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Protein Disulfide Isomerase Erp57 Protects Early Muscle Denervation

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Protein Disulfide Isomerase Erp57 Protects Early Muscle Denervation Rozas et al. acta neuropathol commun (2021) 9:21 https://doi.org/10.1186/s40478-020-01116-z RESEARCH Open Access Protein disulfde isomerase ERp57 protects early muscle denervation in experimental ALS Pablo Rozas1,2,3, Cristina Pinto4, Francisca Martínez Traub1,2,3, Rodrigo Díaz1,2,3, Viviana Pérez4, Daniela Becerra1,2,3, Patricia Ojeda1,2,3, Jorge Ojeda4, Madison T. Wright5, Jessica Mella4, Lars Plate5, Juan Pablo Henríquez4, Claudio Hetz1,2,3,6* and Danilo B. Medinas1,2,3* Abstract Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease that afects motoneurons. Muta- tions in superoxide dismutase 1 (SOD1) have been described as a causative genetic factor for ALS. Mice overexpress- ing ALS-linked mutant SOD1 develop ALS symptoms accompanied by histopathological alterations and protein aggregation. The protein disulfde isomerase family member ERp57 is one of the main up-regulated proteins in tissue of ALS patients and mutant SOD1 mice, whereas point mutations in ERp57 were described as possible risk factors to develop the disease. ERp57 catalyzes disulfde bond formation and isomerization in the endoplasmic reticulum (ER), constituting a central component of protein quality control mechanisms. However, the actual contribution of ERp57 to ALS pathogenesis remained to be defned. Here, we studied the consequences of overexpressing ERp57 in experimental ALS using mutant SOD1 mice. Double transgenic SOD1 G93A/ERp57WT animals presented delayed deterioration of electrophysiological activity and maintained muscle innervation compared to single transgenic SOD1G93A littermates at early-symptomatic stage, along with improved motor performance without afecting survival. The overexpression of ERp57 reduced mutant SOD1 aggregation, but only at disease end-stage, dissociating its role as an anti-aggregation factor from the protection of neuromuscular junctions. Instead, proteomic analysis revealed that the neuroprotective efects of ERp57 overexpression correlated with increased levels of synaptic and actin cytoskel- eton proteins in the spinal cord. Taken together, our results suggest that ERp57 operates as a disease modifer at early stages by maintaining motoneuron connectivity. Keywords: Amyotrophic lateral sclerosis, Mutant SOD1, ERp57, Protein aggregation, Neuromuscular junction Introduction to the identifcation of causative mutations in several Amyotrophic lateral sclerosis (ALS) is a progressive and genes. Te most common genetic alterations in ALS are fatal late-onset neurodegenerative disease character- the GGG GCC (G4C2) hexanucleotide repeat expansion ized by loss of motoneurons leading to muscle weak- in C9ORF72, nonsynonymous mutations in the genes ness, paralysis and death [1]. Although most of ALS cases encoding superoxide dismutase 1 (SOD1), trans-active have no familial records being referred to as sporadic response (TAR) DNA-binding protein 43 (TDP-43), (sALS), around 10% are inherited and termed familial and fused in sarcoma (FUS) [2, 3]. Mice overexpress- ALS (fALS) [2]. Genetic studies of fALS cases have led ing distinct ALS-linked mutant SOD1 develop progres- sive motor impairment with diferent degrees of severity depending on the specifc mutation and transgene copy *Correspondence: [email protected]; [email protected]; dmedinas@med. G93A uchile.cl number [4]. Mutant SOD1 transgenic mouse line 1 Biomedical Neuroscience Institute, Faculty of Medicine, University is the most well-characterized preclinical ALS model of Chile, Independencia 1027, P.O. Box 70086, Santiago, Chile because it recapitulates key disease features such as pro- Full list of author information is available at the end of the article gressive decrease of motor performance, neuromuscular © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Rozas et al. acta neuropathol commun (2021) 9:21 Page 2 of 17 junction (NMJ) denervation, loss of spinal motoneurons ALS progression, preserving NMJ structure and delaying concomitantly with astrogliosis and microgliosis, orga- motor and electrophysiological impairment of afected nelle dysfunction and presence of intracellular mutant muscles. Contrary to expectations, this motor unit