Academic literature on the topic 'SOD1G93A mouse model'

Mice with transgenic expression of human SOD1G93A are a widely used model of ALS, with a caudal–rostral progression of motor impairment. Previous studies have quantified the progression of motoneuron (MN) degeneration based on size, even though alpha (α-) and gamma (γ-) MNs overlap in size. Therefore, using molecular markers and synaptic inputs, we quantified the survival of α-MNs and γ-MNs at the lumbar and cervical spinal segments of 3- and 4-month SOD1G93A mice, to investigate whether there is a caudal–rostral progression of MN death. By 3 months, in the cervical and lumbar spinal cord, there was α-MN degeneration with complete γ-MN sparing. At 3 months, the cervical spinal cord had more α-MNs per ventral horn than the lumbar spinal cord in SOD1G93A mice. A similar spatial trend of degeneration was observed in the corticospinal tract, which remained intact in the cervical spinal cord at 3- and 4- months of age. These findings agree with the corticofugal synaptopathy model that α-MNs and CST of the lumbar spinal cord are more susceptible to degeneration in SOD1G93A mice. Hence, there is a spatial and temporal caudal–rostral progression of α-MN and CST degeneration in SOD1G93A mice.

Glutamate (Glu)-mediated excitotoxicity is a major cause of amyotrophic lateral sclerosis (ALS) and our previous work highlighted that abnormal Glu release may represent a leading mechanism for excessive synaptic Glu. We demonstrated that group I metabotropic Glu receptors (mGluR1, mGluR5) produced abnormal Glu release in SOD1G93A mouse spinal cord at a late disease stage (120 days). Here, we studied this phenomenon in pre-symptomatic (30 and 60 days) and early-symptomatic (90 days) SOD1G93A mice. The mGluR1/5 agonist (S)-3,5-Dihydroxyphenylglycine (3,5-DHPG) concentration dependently stimulated the release of [3H]d-Aspartate ([3H]d-Asp), which was comparable in 30- and 60-day-old wild type mice and SOD1G93A mice. At variance, [3H]d-Asp release was significantly augmented in 90-day-old SOD1G93A mice and both mGluR1 and mGluR5 were involved. The 3,5-DHPG-induced [3H]d-Asp release was exocytotic, being of vesicular origin and mediated by intra-terminal Ca2+ release. mGluR1 and mGluR5 expression was increased in Glu spinal cord axon terminals of 90-day-old SOD1G93A mice, but not in the whole axon terminal population. Interestingly, mGluR1 and mGluR5 were significantly augmented in total spinal cord tissue already at 60 days. Thus, function and expression of group I mGluRs are enhanced in the early-symptomatic SOD1G93A mouse spinal cord, possibly participating in excessive Glu transmission and supporting their implication in ALS. Please define all abbreviations the first time they appear in the abstract, the main text, and the first figure or table caption.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron degeneration and muscle paralysis. The early presymptomatic onset of abnormal processes is indicative of cumulative defects that ultimately lead to a late manifestation of clinical symptoms. It remains of paramount importance to identify the primary defects that underlie this condition and to determine how these deficits lead to a cycle of deterioration. We recently demonstrated that prenatal E17.5 lumbar spinal motoneurons (MNs) from SOD1G93A mice exhibit a KCC2-related alteration in chloride homeostasis, i.e., the EGABAAR is more depolarized than in WT littermates. Here, using immunohistochemistry, we found that the SOD1G93A lumbar spinal cord is less enriched with 5-HT descending fibres than the WT lumbar spinal cord. High-performance liquid chromatography confirmed the lower level of the monoamine 5-HT in the SOD1G93A spinal cord compared to the WT spinal cord. Using ex vivo perforated patch-clamp recordings of lumbar MNs coupled with pharmacology, we demonstrated that 5-HT strongly hyperpolarizes the EGABAAR by interacting with KCC2. Therefore, the deregulation of the interplay between 5-HT and KCC2 may explain the alteration in chloride homeostasis detected in prenatal SOD1G93A MNs. In conclusion, 5-HT and KCC2 are two likely key factors in the presymptomatic phase of ALS, particular in familial ALS involving the SOD1G93A mutation.

