Neurobiology of Disease 137 (2020) 104793 Contents lists available at ScienceDirect Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi Gene therapy for overexpressing Neuregulin 1 type I in skeletal muscles T promotes functional improvement in the SOD1G93A ALS mice Guillem Mòdol-Caballeroa,b,1, Mireia Herrando-Grabulosaa,b,1, Belén García-Lareub,c, Neus Solanesa,b, Sergi Verdésc, Rosario Ostab,d, Isaac Francos-Quijornaa,b,2, Rubèn López-Valesa,b, ⁎ ⁎⁎ Ana Cristina Calvob,d, Assumpció Boschb,c,e, , Xavier Navarroa,b, a Department of Cell Biology, Physiology and Immunology, Institute of Neurosciences, Universitat Autonoma de Barcelona, Bellaterra, Spain b Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Bellaterra, Spain c Department of Biochemistry and Molecular Biology, Institute of Neurosciences, Universitat Autònoma de Barcelona, Bellaterra, Spain d Laboratory of Genetics and Biochemistry (LAGENBIO), Faculty of Veterinary-IIS, University of Zaragoza, Zaragoza, Spain e Vall d'Hebron Research Institute (VHIR), Barcelona, Spain A R T I C LE I N FO A B S T R A C T Keywords: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting motoneurons (MNs), with no ef- Neuregulin 1 fective treatment currently available. The molecular mechanisms that are involved in MN death are complex and ErbB receptors not fully understood, with partial contributions of surrounding glial cells and skeletal muscle to the disease. Motoneuron Neuregulin 1 (NRG1) is a trophic factor highly expressed in MNs and neuromuscular junctions. Recent studies Motor function have suggested a crucial role of the isoform I (NRG1-I) in the collateral reinnervation process in skeletal muscle, Amyotrophic lateral sclerosis Spinal cord and NRG1-III in the preservation of MNs in the spinal cord, opening a window for developing novel therapies for Neuromuscular junction neuromuscular diseases like ALS. In this study, we overexpressed NRG1-I widely in the skeletal muscles of the SOD1G93A transgenic mouse. The results show that NRG1 gene therapy activated the survival pathways in muscle and spinal cord, increasing the number of surviving MNs and neuromuscular junctions and reducing the as- troglial reactivity in the spinal cord of the treated SOD1G93A mice. Furthermore, NRG1-I overexpression pre- served motor function and delayed the onset of clinical disease. In summary, our data indicates that NRG1 plays an important role on MN survival and muscle innervation in ALS, and that viral-mediated overexpression of NRG1 isoforms may be considered as a promising approach for ALS treatment. 1. Introduction frequent. In both sALS and fALS the loss of MNs leads to rapid pro- gressive muscle atrophy and weakness, accompanied with fascicula- Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease tions and spasticity (Robberecht and Philips, 2013). The pathophysio- characterized by loss of motoneurons (MNs) of primary motor cortex, logical mechanisms underlying the development of ALS are brainstem and spinal cord (Wijesekera and Leigh, 2009). The vast multifactorial, with rising evidence of a complex interaction between majority of the cases are sporadic (sALS) in which the etiology is un- genetic and molecular pathways (Mancuso and Navarro, 2015). The known, and about 10% correspond to familiar forms (fALS), associated particular molecular mechanisms that specifically affect the MN to with inherited alterations in genes, such as superoxide dismutase 1 cause its death are still to be elucidated. Moreover, neighboring cells, (SOD1) (Rosen et al., 1993), TAR-DNA binding protein (TDP-43) such as microglia, astrocytes and interneurons, may contribute to the (Kabashi et al., 2008; Yokoseki et al., 2008), hexanucleotide repeat disease (Brites and Vaz, 2014). On the other hand, MN degeneration is expansions in chromosome 9 open reading frame 72 (C9orf72) (De preceded by alterations and disjunction of neuromuscular junctions Jesus-Hernandez et al., 2011; Renton et al., 2011), and others less (NMJs) (Fischer et al., 2004; Mancuso et al., 2011), and a toxic role for ⁎ Correspondence to: A. Bosch, Institute of Neurosciences, Edifici H, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain. ⁎⁎ Correspondence to: X. Navarro, Unitat de Fisiologia Mèdica, Facultat de Medicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain. E-mail addresses: assumpcio.bosch@uab.cat (A. Bosch), xavier.navarro@uab.cat (X. Navarro). 1 First Authors 2 Present address: Regeneration Group, The Wolfson Centre for Age-Related Diseases, Institute of Psychiatry, Psychology and Neuroscience (IoPPN), King's College London, London, UK https://doi.org/10.1016/j.nbd.2020.104793 Received 30 October 2019; Received in revised form 10 January 2020; Accepted 3 February 2020 Available online 04 February 2020 0969-9961/ © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). G. Mòdol-Caballero, et al. Neurobiology of Disease 137 (2020) 104793 skeletal muscle has also been reported (Moloney et al., 2014). 