKV8kBpY9K1JDYQrcWe/ezDj18q7qRvuN38TIKhoo4IW3jSjaVlzjzL3XDnmJkEw5nDxDgsjUFasu1w7oYbCcYGjG0HiNDC/4z5N1BLusGN9M8GjP+IcZhEGpvkx7bDCQY3cgwfMDYwE6HlxjM2a6AXDGfeyCl8kHCMCL+w96cx3/xRcVie70b6hgMfaohwGAgwwyKHIYE4DQwMjD+IVTkKRsEoGAUjEwAA5C05RBKlgCIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-1700-2729","institution":"Center of Applied Medical Research, University of Navarra","correspondingAuthor":true,"prefix":"","firstName":"Tomás","middleName":"","lastName":"Aragón","suffix":""}],"badges":[],"createdAt":"2024-06-07 07:00:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4544133/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4544133/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59397775,"identity":"b0698927-ac78-436a-b449-00c8e53dd311","added_by":"auto","created_at":"2024-07-01 09:28:54","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1947460,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eHEK293 cells expressing mutant SOD1 (immunofluorescence anti-SOD1). Percentage of cells with foci. \u003cstrong\u003eb. \u003c/strong\u003eEffect of HRI, PKR, PERK and GCN2 knock-down; SOD1 foci quantification in co-transfected HEK293 cells with SOD1 and CRISPR/Cas9 plasmids. \u003cstrong\u003ec-d. \u003c/strong\u003eEffect of PERK and GCN2 pharmacological inhibition (PERKib, GCN2ib) in SOD1 foci formation in transfected HEK293. \u003cstrong\u003ea-d.\u003c/strong\u003e FociCount analysis (FigS2) , n=3, ≥100 cells/condition/experiment, One-way ANOVA and Sidak´s post-hoc test. \u003cstrong\u003ee. \u003c/strong\u003ePrimary neurons expressing mutant SOD1 +/- ISRIB and GCN2ib (anti-SOD1 and anti-MAP2). Clustered distribution pattern (CP) quantification in neurons. One-way ANOVA and Sidak´s post hoc test. n=3, \u0026gt;25 neurons/condition. \u003cstrong\u003ef.\u003c/strong\u003e Longitudinal tracking of neurons expressing Ch and Ch-tagged mutant SOD1 (G93ACh). Green arrows point to neurons tracked until the end of the experiment. Red arrows indicate neurons that died before. \u003cstrong\u003eg.\u003c/strong\u003e Cumulative death hazard of neurons co-expressing G93ACh or Ch and gRNAs containing for GCN2 targeting (GCN2g1, GCN2g2) or px458 (empty vector (ctrl)). \u003cstrong\u003eh.\u003c/strong\u003e Cumulative death hazard of neurons expressing G93ACh +/- GCN2 pharmacological inhibitor (A92). \u003cstrong\u003eg-h. \u003c/strong\u003eCox Proportional Hazard analysis; pooled data from 3-5 experiments (Tables S1, S3). n; number of neurons). \u003cstrong\u003ei. \u003c/strong\u003eEMGs activations from Tibialis Anterior (TA) from WT and G93A mice +/- GCN2ib (10 and 12 weeks). \u003cstrong\u003ej. \u003c/strong\u003eLongitudinal EMG analysis in TA and Soleus (SOL) in WT and G93A mice +/-GCN2ib. Number (#) activations/second (s). Red/green boxes highlight differences in GCN2ib-treated G93A mice. (Two-way Repeated Measures (RM) ANOVA, statistical analysis in Supplementary Information). Median and 25\u003csup\u003eth\u003c/sup\u003e-75\u003csup\u003eth\u003c/sup\u003e percentile (“+”outlier). \u003cstrong\u003ek-m. \u003c/strong\u003eGCN2 inhibition effect in G93A mice clinical score, motor phenotype and weight gain.\u003cstrong\u003e \u003c/strong\u003eWT n= 4, WT+GCN2ib=6, G93A n=6, G93A+GCN2ib n=6. Two-way RM ANOVA and Tukey´s post-hoc test. Statistical analysis in Supplementary Information. \u003cstrong\u003el-m\u003c/strong\u003e. Differences apply to 16 weeks in comparison to G93A+veh. SD: standard deviation; SEM; standard error of the mean. ns, not statistically different; *p\u0026lt; 0.05, **p\u0026lt; 0.01, **p\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1LetterTNEU.