Mutations in autophagy genes can cause familial and sporadic amyotrophic lateral sclerosis (ALS). However, the role of autophagy in ALS pathogenesis is poorly understood, in part due to the lack of cell type-specific manipulations of this pathway in animal models. Using a mouse model of ALS expressing mutant superoxide dismutase 1 (SOD1G93A), we show that motor neurons form large autophagosomes containing ubiquitinated aggregates early in disease progression. To investigate whether this response is protective or detrimental, we generated mice in which the critical autophagy gene Atg7 was specifically disrupted in motor neurons (Atg7 cKO). Atg7 cKO mice were viable but exhibited structural and functional defects at a subset of vulnerable neuromuscular junctions. By crossing Atg7 cKO mice to the SOD1G93A mouse model, we found that autophagy inhibition accelerated early neuromuscular denervation of the tibialis anterior muscle and the onset of hindlimb tremor. Surprisingly, however, lifespan was extended in Atg7 cKO; SOD1G93A double-mutant mice. Autophagy inhibition did not prevent motor neuron cell death, but it reduced glial inflammation and blocked activation of the stress-related transcription factor c-Jun in spinal interneurons. We conclude that motor neuron autophagy is required to maintain neuromuscular innervation early in disease but eventually acts in a non–cell-autonomous manner to promote disease progression.

Amyotrophic Lateral Sclerosis (ALS) is an adult-onset, progressive, motor neuron degenerative disease, in which the role of inflammation is not well established. Innate and adaptive immunity were investigated in the CNS of the Superoxide Dismutase 1 (SOD1)G93A transgenic mouse model of ALS. CD4+ and CD8+ T cells infiltrated SOD1G93A spinal cords during disease progression. Cell-specific flow cytometry and gene expression profiling showed significant phenotypic changes in microglia, including dendritic cell receptor acquisition, and expression of genes linked to neuroprotection, cholesterol metabolism and tissue remodeling. Microglia dramatically up-regulated IGF-1 and down-regulated IL-6 expression. When mutant SOD1 mice were bred onto a TCRβ deficient background, disease progression was significantly accelerated at the symptomatic stage. In addition, microglia reactivity and IGF-1 levels were reduced in spinal cords of SOD1G93A (TCRβ−/−) mice. These results indicate that T cells play an endogenous neuroprotective role in ALS by modulating a beneficial inflammatory response to neuronal injury.

Amyothrophic lateral sclerosis (ALS) is a progressive, lethal neuromuscular disease that is associated with the degeneration of cortical and spinal motoneurons, leading to atrophy of limb, axial, and respiratory muscles. Patients with ALS invariably develop respiratory muscle weakness and most die from pulmonary complications. Overexpression of superoxide dismutase 1 ( SOD1) gene mutations in mice recapitulates several of the clinical and pathological characteristics of ALS and is therefore a valuable tool to study this disease. The present study is intended to evaluate an age-dependent progression of respiratory complications in SOD1G93A mutant mice. In each animal, baseline measurements of breathing pattern [i.e., breathing frequency and tidal volume (Vt)], minute ventilation (Ve), and metabolism (i.e., oxygen consumption and carbon dioxide production) were repeatedly sampled at variable time points between 10 and 20 wk of age with the use of whole-body plethysmographic chambers. To further characterize the neurodegeneration of breathing, Ve was also measured during 5-min challenges of hypercapnia (5% CO2) and hypoxia (10% O2). At baseline, breathing characteristics and metabolism remained relatively unchanged from 10 to 14 wk of age. From 14 to 18 wk of age, there were significant ( P < 0.05) increases in baseline Vt, Ve, and the ventilatory equivalent (Ve/oxygen consumption). After 18 wk of age, there was a rapid decline in Ve due to significant ( P < 0.05) reductions in both breathing frequency and Vt. Whereas little change in hypoxic Ve responses occurred between 10 and 18 wk, hypercapnic Ve responses were significantly ( P < 0.05) elevated at 18 wk due to an augmented Vt response. Like baseline breathing characteristics, hypercapnic Ve responses also declined rapidly after 18 wk of age. The phenotypic profile of SOD1G93A mutant mice was apparently unique because similar changes in respiration and metabolism were not observed in SOD1 controls. The present results outline the magnitude and time course of respiratory complications in SOD1G93A mutant mice as the progression of disease occurs in this mouse model of ALS.