2. Materials and methods During the last decades several animal models carrying ALS-related mutations have been developed. The most widely used ALS model is a 2.1. Animals transgenic mouse expressing the human mutated form of the SOD1 gene with a glycine to alanine conversion at the 93rd amino acid (SOD1G93A) Transgenic mice carrying the mutation G93A in the SOD1 gene and (Gurney et al., 1994; Ripps et al., 1995), which outlines the most im- non-transgenic wild-type (WT) littermates as controls were used. portant clinical and pathological features of ALS in a rapid and severe SOD1G93A high copy mice (Tg[SOD1-G93A]1Gur) were obtained from progression of the disease (Ripps et al., 1995; Mancuso et al., 2011). the Jackson Laboratory (Bar Harbor, ME). These mice were bred and Alterations in SOD1 protein have been also found in sporadic ALS pa- maintained as hemizygotes by mating transgenic males with F1 hybrid tients (Bosco and Landers, 2010), and accumulation of wild-type SOD1 females obtained from Janvier Laboratories (France). Transgenic mice was shown to produce ALS in mice (Graffmo et al., 2013), thus, in- were identified by polymerase chain reaction amplification of DNA creasing the potential interest of the SOD1-linked ALS models. extracted from the tail. Mice were maintained in standard conditions Unfortunately, there is no cure for ALS, with only two drugs ap- with access to food and water ad libitum at the Animal Service of the proved for use in ALS patients, riluzole, that slightly prolongs survival Universitat Autònoma de Barcelona and were cared for and handled in for 3–4 months, and edaravone, a drug that presumably works to mi- accordance with the guidelines of the European Union Council tigate oxidative injury in central neurons. Symptomatic and palliative (Directive 2010/63/EU) and Spanish regulations on the use of labora- measures (including feeding and respiratory support) are the mainstay tory animals. The experimental procedures had been approved by the of patients' management (Pasinelli and Brown, 2006). Recently, gene Ethics Committee of the Universitat Autònoma de Barcelona. therapy for ALS has emerged as an alternative therapeutic strategy. The Sex-balanced therapeutic studies were performed using mice with main advantage of this approach is that, by one single administration, it B6xSJL background. The animals were divided in a group of WT mice allows continuous expression of the therapeutic gene in specific tissues and two groups of SOD1G93A mice, that were administered at 6 weeks of (Federici and Boulis, 2006). Among the different viral vectors, adeno- age with either AAV8 coding for NRG1-I or mock vector. For the associated vectors (AAV) have some advantages over other viral vec- functional studies we used the following number of mice per group: WT tors, such as the ability to provide sustained gene expression in term- mice (n = 6 females, n = 5 males), SOD Mock mice (n = 14 females, inally differentiated cells, avoiding the risk for insertional mutagenesis. n = 12 males), SOD NRG1-I mice (n = 12 females, n = 5 males). These Viral transgene expression can also be restricted depending on the viral animals were then further distributed in two different subgroups at tropism and using promoters with selective and defined expression 16 weeks. One subgroup to analyze mRNA expression: WT (n = 3 fe- patterns (Kügler, 2016). males), SOD Mock (n = 4 females), SOD NRG1-I (n = 4 females); the Neurotrophic factors derived from the spliced forms of Neuregulin 1 second subgroup for histological analysis: WT (n = 3 females), SOD (NRG1) have been shown to be critical for MN survival, supporting Mock (n = 4 females), SOD NRG1-I (n = 7 females). axonal and neuromuscular development and maintenance (Syroid et al., 1996; Sandrock Jr et al., 1997; Loeb et al., 1999; Wolpowitz et al., 2.2. Virus production and administration 2000; Nave and Salzer, 2006). NRG1 has been localized in spinal MNs, concentrated in the subsurface cisterns underlying postsynaptic sites of The cDNA of the extracellular domain of NRG1-I isoform (provided cholinergic C boutons (Issa et al., 2010; Gallart-Palau et al., 2014). by G. Corfas, Harvard Medical School, Boston, MA, USA) was cloned NRG1 acts through the EGF domain of ErbB receptors, and NRG1-ErbB between AAV2 ITRs under the regulation of the human desmin pro- system alterations have been related to ALS, since loss-of-function moter (provided by G. Lemke, Salk Institute, La Jolla, CA, USA). The mutations of ErbB4 produce late-onset, autosomal-dominant ALS in woodchuck hepatitis virus responsive element (WPRE) was added at 3′ human patients (Takahashi et al., 2013). We have recently reported to stabilize mRNA expression. AAV2/8 stocks were produced by the that ErbB4 ectodomain fragments were reduced in cerebrospinal fluid Viral Production Unit of UAB-VHIR (http://viralvector.eu). Briefly, and plasma of ALS patients, suggesting impaired NRG1-ErbB signaling vectors were generated using the triple transfection system in HEK293- (Lopez-Font et al., 2019). Interestingly, several studies support that AAV cells of the expression plasmids, Rep2Cap8 plasmid containing NRG1 type III expression is reduced in the spinal cord of both ALS AAV genes (provided by J.M. Wilson, University of Pennsylvania, patients and SOD1G93A mice, whereas NRG1 type I appears to be up- Philadelphia, USA) and pXX6 plasmid containing adenoviral genes, regulated (Song et al., 2012; Mòdol-Caballero et al., 2019). Further- needed as helper virus (Zolotukhin et al., 1999). After 48 h, AAV vec- more, viral-mediated delivery of NRG1-III in the spinal cord par- tors were harvested, treated with benzonase, purified in an iodixanol enchyma restored the number of C-boutons and produced an increase of gradient, and titred using the Picogreen (Invitrogen) system quantifi- survival of the SOD1G93A mouse (Lasiene et al., 2016). Also, intrathecal cation (Piedra et al., 2015) and calculated as viral genomes per milli- administration of an AAV coding for NRG1-III improves the progressive liter (vg/ml). AAV2/8 vectors containing the same regulatory se- decline in motor function and MN number and reduces glial reactivity quences without the therapeutic gene (empty or mock vector) were in SOD1G93A mice (Mòdol-Caballero et al., 2019). However, the exact used as control. A suspension of 7.6 × 1013 vg of AAV2/8 vectors in role of NRG1-I is not fully known. It has been reported that NRG1-I 250 μl was injected intravenously in 6 weeks- old mice via tail vein. expression by Schwann cells is essential for promoting axonal re- generation and remyelination (Stassart et al., 2013). Indeed, over- 2.3. Electrophysiological tests expression of NRG1-I by means of an AAV vector, locally injected in the gastrocnemius muscle produced functional improvement by enhancing Motor nerve conduction tests were performed by stimulation of the motor axons collateral sprouting in SOD1G93A mice (Mancuso et al., sciatic nerve with single pulses of 20 μs duration (Grass S88) delivered 2016). Therefore, NRG-I plays a crucial role in the collateral re- by two needle electrodes placed at the sciatic notch. The compound innervation process. Considering these recent findings, our aim here muscle action potential (CMAP) was recorded from tibialis anterior was to overexpress NRG1-I in all the skeletal muscles using gene (TA), gastrocnemius (GM) and plantar interossei (PL) muscles with therapy vectors to maintain motor innervation in the SOD1G93A mouse. microneedle electrodes (Mancuso et al., 2011) at 9, 12, 14 and 16 weeks For this reason, we administered intravenously AAV vectors under the of age. The recorded potentials were amplified and displayed on a di- regulation of the human desmin promoter, in order to restrict NRG1-I gital oscilloscope (Tektronix 450S), measuring the latency and ampli- expression in skeletal and cardiac muscles. tude of the CMAP. The mouse body temperature was maintained con- stant by means of a thermostated heating pad. Motor unit number estimation (MUNE) was performed using the 2 G. Mòdol-Caballero, et al. Neurobiology of Disease 137 (2020) 104793 incremental technique (Mancuso et al., 2011) with the same setting as 2.6. Western blot analysis for motor nerve conduction tests. The sciatic nerve was stimulated with pulses of gradual increasing intensity, starting from a subthreshold in- Fresh gastrocnemius muscle and lumbar spinal cord tissues were tensity. Then, quantal increases in the CMAP were recorded. The in- sonicated and homogenized in RIPA lysis buffer (50 mM Tris-Cl pH 7.4, crements higher than 50 μV were considered as due to the recruitment 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodiumdeoxycholate) of an additional motor unit. The mean amplitude of single motor units containing a mixture of protease inhibitors (Millipore). Protein con- was calculated as the average of consistent increases. The MUNE was centration was determined by PierceTM BCA Protein Assay Kit (Thermo calculated as the ratio between the CMAP maximal amplitude and the Scientific). Between 25 and 35 μg of protein were separated on 10% mean amplitude of single motor unit action potentials. SDS-polyacrylamide gel electrophoresis gel (VWR Life Science), trans- To evaluate the central descending pathways, motor evoked po- ferred to polyvinylidene difluoride membranes (GE Healthcare) and tentials (MEP) were recorded from TA and GM muscles following immunoblotted. The following antibodies were used: rabbit anti- electrical stimulation of the motor cortex with pulses of 0.1 ms duration phospho Akt (1:500, Ser473 #9271, Cell Signaling), total Akt (1:500; and supramaximal intensity, delivered with needle electrodes placed #9272 Cell Signaling), rabbit anti-phospho Erk1/2 (1:500, #9101 subcutaneously over the skull overlaying the sensorimotor cortex Thr202/Tyr204, Cell Signaling), total Erk (1:500; #9102, Cell (Mancuso et al., 2011). Signaling), rabbit anti-GAPDH (1:1000; #14C10, Cell Signaling), and mouse anti-gamma tubulin T6557 (1:1000; Sigma-Aldrich). Detection was performed with swine anti-rabbit HRP-conjugated secondary an- 2.4. Locomotion tests tibody (1:10,000; Dako) and Westar Eta C Ultra 2.0 ECL substrate (CYANAGEN). Image LabTM software (Bio-Rad) was used for image The Rotarod test was performed to evaluate motor coordination, density quantification. Minimum of 3 animals per group and treatment strength and balance of the animals. Mice were placed onto the rod were used and at least 3 different blots were quantified for each marker. rotating at a constant speed of 14 rpm. The time that the animal re- mained on the rotating rod was measured. Each mouse was given three 2.7. Measurement of muscle biomarkers trials and the longest time until falling recorded; 180 s was chosen as the arbitrary cut-off time. The test was performed from 9 to 16 weeks of The gene expression analysis was performed by real-time PCR (7500 age. Clinical disease onset for each mouse was determined as the first Real-Time PCR System, Applied Biosystem, Madrid, Spain). Two target week when the maintenance time was lower than 180 s. genes related to skeletal muscle homeostasis and regeneration were included in this analysis: