Persistent NRG1 Type III Overexpression in Spinal Motor Neurons Has No Therapeutic Effect on ALS-Related Pathology in SOD1G93A Mice.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting upper and lower motor neurons (MNs). Neuregulin-1 (NRG1) is a pleiotropic growth factor that has been shown to be potentially valuable for ALS when supplemented by means of viral-mediated gene therapy. However, these results are inconsistent with other reports. An alternative approach for investigating the therapeutic impact of NRG1 on ALS is the use of transgenic mouse lines with genetically defined NRG1 overexpression. Here, we took advantage of a mouse line with NRG1 type III overexpression in spinal cord α motor neurons (MN) to determine the impact of steadily enhanced NRG1 signalling on mutant superoxide dismutase 1 (SOD1)-induced disease. The phenotype of SOD1G93A-NRG1 double transgenic mice was analysed in detail, including neuropathology and extensive behavioural testing. At least 3 animals per condition and sex were histopathologically assessed, and a minimum of 10 mice per condition and sex were clinically evaluated. The accumulation of misfolded SOD1 (mfSOD1), MN degeneration, and a glia-mediated neuroinflammatory response are pathological hallmarks of ALS progression in SOD1G93A mice. None of these aspects was significantly improved when examined in double transgenic NRG1-SOD1G93A mice. In addition, behavioural testing revealed that NRG1 type III overexpression did not affect the survival of SOD1G93A mice but accelerated disease onset and worsened the motor phenotype.

NOD mouse dorsal root ganglia display morphological and gene expression defects before and during autoimmune diabetes development.

Introduction: During the development of Autoimmune Diabetes (AD) an autoimmune attack against the Peripheral Nervous System occurs. To gain insight into this topic, analyses of Dorsal Root Ganglia (DRG) from Non-Obese Diabetic (NOD) mice were carried out. Methods: Histopathological analysis by electron and optical microscopy in DRG samples, and mRNA expression analyzes by the microarray technique in DRG and blood leukocyte samples from NOD and C57BL/6 mice were performed. Results: The results showed the formation of cytoplasmic vacuoles in DRG cells early in life that could be related to a neurodegenerative process. In view of these results, mRNA expression analyses were conducted to determine the cause and/or the molecules involved in this suspected disorder. The results showed that DRG cells from NOD mice have alterations in the transcription of a wide range of genes, which explain the previously observed alterations. In addition, differences in the transcription genes in white blood cells were also detected. Discussion: Taken together, these results indicate that functional defects are not only seen in beta cells but also in DRG in NOD mice. These results also indicate that these defects are not a consequence of the autoimmune process that takes place in NOD mice and suggest that they may be involved as triggers for its development.

Accumulation of misfolded SOD1 outlines distinct patterns of motor neuron pathology and death during disease progression in a SOD1G93A mouse model of amyotrophic lateral sclerosis.

Early misfolded superoxide dismutase 1 (mfSOD1) accumulation, motor neuron (MN) degeneration, and microgliosis are hallmark pathological features in SOD1G93A amyotrophic lateral sclerosis (ALS) mice. Because of the different vulnerabilities of distinct MN subtypes, degenerating and surviving MNs coexist in different proportions during disease progression. By examining the expression of misfolded conformers of SOD1 using specific antibodies, we defined distinct MN phenotypes that were evaluated during disease progression and the local neuroinflammatory reaction. The most severe phenotype corresponded to somata of fast-twitch subtype MNs, which exhibited highly positive mfSOD1 immunostaining and an extreme degree of vacuolar degeneration. Vacuoles, which are of mitochondrial origin, contain mfSOD1 in conjunction with nonmitochondrial proteins, such as chromogranin, CD81, and flotillin. The fusion of ER-derived vesicles enriched in mfSOD1 with outer mitochondrial membranes is thought to be the primary mechanism for vacuole formation. In addition, the ulterior coalescence of enlarged mitochondria may lead to the formation of giant vacuoles. Vacuolar degeneration is a transient degenerative process occurring early during the presymptomatic stages of the disease in ALS mice. Some vacuolated MNs are also positive for pMLKL, the effector protein of necroptosis. This indicates a newly described mechanism in which extracellular vesicles derived from damaged MNs, via cellular secretion or necroptotic disruption, may be the triggers for initiating neuroinflammation, glial-mediated neurotoxicity, and disease spreading. Furthermore, as MN degeneration in mutant SOD1 mice is noncell autonomous, the effects of experimentally increasing or decreasing the microglial response on the expression of MN phenotypes were also evaluated, demonstrating bidirectional cross talk signaling between the degree of expression of mfSOD1 and local neuroinflammation. More detailed knowledge regarding these processes occurring long before the end stages of the disease is necessary to identify novel molecular targets for future preclinical testing.

Beneficial effects of dietary supplementation with green tea catechins and cocoa flavanols on aging-related regressive changes in the mouse neuromuscular system.

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.

Microglial recruitment and mechanisms involved in the disruption of afferent synaptic terminals on spinal cord motor neurons after acute peripheral nerve injury.

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.

Investigative monitoring of pesticide and nitrogen pollution sources in a complex multi-stressed catchment: The lower Llobregat River basin case study (Barcelona, Spain).