Neuroinflammation plays a significant role in amyotrophic lateral sclerosis (ALS) pathology, leading to the development of therapies targeting inflammation in recent years. Our group has studied the tetanus toxin C-terminal fragment (TTC) as a therapeutic molecule, showing neuroprotective properties in the SOD1G93A mouse model. However, it is unknown whether TTC could have some effect on inflammation. The objective of this study was to assess the effect of TTC on the regulation of inflammatory mediators to elucidate its potential role in modulating inflammation occurring in ALS. After TTC treatment in SOD1G93A mice, levels of eotaxin-1, interleukin (IL)-2, IL-6 and macrophage inflammatory protein (MIP)-1 alpha (α) and galectin-1 were analyzed by immunoassays in plasma samples, whilst protein expression of caspase-1, IL-1β, IL-6 and NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) was measured in the spinal cord, extensor digitorum longus (EDL) muscle and soleus (SOL) muscle. The results showed reduced levels of IL-6 in spinal cord, EDL and SOL in treated SOD1G93A mice. In addition, TTC showed a different role in the modulation of NLRP3 and caspase-1 depending on the tissue analyzed. In conclusion, our results suggest that TTC could have a potential anti-inflammatory effect by reducing IL-6 levels in tissues drastically affected by the disease. However, further research is needed to study more in depth the anti-inflammatory effect of TTC in ALS.

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) affects the spinal cord, brain stem, and cerebral cortex. In this pathology, both neurons and glial cells are affected. However, few studies have analyzed retinal microglia in ALS models. In this study, we quantified the signs of microglial activation and the number of retinal ganglion cells (RGCs) in an SOD1G93A transgenic mouse model at 120 days (advanced stage of the disease) in retinal whole-mounts. For SOD1G93A animals (compared to the wild-type), we found, in microglial cells, (i) a significant increase in the area occupied by each microglial cell in the total area of the retina; (ii) a significant increase in the arbor area in the outer plexiform layer (OPL) inferior sector; (iii) the presence of cells with retracted processes; (iv) areas of cell groupings in some sectors; (v) no significant increase in the number of microglial cells; (vi) the expression of IFN-γ and IL-1β; and (vii) the non-expression of IL-10 and arginase-I. For the RGCs, we found a decrease in their number. In conclusion, in the SOD1G93A model (at 120 days), retinal microglial activation occurred, taking a pro-inflammatory phenotype M1, which affected the OPL and inner retinal layers and could be related to RGC loss.

Degeneration of cortical and spinal motor neurons is the typical feature of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease for which a pathogenetic role for the Cu/Zn superoxide dismutase (SOD1) has been demonstrated. Mice overexpressing a mutated form of the SOD1 gene (SOD1G93A) develop a syndrome that closely resembles the human disease. The SOD1 mutations confer to this enzyme a “gain-of-function,” leading to increased production of reactive oxygen species. Several oxidants induce tyrosine phosphorylation through direct stimulation of kinases and/or phosphatases. In this study, we analyzed the activities of src and fyn tyrosine kinases and of protein tyrosine phosphatases in synaptosomal fractions prepared from the motor cortex and spinal cord of transgenic mice expressing SOD1G93A. We found that (i) protein phosphotyrosine level is increased, (ii) src and fyn activities are upregulated, and (iii) the activity of tyrosine phosphatases, including the striatal-enriched tyrosine phosphatase (STEP), is significantly decreased. Moreover, the NMDA receptor (NMDAR) subunit GluN2B tyrosine phosphorylation was upregulated in SOD1G93A. Tyrosine phosphorylation of GluN2B subunits regulates the NMDAR function and the recruitment of downstream signaling molecules. Indeed, we found that proline-rich tyrosine kinase 2 (Pyk2) and ERK1/2 kinase are upregulated in SOD1G93A mice. These results point out an involvement of tyrosine kinases and phosphatases in the pathogenesis of ALS.