Collagen type XIX alpha 1 (Col19a1), Myocyte enhancer factor 2C (Mef2c) and glutathione reductase (Gsr). The 2.5. RNA extraction and real time PCR Taqman probes (Applied Biosystems, Madrid, Spain) (Col19a1: Mm00483576_m1; Mef2c: Mm00600423_m1; Gsr: Mm00833903_m1; To obtain DNA or RNA samples, mice were sacrificed at 16 weeks of see Supplementary Table 1) used in this study were tested and in all the age by decapitation after deep anesthesia. Gastrocnemius muscle and cases the efficiency was near 100%. All the real-time PCR reactions L4-L5 spinal cord segments were rapidly dissected. DNA was extracted were done in triplicate and two endogenous genes (beta actin, β-actin, from spinal cord with 0.1 mg/ml of proteinase K (Roche Diagnostics), 4352932E, and glyceraldehyde 3-phosphate dehydrogenase, Gapdh, followed by phenol/chloroform extraction. Real time primers for cy- 4352933E) were used to normalize the target gene expression. The clophilin B, as housekeeping gene, or Desmin-NRG1.1 construct, where ΔΔCt method was used to determine relative changes in transcriptional the forward primer is located in the desmin promoter sequence and the expression. Statistical analyses of the data were performed using the reverse primer in the Nrg1.1 cDNA sequence, are listed in fold change 2-ΔΔCt. Supplementary Table 1. Viral genome copies per cell were calculated using a standard curve generated from known amounts of a plasmid 2.8. Histological analysis DNA containing the Desmin-NRG1.1 sequence or a 525 bp cyclophilin PCR product purified by Geneclean (Q-Biogene) in 10 ng per ml of At 16 weeks of age, subgroups of mice were transcardially perfused salmon's sperm DNA (Sigma) and assuming that 1 mg of mouse genomic with 4% paraformaldehyde in PBS, and the lumbar spinal cord and the DNA contains 3 × 105 haploid genomes. gastrocnemius muscle were harvested. Spinal cords were post-fixed For mRNA analyses, tissues were homogenized in Qiazol (Qiagen) during 2 h, then cryopreserved in PBS with 30% sucrose. GM muscles using a Tissue Lyser LT (Qiagen) at 50 Hz twice. One μg of RNA was were directly cryopreserved. reverse-transcribed using 10 μmol/l DTT, 200 U M-MuLV reverse tran- For spinal MN counting, cord samples were cut in 20 μm thick scriptase (New England BioLabs), 10 U RNase Out Ribonuclease transverse sections and serially collected in gelatinized slides. For each Inhibitor (Invitrogen), 1 μmol/l oligo(dT), and 1 μmol/l of random animal, slides corresponding to L4-L5 spinal cord sections with a se- hexamers (New England BioLabs). The reverse transcription cycle paration of 100 μm were stained with cresyl violet. MNs were identified conditions were 25 °C for 10 min, 42 °C for 1 h and 72 °C for 10 min. We by their localization in the ventral horn and strict size and morpholo- analyzed the mRNA expression of Nrg1.1, ErbB2, ErbB3 and ErbB4, by gical criteria (Mancuso et al., 2011). means of specific primer sets (Supplementary Table 1). Mouse 36B4 For glial cell reactivity, L4-L5 spinal sections were blocked with was used to normalize the expression levels of the different genes of PBS-0.3%Triton-10% Normal Donkey serum and 20 mM glycine, and interest for mouse samples. Gene-specific mRNA analysis was per- incubated 24 h at 4 °C with primary antibodies anti-ionized calcium formed by SYBR-green real-time PCR using the MyiQ5 real-time PCR binding adapter molecule 1 (Iba-1, 1:1000; 019–19,741, Wako, Japan), detection system (Bio-Rad). The thermal cycling conditions comprised and anti-glial fibrillary acidic protein (GFAP, 1:1000; 130,300, 5 min polymerase activation at 95 °C, 45 cycles of 15 s at 95 °C, 30s at Invitrogen, USA) to label microglia and astroglia respectively. After 60 °C, 30s at 72 °C and 5 s at 65 °C to 95 °C (increasing 0.5 °C every 5 s). washes, sections were incubated overnight with Alexa 488-conjugated Fluorescence detection was performed at the end of the PCR extension, secondary antibody (1:200; A21206, Invitrogen, USA) or Cy3-con- and melting curves were analyzed by monitoring the continuous de- jugated secondary antibody (1:200; 712–165-150, Jackson IR, USA). crease in fluorescence of the SYBR Green signal. Quantification relative DAPI (1:2000; D9563-10MG, Sigma, USA) was used to stain cell nu- to 36B4 controls for mRNA or Cyclophilin for DNA was calculated using cleus. Slides were mounted in Fluoromount-G (Southern Biotech, USA) the Pfaffl method (Pfaffl, 2001). and viewed under a fluorescence microscope (Nikon Eclipse NI, Japan). A region of interest centered in the ventral horn was selected to 3 G. Mòdol-Caballero, et al. Neurobiology of Disease 137 (2020) 104793 measure the integrated density of Iba1 and GFAP labeling using ImageJ software (NIH, Bethesda, MD). For muscle immunohistochemistry, 60 μm longitudinal sections were serially cut and collected in 24-well plates in sequential series of 4 slices per well in Olmos solution. Sections were blocked with PBS- 0.3%Triton-5%Normal Donkey serum and incubated 48 h at 4 °C with primary antibodies anti-synaptophysin (1:500; AB130436, Abcam, UK) and anti-neurofilament 200 (NF200, 1:1000; AB5539, Millipore, USA). After washes, sections were incubated overnight with Alexa 594-con- jugated secondary antibody (1:200; A11042, Invitrogen, USA) and Alexa 488 conjugated alfa-bungarotoxin (1:200; B13422, Life Technologies, USA). Confocal images were captured with a scanning confocal microscope (LSM 700 Axio Observer, Carl Zeiss 40×/1.3 Oil DIC M27, Germany). Maximum projections images shown in this study were generated from 1.5 μm z projections. For NMJ analysis, the pro- portion of innervated endplates was calculated by classifying each endplate as fully occupied (when presynaptic terminals overlie the endplate), partially occupied (when presynaptic terminals were not clearly within the endplate) or vacant (no presynaptic label in contact with the endplate). At least 4 fields with more than 100 endplates were analyzed per muscle. 