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Peripheral nerve section with subsequent disconnection of motor neuron (MN) cell bodies from their skeletal muscle targets leads to a rapid reactive response involving the recruitment and activation of microglia. In addition, the loss of afferent synapses on MNs occurs in concomitance with microglial reaction by a process described as synaptic stripping. However, the way in which postaxotomy‐activated microglia adjacent to MNs are involved in synaptic removal is less defined. Here, we used confocal and electron microscopy to examine interactions between recruited microglial cells and presynaptic terminals in axotomized MNs between 1 and 15 days after sciatic nerve transection in mice. We did not observe any bulk engulfment of synaptic boutons by microglia. Instead, microglial cells internalized small membranous‐vesicular fragments which originated from the acute disruption of synaptic terminals involving the activation of the necroptotic pathway. The presence of abundant extracellular vesicles in the perineuronal space after axotomy, together with the increased expression of phospho‐mixed lineage kinase domain‐like protein and, later, of extracellular vesicle markers, such as CD9, CD63, and flotillin, indicate that the vesicles mainly originated in synapses and were transferred to microglia. The upregulation of Rab7 and Rab10 in microglia interacting with injured MNs, indicated the activation of endocytosis. As activated microglia and synaptic boutons displayed positive C1q immunoreactivity, a complement‐mediated opsonization may also contribute to microglial‐mediated synaptic disruption. In addition to the relevance of our data in the context of neuroinflammation and MN disease, they should also be taken into account for understanding functional recovery after peripheral nerve injury., Main Points Early after axotomy, microglia recruited near injured motor neurons, emit processes that tend to contact their afferent synaptic terminals.Extracellular vesicles resulting from necroptotic synaptic disruption are removed by microglia.

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. This work was supported by Abbott and a grant from the Ministerio de Ciencia, Innovación y Universidades cofinancedby Fondo Europeo de Desarrollo Regional (RTI2018-099278-B-I00 to J.C. and J.E.)

Early misfolded superoxide dismutase 1 (mfSOD1) accumulation, motor neuron (MN) degeneration, and microgliosis are hallmark pathological features in SOD1

This work was supported by Abbott and a grant from the Spanish Ministerio de Ciencia, Innovacion y Universidades cofinanced by Fondo Europeo de Desarrollo Regional (RTI2018-099278-B-I00 to JC and JE) ., Besides skeletal muscle wasting, sarcopenia entails morphological and molecular changes in distinct components of the neuromuscular system, including spinal cord motoneurons (MNs) and neuromuscular junctions (NMJs); moreover, noticeable microgliosis has also been observed around aged MNs. Here we examined the impact of two flavonoid-enriched diets containing either green tea extract (GTE) catechins or cocoa flavanols on age-associated regressive changes in the neuromuscular system of C57BL/6J mice. Compared to control mice, GTE- and cocoa-supplementation significantly improved the survival rate of mice, reduced the proportion of fibers with lipofuscin aggregates and central nuclei, and increased the density of satellite cells in skeletal muscles. Additionally, both supplements significantly augmented the number of innervated NMJs and their degree of maturity compared to controls. GTE, but not cocoa, prominently increased the density of VAChT and VGluT2 afferent synapses on MNs, which were lost in control aged spinal cords; conversely, cocoa, but not GTE, significantly augmented the proportion of VGluT1 afferent synapses on aged MNs. Moreover, GTE, but not cocoa, reduced aging-associated microgliosis and increased the proportion of neuroprotective microglial phenotypes. Our data indicate that certain plant flavonoids may be beneficial in the nutritional management of age-related deterioration of the neuromuscular system., Abbott Laboratories, Spanish Ministerio de Ciencia, Innovacion y Universidades, European Commission RTI2018-099278-B-I00

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 Smn2B/− mouse, in which we performed a detailed characterization of the histopathological changes that occur throughout the disease. We report that Smn2B/− mice exhibited qualitative differences in major alterations found in mouse models of severe SMA: Smn2B/− 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 Smn2B/− 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. This work was supported by grants from the Ministerio de Economía y Competitividad co-financed by FEDER (SAF2015-70801).