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of corticospinal tract motor neurons. Previous studies showed that adenosine-mediated neuromodulation is disturbed in ALS and that vascular endothelial growth factor (VEGF) has a neuroprotective function in ALS mouse models. We evaluated how adenosine (A1R and A2AR) and VEGF (VEGFA, VEGFB, VEGFR-1 and VEGFR-2) system markers are altered in the cortex and spinal cord of pre-symptomatic and symptomatic SOD1G93A mice. We then assessed if/how chronic treatment of SOD1G93A mice with a widely consumed adenosine receptor antagonist, caffeine, modulates VEGF system and/or the levels of Brain-derived Neurotrophic Factor (BDNF), known to be under control of A2AR. We found out decreases in A1R and increases in A2AR levels even before disease onset. Concerning the VEGF system, we detected increases of VEGFB and VEGFR-2 levels in the spinal cord at pre-symptomatic stage, which reverses at the symptomatic stage, and decreases of VEGFA levels in the cortex, in very late disease states. Chronic treatment with caffeine rescued cortical A1R levels in SOD1G93A mice, bringing them to control levels, while rendering VEGF signaling nearly unaffected. In contrast, BDNF levels were significantly affected in SOD1G93A mice treated with caffeine, being decreased in the cortex and increased in spinal the cord. Altogether, these findings suggest an early dysfunction of the adenosinergic system in ALS and highlights the possibility that the negative influence of caffeine previously reported in ALS animal models results from interference with BDNF rather than with the VEGF signaling molecules.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder due to upper and lower motor neurons (MNs) death. Recognized as a non-cell autonomous disease, ALS is also characterized by damage and degeneration of glial cells, such as astrocytes, microglia, and oligodendrocytes. In particular, astrocytes acquire a reactive and toxic phenotype defined by an abnormal proliferation and by the release of neurotoxic factors. One major cause for MN degeneration in ALS is represented by the glutamate-mediated excitotoxicity, due to the alteration of glutamate transmission mechanisms, including glutamate receptor function. In this context, the Group I metabotropic glutamate receptor 5 (mGluR5) has been proposed to play an important role in ALS, since it is largely overexpressed during disease progression and is involved in the altered neuronal and glial cellular processes. We previously demonstrated that mGluR5 produces abnormal glutamate release in the spinal cord of the SOD1G93A mouse model of ALS and that halving its expression has a positive impact on in-vivo disease progression, including motor neuron survival, astrogliosis and microgliosis. We here investigated the consequences of reducing the mGluR5 expression in SOD1G93A mice on the reactive phenotype of spinal cord astrocytes cultured from late symptomatic (120 days old) SOD1G93A, age matched WT and SOD1G93AGrm5-/+ mice. SOD1G93A astrocytes displayed a higher cytoplasmic calcium concentration respect to WT cells and knocking down of mGluR5 reduced calcium level, both under basal and 3,5-DHPG-stimulated conditions. GFAP and S100β, two markers of astrogliosis, were increased in SOD1G93A astrocytes, whereas their overexpression was reduced in SOD1G93AGrm5-/+ cells. The same positive effect was obtained in the case of NLRP3, a marker strictly related to inflammation, which was upregulated in SOD1G93A astrocytes and less expressed in double mutant astrocytes. The partial ablation of mGluR5 also resulted in a lower cellular presence of misfolded SOD1. Both the expression and secretion of pro-inflammatory cytokines were strongly reduced in SOD1G93AGrm5-/+ respect to SOD1G93A astrocytes. The uncoupling between oxygen consumption and ATP synthesis and the impairment of mitochondria function, present in SOD1G93A astrocytes, were recovered in double mutant astrocytes. Notably, the viability of spinal MNs co-cultured with SOD1G93AGrm5-/+ astrocytes was significantly increased respect to MNs co-cultured with SOD1G93A astrocytes. The acute in-vitro treatment of SOD1G93A astrocytes with an antisense nucleotide (ASO) specific for mGluR5 decreased the mRNA and protein expression of mGluR5 in these cells and led to the reduction of GFAP and S100β. The in-vitro pharmacological treatment with the negative allosteric modulator of mGluR5, CTEP, also reduced the expression of GFAP and S100β in SOD1G93A astrocytes. Altogether, these results indicate that mGluR5 ablation has a positive impact on astrocytes in SOD1G93A mice, supporting the idea that the in-vivo amelioration of the disease progression, registered after mGluR5 genetical or pharmacological silencing, involve astrocyte phenotype improvement. As a whole, it may be outlined that mGluR5 may represent a potential therapeutic target able to preserve MNs from death, also by modulating the reactive astroglia phenotype in ALS. Due to the active contribution of microglia to ALS pathogenesis, the effect of mGluR5 partial ablation in SOD1G93A mice on the balance between the pro- and anti-inflammatory profile of microglia acutely purified from the brain and spinal cord of WT, Grm5-/+, SOD1G93A and SOD1G93AGrm5-/+ mice has been investigated at the early (90 days) and late symptomatic (120 days) stages of ALS by detecting the mRNA and protein levels of some relevant markers involved in neuroinflammation, such as IL-1β, CD86, iNOS, TNF-α (pro-inflammatory), Arginase 1, IL-10, CD206 and IL-4 (anti-inflammatory). Experiments are in progress to complete this part of the project.

