Jordi Calderó

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by loss of the SMN1 gene, reduced levels of SMN protein, and motor neuron degeneration. However, increasing evidence shows that SMA is a multisystemic disease with immune system involvement. We investigated how SMN deficiency affects lymphoid organ development and function using a severe SMA mouse model (SMNΔ7) and postmortem human fetal and postnatal tissues lacking SMN1 and carrying one or two SMN2 copies, consistent with type 0–I SMA. Histology, immunostaining, and flow cytometry were used to examine tissue architecture and immune cell composition. SMNΔ7 mice displayed thymus, spleen, and bone marrow abnormalities, including mislocalization of T- and B-cells and expansion of resident macrophages. Bone marrow analysis revealed impaired B-cell development, suggesting intrinsic hematopoietic defects rather than apoptosis. Early treatment with a nusinersen-like antisense oligonucleotide, administered intracerebroventricularly or subcutaneously, restored SMN2 splicing, improved survival, motor function, and prevented lymphoid pathology. Human SMA samples exhibited similar, though milder, splenic alterations compared to SMNΔ7 mice, while thymic organization remained largely preserved. These findings demonstrate that SMN deficiency disrupts lymphoid organ development through defective bone marrow output and impaired immune cell maturation. Early SMN restoration prevents these abnormalities, highlighting immune dysfunction as a key component of SMA pathology.

Neuregulins (NRGs) are ligands of tyrosine kinase receptors from the ErbB family and play multiple developmental roles. NRG1–ErbB signaling regulates myelination and has been associated with amyotrophic lateral sclerosis (ALS) pathology. Given the potential therapeutic relevance of this pathway for motor neuron (MN) diseases, we employed a transgenic (TG) mouse with persistent neuronal overexpression of neuregulin type III (NRG1-III) to investigate its impact on the neuromuscular system. We performed an analysis of phenotypic changes in this TG model, including motor behavior, neuropathological evaluation by immunocytochemistry and ultrastructural examination of the spinal cord, peripheral nerves, and neuromuscular junctions (NMJs). Calcium dynamics in cultured MNs were also examined. We found that cholinergic C-boutons on TG MNs, where NRG1-III typically accumulates, exhibited upregulation of C-bouton-associated proteins and expansion of the subsynaptic cistern (SSC)-associated endoplasmic reticulum. Calcium imaging revealed altered homeostasis in TG MNs, accompanied by the upregulation of molecules linked to axonal plasticity. At NMJs, regressive changes involving autophagic dysregulation were observed. These alterations were accompanied by increased motor activity in behavioral tests. Overall, our findings indicate that persistently elevated NRG1-III signaling compromises MN connectivity and long-term health, a factor to consider when developing therapeutic strategies for neurodegenerative diseases such as ALS.

Neuregulins (NRGs) are ligands for tyrosine kinase receptors from the ErbB family and serve multiple developmental functions in the nervous system. NRG1-ErbB signaling regulates myelination and is associated with amyotrophic lateral sclerosis (ALS) pathology. Given the potential therapeutic relevance of this pathway for motor neuron (MN) diseases, we employed a transgenic (TG) mouse model with persistent neuronal overexpression of NRG type III (NRG1-III) to investigate its long-term impact on the neuromuscular system. We examined MNs and their synaptic inputs, including cholinergic C-boutons, at which NRG1-III typically accumulates. MNs from TG mice exhibited persistent upregulation of C-bouton-associated postsynaptic proteins, as well as increased biogenesis of subsynaptic cistern-like ER-plasma membrane contacts. Following nerve injury, NRG1-III overexpression resulted in exacerbated neuroinflammation and disruption of enlarged NRG1 clusters. Calcium imaging revealed altered homeostasis in TG MNs, which upregulated molecules linked to axonal plasticity. At neuromuscular junctions, we observed regressive changes involving autophagic dysregulation. These alterations were accompanied by increased motor activity in behavioral tests. Overall, our findings indicate that persistently elevated NRG1-III signaling diminishes MN connectivity and long-term health, which must be considered when developing therapeutic strategies for neurodegenerative diseases, such as ALS.