The management of the anthropogenic water cycle must ensure the preservation of the quality and quantity of water resources and their careful allocation to the different uses. Protection of water resources requires the control of pollution sources that may deteriorate them. This is a challenging task in multi-stressed catchments. This work presents an approach that combines pesticide occurrence patterns and stable isotope analyses of nitrogen (δ15N-NO3-, δ15N-NH4+), oxygen (δ18O-NO3-), and boron (δ11B) to discriminate the origin of pesticides and nitrogen-pollution to tackle this challenge. The approach has been applied to a Mediterranean sub-catchment subject to a variety of natural and anthropogenic pressures. Combining the results from both analytical approaches in selected locations of the basin, the urban/industrial activity was identified as the main pressure on the quality of the surface water resources, and to a large extent also on the groundwater resources, although agriculture may play also an important role, mainly in terms of nitrate and ammonium pollution. Total pesticide concentrations in surface waters were one order of magnitude higher than in groundwaters and believed to originate mainly from soil and/or sediments desorption processes and urban and industrial use, as they were mainly associated with treated wastewaters. These findings were supported by the stable isotope results that pointed to an organic origin of nitrate in surface waters and most groundwater samples. Ammonium pollution observed in some aquifer locations is probably generated by nitrate reduction. Overall, no significant attenuation processes could be inferred for nitrate pollution. The approach presented here exemplifies the investigative monitoring envisioned in the Water Framework Directive.

Motoneuron deafferentation and gliosis occur in association with neuromuscular regressive changes during ageing in mice.

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-ß1. 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.

Localization and dynamic changes of neuregulin-1 at C-type synaptic boutons in association with motor neuron injury and repair.

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.

The Y172 Monoclonal Antibody Against p-c-Jun (Ser63) Is a Marker of the Postsynaptic Compartment of C-Type Cholinergic Afferent Synapses on Motoneurons.

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.

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.

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.

Cryostat Slice Irregularities May Introduce Bias in Tissue Thickness Estimation: Relevance for Cell Counting Methods.

Stereological techniques using the optical disectors require estimation of final section thickness, but frozen tissue irregularities may interfere with this estimation. Cryostat slices from rodent nerve tissues (dorsal root ganglia, spinal cord, and brain), cut at 16, 40, and 50 µm, were digitized with a confocal microscope and visualized through 3D software. Geometric section thickness of tissue (T geom) was defined as tissue volume/area. Maximal section thicknesses (T max), from the top to the bottom of the section, were measured in a random sample of vertical ZX planes. Irregularities were mostly related to blood vessels traversing the tissue and neuronal somas protruding over the cut surfaces, with other neuron profiles showing a fragmented appearance. Irregularities contributed to increasing the distance between the tops and bottoms of slices sectioned in different laboratories. Significant differences were found between T max and T geom for all thickness studies and counting frames (p<0.01). The T geom/T max average rate was 68.4-85.7% in volumes around cell profiles (∼600-1,200 µm2) and 83.3-91.8% in subcellular samples (∼25-160 µm2). Confocal microscopy may help to assess tissue irregularities, which might lead to an overestimation of tissue volume if section thickness is estimated by focusing on the top and bottom of the sections.

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 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.

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 (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.

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.

Dyrk1A is dynamically expressed on subsets of motor neurons and in the neuromuscular junction: possible role in Down syndrome.

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.

[Identification of occupational risks associated with diseases suspected to be of possible occupational origin seen in the National Health System]. / Identificación de los riesgos laborales asociados a enfermedad sospechosa de posible origen laboral atendida en el Sistema Nacional de Salud.

In an earlier study, we identified 26 diseases of possible occupational origin, seen in different settings within the national health system in Catalonia. In this literature review we identify those occupational risk factors most strongly associated with these pathologies. After applying inclusion/exclusion criteria to 754 reviews, meta-analyses and/or practice guidelines, 37 articles remained that were rated for study quality by 3 reviewers. Of these, the 31 studies of highest quality were examined in depth, and summarised in a final table. Ergonomic risk factors (manual handling of materials, highly repetitive movements and awkward postures), especially for musculoskeletal disorders of the upper extremity, and exposures to chemical products and physical agents, were prominent. These results provide an additional resource for primary care physicians to assist them with the identification of possible occupational illness and to improve communication between the National Health Service and social security system.

Sera from amyotrophic lateral sclerosis patients induce the non-canonical activation of NMDA receptors "in vitro".

Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by the selective loss of both upper and lower motoneurons (MNs). The familial form of the illness is associated with mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SOD-1) enzyme, but it accounts for fewer than 10% of cases; the rest, more than 90%, correspond to the sporadic form of ALS. Although many proposals have been suggested over the years, the mechanisms underlying the characteristic selective killing of MN in ALS remain unknown. In this study we tested the effect of sera from sporadic ALS patients on NMDA receptors (NMDAR). We hypothesize that an endogenous seric factor is implicated in neuronal death in ALS, mediated by the modulation of NMDAR. Sera from ALS patients and from healthy subjects were pretreated to inactivate complement pathways and dialyzed to remove glutamate and glycine. IgGs from ALS patients and healthy subjects were obtained by affinity chromatography and dialyzed against phosphate-buffered saline. Human NMDAR were expressed in Xenopus laevis oocytes, and ionic currents were recorded using the two-electrode voltage clamp technique. Sera from sporadic ALS patients induced transient oscillatory currents in oocytes expressing NMDAR with a significantly higher total electrical charge than that induced by sera from healthy subjects. Sera from patients with other neuromuscular diseases did not exert this effect. The currents were inhibited by MK-801, a noncompetitive blocker of NMDAR. The PLC inhibitor, U-73122, and the IP(3) receptor antagonist, 2-APB, also inhibited the sera-induced currents. The oscillatory signal recorded was due to internal calcium mobilization. Isolated IgGs from ALS patients significantly affected the activity of oocytes injected with NMDAR, causing a 2-fold increase over the response recorded for IgGs from healthy subjects. Our data support the notion that ALS sera contain soluble factors that mobilize intracellular calcium, not opening directly the ionic conductance, but through the non-canonical activation of NMDAR.

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.

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.