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative fatale de l’adulte caractérisée par la dégénérescence des motoneurones (MNs) et ayant une étiologie multifactorielle. La plupart des études sur la SLA se sont focalisées aux stades symptomatiques selon l’hypothèse que la pathogénicité apparaît lorsque la maladie devient symptomatique. Cependant, un nombre grandissant d’évidences indique que la pathogénicité se développerait bien avant les symptômes. Mon travail de thèse de Doctorat a été basé sur l’hypothèse selon laquelle la SLA – familiale et sporadique – découlerait de déficits présents dès le développement précoce. La première partie de ma thèse a consisté à analyser les courants post-synaptiques GABA/glycine (IPSCs) au niveau des MNs embryonnaire (E) E17,5, localisés dans la colonne motrice ventro-laterale, chez la souris SOD1G93A (SOD) modèle de la SLA, en parallèle à l’analyse de l’homéostasie chlorure. Nos résultats ont montré que les IPSCs sont moins fréquents chez les animaux SOD en accord avec une réduction des terminaisons synaptiques VIAAT autour des MNs. Les MNs SODs avaient un ECI 10 mV plus positif que les MNs sauvages (WT) de la même portée. Ce déficit était lié à une réduction du co-transporteur chlorure KCC2. Les IPSCs évoqués et spontanés présentaient une relaxation plus longue chez les MNs SOD, en corrélation à une [Cl-]i plus élevée. La modélisation a montré que cet excès de relaxation permettait de compenser la moindre efficacité de l’inhibition GABA/glycine liée au ECI dépolarisé. De manière intéressante, les simulations ont révélé la nature excitatrice des potentiels dépolarisants post-synaptiques GABA/glycine (dGPSPs) survenant à basse fréquence (<50Hz) sur les MNs SOD mais pas sur les MNs WT. A plus haute fréquence, les dGPSPs basculaient vers une inhibition du MN liée à une sommation de composantes « shuntantes ». La seconde partie de ma thèse a donc focalisé sur les effets de dGPSPs évoqués électriquement at différentes fréquences (7,5 - 100 Hz) sur de vrais MNs E17,5 au niveau desquels un ECl dépolarisant (sous le seuil du PA) était imposé. Le but était d’examiner si l’effet excitateur pouvait être lié aux changements morphologiques des MNs E17,5 décrits précédemment. Les résultats ont montré que certains MNs étaient bien excités par les dGPSPs basse fréquence et inhibés à plus forte fréquence (MNs bi-effet) alors que d’autres MNs étaient inhibés quelles que soient les fréquences (MNs inhibés). L’effet double était plus souvent détecté au niveau des MNs SOD. Les MNs WT ont été classés en deux groupes en fonction de leur résistance d’entrée (Rin), les MNs bi-effet ayant une Rin élevée et les MNs inhibés une Rin basse. Les données morphométriques ont mis en avant un arbre dendritique réduit pour les MNs WT bi-effet (Rin élevée) et un arbre dendritique étendu pour les MNs inhibés (Rin basse). Ce n’était pas le cas des MNs SOD excités ou inhibés indépendamment de leur morphologie. En accord avec les simulations montrant qu’une baisse de la densité des courants inhibiteurs sur le soma du MN favorise l’excitation des dGPSPs, nous avons trouvé moins de terminaisons synaptiques VIAAT sur le soma et dendrites proximales des MNs SOD, et une fréquence réduite des dGPSPs spontanés. Dans leur ensemble, les données de ma thèse soulignent une altération précoce de l’homéostasie chlorure et de l’innervation GABA/glycine des MNs SOD1G93A. Avant la naissance, une population dominante de MNs avec Rin basse émerge chez les animaux WT. Ces MNs qui sont inhibés par les dGPSPs pourraient correspondre aux futures MNs vulnérables (rapides, FF). Ces MNs ne sont pas inhibés chez les animaux SOD. Le dysfonctionnement de l’inhibition pourrait être attribué à deux facteurs distincts : la morphologie et la densité des synapses inhibitrices péri-somatiques. Parmi ces facteurs, le deuxième joue un rôle majeur en contrôlant la capacité des neurones GABA/glycine à façonner la sortie motrice spinale
Amyotrophic lateral sclerosis (ALS) is a fatal and adult-onset neurodegenerative disease characterized by a progressive degeneration of motoneurons (MNs) with complex multifactorial aetiology. Most ALS studies have focused on symptomatic stages based on the hypothesis that ALS pathogenesis occurs when the disease becomes symptomatic. However, growing evidence indicates that ALS pathogenesis might start long before symptom onset. My PhD thesis work was based on the hypothesis that ALS - familial and sporadic - stems from deficits taking place during early development. With the aim of identifying early changes underpinning ALS neurodegeneration, the first part of my thesis analysed the GABAergic/glycinergic inhibitory postsynaptic currents (IPSCs) to embryonic (E) E17.5 MNs located in the ventro-lateral motor column from SOD1G93A (SOD) mice, in parallel with the analyse of chloride homeostasis. Our results showed that IPSCs are less frequent in SOD animals in accordance with a