Olga Tarabal

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.

Regulation of Motoneuronal Calcitonin Gene–related Peptide (CGRP) During Axonal Growth and Neuromuscular Synaptic Plasticity Induced by Botulinum Toxin in Rats

The aim of this study was to examine whether changes in rat motoneuronal calcitonin gene–related peptide (CGRP) can be correlated with axonal growth and plasticity of neuromuscular synapses. Nerve terminal outgrowth was induced by local paralysis with botulinum toxin. Normal adult soleus and tibialis anterior did not show detectable CGRP content at the motor endplates. Following botulinum toxin injection there was a progressive, transient and bimodal increase in CGRP in both motoneuron cell bodies which innervated poisoned muscles and their motor endplates. CGRP content was moderately increased 1 day after paralysis and, after an initial decline, reached a peak 20 days after injection. This was followed by a gradual decrease and a return to normal levels at the 200th day. CGRP changes in intoxicated endplates were less evident in the tibialis anterior than in the soleus muscle. The CGRP content in motoneurons was positively correlated with the degree of intramuscular nerve sprouting found by silver staining. In situ hybridization revealed an increase in CGRP mRNA in spinal cord motoneurons 20 days after toxin administration. We conclude that motoneurons regulate their CGRP in situations in which peripheral synapse remodelling and plasticity occur.

Protein retention in the endoplasmic reticulum, blockade of programmed cell death and autophagy selectively occur in spinal cord motoneurons after glutamate receptor-mediated injury

We previously showed that, in contrast to the acute administration of NMDA, chronic treatment of chick embryos from embryonic day (E) 5 to E9 with this excitotoxin rescues motoneurons (MNs) from programmed cell death. Following this protocol, MNs are also protected against later acute excitotoxic cell death. Previously, we found that MNs treated from E5 to E9 develop long-lasting changes involving vesicular trafficking and other organelle pathology similar to the abnormalities observed in certain chronic neurological diseases including amyotrophic lateral sclerosis (ALS). Here we extend these previous results by showing that protein aggregation within the endoplasmic reticulum (ER) takes place selectively in MNs as an early event of chronic excitotoxicity. Although protein aggregates do not induce appreciable MN death, they foreshadow the activation of a conspicuous autophagic response leading to long-lasting degenerative changes that causes dysfunction but not immediate cell death. Chronic early treatment with NMDA results in a transient (between E6 and E10) lack of vulnerability to undergo cell death induced by different types of stimuli. It is suggested that blockade of protein translation in stressed ER may inhibit apoptosis in NMDA-treated MNs. However, in embryos older than E12, degenerating MNs are sensitized to die after limb ablation (axotomy) and accumulate hyperphosphorylated neurofilaments. Moreover, chronic NMDA treatment does not induce the upregulation of molecular chaperones in spinal cord. These results represent a new model of glutamate receptor-mediated neurotoxicity that selectively occurs in spinal cord MNs and also demonstrate an experimental system that may be valuable for understanding the mechanisms involved in chronic MN degeneration and in certain cytological hallmarks of ALS-diseased MNs such as inclusion bodies.

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.

Survival and death of mature avian motoneurons in organotypic slice culture: trophic requirements for survival and different types of degeneration.

We have developed an organotypic culture technique that uses slices of chick embryo spinal cord, in which trophic requirements for long‐term survival of mature motoneurons (MNs) were studied. Slices were obtained from E16 chick embryos and maintained for up to 28 days in vitro (DIV) in a basal medium. Under these conditions, most MNs died. To promote MN survival, 14 different trophic factors were assayed. Among these 14, glial cell line‐derived neurotrophic factor (GDNF) and vascular endothelial growth factor were the most effective. GDNF was able to promote MN survival for at least 28 DIV. K+ depolarization or caspase inhibition prevented MN death but also induced degenerative‐like changes in rescued MNs. Agents that elevate cAMP levels promoted the survival of a proportion of MNs for at least 7 DIV. Examination of dying MNs revealed that, in addition to cells exhibiting a caspase‐3‐dependent apoptotic pattern, some MNs died by a caspase‐3‐independent mechanism and displayed autophagic vacuoles, an extremely convoluted nucleus, and a close association with microglia. This organotypic spinal cord slice culture may provide a convenient model for testing conditions that promote survival of mature‐like MNs that are affected in late‐onset MN disease such as amyotrophic lateral sclerosis. J. Comp. Neurol. 501:669–690, 2007.

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.

Mechanisms involved in spinal cord central synapse loss in a mouse model of spinal muscular atrophy.

Motoneuron (MN) cell death is the histopathologic hallmark of spinal muscular atrophy (SMA), although MN loss seems to be a late event. Conversely, disruption of afferent synapses on MNs has been shown to occur early in SMA. Using a mouse model of severe SMA (SMNΔ7), we examined the mechanisms involved in impairment of central synapses. We found that MNs underwent progressive degeneration in the course of SMA, with MN loss still occurring at late stages. Loss of afferent inputs to SMA MNs was detected at embryonic stages, long before MN death. Reactive microgliosis and astrogliosis were present in the spinal cord of diseased animals after the onset of MN loss. Ultrastructural observations indicate that dendrites and microglia phagocytose adjacent degenerating presynaptic terminals. Neuronal nitric oxide synthase was upregulated in SMNΔ7 MNs, and there was an increase in phosphorylated myosin light chain expression in synaptic afferents on MNs; these observations implicate nitric oxide in MN deafferentation and suggest that the RhoA/ROCK pathway is activated. Together, our observations suggest that the earliest change occurring in SMNΔ7 mice is the loss of excitatory glutamatergic synaptic inputs to MNs; reduced excitability may enhance their vulnerability to degeneration and death.

Intramuscular nerve sprouting induced by CNTF is associated with increases in CGRP content in mouse motor nerve terminals

It is known that motor nerve terminal sprouting induced by either nerve injury or muscle paralysis is associated with an increase in calcitonin gene-related peptide (CGRP) content in the soma of motoneurons and in motor endplates. In the present study, CGRP-like immunoreactivity (CGRP-LI) was determined in motor endplates of animals in which nerve terminal sprouting had been induced by exogenous application of ciliary neurotrophic factor (CNTF). After 18 days of CNTF treatment we observed a significant increase in CGRP-LI in motor endplates. The results indicate that CGRP is upregulated when motor nerve outgrowth is induced, even in the absence of muscle paralysis or nerve lesion.

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.