2.9. Statistical analysis All experiments were performed by researchers blinded with respect to treatment received by each mouse group, and random allocation of animals in groups taking into account weight and litter. Data are ex- pressed as mean ± SEM. Electrophysiological and locomotion tests results were statistically analyzed using one-way or repeated mea- surements ANOVA with Tukey's post-hoc test. For the MUNEs and MEPs electrophysiological results Student's t-test was applied. For clinical disease onset data Log-rank (Mantel-Cox) test was applied. Histological and molecular biology data were analyzed using ANOVA and t-Student. 3. Results Fig. 1. AAV8 under the desmin promoter coding for NRG1-I enhances NRG1-I ex- 3.1. Viral-delivery of NRG-I in skeletal muscles pression in the hindlimb muscles of SOD1G93A mice. A) mRNA expression analysis shows that NRG1-I is overexpressed in the GM muscle of treated mice compared We first tested the transduction of the AAV8-desmin-NRG1-I vector to the control SOD1G93A and WT mice. B) Viral genome analysis reveals that in the skeletal muscles based on a single intravenous injection of intravenous AAV injection was effective in the treated mice. (n = 5 WT, 4 SOD 7.6 × 1013 vg in 250 μl in 6 weeks-old mice, in order to obtain a high Mock, 4 SOD NRG1-I mice per group, one-way ANOVA, ***p < .001, *p < .05 expression of NRG1-I during the symptomatic stage, starting between 8 vs SOD Mock mice). Data are shown as mean ± SEM. and 10 weeks of age in this model. At the end stage of the disease (16 weeks) we confirmed that the treated SOD1G93A mice had a higher 3.3. NRG1-I overexpression preserves spinal MNs and reduces expression of NRG1-I in the GM muscle (18.64 ± 7.09 fold) compared neuroinflammation to the SOD1G93A mock mice (0.76 ± 0.41) (Fig. 1A). This result agreed with the detection of 0.24 ± 0.05 viral genomes of Desmin-NRG1.1/ We then performed the histological analysis of the SOD1G93A mice cell detected in the treated SOD1G93A mice (Fig. 1B). at 16 weeks. The results showed that the NRG1-I overexpression in skeletal muscles preserved the number of MNs (12.1 ± 0.5 mean 3.2. NRG1-I overexpression preserves neuromuscular function of the treated number of MNs per ventral horn ± SEM) compared to the SOD1G93A SOD1G93A mice mock mice (6.0 ± 0.5) (Fig. 3A,B) in line with our electrophysiological findings. The increased CMAP and motor unit number in the hindlimb To study the effect of the NRG1-I overexpression, we evaluated by muscles at 16 weeks was further confirmed by the NMJ analysis, since means of electrophysiological tests the neuromuscular function in the NRG1-I gene therapy significantly preserved the number of occu- hindlimb muscles of SOD1G93A mice. The results showed that there was pied motor endplates (68.8 ± 5.1 percentage of occupied end- a significant preservation of the PL, TA and GM CMAP amplitude at plates ± SEM) compared to the mock SOD1G93A group (35.9 ± 7.2) 16 weeks in the female (Fig. 2A,B,C) and male treated SOD1G93A mice (Fig. 3A,B). We also assessed the glial immunoreactivity in the spinal (Supplementary Fig. 1A,B,C) compared to the control mice. This higher cord and we found a significant decrease of astrocyte and microglial CMAPs amplitude of hindlimb muscles was explained by an increase of activation (3.89 × 107 ± 6.60 × 106 and 5.52 × 107 ± 1.08 × 107 the mean amplitude and the number of surviving motor units in female respectively; integrated density ± SEM) compared to the SOD1G93A (Fig. 2D,E,F) and also male mice (Supplementary Fig. 1D,E,F). The mock mice (1.825 × 108 ± 2.67 × 107 and 2.25 × 108 ± 9.07 × 107 distribution of TA motor units grouped by amplitude of the action po- respectively) (Fig. 3C,D). tential show a higher number in most of the intervals for female (Fig. 2F) and male mice (Supplementary Fig. 1F). The amplitude of the MEPs recorded in the GM and TA muscles was also significantly higher in treated SOD1G93A mice (Fig. 2G and Supplementary Fig. 1G). 4 G. Mòdol-Caballero, et al. Neurobiology of Disease 137 (2020) 104793 Fig. 2. NRG1-I overexpression promotes motor functional improvement of the SOD1G93A mice. A) Electrophysiological tests show that AAV-NRG1-I injection produced a significant preservation of the CMAP amplitude of PL (A), TA (B), and GM (C) muscles in the SOD1G93A mice along time (n = 6 WT, 6–13 SOD Mock, 10–11 SOD NRG1-I mice per group, two-way ANOVA, **p < .01, *p < .05 vs SOD Mock mice; ####p < .0001, ###p < .001, #p < .05 vs WT mice). Electrophysiological estimation of motor unit number (MUNE) and mean amplitude of single motor unit potential (SMUA) of the PL (D) and TA (E) muscles show that the increased CMAP amplitude was the result of preservation of more motor units and of larger size, confirmed by the frequency distribution (F). G) NRG1-I treatment increased the amplitude of