pro- SOD1 inclusions [5, 6]. In addition, misfolding and aggre- tection did not correlate with a reduction in SOD1 aggre- gation of wild-type SOD1 have been also reported in gation. We speculate that experimental strategies to sALS cases [7, 8]. improve ER folding may translate into important benef- Independent studies identifed two protein disulfde cial efects to ALS patients. isomerase family members (PDIs), PDI and ERp57 (also known as PDIA3 or GRP58), among the main proteins Materials and methods induced in spinal cord of ALS rodents at diferent disease Animals stages, suggesting that dysregulation of redox folding in SOD1G93A ALS mouse model carrying high copy num- the endoplasmic reticulum (ER) contributes to disease ber in C57BL/6 strain (B6.Cg-Tg (SOD1*G93A)1Gur/J) pathogenesis [9–11]. PDI and ERp57 were also found from Jackson Laboratory (strain number: 004435) was up-regulated in post-mortem spinal cord tissue of sALS employed. SOD1G93A transgene has human SOD1 pro- patients [10, 12]. In addition, increased levels of PDI moter and approximately 25 copies inserted in tan- were also detected in cerebrospinal fuid of sALS patients dem at mouse non-sexual chromosome 12 [4, 5, 23]. [10], while a proteomic screening in blood cells revealed Tis promoter assures ubiquitous expression of human ERp57 as the most reliable biomarker of sALS progres- SOD1G93A. Importantly, this transgenic line is used sion [13]. in a heterozygous fashion recapitulating ALS features We have previously reported missense mutations in described previously [6]. Symptomatic mice were pro- PDIA1 and PDIA3 genes, which encode for PDI and vided with pellet food on the foor of the cages in order to ERp57, respectively, as risk factors to develop ALS [14]. facilitate food intake. Tis was performed to reduce non- Moreover, intronic SNPs in PDIA1 gene have been asso- motor-related noise in disease progression parameters ciated to decreased survival of two diferent population such as body weight and clinical score. of ALS patients [15, 16]. We discovered that these PDIs Mice from C57BL/6 strain that overexpress human enhance neuritogenesis in motoneurons, a function form of wild-type ERp57 (termed ERp57WT) were gen- impaired by the ALS-linked mutations [17]. Further- erated in Centro de Estudios Científcos (CECs), Val- more, ERp57 defciency in the nervous system resulted divia, Chile and characterized previously [24, 25]. Tis in altered NMJs and impaired motor function in mice transgenic line employs the Prion protein promoter to [17]. In addition, PDI nitrosylation and co-localization express human ERp57 conjugated with a FLAG tag at the with protein inclusions in spinal motoneurons of sALS C-terminus. cases suggest compromised chaperone function possibly All mice were housed in cages supplied with water and due to protein oxidation and aggregation [10, 18]. Over- pellet food ad libitum in a light/dark cycle of 12 h/12 h at expression of PDI and ERp57 has been proposed to pro- 22 ± 2 °C. General guidelines from the National Institutes vide protection in cell culture models of ALS by reducing of Health guide for the care and use of laboratory ani- aggregation of SOD1 and TDP-43, an activity lost by the mals and from preclinical animal research in ALS/MND ALS-linked PDI mutants [11, 19]. Finally, PDIs have been were followed [26]. Te experimental procedures involv- linked to the progression of other neurodegenerative dis- ing these mouse lines were approved by the Institutional eases linked to protein misfolding including Alzheimer’s, Review Board for Animal Care of the Faculty of Medi- Huntington’s, Parkinson’s and Prion-related diseases cine of the University of Chile (approved protocol CBA (reviewed in [20]), highlighting the protective efects of #0821-FMUCH). To generate double transgenic animals, overexpressing ERp57 in cell culture and mice infected heterozygous female mice from ERp57WT colony were with prions [21, 22]. crossed with heterozygous male mice from SOD1G93A Despite the accumulating evidence linking PDIs to line (see Additional fle 1: Table S1). ALS pathogenesis, the actual contribution of these ER foldases to the disease process in vivo remains specu- Phenotypic characterization lative. To date, no pharmacological or genetic studies SOD1G93A ALS model is characterized by progressive loss have been
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