C boutons are large, cholinergic, synaptic terminals that arise from local interneurons and specifically contact spinal α-motoneurons (MNs). C boutons characteristically display a postsynaptic specialization consisting of an endoplasmic reticulum-related subsurface cistern (SSC) of unknown function. In the present work, by using confocal microscopy and ultrastructural immunolabeling, we demonstrate that neuregulin-1 (NRG1) accumulates in the SSC of mouse spinal MNs. We also show that the NRG1 receptors erbB2 and erbB4 are presynaptically localized within C boutons, suggesting that NRG1-based retrograde signaling may occur in this type of synapse. In most of the cranial nuclei, MNs display the same pattern of NRG1 distribution as that observed in spinal cord MNs. Conversely, MNs in oculomotor nuclei, which are spared in amyotrophic lateral sclerosis (ALS), lack both C boutons and SSC-associated NRG1. NRG1 in spinal MNs is developmentally regulated and depends on the maintenance of nerve-muscle interactions, as we show after nerve transection experiments. Changes in NRG1 in C boutons were also investigated in mouse models of MN diseases: i.e., spinal muscular atrophy (SMNΔ7) and ALS (SOD1(G93A)). In both models, a transient increase in NRG1 in C boutons occurs during disease progression. These data increase our understanding of the role of C boutons in MN physiology and pathology.

In the chick embryo, in ovo application of NMDA from embryonic day (E) 5 to E9 results in selective damage to spinal cord motoneurons (MNs) that undergo a long-lasting degenerative process without immediate cell death. This contrasts with a single application of NMDA on E8, or later, which induces massive necrosis of the whole spinal cord. Chronic MN degeneration after NMDA implies transient incompetence to develop programmed cell death, altered protein processing within secretory pathways, and late activation of autophagy. Chronic NMDA treatment also results in an enlargement of thapsigargin-sensitive Ca(2+) stores. In particular MN pools, such as sartorius-innervating MNs, the neuropeptide CGRP is accumulated in somas, peripheral axons and neuromuscular junctions after chronic NMDA treatment, but not in embryos paralyzed by chronic administration of curare. Intramuscular axonal branching is also altered severely after NMDA: it usually increases, but in some cases a marked reduction can also be observed. Moreover, innervated muscle postsynaptic sites increase by NMDA, but to a lesser extent than by curare. Because some of these results show interesting homologies with MN pathology in human sporadic ALS, the model presented here provides a valuable tool for advancing in the understanding of some cellular and molecular processes particularly involved in this disease.

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disease affecting upper and lower motoneurons (MNs). Although the motor phenotype is a hallmark for ALS, there is increasing evidence that systems other than the efferent MN system can be involved. Mutations of superoxide dismutase 1 (SOD1) gene cause a proportion of familial forms of this disease. Misfolding and aggregation of mutant SOD1 exert neurotoxicity in a noncell autonomous manner, as evidenced in studies using transgenic mouse models. Here, we used the SOD1(G93A) mouse model for ALS to detect, by means of conformational-specific anti-SOD1 antibodies, whether misfolded SOD1-mediated neurotoxicity extended to neuronal types other than MNs. We report that large dorsal root ganglion (DRG) proprioceptive neurons accumulate misfolded SOD1 and suffer a degenerative process involving the inflammatory recruitment of macrophagic cells. Degenerating sensory axons were also detected in association with activated microglial cells in the spinal cord dorsal horn of diseased animals. As large proprioceptive DRG neurons project monosynaptically to ventral horn MNs, we hypothesise that a prion-like mechanism may be responsible for the transsynaptic propagation of SOD1 misfolding from ventral horn MNs to DRG sensory neurons. The authors would like to thank Montse Ortega for her technical assistance and Claudia Cervero, Alexandra Eritja and Ariadna Salvador for their help with some of the experiments in this study. This work was supported by grants from the Ministerio de Ciencia y Tecnologia and Ministerio de Economia y Competitividad and cofinanced by FEDER (SAF2011-22908; SAF2012-31831).

Motoneurons respond to peripheral nerve transection by either regenerative or degenerative events depending on their state of maturation. Since the expression of c-Jun has been involved in the early signalling of the regenerative process that follows nerve transection in adults, we have investigated c-Jun on rat neonatal axotomized motoneurons during the period in which neuronal death is induced. Changes in levels of c-Jun protein and its mRNA were determined by means of quantitative immunocytochemistry and in situ hybridization. Three hours after nerve transection performed on postnatal day (P)3, c-Jun protein and mRNA is induced in axotomized spinal cord motoneurons, and high levels were reached between 1 and 10 days after. This response is associated with a detectable c-Jun activation by phosphorylation on serine 63. No changes were found in the levels of activating transcription factor -2. Most of dying motoneurons were not labelled by either a specific c-Jun antibody or a c-jun mRNA probe. However, dying motoneurons were specifically stained by a polyclonal anti c-Jun antibody, indicating that some c-Jun antibodies react with unknown epitopes, probably distinct from c-Jun p39, that are specifically associated with apoptosis. J. Neurosci. Res. 63:469–479, 2001. © 2001 Wiley-Liss, Inc.