the MEPs (n = 6–14 SOD Mock, 10–12 SOD NRG1-I mice per group, T-student, ***p < .001, *p < .05 vs SOD Mock mice). Data are mean ± SEM. 3.4. NRG1-I signaling through ErbB receptors produces motor functional Mòdol-Caballero et al., 2019). Nrg1-I treatment significantly reduced improvement in the treated SOD1G93A mice the ratio of phosphorylation of Erk2 (Fig. 4D). We also evaluated ac- tivation of cell survival pathways in the spinal cord, where we found We evaluated the expression of the ErbB 2, 3 and 4 receptors in GM similar results than in muscle: NRG1-I overexpression in muscle acti- muscle samples of SOD1G93A mice. While no changes were detected vated Akt phosphorylation but decreased ERK activation, particularly of between WT and mock-treated SOD mice for the ErbB receptors mRNA, p42, returning to control levels (Fig. 4E). upon NRG1-I overexpression, mRNA levels of ErbB2 (1.7 ± 0.42-fold In order to better characterize muscle homeostatic maintenance, we increase compared to WT levels) (Fig. 4A) and ErbB3 (3.36 ± 0.26) analyzed the expression of genes related to ALS disease progression in (Fig. 4B) were significantly upregulated compared to SOD1G93A mock the muscle by quantitative real time PCR. Two target genes, Col19a1 and WT mice (0.59 ± 0.12 and 1.57 ± 0.43 respectively). The ErbB4 and Mef2c, were found increased in the GM muscle of SOD1G93A mice, receptor levels were also increased in NRG1-I SOD1G93A treated mice as previously reported (Calvo et al., 2012). Albeit no statistical sig- (1.48 ± 0.66) compared to the SOD1G93A mock group (0.79 ± 0.13) nificant difference was found between untreated SOD1G93A mice and but the differences were not statistically significant (Fig. 4C). NRG1-I treated mice regarding Col19a1 and Mef2c gene levels (Fig. 4F), We also explored cell signaling pathways to corroborate functional probably due to the variability of the samples, NRG1-I treated mice activity of the Nrg1/ErbB axis by western blot analysis of GM muscle. showed a tendency to have lower Col19a1 and Mef2c mean levels, We found that activation of Akt was strongly downregulated in suggesting that NRG1-I could ameliorate muscle damage during pro- SOD1G93A mice and NRG1-I treatment completely restored Akt phos- gressive denervation. In addition, we aimed to investigate if NRG1-I phorylation compared to the mock treated group (Fig. 4D). On the treatment was able to reduce oxidative stress in the muscles, as pre- contrary, activation of Erk in SOD1 animals is high, particularly in the viously described in adult rat cardiomyocytes (Timolati et al., 2006). p42 isoform, in agreement with previous reports (Bhinge et al., 2017; Therefore, we investigated the gene expression levels of glutathione 5 G. Mòdol-Caballero, et al. Neurobiology of Disease 137 (2020) 104793 Fig. 3. Viral-delivery of NRG1-I preserves spinal MN number and decreases glial reactivity. A) Representative images of L4 spinal cord and GM neuromuscular junctions of WT and SOD1G93A mice, treated with AAV-NRG1-I or with mock vector (scale bar = 100 μm). B) Histological analysis showed higher number of spinal MNs, as well as increased proportion of innervated NMJs in the GM muscle in the treated SOD1G93A mice compared to the mock treated mice (n = 3 WT, 3–4 SOD Mock, 7 SOD NRG1-I mice per group, one-way ANOVA, ****p < .0001, ***p < .001, **p < .01 vs SOD Mock mice). C) Representative confocal images of astrocytes labeled against GFAP, and microglia labeled against Iba-1, in the spinal cord ventral horn of SOD1G93A mice (scale bar = 100 μm). D) The viral-mediated delivery of NRG1-I reduced the astrocyte and microglia reactivity in the treated SOD1G93A mice (n = 3–4 SOD Mock, 5–6 SOD NRG1-I mice per group, T-student, **p < .01, *p < .05). Data are shown as mean ± SEM. reductase (Gsr), previously identified altered in SOD1G93A mice and Altogether these data suggest a mechanism of protection from ex- closely related to the oxidative stress metabolism (Calvo et al., 2012). citotoxicity and inflammation and activation of the cell survival path- Interestingly, Gsr levels were found significantly decreased in NRG1-I ways both in muscle and in spinal cord. Consequently, NRG1-I/ErbB treated mice compared to mock treated SOD1G93A mice (Fig. 4F), in- signaling in the skeletal muscles was able to produce also functional dicating that NRG1-I reduced oxidative stress in the GM muscle. improvement in the rotarod performance in the treated SOD1G93A mice 6 G. Mòdol-Caballero, et al. Neurobiology of Disease 137 (2020) 104793 (caption on next page) 7 G. Mòdol-Caballero, et al. Neurobiology of Disease 137 (2020) 104793 Fig. 4. Increased NRG1-I/ErbB signaling activates survival pathways in muscle and spinal cord and produces functional improvement in the treated mice. mRNA expression analysis of ErbB receptors shows a significant upregulation of ErbB2 (A) and ErbB3 (B) upon NRG1-I overexpression in the treated SOD1G93A mice compared to the mock mice, whereas the levels of ErbB4 receptor (C) showed a non-significant increase (n = 3 WT, 3–4 SOD Mock, 4 SOD NRG1-I mice, one-way ANOVA, **p < .01, *p < .05 vs SOD Mock mice, ##p < .01 vs WT mice). D) NRG1-I increases Akt phosphorylation and diminishes Erk2 activation in the muscle of SOD1G93A treated animals, as demonstrated by Western blot. E) Muscle NRG1-I increases Akt phosphorylation and normalizes ERK2 activity in the spinal cord of treated