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4544133/v1/eab575c112f8965b3db9831d.jpg"},{"id":59459504,"identity":"df462404-e1d0-484e-a195-f50574b35de4","added_by":"auto","created_at":"2024-07-02 04:46:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2235721,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4544133/v1/5b9286a8-a716-41c2-9de0-b10a42822a07.pdf"},{"id":59397774,"identity":"b9b2c15f-253e-48b3-874a-ebc6d29c6d95","added_by":"auto","created_at":"2024-07-01 09:28:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1774590,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialTNEULetter040624FINAL.docx","url":"https://assets-eu.researchsquare.com/files/rs-4544133/v1/5ab30ce7e494139d443e7b2d.docx"}],"financialInterests":"","formattedTitle":"GCN2 inhibition reduces mutant SOD1 clustering and toxicity and delays disease progression in an Amyotrophic Lateral Sclerosis mouse model","fulltext":[{"header":"Full Text","content":"\u003cp\u003eThe disruption of protein folding homeostasis in motoneurons (MNs) (and the ensuing accumulation of protein aggregates) is one of the main molecular hallmarks of amyotrophic lateral sclerosis (ALS). Both in sporadic ALS patients, in patients with inheritable forms of the disease (familial ALS, or fALS), or in neuronal and animal transgenic disease models, protein aggregation correlates and \u0026ndash; in many experimental models \u0026ndash; directly participates in neurodegeneration. In turn, the loss of motoneuron homeostasis triggers a coping mechanism, the integrated stress response (ISR) [1]. The ISR can be initiated by four independent stress-sensing kinases (PKR, PERK, GCN2, and HRI), each of which is activated by distinct stresses: GCN2 by aminoacid and glucose starvation or bribosome collision, HRI by heme deprivation, mitochondrial stress and proteasome deficiency, PERK by protein misfolding at the endoplasmic reticulum (ER) and PKR by double-strand RNA. Once activated, either of them phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2a), leading to 1) the general reduction in translation of most mRNAs and 2) the enhanced translation of transcripts encoding stress response factors, such as the activating transcription factor 4 (ATF4). While these changes in gene expression are in principle neuroprotective, under chronic stress the ISR can also drive apoptosis\u0026nbsp;[1]. In most ALS studies, the ISR kinase PERK was proposed to be the ISR trigger. Importantly, in ALS animal models ISR activation is most prominent in fast-fatigable MNs, the most vulnerable MN subpopulation\u0026nbsp;[2]. Driven by this compelling evidence, many groups have developed pharmacological ISR activators or suppressors to treat ALS, some of which have reached clinical trials\u0026nbsp;[3]; yet, it is still unclear what stress/es drives ISR activation in ALS and which is the most robust therapeutic strategy.\u003c/p\u003e\n\u003cp\u003eMutations in the SOD1 gene cause fALS and SOD1 protein aggregation in patient postmortem samples and transgenic mouse models. \u0026nbsp;We previously developed a neuronal ALS model based on the expression of the mutant allele SOD1 G93A, where we can quantitatively score the risk of neuronal death. Importantly, this experimental setting recapitulates both ISR activation and neuronal death. In this model, the ISR downstream inhibitor (ISRIB,\u0026nbsp;[4]) tuned neuronal ISR and enhanced neuronal survival\u0026nbsp;[5]. Intriguingly, ISRIB also mitigated a distinct proteostatic mechanism, the unfolded protein (UPR), suggesting that ISR dampening could somehow reduce neurotoxicity\u0026nbsp;[5]. The neuroprotective effect of ISR inhibition could not be recapitulated by the inhibition of PERK, suggesting that ISR stress sensing kinase/s other than PERK trigger ISR activation in ALS.\u003c/p\u003e\n\u003cp\u003eTo investigate if ISR modulation affects the localization and neurotoxicity of mutant SOD1 protein we explored the effect of ISRIB in mutant SOD1 clustering when overexpressed in HEK293 cells.\u0026nbsp;Consistent with previous studies, SOD1 overexpression led to the formation of protein clusters in approximately 50% of cells (FigS1B-C). In this setting, ISRIB prevented mutant SOD1 clustering (Fig1A, FigS3A). This effect was not associated to changes in SOD1 steady-state levels (FigS3B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBy relieving the translational inhibition imposed by eIF2a phosphorylation, ISRIB prevents the formation of stress granules (SGs), dynamic assemblies of untranslated mRNPs formed upon abrupt translational shutdown\u0026nbsp;[4]. Since SGs may contribute to the aggregation of ALS neurotoxic proteins such as FUS and TDP43\u0026nbsp;[6],\u0026nbsp;we asked whether SOD1 foci were related to SGs. Mutant\u0026nbsp;SOD1 failed to promote the clustering of the SG marker G3BP2 in HEK293 cells. Moreover, when formation of SGs was pharmacologically induced by arsenite treatment (ARS), SG foci did not colocalize with SOD1 clusters, confirming that SOD1 foci are not SG-related (FigS3C-D). We concluded that basal or stress induced ISR is necessary for SOD1 clustering.