Individuals with Down syndrome (DS) present important motor deficits that derive from altered motor development of infants and young children. DYRK1A, a candidate gene for DS abnormalities has been implicated in motor function due to its expression in motor nuclei in the adult brain, and its overexpression in DS mouse models leads to hyperactivity and altered motor learning. However, its precise role in the adult motor system, or its possible involvement in postnatal locomotor development has not yet been clarified. During the postnatal period we observed time-specific expression of Dyrk1A in discrete subsets of brainstem nuclei and spinal cord motor neurons. Interestingly, we describe for the first time the presence of Dyrk1A in the presynaptic terminal of the neuromuscular junctions and its axonal transport from the facial nucleus, suggesting a function for Dyrk1A in these structures. Relevant to DS, Dyrk1A overexpression in transgenic mice (TgDyrk1A) produces motor developmental alterations possibly contributing to DS motor phenotypes and modifies the numbers of motor cholinergic neurons, suggesting that the kinase may have a role in the development of the brainstem and spinal cord motor system. The laboratory of MD is supported by Departament d’Universitats, Recerca i Societat de la Informació (DURSI) (Grups consolidats 09 2009SGR1313). This work was supported by FCT-08-0782, SAF2007-60827, SAF2010-16427, SAF2007-31093-E, Fondo de Investigación Sanitaria (FIS) (PI 082038), Marató TV3, Jerome Lejeune, Reina Sofia and Areces Foundations and EU (LSHG-CT-2006-037627; CureFXS ERare-EU/FIS PS09102673). The CIBER of Enfermedades Raras is an initiative of the ISCIII. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Massive programmed cell death (PCD) of developing chick embryo motoneurons (MNs) occurs in a well defined temporal and spatial sequence between embryonic day (E) 6 and E10. We have found that, when administered in ovo, either circulating immunoglobulins G (IgGs) or cerebrospinal fluid from patients with MN disease can rescue a significant number of chick embryo MNs from normally occurring PCD. An increase of branching of intramuscular nerves was also observed that may account for the rescuing effects of pathologic IgGs. Proteomic analysis and further analysis by ELISA indicated that these effects may be mediated by the interaction of circulating human immunoglobulins with proteins of the semaphorin family. (C) 2010 Elsevier B.V. All rights reserved.

The distribution of the P2X family of ATP receptors was analyzed in a rat model for amyotrophic lateral sclerosis (ALS) expressing mutated human superoxide dismutase (mSOD1G93A). We showed that strong P2X4 immunoreactivity was selectively associated with degenerating motoneurons (MNs) in spinal cord ventral horn. Degenerating P2X4-positive MNs did not display apoptotic features such as chromatin condensation, positive TUNEL reaction, or active caspase 3 immunostaining. In contrast, these neurons showed other signs of abnormality, such as loss of the neuronal marker NeuN and recruitment of microglial cells with neuronophagic activity. Similar changes were observed in MNs from the cerebral cortex and brainstem in mSOD1G93A in both rat and mice. In addition, P2X4 immunostaining demonstrated the existence of neuronal degeneration in the locus coeruleus, reticular formation, and Purkinje cells of the cerebellar cortex. It is suggested that abnormal trafficking and proteolytic processing of the P2X4 receptor protein may underlie these changes. J. Comp. Neurol. 506:75–92, 2008. © 2007 Wiley-Liss, Inc.

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. We would like to thank Dr. Ronald W. Oppenheim for his critical reading of the manuscript and for helpful comments and suggestions. We would also like to thank Marta Hereu for her technical assistance, Lidia Delgado, Gema Marta Martínez and M. Yolanda Muela, from the Unitat de Criomicroscòpia Electrònica (Centres Científics i Tecnològics de la Universitat de Barcelona), for their technical support with ultrastructural immunolabelling, Daniel Cabezas for his help in some experiments, and the SCT animal facility of the Universitat de Lleida for mouse care and housing. This work was supported by grants from the Ministerio de Economía y Competitividad cofinanced by FEDER (SAF2015–70801-R to J.E.E. and J.C.), and from Jack Van den Hoek a la investigació de l’ELA - Fundació Miquel Valls (to J.E.E.).