Spinal muscular atrophy (SMA) is caused by a deficiency of the ubiquitously expressed survival motor neuron (SMN) protein. The main pathological hallmark of SMA is the degeneration of lower motor neurons (MNs) with subsequent denervation and atrophy of skeletal muscle. However, increasing evidence indicates that low SMN levels not only are detrimental to the central nervous system (CNS) but also directly affect other peripheral tissues and organs, including skeletal muscle. To better understand the potential primary impact of SMN deficiency in muscle, we explored the cellular, ultrastructural, and molecular basis of SMA myopathy in the SMNΔ7 mouse model of severe SMA at an early postnatal period (P0-7) prior to muscle denervation and MN loss (preneurodegenerative [PND] stage). This period contrasts with the neurodegenerative (ND) stage (P8-14), in which MN loss and muscle atrophy occur. At the PND stage, we found that SMN∆7 mice displayed early signs of motor dysfunction with overt myofiber alterations in the absence of atrophy. We provide essential new ultrastructural data on focal and segmental lesions in the myofibrillar contractile apparatus. These lesions were observed in association with specific myonuclear domains and included abnormal accumulations of actin-thin myofilaments, sarcomere disruption, and the formation of minisarcomeres. The sarcoplasmic reticulum and triads also exhibited ultrastructural alterations, suggesting decoupling during the excitation–contraction process. Finally, changes in intermyofibrillar mitochondrial organization and dynamics, indicative of mitochondrial biogenesis overactivation, were also found. Overall, our results demonstrated that SMN deficiency induces early and MN loss-independent alterations in myofibers that essentially contribute to SMA myopathy. This strongly supports the growing body of evidence indicating the existence of intrinsic alterations in the skeletal muscle in SMA and further reinforces the relevance of this peripheral tissue as a key therapeutic target for the disease.

Survival motor neuron (SMN) is an essential and ubiquitously expressed protein that participates in several aspects of RNA metabolism. SMN deficiency causes a devastating motor neuron disease called spinal muscular atrophy (SMA). SMN forms the core of a protein complex localized at the cytoplasm and nuclear gems and that catalyzes spliceosomal snRNP particle synthesis. In cultured motor neurons, SMN is also present in dendrites and axons, and forms part of the ribonucleoprotein transport granules implicated in mRNA trafficking and local translation. Nevertheless, the distribution, regulation, and role of SMN at the axons and presynaptic motor terminals in vivo are still unclear. By using conventional confocal microscopy and STED super-resolution nanoscopy, we found that SMN appears in the form of granules distributed along motor axons at nerve terminals. Our fluorescence in situ hybridization and electron microscopy studies also confirmed the presence of β-actin mRNA, ribosomes, and polysomes in the presynaptic motor terminal, key elements of the protein synthesis machinery involved in local translation in this compartment. SMN granules co-localize with the microtubule-associated protein 1B (MAP1B) and neurofilaments, suggesting that the cytoskeleton participates in transporting and positioning the granules. We also found that, while SMN granules are physiologically downregulated at the presynaptic element during the period of postnatal maturation in wild-type (non-transgenic) mice, they accumulate in areas of neurofilament aggregation in SMA mice, suggesting that the high expression of SMN at the NMJ, together with the cytoskeletal defects, contribute to impairing the bi-directional traffic of proteins and organelles between the axon and the presynaptic terminal.