\u003c/p\u003e\n\u003cp\u003eTo identify the ISR kinase/s required for mutant SOD1 clustering, we generated CRISPR-Cas9 gene editing constructs that prevent the expression of human ISR kinases (FigS4C), and analyzed the clustering of WT and mutant SOD1 whenever either of these kinases was knocked down. This approach provides the effective shutdown of each of the ISR kinases and enables to address the role of ISR kinases in neuronal survival (see below). Surprisingly, the clustering ability of WT and mutant SOD1 was affected by distinct ISR kinases. In the case of WT SOD1, gene editing of PERK increased the fraction of SOD1-expressing cells containing foci. On the other hand, mutant SOD1 clustering ability was strongly reduced by GCN2 gene editing (Fig1B). We confirmed that these effects were indeed due to the loss of their kinase activity by using two well-established pharmacological inhibitors of GCN2 or PERK kinase activity: PERKib (GSK2606414), and GCN2ib (GCN2iB). As anticipated, PERKib was able to prevent PERK autophosphorylation/activation and ATF4 translation in cells treated with the ER stress pharmacological inducer thapsigargin (Thap) (Fig1B). Similarly, upon treatment with the histidine analog histidinol (HisOH), GCN2ib prevented the autophosphorylation of GCN2 and the ensuing translation of ATF4 protein. These effects were specific since neither of the inhibitors prevented the activation of the non-cognate ISR kinase (FigS4B). In line with the CRISPR/Cas9 observations, PERKib enhanced the appearance of WT SOD1 foci and displayed a tendency to increase the number of cells with SOD1G93A foci (Fig1C), while GCN2ib strongly reduced mutant SOD1 clustering (Fig1D). Next, we tested if PERK and GCN2 inhibition would affect SOD1 distribution in primary neurons. In neurons overexpressing WT SOD1, the protein is evenly distributed through soma and processes; quite differently, mutant SOD1 protein is discontinuously distributed through soma, dendrites, and axons. Treatment of WT SOD1-expressing neurons with PERKib subverted the protein distribution into a discontinuous pattern (FigS5), while blocking the ISR with ISRIB or GCN2ib restored the homogeneous distribution of mutant SOD1 (Fig1E). These results put forward an unanticipated role of GCN2 in mutant SOD1 behaviour.\u003c/p\u003e\n\u003cp\u003eWe determined the effect of GCN2 in mutant SOD1-induced neurodegeneration by using longitudinal survival analysis (Fig1F)\u0026nbsp;[5]. We generated and validated constructs expressing the endonuclease Cas9 with three different guide RNAs (gRNAs) targeting the coding sequence of rat GCN2 (FigS4E). Then, we co-transfected into primary neuronal cultures these constructs (or a control plasmid expressing a non-targeting gRNA) with plasmids expressing a recombinant version of SOD1G93A bearing a C-terminal mCherry tag (G93ACh)\u0026nbsp;[5]. To assess the effect of GCN2 gene editing in neuronal survival in a non-ALS context, CRISPR-Cas9 constructs were co-transfected with a plasmid expressing mCherry (Ch). As shown in Fig1G, GCN2 knock-down with two different gRNAs significantly enhanced the survival of neurons overexpressing G93ACh, while it did not affect the survival of Ch-expressing neurons (FigS6A). In line with these results, pharmacological inhibition of GCN2 also reduced the risk of death of G93ACh-expressing neurons (Fig1H). Therefore, GCN2 plays an important role in mutant SOD1 neuronal distribution and toxicity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, we evaluated the therapeutic potential of GCN2 inhibition by treating the SOD1\u003csup\u003eG93A\u003c/sup\u003e (G93A) transgenic ALS mouse model with a pharmacological GCN2 inhibitor. The