SOD1G93A mice. Quantifications show relative phosphorylation compared to total protein, normalized by GAPDH and are represented by fold-change compared to WT animals; **p < .01 vs SOD Mock mice, ###p < .0005, #p < .05 vs WT mice). F) mRNA expression of muscle biomarkers Col19a1, Mef2c and Gsr (n = 3 for each group; #p < .05 vs WT mice and ⁎p < .05 vs SOD Mock mice). G) NRG1-I overexpression produced an improvement in the rotarod performance of treated SOD1G93A mice from 14 to 16 weeks (n = 12 SOD Mock, 7 SOD NRG1-I mice per group, two-way ANOVA, ****p < .0001, **p < .01, *p < .05 vs SOD Mock mice). Data are shown as mean ± SEM. H) Clinical onset of disease was significantly delayed (n = 12 SOD Mock, 9 SOD NRG1-I mice per group, Mantel-Cox test, **p < .01, vs SOD Mock mice). (Fig. 4G) and a significant delay of the clinical disease onset (Fig. 4H) development and after nerve injury (Esper and Loeb, 2004). NRG1-I compared to the control SOD1G93A mice. overexpression promotes axonal regeneration and remyelination in a transient autocrine/paracrine signaling loop with Schwann cells ex- 4. Discussion pressing ErbB2 and 3 receptors (Stassart et al., 2013). Moreover, con- stitutively active ErbB2 in Schwann cells resulted in proliferation and We previously showed that NRG1-I overexpression localized in the process extension from these cells (Hayworth et al., 2006), and loss of GM muscle produced functional compensation by enhancing collateral NRG1/ErbB signaling destabilizes the anchoring of acetylcholine re- sprouting in the SOD1G93A mouse and in WT mouse after partial muscle ceptors in the postsynaptic muscle membrane (Schmidt et al., 2011). denervation (Mancuso et al., 2016). However, an increased expression Also, considering that only the extracellular domain of NRG1-I was of NRG1-I in a single muscle was only a proof of principle, since it was overexpressed in our study, we cannot discard an intracrine signaling of not able to globally improve ALS disease progression. Therefore, we NRG1-I by the muscle cells that could explain the NMJ maintenance attempted to promote a general overexpression of NRG1-I in most (Wiley et al., 1998). Therefore, NRG1-I expressed at the NMJ may act skeletal muscles to produce global functional improvement by in- on terminal Schwann cells and on muscle fibers, promoting main- travenous administration of an AAV vector under the regulation of the tenance of muscle innervation and motor axon survival. This is in ac- desmin promoter. Indeed, NRG1-I acting through the ErbB receptors in cordance with the findings obtained in target biomarkers of muscle the periphery causes neuromuscular functional improvement, main- degeneration and oxidative stress. NRG1-I gene therapy promoted tains NMJ innervation and increases spinal MN survival in both female lower mean values of Col19a1 and Mef2c mRNA with respect to un- and male mice, while reducing glial cell reactivity around MNs in the treated mice, although not significantly (Calvo et al., 2012). Col19a1 is SOD1G93A mice that received a single administration of the viral vector. involved in the maintenance of muscle integrity, albeit it has been also NRG1 isoforms are important trophic factors that play multiple roles described to play a role in differentiation of muscle cells, in a similar in the nervous system development and maintenance (Esper et al., way to Mef2c gene. Structural or functional alterations of skeletal 2006). Alterations of the NRG1/ErbB system have been related to ALS muscle in MN disease can alter the transmission of chemical and elec- (Takahashi et al., 2013; Lopez-Font et al., 2019). NRG1-III expression trical signals between motor axons and NMJ, favoring the dying-back was found reduced in the spinal cord of both ALS patients and phenomenon and therefore contributing to disease pathology SOD1G93A mice in parallel with MN loss (Song et al., 2012; Lasiene (Ferraiuolo et al., 2009). Interestingly, transcript levels of collagen type et al., 2016; Mòdol-Caballero et al., 2019). Indeed, viral-mediated de- I and III were found reduced in the myocardium of a murine model of livery of NRG1-III in the CNS restored the number of C-boutons and diabetic cardiomyopathy after NRG1 administration, promoting an produced a slight increase of survival of the SOD1G93A mice (Lasiene improvement of cardiac function (Li et al., 2011). In this study, the et al., 2016). We recently demonstrated that addition of NRG1 exerts tendency to lower Col19a1 and Mef2c mean levels suggests an en- neuroprotective effects on MNs under chronic excitotoxicity in an in hancement of muscle integrity and homeostasis. In addition, the sig- vitro model (Mòdol-Caballero et al., 2018), and that overexpression of nificant reduction in Glutathione reductase (Gsr) levels in NRG1-I NRG1-III along the spinal cord of SOD1G93A mice improves motor treated mice also suggests that NRG1-I treatment ameliorates oxidative function and MNs survival (Mòdol-Caballero et al., 2019). stress in the skeletal muscle. Glutathione reductase maintains the However, the role of NRG1-I isoform in MN degenerative diseases supply of reduced glutathione, which is one of the most abundant re- remains unclear. NRG1-I is critical for the axoglial intercommunication ducing thiols in the majority of cells, modulating the reactive oxygen during development (Loeb et al., 1999; Esper and Loeb, 2004), the species (Couto et al., 2016). The reduction of Gsr levels in NRG1-I formation of muscle spindles (Andrechek et al., 2002; Hippenmeyer treated mice would indicate a better modulation of the redox home- et al., 2002) and the maintenance of the mature NMJ (Sandrock Jr ostasis, that depends on ErbB2 and Akt receptors, as was described in et al., 1997; Loeb et al., 2002; Loeb, 2003). Indeed, NRG1/ErbB sig- rat cardiomyocytes (Timolati et al., 2006). Taking together these find- naling is important for the stability of acetylcholine receptors at the ings, NRG1-I appear to positively influence the response to muscle NMJ and the structural integrity of the postsynaptic site (Schmidt et al., denervation and secondary damage. 