reduction of synaptic VIAAT-positive terminals in the close proximity of MN somata. SOD MNs exhibited an ECI 10 mV more depolarized than wild type (WT) MNs. This deficit in GABA/glycine inhibition was due to a reduction of the neuronal chloride transporter KCC2. SOD spontaneous IPSCs and evoked GABAAR-currents exhibited a slower decay correlated to elevated [Cl-]i. Using computer modelling approach, we revealed that the slower relaxation of synaptic inhibitory events acts as a compensatory mechanism to strengthen or increase the efficacy of GABA/glycine inhibition when ECI is more depolarized. Interestingly, simulations revealed an excitatory effect of low frequency (<50Hz) depolarizing GABA/glycine post-synaptic potentials (dGPSPs) in SOD-like MNs but not in WT-like littermates. At high frequency, dGPSPs switched to inhibitory effect resulting from the summation of the shunting components. The second part of my PhD thesis focussed on the effect of electrically evoked-dGPSPs, at different frequencies (7.5 to 100 Hz), on real lumbar E17.5 MNs in which a depolarized ECI (below spike threshold) was imposed. The aim was to examine whether the excitatory effect could be linked to morphological changes previously described in E17.5 SOD MNs. Results showed that some MNs were excited by low frequency dGPSPs and inhibited by high frequency dGPSPs (Dual MNs) and others were inhibited at all frequencies (Inhibited MNs). Dual effect was more often detected in SOD MNs. WT MNs were classified into two clusters according to their input resistance (Rin), Dual MNs being specific to high Rin and Inhibited MNs to low Rin. Morphometric data pointed out a reduced dendritic tree in high Rin WT Dual MNs and a large dendritic tree in low Rin Inhibited MNs. This was not the case in SOD MNs that were excited or inhibited whatever their morphology and Rin. In agreement with simulation showing that a less density of inhibitory current on MNs soma favours excitatory dGPSPs, we found less synaptic VIAAT terminals on the soma and proximal dendrites of SOD MNs, compared to littermate WT MNs, as well as a lower frequency of spontaneous dGPSPs. Altogether, my thesis data emphasize a prenatal defect in the CI- homeostasis and GABA/glycine innervation in the SOD1G93A ALS MNs. Before birth, a dominant population of MNs with low Rin emerges in WT animals. These MNs that are inhibited by dGPSPs could represent future ALS vulnerable fast MNs (putative FF). Interestingly, those MNs are not inhibited in SOD animals. The inhibitory dysfunction could be attributed to two distinct factors: morphology and perisomatic inhibitory synapse density. Of these two factors, the latter plays a major role by controlling capability of GABAergic/glycinergic neurons for shaping spinal motor output

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the pro-gressive loss of motoneurons (MN). Increasing evidence points glial cells as key players for ALS onset and progression. Indeed, MN-glia signalling pathways involving either neuroprotection or inflammation are likely to be altered in ALS. We aimed to study the molecules related with glial function and/or reactivity by evaluating glial markers and hemichannels, mainly present in astrocytes. We also studied molecules involved in mi-croglia-MN dialogue (CXCR3/CCL21; CX3CR1/CX3CL1; MFG-E8), as well as proliferation (Ki-67) and inflammatory-related molecules (TLR2/4, NLRP3; IL-18) and alarming/calming signals (HMGB1/autotaxin). We used lumbar spinal cord (SC) homogenates from mice expressing a mutant human-SOD1 protein (mSOD1) at presymptomatic and late-symptomatic ALS stages. SJL (WT) mice at same ages were used as controls. We observed decreased expression of genes associated with astrocytic (GFAP and S100B) and microglial (CD11b) markers in mSOD1 at the presymptomatic phase, as well as diminished levels of gap junction components pannexin1 and connexin43 and expression of Ki-67 and decreased autotax-in. In addition, microglial-MN communication was negatively affected in mSOD1 mice as well as in-flammatory response. Interestingly, we observed astrocytic (S100B) and microglial (CD11b) reactivity, increased proliferation (Ki-67) and increased autotaxin expression in symptomatic mSOD1 mice. In-creased MN-microglial dialogue (CXCR3/CCL21; CX3CR1/CX3CL1; MFG-E8) and hemichannel activ-ity, namely connexin43 and pannexin1, were also observed in mSOD1 at the symptomatic phase, along with an elevated inflammatory response as indicated by increased levels of HMGB1 and NLRP3. Our results suggest that decreased autotaxin expression is a feature of the presymptomatic stage, and precede the network of pro-inflammatory-related symptomatic determinants, including HMGB1, CCL21, CX3CL1, and NLRP3. The identification of the molecules and signaling pathways that are dif-ferentially activated along ALS progression will contribute for a better design of therapeutic strategies for disease onset and progression.