small molecule GCN2ib was intraperitoneally delivered to WT and G93A mice at 10mg/kg (twice a day) from 6 to 16 weeks of age. To track the denervation process, we made electromyographic (EMG) recordings, where the detection of spontaneous activation potentials (SAPs) serves as a non-invasive readout of muscle denervation. EMG analysis serves to document the higher vulnerability of fast-fatigable MNs in the tibialis anterior (TA, a muscle mainly innervated by fast-fatigable MNs), when compared to slow-resistant MNs (more represented in the soleus, SOL). Starting at 10 weeks of age, a significant number of SAPs was detected in G93A TA muscles compared to WT mice. GCN2ib treatment of G93A mice delayed the appearance of SAP in TA muscles, only becoming significant at 14 weeks. A similar trend was observed in SOL recordings from G93A mice. No effects in EMG recordings whatsoever were found in GCN2ib-treated WT mice (Fig1I-J) (Supplementary Material). Accordingly, GCN2ib treatment delayed the clinical score and the motor phenotype of G93A mice until 14 weeks old (Fig1K-L). Moreover, GCN2ib treatment increased the weight gain of both treated WT and G93A mice (Fig1M). To directly test if GCN2 enhanced survival of spinal cord MNs, independent WT and G93A mice cohorts were treated and sacrificed at 12 weeks of age (FigS7) and surviving anti-choline acetyltransferase (ChAT+) neurons (MNs) were counted after immunohistochemistry. Importantly, in GCN2ib-treated ALS mice, the number of MNs tended to be higher than in vehicle-treated G93A mice (Figure S7C). Altogether, these results indicate that GCN2 pharmacological inhibition delays disease progression in ALS mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur findings support the notion that stress-induced ISR contributes to the neurotoxicity of fALS neurotoxic proteins. Indeed, ISR activation has been proposed to facilitate ALS neurotoxicity in other fALS experimental models: In the case of the pathological (GGGGCC) expansions in C9ORF72 intron, RAN translation of dipeptide repeats (DpR) is enhanced by ISR activation\u0026nbsp;[7]; in turn, DpR peptides enhance ISR activation\u0026nbsp;[8]. In the case of TDP43 or FUS fALS, ISR-induced assembly of SGs may act as \u0026ldquo;seeds\u0026rdquo; for protein aggregation, promoting cytosolic toxicity and/or nuclear loss-of-function neurotoxicity\u0026nbsp;[6].\u0026nbsp;Together with these works, our study demonstrates that ISR inhibition can change the behavior/toxicity of fALS neurotoxic proteins, and thereby dampen the initial trigger of motoneuron death.\u003c/p\u003e\n\u003cp\u003eRemarkably, PERK inhibition promotes the clustering of WT SOD1 but does not affect mutant SOD1 aggregation. Tampering with basal or ALS-induced PERK activity may alter ER homeostasis, affecting the redox regulation of WT SOD1 and promoting its aggregation as described previously\u0026nbsp;[9]. That said, the fact that PERK inhibition\u0026nbsp;[5]\u0026nbsp;cannot improve the survival of mutant SOD1-expressing neurons suggests that PERK is not the main/sole driver of ISR in ALS\u0026nbsp;[9]. In this regard, our results agree with a recent work where PERK inhibition did not improve disease progression in transgenic mutant SOD1 mice\u0026nbsp;[10].\u003c/p\u003e\n\u003cp\u003eGCN2 can be activated by either uncharged tRNAs or by ribosome stalling/collision \u0026nbsp;[11]. \u0026nbsp;The low amino acid levels documented in the serum and cerebrospinal fluid of ALS patients may be a plausible GCN2 trigger in ALS. A recent report describes in a C9ORF72 model that glucose hypometabolism induces GCN2 activation, and plays a neurotoxic role\u0026nbsp;[12]. GCN2 was also identified as a modulator of DpR toxicity\u0026nbsp;[8]. Based on these evidences we postulate that GCN2 inhibition could serve to treat (at least) C9orf72 and SOD1 fALS patients, that account for 50% of all fALS cases.\u003c/p\u003e\n\u003cp\u003eThe discovery of GCN2 as a key determinant of mutant SOD1 behavior and neurotoxicity opens up a new perspective, where understanding how ISR determines proteostasis, and exploring the neuroprotective effect of GCN2 in other ALS models will serve to establish its therapeutic potential.