2011). While Song et al. (2012) found that NRG1-I isoform was in- Furthermore, NRG1-I therapy restored Akt phosphorylation in creased and associated with glial activation in the spinal cord of muscle and spinal cord of ALS mice. On the contrary, SOD1G93A animals SOD1G93A mice in agreement with our recently published results with B6xSJL background show activation of Erk1/2 as we previously (Mòdol-Caballero et al., 2019), Lasiene et al. (2016) showed decreased described (Modol-Caballero et al., 2019), as well as do MNs derived NRG1-I mRNA levels. However, when they applied a virus-mediated from iPS cells from SOD1 ALS patients (Bhinge et al., 2017). NRG1-I delivery to overexpress this isoform in the lumbar spinal cord par- treatment significantly reduced Erk activation, so the imbalance be- enchyma, their results were less beneficial than with NRG1-III (Lasiene tween Akt and Erk1/2 was restored. A similar imbalance was described et al., 2016). in rodent models of CMT1A and soluble NRG1–1 overexpression re- In this study, we found that overexpression of NRG1-I in skeletal stored such dysregulation and preserved peripheral nerve axons muscles produced an upregulation of the ErbB receptors, which in turn, (Fledrich et al., 2014). Nevertheless, it has been reported that soluble are expressed by the terminal Schwann cells in the NMJs (Syroid et al., NRG1-I overexpression may promote demyelination (Zanazzi et al., 1996). Indeed, bidirectional communication between Schwann cells 2001; Syed et al., 2010; Fledrich et al., 2019). The adverse effect on and MNs supports their mutual survival and differentiation during demyelination is driven by the activation of Erk pathway, which was 8 G. Mòdol-Caballero, et al. Neurobiology of Disease 137 (2020) 104793 found reduced when our NRG1-I therapy was applied. Moreover, our References electrophysiology data showed that for both TA and PL muscles the CMAP latencies were similar in SOD untreated and treated groups (data Alizadeh, A., Dyck, S.M., Kataria, H., Shahriary, G.M., Nguyen, D.H., Santhosh, K.T., not shown). The latency time reflects the conduction velocity and thus, Karimi-Abdolrezaee, S., 2017. Neuregulin-1 positively modulates glial response and improves neurological recovery following traumatic spinal cord injury. Glia 65, shows if myelinated fibers are affected in ALS (Cornblath et al., 1992; 1152–1175. de Carvalho and Swash, 2000) and in other diseases. Therefore, we can Alizadeh, A., Santhosh, K.T., Kataria, H., Gounni, A.S., Karimi-Abdolrezaee, S., 2018. discard the potential side effect of demyelination induced by the NRG1- Neuregulin-1 elicits a regulatory immune response following traumatic spinal cord injury. J. Neuroinflammation 15, 53. I overexpression therapy. 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Soluble neuregulin-1 modulates disease pathogenesis in rodent models Supplementary data to this article can be found online at https:// of Charcot-Marie-Tooth disease 1A. Nat. Med. 20, 1055–1061. doi.org/10.1016/j.nbd.2020.104793. Fledrich, R., Akkermann, D., Schütza, V., Abdelaal, T.A., Hermes, D., Schäffner, E., Soto- Bernardini, M.C., Götze, T., Klink, A., Kusch, K., Krueger, M., Kungl, T., Frydrychowicz, C., Möbius, W., Brück, W., Mueller, W.C., Bechmann, I., Sereda, M.W., Schwab, M.H., Nave, K.A., Stassart, R.M., 2019. NRG1 type I dependent au- Declaration of Competing Interest toparacrine stimulation of Schwann cells in onion bulbs of peripheral neuropathies. Nat. Commun. 10 (1), 1467. Gallart-Palau, X., Tarabal, O., Casanovas, A., Sábado, J., Correa, F.J., Hereu, M., The authors report no conflicts of interest. The authors alone are Piedrafita, L., Calderó, J., Esquerda, J.E., 2014. Neuregulin-1 is concentrated in the responsible for the content and writing of the paper. postsynaptic subsurface cistern of C-bouton inputs to α-motoneurons and altered during motoneuron diseases. FASEB J. 28, 3618–3632. Graffmo, K.S., Forsberg, K., Bergh, J., Birve, A., Zetterström, P., Andersen, P.M., Marklund, S.L., Brännström, T., 2013. Expression of wild-type human superoxide Acknowledgements dismutase-1 in mice causes amyotrophic lateral sclerosis. Hum. Mol. Genet. 22, 51–60. Gurney, M.E., Pu, H., Chiu, A.Y., Dal Canto, M.C., Polchow, C.Y., Alexander, D.D., This work was supported by grant TV3201428-10 of Fundació La Caliendo, J., Hentati, A., Kwon, Y.W., Deng, H.X., 1994. Motor neuron degeneration Marato-TV3, grant #20289 of AFM-Telethon, cooperative project 2015- in mice that express a human cu,Zn superoxide dismutase mutation. Science 264, 01 from CIBERNED, TERCEL funds (RD16/0011/0014) and grant PS09- 1772–1775. 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