Background: The cellular mechanisms underlying the age‐associated loss of muscle mass and function (sarcopenia) are poorly understood, hampering the development of effective treatment strategies. Here, we performed a detailed characterization of age‐related pathophysiological changes in the mouse neuromuscular system.Methods: Young, adult, middle‐aged, and old (1, 4, 14, and 24–30 months old, respectively) C57BL/6J mice were used. Motor behavioural and electrophysiological tests and histological and immunocytochemical procedures were carried out to simultaneously analyse structural, molecular, and functional age‐related changes in distinct cellular components of the neuromuscular system.Results: Ageing was not accompanied by a significant loss of spinal motoneurons (MNs), although a proportion (~15%) of them in old mice exhibited an abnormally dark appearance. Dark MNs were also observed in adult (~9%) and young (~4%) animals, suggesting that during ageing, some MNs undergo early deleterious changes, which may not lead to MN death. Old MNs were depleted of cholinergic and glutamatergic inputs (~40% and ~45%, respectively, P < 0.01), suggestive of age‐associated alterations in MN excitability. Prominent microgliosis and astrogliosis [~93% (P < 0.001) and ~100% (P < 0.0001) increase vs. adults, respectively] were found in old spinal cords, with increased density of pro‐inflammatory M1 microglia and A1 astroglia (25‐fold and 4‐fold increase, respectively, P < 0.0001). Ageing resulted in significant reductions in the nerve conduction velocity and the compound muscle action potential amplitude (~30%, P < 0.05, vs. adults) in old distal plantar muscles. Compared with adult muscles, old muscles exhibited significantly higher numbers of both denervated and polyinnervated neuromuscular junctions, changes in fibre type composition, higher proportion of fibres showing central nuclei and lipofuscin aggregates, depletion of satellite cells, and augmented expression of different molecules related to development, plasticity, and maintenance of neuromuscular junctions, including calcitonin gene‐related peptide, growth associated protein 43, agrin, fibroblast growth factor binding protein 1, and transforming growth factor‐β1. Overall, these alterations occurred at varying degrees in all the muscles analysed, with no correlation between the age‐related changes observed and myofiber type composition or muscle topography.Conclusions: Our data provide a global view of age‐associated neuromuscular changes in a mouse model of ageing and help to advance understanding of contributing pathways leading to development of sarcopenia.

C-bouton-type cholinergic afferents exert an important function in controlling motoneuron (MN) excitability. During the immunocytochemical analysis of the role of c-Jun in MNs with a monoclonal (clone Y172) antibody against phospho (p)-c-Jun (serine [Ser]63), unexpected labeling was identified in the cell body cytoplasm. As predicted for c-Jun in adult spinal cord, very few, if any MNs exhibited nuclear immunoreactivity with the Y172 antibody; conversely, virtually all MNs displayed strong Y172 immunostaining in cytoplasmic structures scattered throughout the soma and proximal dendrites. The majority of these cytoplasmic Y172-positive profiles was closely associated with VAChT-positive C-boutons, but not with other types of nerve afferents contacting MNs. Ultrastructural analysis revealed that cytoplasmic Y172 immunostaining was selectively located at the subsurface cistern (SSC) of C-boutons and also in the inner areas of the endoplasmic reticulum (ER). We also described changes in cytoplasmic Y172 immunoreactivity in injured and degenerating MNs. Moreover, we noticed that MNs from NRG1 type III-overexpressing transgenic mice, which show abnormally expanded SSCs, exhibited an increase in the density and size of peripherally located Y172-positive profiles. A similar immunocytochemical pattern to that of the Y172 antibody in MNs was found with a polyclonal antibody against p-c-Jun (Ser63) but not with another polyclonal antibody that recognizes c-Jun phosphorylated at a different site. No differential band patterns were found by western blotting with any of the antibodies against c-Jun or p-c-Jun used in our study. In cultured MNs, Y172-positive oval profiles were distributed in the cell body and proximal dendrites. The in vitro lentiviral-based knockdown of c-Jun resulted in a dramatic decrease in nuclear Y172 immunostaining in MNs without any reduction in the density of cytoplasmic Y172-positive profiles, suggesting that the synaptic antigen recognized by the antibody corresponds to a C-bouton-specific protein other than p-c-Jun. Our results lay the foundation for further studies aimed at identifying this protein and determining its role in this particular type of synapse.