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eFUS: Fused in sarcoma\u003c/p\u003e\n\u003cp\u003eGCN2: general control nonderepressible 2\u003c/p\u003e\n\u003cp\u003eHRI: heme-regulated inhibitor\u003c/p\u003e\n\u003cp\u003eiPSC: Induced pluripotent stem cells\u003c/p\u003e\n\u003cp\u003ePERK: Protein kinase RNA- like endoplasmic reticulum kinase\u003c/p\u003e\n\u003cp\u003ePKR: protein kinase R\u003c/p\u003e\n\u003cp\u003eRAN: Repeat Associated Non-AUG\u003c/p\u003e\n\u003cp\u003eSOD1: superoxide dismutase type 1\u003c/p\u003e\n\u003cp\u003eTDP43: TAR DNA-binding protein 43\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThis work was supported by PID2020-120497RB-I00 MCIU/AEI/10.13039/501100011033, BFU2017-90043-P MCINN/AEI/10.13039/501100011033/ and by FEDER \u0026ldquo;Una manera de hacer Europa\u0026rdquo; (MA and TA), Proyecto Intramural IdisNa 2022 (MA) and Fundaci\u0026oacute;n para la Investigaci\u0026oacute;n M\u0026eacute;dica Aplicada (FIMA) Proyectos I+D, 2017 (TA).\u0026nbsp;DO was supported by Rep\u0026uacute;blica de Panam\u0026aacute;, Programa de Becas IFARHU-SENACYT (reference number 270-2018-922), and NP by AC FIMA pre-doctoral fellowship.\u003c/p\u003e\n\u003ch3\u003eAuthor\u0026acute;s contributions\u003c/h3\u003e\n\u003cp\u003eConception and design: MA, TA. Acquisition of data: DO, NP, EMO, RVG, RF, MJN. Analysis of data: DO, NP, MV, EMO, RVG, RF, MA, TA. Interpretation of data: DO, NP, MV, EMO, RVG, RF, MA, TA. Drafting of the manuscript: DO, NP, MA, TA. Substantial revision of the manuscript: DO, NP, MV, MA, TA. Critical revision of the data: GGA. Each author has approved the submitted version of this manuscript as well as subsequent modifications.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eAcknowledgments\u003c/h3\u003e\n\u003cp\u003eWe would like to acknowledge Dr. Julio Artieda (Neurophysiology Unit, Clinica Universidad de Navarra) and Dr. Ivone Jeric\u0026oacute; (Motoneuron Disease Unit, Hospital Universitario de Navarra) \u0026nbsp;for their continuous support and advice in this project.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eAvailability of data and materials\u003c/h3\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch3\u003eEthics approval and consent to participate\u003c/h3\u003e\n\u003cp\u003eAnimal handling was carried out in accordance with the European Community Council Directive 2010/63/EU and Spanish legislation (Real Decreto 53/2013). All protocols were approved by the Ethics Committee of the University of Navarra (CEEA): rat primary neuronal cultures (CEEA 038-18, CEEA 018-23), breeding SOD1\u003csup\u003eG93A\u003c/sup\u003e transgenic mouse colony (CEEA104c-17, CEEA 111c-22) and procedures in SOD1\u003csup\u003eG93A\u003c/sup\u003e transgenic mouse (CEEA 005-18, CEEA E49-19(005-18E1), CEEA E30-23 (019-23E1)).\u003c/p\u003e\n\u003ch3\u003eConsent for publication\u003c/h3\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch3\u003eCompeting interests\u003c/h3\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCosta-Mattioli M, Walter P. The integrated stress response: From mechanism to disease. Science. 2020;368.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaxena S, et al. A role for motoneuron subtype-selective ER stress in disease manifestations of FALS mice. Nat Neurosci. 2009;12:627\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarlin E et al. The Role and Therapeutic Potential of the Integrated Stress Response in Amyotrophic Lateral Sclerosis. Int J Mol Sci. 2022;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSidrauski C et al. The small molecule ISRIB reverses the effects of eIF2alpha phosphorylation on translation and stress granule assembly. Elife 2015;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBugallo R, et al. Fine tuning of the unfolded protein response by ISRIB improves neuronal survival in a model of amyotrophic lateral sclerosis. Cell Death Dis. 2020;11:397.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong J. Molecular mechanisms of phase separation and amyloidosis of ALS/FTD-linked FUS and TDP-43. Aging Dis. 2023;0.