Lídia Piedrafita

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.

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.

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.

Immunodetection of disease-associated conformers of mutant cu/zn superoxide dismutase 1 selectively expressed in degenerating neurons in amyotrophic lateral sclerosis.

We previously showed that some antipurinergic receptor P2X4 antibodies cross react with misfolded forms of amyotrophic lateral sclerosis (ALS)-linked mutant Cu/Zn superoxide dismutase (SOD1). Cross reactivity might be caused by abnormal exposure of an epitope in the inner hydrophobic region of SOD1 that shares structural homology with the P2X4-immunizing peptide. Here, we raised antibodies against the human SOD1 epitope mimicked by the P2X4 immunizing peptide. One of these antibodies, AJ10, is a recognized mutant/misfolded form of ALS-linked mutant SOD1. This was demonstrated in the hybrid motoneuron cell line NSC34 expressing enhanced green fluorescent protein-tagged G943A or A4V mutant SOD1. We also found AJ10 immunoreactivity to be selectively associated with degenerating neurons but not with glial cells in mice overexpressing either SOD1 or SOD1 mutants. Neurons with strongly positive AJ10 immunostaining were often associated with activated microglia displaying neuronophagic activity. AJ10-immunopositive SOD1 aggregates were also found in spinal cord tissue from a patient with a SOD1-linked familial ALS. AJ10-immunoreactive mutant SOD1 conformers were localized in large intracellular protein aggregates with a filamentous amyloid-like organization by ultrastructural immunolabeling and were also detected in neuronal organelles. These data are consistent with the ability of the AJ10 antibody to recognize misfolded conformations of SOD1 shared by different ALS-linked SOD1 mutations but not with the native protein. The neuronal mutant SOD1 conformers detected with AJ10 may promote neuroinflammation and may define a new epitope in SOD1 for ALS research.

Chronic Treatment with the AMPK Agonist AICAR Prevents Skeletal Muscle Pathology but Fails to Improve Clinical Outcome in a Mouse Model of Severe Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder characterized by spinal and brainstem motor neuron (MN) loss and skeletal muscle paralysis. Currently, there is no effective treatment other than supportive care to ameliorate the quality of life of patients with SMA. Some studies have reported that physical exercise, by improving muscle strength and motor function, is potentially beneficial in SMA. The adenosine monophosphate-activated protein kinase agonist 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) has been reported to be an exercise mimetic agent that is able to regulate muscle metabolism and increase endurance both at rest and during exercise. Chronic AICAR administration has been shown to ameliorate the dystrophic muscle phenotype and motor behavior in the mdx mouse, a model of Duchenne muscular dystrophy. Here, we investigated whether chronic AICAR treatment was able to elicit beneficial effects on motor abilities and neuromuscular histopathology in a mouse model of severe SMA (the SMNΔ7 mouse). We report that AICAR improved skeletal muscle atrophy and structural changes found in neuromuscular junctions of SMNΔ7 animals. However, although AICAR prevented the loss of glutamatergic excitatory synapses on MNs, this compound was not able to mitigate MN loss or the microglial and astroglial reaction occurring in the spinal cord of diseased mice. Moreover, no improvement in survival or motor performance was seen in SMNΔ7 animals treated with AICAR. The beneficial effects of AICAR in SMA found in our study are SMN-independent, as no changes in the expression of this protein were seen in the spinal cord and skeletal muscle of diseased animals treated with this compound.

Increased intramuscular nerve branching and inhibition of programmed cell death of chick embryo motoneurons by immunoglobulins from patients with motoneuron disease.

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.

Neuregulin 1-ErbB module in C-bouton synapses on somatic motor neurons: molecular compartmentation and response to peripheral nerve injury

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.

Glial Activation and Central Synapse Loss, but Not Motoneuron Degeneration, Are Prevented by the Sigma-1 Receptor Agonist PRE-084 in the Smn2B/− Mouse Model of Spinal Muscular Atrophy

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.

Synaptic localization of a 66-kDa soluble protein from skeletal muscle: Evidence for its developmental and neural regulation

Soluble proteins from normal, adult denervated, and developing rat muscles were studied in order to identify common molecular species undergoing developmental regulation and nerve dependence. Significant increases in 66- and 30-kDa proteins were found as a consequence of 14 days of denervation. Subsequent reinnervation restores normal adult levels. During development, high levels of the 66-kDa protein were found in neonatal muscles but slowly decreased concomitant with the following postnatal maturation period; the adult levels were reached at Postnatal Day (P) 21. From the immunocytochemical studies it is deduced that both proteins were concentrated mainly at the end-plate region in adult normal muscle. Following denervation, the proteins were found distributed over the entire cell. For the 66-kDa protein, a similar pattern of extensive distribution was seen in immature muscle. Although no data for functional implications for these proteins are available at present, the properties described here make them of interest in understanding nerve-muscle interactions.