C‐type synaptic boutons (C‐boutons) provide cholinergic afferent input to spinal cord motor neurons (MNs), which display an endoplasmic reticulum (ER)–related subsurface cistern (SSC) adjacent to their postsynaptic membrane. A constellation of postsynaptic proteins is clustered at C‐boutons, including M2 muscarinic receptors, potassium channels, and σ‐1 receptors. In addition, we previously found that neuregulin (NRG)1 is associated with C‐boutons at postsynaptic SSCs, whereas its ErbB receptors are located in the presynaptic compartment. C‐bouton–mediated regulation of MN excitability has been implicated in MN disease, but NRG1‐mediated functions and the impact of various pathologic conditions on C‐bouton integrity have not been studied in detail. Here, we investigated changes in C‐boutons after electrical stimulation, pharmacological treatment, and peripheral nerve axotomy. SSC‐linked NRG1 clusters were severely disrupted in acutely stressed MNs and after tunicamycin‐induced ER stress. In axotomized MNs, C‐bouton loss occurred in concomitance with microglial recruitment and was prevented by the ER stress inhibitor salubrinal. Activated microglia displayed a positive chemotaxis to C‐boutons. Analysis of transgenic mice overexpressing NRG1 type I and type III isoforms in MNs indicated that NRG1 type III acts as an organizer of SSC‐like structures, whereas NRG1 type I promotes synaptogenesis of presynaptic cholinergic terminals. Moreover, MN‐derived NRG1 signals may regulate the activity of perineuronal microglial cells. Together, these data provide new insights into the molecular and cellular pathology of C‐boutons in MN injury and suggest that distinct NRG1 isoform–mediated signaling functions regulate the complex matching between pre‐ and postsynaptic C‐bouton elements.—Salvany, S., Casanovas, A., Tarabal, O., Piedrafita, L., Hernández, S., Santafé, M., Soto‐Bernardini, M. C., Calderó, J., Schwab, M. H., Esquerda, J. E. Localization and dynamic changes of neuregulin‐1 at C‐type synaptic boutons in association with motor neuron injury and repair. FASEB J. 33, 7833–7851 (2019). www.fasebj.org

Spinal muscular atrophy (SMA) is characterized by the loss of α-motoneurons (MNs) with concomitant muscle denervation. MN excitability and vulnerability to disease are particularly regulated by cholinergic synaptic afferents (C-boutons), in which Sigma-1 receptor (Sig1R) is concentrated. Alterations in Sig1R have been associated with MN degeneration. Here, we investigated whether a chronic treatment with the Sig1R agonist PRE-084 was able to exert beneficial effects on SMA. We used a model of intermediate SMA, the Smn^2B/− mouse, in which we performed a detailed characterization of the histopathological changes that occur throughout the disease. We report that Smn^2B/− mice exhibited qualitative differences in major alterations found in mouse models of severe SMA: Smn^2B/− animals showed more prominent MN degeneration, early motor axon alterations, marked changes in sensory neurons, and later MN deafferentation that correlated with conspicuous reactive gliosis and altered neuroinflammatory M1/M2 microglial balance. PRE-084 attenuated reactive gliosis, mitigated M1/M2 imbalance, and prevented MN deafferentation in Smn^2B/− mice. These effects were also observed in a severe SMA model, the SMNΔ7 mouse. However, the prevention of gliosis and MN deafferentation promoted by PRE-084 were not accompanied by any improvements in clinical outcome or other major pathological changes found in SMA mice.

The electric activity of lower motor neurons (MNs) appears to play a role in determining cell-vulnerability in MN diseases. MN excitability is modulated by cholinergic inputs through C-type synaptic boutons, which display an endoplasmic reticulum-related subsurface cistern (SSC) adjacent to the postsynaptic membrane. Besides cholinergic molecules, a constellation of proteins involved in different signal-transduction pathways are clustered at C-type synaptic sites (M2 muscarinic receptors, Kv2.1 potassium channels, Ca²⁺ activated K⁺ [SK] channels, and sigma-1 receptors [S1R]), but their collective functional significance so far remains unknown. We have previously suggested that neuregulin-1 (NRG1)/ErbBs-based retrograde signalling occurs at this synapse. To better understand signalling through C-boutons, we performed an analysis of the distribution of C-bouton-associated signalling proteins. We show that within SSC, S1R, Kv2.1 and NRG1 are clustered in highly specific, non-overlapping, microdomains, whereas ErbB2 and ErbB4 are present in the adjacent presynaptic compartment. This organization may define highly ordered and spatially restricted sites for different signal-transduction pathways. SSC associated proteins are disrupted in axotomised MNs together with the activation of microglia, which display a positive chemotactism to C-bouton sites. This indicates that C-bouton associated molecules are also involved in neuroinflammatory signalling in diseased MNs, emerging as new potential therapeutic targets.