Anna Casanovas

Defective Neuromuscular Junction Organization and Postnatal Myogenesis in Mice With Severe Spinal Muscular Atrophy

A detailed pathologic analysis was performed on Smn−/−;SMN2+/+ mice as a mouse model for human type I spinal muscular atrophy (SMA). We provide new data concerning changes in the spinal cord, neuromuscular junctions and muscle cells, and in the organs of the immune system. The expression of 10 synaptic proteins was analyzed in 3-dimensionally reconstructed neuromuscular junctions by confocal microscopy. In addition to defects in postsynaptic occupancy, there was a marked reduction in calcitonin gene-related peptide and Rab3A in the presynaptic motor terminals of some, but not all, of the skeletal muscles analyzed. Defects in the organization of presynaptic nerve terminals were also detected by electron microscopy. Moreover, degenerative changes in muscle cells, defective postnatal muscle growth, and prominent muscle satellite cell apoptosis were also observed. All of these changes occurred in the absence of massive loss of spinal cord motoneurons. On the other hand, astroglia, but not microglia, increased in the ventral horn of newborn SMA mice. In skeletal muscles, the density of interstitial macrophages was significantly reduced, and monocyte chemotactic protein-1 was downregulated. These findings raise questions regarding the primary contribution of a muscle cell defect to the SMA phenotype.

Nitric oxide synthase in rat neuromuscular junctions and in nerve terminals of torpedo electric organ: Its role as regulator of acetylcholine release

The distribution of nitric oxide synthase on peripheral motor system was studied using a specific antibody against the neuronal isoform of nitric oxide synthase (nNOS). The immunoreactivity for nNOS was detected on the sarcolemmal surface of muscle cells, in intramuscular axons and in neuromuscular synapses. At the neuromuscular junctions, ultrastructural immunolabeling demonstrated that nNOS immunoreactivity was localized mainly into the presynaptic nerve terminals as well as adjacent postsynaptic muscle membrane. Similar immunostaining pattern was present in frog muscles and Torpedo electric organs. After chronic muscle denervation, nNOS immunoreactity at endplate level decreased during the first week but it was upregulated after 30 days of denervation. In denervated endplates, nNOS immunoreactivity was localized in the terminal Schwann cells covering the degenerated neuromuscular junctions whereas nNOS was not detected in Schwann cells under normal conditions. In Torpedo synaptosomes, acetylcholine (ACh) release elicited by potassium depolarization was inhibited by NO donors such as sodium nitroprusside. In contrast, application of inhibitors of NOS activity, aminoguanidine (AMG) and Nω‐Nitro‐L‐arginine methyl esther (L‐NAME) increased acetylcholine release. These results indicate that nNOS is present at the motor nerve terminals in a variety of vertebrates and that it may be involved in the physiological modulation of ACh release and in the regulation of muscle response to nerve injury.

Strong P2X4 purinergic receptor-like immunoreactivity is selectively associated with degenerating neurons in transgenic rodent models of amyotrophic lateral sclerosis.

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.

c-Jun regulation in rat neonatal motoneurons postaxotomy.

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.

Neuregulin-1 is concentrated in the postsynaptic subsurface cistern of C-bouton inputs to α-motoneurons and altered during motoneuron diseases

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 (SOD1G93A). 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.—Gallart‐Palau, X., Tarabal, O., Casanovas, A., Sábado, J., Correa, F. J., Hereu, M., Piedrafita, L., Calderó, J., Esquerda, J. E. Neuregulin‐1 is concentrated in the postsynaptic subsurface cistern of C‐bouton inputs to α‐motoneurons and altered during motoneuron diseases. FASEB J. 28, 3618–3632 (2014). www.fasebj.org

Accumulation of Misfolded SOD1 in Dorsal Root Ganglion Degenerating Proprioceptive Sensory Neurons of Transgenic Mice with Amyotrophic Lateral Sclerosis

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 SOD1G93A 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.