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoodman LD, Bonini NM. Repeat-associated non-AUG (RAN) translation mechanisms are running into focus for GGGGCC-repeat associated ALS/FTD. Prog Neurobiol. 2019;183:101697.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKramer NJ, et al. CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity. Nat Genet. 2018;50:603\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMedinas DB, et al. Endoplasmic reticulum stress leads to accumulation of wild-type SOD1 aggregates associated with sporadic amyotrophic lateral sclerosis. PNAS. 2018;115:8209\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDzhashiashvili Y, et al. The UPR-PERK pathway is not a promising therapeutic target for mutant SOD1-induced ALS. Neurobiol Dis. 2019;127:527\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu CCC, et al. Ribosome Collisions Trigger General Stress Responses to Regulate Cell Fate. Cell. 2020;182:404\u0026ndash;e41614.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNelson AT, et al. Glucose hypometabolism prompts RAN translation and exacerbates C9orf72-related ALS/FTD phenotypes. EMBO Rep. 2024;25:2479\u0026ndash;510.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Amyotrophic Lateral Sclerosis, Integrated Stress Response, GCN2, SOD1 clustering, neuronal models, SOD1G93A transgenic mouse, electromyographic analysis","lastPublishedDoi":"10.21203/rs.3.rs-4544133/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4544133/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The disruption of protein folding homeostasis in motoneurons (MNs), and the ensuing accumulation of protein aggregates, is one of the main molecular hallmarks of amyotrophic lateral sclerosis (ALS) pathology, and has been recapitulated in cellular and animal disease models. The loss of proteostasis and other stresses in the MN trigger the activation of a general stress mechanism, the integrated stress response (ISR). The ISR is initiated by either of four stress-sensing kinases (GCN2, HRI, PERK and PKR) which, upon activation by distinct insults, promote a dramatic remodeling of gene expression to combat stress and promote survival. Paradoxically, in pathologies where stress is chronic or overwhelming, the ISR can also promote neuronal death. In ALS experimental models, extensive evidence demonstrates a key role of this mechanism in the progression of disease, and has inspired many attempts to develop ALS therapies based on ISR modulation.\nIn our group, we recently discovered that the downstream ISR inhibitor ISRIB increases survival of a neuronal ALS model based on the expression of the neurotoxic ALS allele, SOD1 G93A. In the current study, we found that ISR inhibition is sufficient to prevent the concentration of mutant SOD1 into cytosolic foci, suggesting that ISR is required for SOD1 protein aggregation. Through a systematic CRISPR Cas9 approach and pharmacological inhibition, we demonstrate that, unexpectedly, the ISR kinase GCN2 is required for SOD1 clustering in cell lines and primary neuronal cultures. Moreover, genetic or pharmacological GCN2 inhibition strongly enhances survival of neurons overexpressing mutant SOD1. Finally, GCN2 pharmacological inhibition in fALS SOD1G93A transgenic mice delayed muscle denervation, strength loss, weight loss, and the appearance of ALS symptoms. Based on these findings, we propose GCN2 as a new potential therapeutic target for ALS.","manuscriptTitle":"GCN2 inhibition reduces mutant SOD1 clustering and toxicity and delays disease progression in an Amyotrophic Lateral Sclerosis mouse model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-01 09:28:49","doi":"10.21203/rs.3.rs-4544133/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"302a7c9b-7bbf-4b92-9af2-e20da353ca38","owner":[],"postedDate":"July 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-12T06:17:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-01 09:28:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4544133","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4544133","identity":"rs-4544133","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}