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.

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.

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.

Accumulation of poly(A) RNA in nuclear granules enriched in Sam68 in motor neurons from the SMNΔ7 mouse model of SMA.

Spinal muscular atrophy (SMA) is a severe motor neuron (MN) disease caused by the deletion or mutation of the survival motor neuron 1 (SMN1) gene, which results in reduced levels of the SMN protein and the selective degeneration of lower MNs. The best-known function of SMN is the biogenesis of spliceosomal snRNPs, the major components of the pre-mRNA splicing machinery. Therefore, SMN deficiency in SMA leads to widespread splicing abnormalities. We used the SMN∆7 mouse model of SMA to investigate the cellular reorganization of polyadenylated mRNAs associated with the splicing dysfunction in MNs. We demonstrate that SMN deficiency induced the abnormal nuclear accumulation in euchromatin domains of poly(A) RNA granules (PARGs) enriched in the splicing regulator Sam68. However, these granules lacked other RNA-binding proteins, such as TDP43, PABPN1, hnRNPA12B, REF and Y14, which are essential for mRNA processing and nuclear export. These effects were accompanied by changes in the alternative splicing of the Sam68-dependent Bcl-x and Nrnx1 genes, as well as changes in the relative accumulation of the intron-containing Chat, Chodl, Myh9 and Myh14 mRNAs, which are all important for MN functions. PARG-containing MNs were observed at presymptomatic SMA stage, increasing their number during the symptomatic stage. Moreover, the massive accumulations of poly(A) RNA granules in MNs was accompanied by the cytoplasmic depletion of polyadenylated mRNAs for their translation. We suggest that the SMN-dependent abnormal accumulation of polyadenylated mRNAs and Sam68 in PARGs reflects a severe dysfunction of both mRNA processing and translation, which could contribute to SMA pathogenesis.

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.

Cellular bases of the RNA metabolism dysfunction in motor neurons of a murine model of spinal muscular atrophy: Role of Cajal bodies and the nucleolus.

Spinal muscular atrophy (SMA) is caused by a homozygous deletion or mutation in the survival motor neuron 1 (SMN1) gene that leads to reduced levels of SMN protein resulting in degeneration of motor neurons (MNs). The best known functions of SMN is the biogenesis of spliceosomal snRNPs. Linked to this function, Cajal bodies (CBs) are involved in the assembly of spliceosomal (snRNPs) and nucleolar (snoRNPs) ribonucleoproteins required for pre-mRNA and pre-rRNA processing. Recent studies support that the interaction between CBs and nucleoli, which are especially prominent in neurons, is essential for the nucleolar rRNA homeostasis. We use the SMN∆7 murine model of type I SMA to investigate the cellular basis of the dysfunction of RNA metabolism in MNs. SMN deficiency in postnatal MNs produces a depletion of functional CBs and relocalization of coilin, which is a scaffold protein of CBs, in snRNP-free perinucleolar caps or within the nucleolus. Disruption of CBs is the earliest nuclear sign of MN degeneration. We demonstrate that depletion of CBs, with loss of CB-nucleolus interactions, induces a progressive nucleolar dysfunction in ribosome biogenesis. It includes reorganization and loss of nucleolar transcription units, segregation of dense fibrillar and granular components, retention of SUMO-conjugated proteins in intranucleolar bodies and a reactive, compensatory, up-regulation of mature 18S rRNA and genes encoding key nucleolar proteins, such as upstream binding factor, fibrillarin, nucleolin and nucleophosmin. We propose that CB depletion and nucleolar alterations are essential components of the dysfunction of RNA metabolism in SMA.

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.

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-ß-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.

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.

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.

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 (SOD1) rodents exhibit immunoreactivity to P2X(4) antibodies. Neurons with strong P2X(4)-like immunoreactivity (P2X(4)-LIR) do not show an apoptotic phenotype and are often associated with microglial cells that display neuronophagic activity. Western blot analysis showed that P2X(4) antibodies recognize not only the P2X(4) adenosine triphosphate receptor protein but also a hitherto unidentified low-molecular weight band. Here, we identify the molecular counterpart of the strong P2X(4)-LIR observed in association with neuronal degeneration in SOD1 animals. After matrix-assisted laser desorption/ionization time-of-flight, we found that the low-molecular weight P2X(4)-immunoreactive protein was SOD1. Further analysis demonstrated that the P2X(4) antibody recognizes a form of misfolded mutant SOD1 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 P2X(4)-immunizing peptide used for raising the antibody. No positive P2X(4) immunostaining was detected in mice overexpressing human wild-type SOD1. Intracerebral injections of affinity chromatography-isolated P2X(4)-immunoreactive SOD1 species promote microglial and astroglial activation. We conclude that neuronal SOD1 conformers with P2X(4)-LIR may have pathogenetic relevance in the promotion of neuroinflammation.

Saccharomyces cerevisiae cells have three Omega class glutathione S-transferases acting as 1-Cys thiol transferases.

The Saccharomyces cerevisiae genome encodes three proteins that display similarities with human GSTOs (Omega class glutathione S-transferases) hGSTO1-1 and hGSTO2-2. The three yeast proteins have been named Gto1, Gto2 and Gto3, and their purified recombinant forms are active as thiol transferases (glutaredoxins) against HED (beta-hydroxyethyl disulphide), as dehydroascorbate reductases and as dimethylarsinic acid reductases, while they are not active against the standard GST substrate CDNB (1-chloro-2,4-dinitrobenzene). Their glutaredoxin activity is also detectable in yeast cell extracts. The enzyme activity characteristics of the Gto proteins contrast with those of another yeast GST, Gtt1. The latter is active against CDNB and also displays glutathione peroxidase activity against organic hydroperoxides such as cumene hydroperoxide, but is not active as a thiol transferase. Analysis of point mutants derived from wild-type Gto2 indicates that, among the three cysteine residues of the molecule, only the residue at position 46 is required for the glutaredoxin activity. This indicates that the thiol transferase acts through a monothiol mechanism. Replacing the active site of the yeast monothiol glutaredoxin Grx5 with the proposed Gto2 active site containing Cys46 allows Grx5 to retain some activity against HED. Therefore the residues adjacent to the respective active cysteine residues in Gto2 and Grx5 are important determinants for the thiol transferase activity against small disulphide-containing molecules.

Prokaryotic and eukaryotic monothiol glutaredoxins are able to perform the functions of Grx5 in the biogenesis of Fe/S clusters in yeast mitochondria.

The Saccharomyces cerevisiae monothiol glutaredoxin Grx5 participates in the mitochondrial biogenesis of iron-sulfur clusters. Grx5 homologues exist in organisms from bacteria to humans. Chicken (cGRX5) and human (hGRX5) homologues contain a mitochondrial targeting sequence, suggesting a mitochondrial localization for these two proteins. We have compartmentalized the Escherichia coli and Synechocystis sp. homologues, and also cGRX5 and hGRX5, in the mitochondrial matrix of a yeast grx5 mutant. All four heterologous proteins rescue the defects of the mutant. The chicken cGRX5 gene was significantly expressed throughout the embryo stages in different tissues. These results underline the functional conservation of Grx5 homologues throughout evolution.