Induction of reactive astrocytosis and prevention of motoneuron cell death by the I2-imidazoline receptor ligand LSL 60101

I2‐imidazoline receptors are mainly expressed on glial cells in the rat brain. This study was designed to test the effect of treatment with the I2‐imidazoline selective receptor ligand LSL 60101 [2‐(2‐benzofuranyl)imidazole] on the morphology of astrocytes in the neonate and adult rat brain, and to explore the putative neuroprotective effects of this glial response. Short‐term (3 days) or chronic (7–10 days) treatment with LSL 60101 (1 mg kg−1, i.p. every 12 h) enhanced the area covered by astroglial cells in sections of facial motor nucleus from neonate rats processed for glial fibrillary acidic protein (GFAP) immunostaining. Facial motoneurons surrounded by positive glial cell processes were frequently observed in sections of LSL 60101‐treated rats. A similar glial response was observed in the parietal cortex of adult rats after chronic (10 days) treatment with LSL 60101 (10 mg kg−1, i.p. every 12 h). Western‐blot detection of the specific astroglial glutamate transporter GLT‐1, indicated increased immunoreactivity after LSL 60101 treatment in the pons of neonate and in the parietoccipital cortex of adult rats. In the facial motor nucleus of neonate rats, the glial response after LSL 60101 treatment was associated to a redistribution of the immunofluorescence of the basic fibroblast growth factor (FGF‐2) from the perinuclear area of motoneurons to cover most of their cytoplasm, suggesting a translocation of this mitogenic and neurotrophic factor towards secretion pathways. The neuroprotective potential of the above effects of LSL 60101 treatment was tested after neonatal axotomy of facial motor nucleus. Treatment with LSL 60101 (1 mg kg−1, i.p. every 12 h from day 0 to day 10 after birth) significantly reduced (38%) motoneuron death rate 7 days after facial nerve axotomy performed on day 3 after birth. It is concluded that treatment with the I2‐imidazoline selective receptor ligand LSL 60101 provokes morphological/biochemical changes in astroglia that are neuroprotective after neonatal axotomy.

Occurrence of glutamate receptor subunit 1–containing aggresome-like structures during normal development of rat spinal cord interneurons

During a developmental study of the expression of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) ‐type glutamate receptor subunits in rat spinal cord, we observed the existence of cytoplasmic inclusion bodies with positive immunoreactivity to glutamate receptor subunit 1 (GluR1) but not to other glutamate receptor subunits. GluR1‐positive bodies have a diameter of between 1 and 3 μm and can be seen widely distributed throughout spinal cord gray matter, with the exception of the ventral horn region. They transiently appear within a definite developmental time‐period between embryonic day 19 and postnatal day 17 and are only associated with neuronal cells. Ultrastructural analysis revealed that these inclusions were located adjacent to the nucleus and consisted of amorphous material without any limiting membrane. Immunocytochemical analysis revealed that the inclusions displayed positive immunoreactivity to ubiquitin, HSP70, and 20S proteasome. All these data indicate that GluR1‐containing inclusions display all the ultrastructural and immunocytochemical characteristics of the recently described structure, which have been given the name aggresomes. Further studies are needed to determine the biological significance of these normally occurring aggresome‐like inclusions, because aggresomes are usually considered in a pathologic context. J. Comp. Neurol. 442:23–34, 2002.

Neurotoxic species of misfolded SOD1G93A recognized by antibodies against the P2X4 subunit of the ATP receptor accumulate in damaged neurons of transgenic animal models of amyotrophic lateral sclerosis.

We recently reported that degenerating motor neurons of superoxide dismutase mutant 1 (SOD1G93A) rodents exhibit immunoreactivity to P2X4 antibodies. Neurons with strong P2X4-like immunoreactivity (P2X4-LIR) do not show an apoptotic phenotype and are often associated with microglial cells that display neuronophagic activity. Western blot analysis showed that P2X4 antibodies recognize not only the P2X4 adenosine triphosphate receptor protein but also a hitherto unidentified low-molecular weight band. Here, we identify the molecular counterpart of the strong P2X4-LIR observed in association with neuronal degeneration in SOD1G93A animals. After matrix-assisted laser desorption/ionization time-of-flight, we found that the low-molecular weight P2X4-immunoreactive protein was SOD1. Further analysis demonstrated that the P2X4 antibody recognizes a form of misfolded mutant SOD1G93A that is expressed in neuronal cells undergoing degeneration but not in glial cells. Cross-reactivity could have been caused by the abnormal exposure of an epitope in the inner hydrophobic region of SOD1 that shared structural homology with the P2X4-immunizing peptide used for raising the antibody. No positive P2X4 immunostaining was detected in mice overexpressing human wild-type SOD1. Intracerebral injections of affinity chromatography-isolated P2X4-immunoreactive SOD1G93A species promote microglial and astroglial activation. We conclude that neuronal SOD1G93A conformers with P2X4-LIR may have pathogenetic relevance in the promotion of neuroinflammation.

Excitotoxic motoneuron disease in chick embryo evolves with autophagic neurodegeneration and deregulation of neuromuscular innervation

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 Ca2+ 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.