Fundação para a Ciência e a Tecnologia - PTDC/SAU-FAR/118787/2010 (DB) and PEst-OE/SAU/UI4013/2013-2014

Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2013
Amyotrophic Lateral Sclerosis (ALS) is an adult-onset progressive neurodegenerative disease characterized by the selective loss of motor neuron function leading to muscle atrophy and weakness. After symptoms onset patients last 4 to 5 years and ultimately die due to bulbar failure. Animal models have been developed to study the neurobiology of ALS, with the SOD1G93A mouse model as the most studied so far. Morphological and functional abnormalities have been reported in both pre-symptomatic and symptomatic stages of ALS progression in this rodent model. Dysfunctions in neuromuscular transmission at phrenic nerve-hemidiaphragm preparations of the SOD1G93A mouse have recently been put in evidence by our group. Since adenosine A2A receptors (A2AR) play a major role in fine-tuning neurotransmitter release in mammalian neuromuscular junctions (NMJs), we decided to evaluate how A2AR modulate acetylcholine (ACh) release in presymptomatic (4-6 weeks old (wo)) and symptomatic (12-14 wo) SOD1G93A mice. Using the selective A2AR agonist CGS 21680, we performed a dose-response study using 3, 5 and 10 nM in pre-symptomatic SOD1G93A mice and age-matched Wild Type (WT) animals. CGS 21680 at 3, 5, 10 and 25 nM was studied in symptomatic SOD1G93A and 12-14 wo WT rodents. 25 nM was used in the previous mentioned group of animals to verify if at concentrations higher than 10nM CGS 21680 effect in NMT would be increased. Intracellular recordings of endplate potentials (EPPs), miniature endplate potentials (MEPPs) and giant miniature endplate potentials (GMEPPs) where performed in high [Mg2+] paralyzed phrenic nerve-hemidiaphragm preparations. Low-frequency stimuli (0.5 Hz) allowed the evaluation of the evoked activity (EPPs) while spontaneous activity (MEPPs and GMEPPs) was measured in gap-free intervals. Quantal content (QC) was measured as the ratio between the mean EPP amplitude and the mean MEPP amplitude recorded in the same period of time. We first validated high [Mg2+] as an useful method to study neuromuscular transmission (NMT) in these mice. The shift in the Ca2+/Mg2+ ratio preserved the previous described features of ACh release in SOD1G93A mice diaphragm fibers. In the pre-symptomatic phase, SOD1G93A mice displayed a significant increase in EPP amplitude and QC in tested concentrations of CGS 21680 when compared to WT mice (p<0.05 Unpaired t-test) except for EPP changes at 3nM (p>0.05 Unpaired t-test). MEPP and GMEPP amplitude were not changed by CGS 21680 (p>0.05 Paired t-test). The A2AR-mediated increase in MEPP frequency was not statistically different between both groups in the presence of 3 and 10nM of the A2AR agonist (p>0.05 Unpaired t-test), but was significantly higher in SOD1G93A mice when perfused at 5nM (p<0.05 Unpaired t-test). We also found that in the pre-symptomatic SOD1G93A fibers GMEPP frequency was statistically higher in the presence of CGS 21680 (5nM) than in age-matched WT NMJs (p<0.05 Unpaired t-test). In the symptomatic phase, CGS 21680 did not elicit any changes in SOD1G93A mice evoked (EPP amplitude and QC) and spontaneous (MEPP and GMEPP frequency and amplitude) activity from SOD1G93A mice (p>0.05 Paired t-test). When 0.5 Hz stimuli were delivered, EPP amplitude, QC and MEPP frequency were increased in 12-14 wo WT mice in the presence of 3, 5, 10 and 25 nM of CGS 21680 (p<0.05 Paired t-test). These parameters together with GMEPP frequency were significantly higher than in symptomatic mice (p<0.05 Unpaired t-test). GMEPP and MEPP amplitude were not changed by CGS 21680 in older WT mice (p>0.05 Paired t-test). The A2AR receptor antagonist SCH 58261 (50nM) was devoid of effect in spontaneous and evoked release (p>0.05 Paired t-test) and effectively blocked CGS 21680 (5nM in 4-6 wo mice and 10 nM in 12-14 wo WT rodents) facilitation (p<0.05 one-way ANOVA followed by Tukey’s pos-hoc). Our results strongly suggest an early functionally enhancement of NMT concerning adenosine modulation before symptoms appear. This dysfunction involves an A2AR functional upregulation at diaphragm NMJs in pre-symptomatic SOD1G93A mice. When symptoms start to develop, A2AR receptor function is lost in symptomatic SOD1G93A mice but remains present in 12-14 wo WT mice. Furthermore, CGS 21680 perfusion triggered a higher A2AR facilitation in 12-14 wo than in 4-6 wo WT mice. This might be attributable to a normal maturation feature of the studied synapse, which contrasted with the observations in transgenic mice. The shift from a functional upregulation of A2AR before symptomatology that arises in SOD1G93A mice towards an apparent loss of A2AR functionality in symptomatic phase, highlights the role of this subtype of P1 receptors in the scope of ALS. A2AR targeted drugs could eventually play an import role in the delay of this disease.
A Esclerose Lateral Amiotrófica (ALS) é uma doença neurodegenerativa progressiva caracterizada pela perda seletiva da função do neurónio motor, levando a atrofia e fraqueza muscular. Após o início dos sintomas a esperança de vida dos doentes é de cerca de 4 a 5 anos, sendo a causa da morte geralmente devida a insuficiência respiratória. Modelos animais têm sido desenvolvidos para estudar a neurobiologia da ALS, sendo o do murganho SOD1G93A o mais estudado. Neste modelo, anormalidades morfológicas e funcionais têm sido relatadas em ambos os estadios pré-sintomático e sintomático da ALS. Disfunções na transmissão neuromuscular (NMT) em preparações de nervo frénico-hemidiafragma de murganho SOD1G93A foram recentemente postas em evidência pelo nosso grupo. Considerando que os recetores A2A de adenosina (A2AR) desempenham um papel importante no controlo da libertação de acetilcolina (ACh) na junção neuromuscular (NMJ) de mamífero, explorámos o papel dos A2AR na modulação da NMT em murganhos SOD1G93A nas fases pré-sintomática (4-6 semanas de idade) e sintomática (12-14 semanas de idade). Usando o agonista seletivo dos A2AR (CGS 21680), foi realizado um estudo de dose-resposta utilizando concentrações de 3, 5 e 10 nM em animais pré-sintomáticos SOD1G93A e Wild Type (WT) (4-6 semanas). CGS 21680 a 3, 5, 10 e 25 nM foi utilizado em murganhos sintomáticos SOD1G93A e WT (12 - 14 semanas). A concentração 25 nM foi testada neste último grupo, de modo a verificar se a concentrações superiores a 10 nM o efeito modulador do CGS 21680 na NMT estaria aumentado. Foram realizados registos intracelulares de potenciais de placa motora (EPPs), potenciais miniatura de placa motora (MEPPs) e potenciais miniatura gigantes de placa motora (GMEPPs) em preparações de nervo frénico-hemidiafragma paralisadas com elevada [Mg2+]. Estímulos de baixa frequência (0.5 Hz) permitiram a avaliação da atividade evocada (EPPs), ao passo que as atividades espontâneas (MEPPs e GMEPPs) foram medidas sem estimulação elétrica. O conteúdo quântico (QC) foi considerado como sendo a razão entre a média da amplitude dos EPPs e média da amplitude dos MEPPs registados no mesmo período de tempo. Inicialmente validámos o aumento da [Mg2+] como um método útil para o estudo da NMT nos murganhos SOD1G93A. A alteração do equilíbrio entre a razão Ca2 +/ Mg2+ preservou as características da NMT anteriormente descritas neste modelo. Na fase pré-sintomática, os animais SOD1G93A apresentaram um aumento na amplitude do EPP e do QC nas concentrações testadas de CGS 21680 quando comparado com murganhos WT (p<0.05 Unpaired t-test), exceto nas variações do EPP a 3nM (p>0.05 Unpaired t-test). A amplitude dos MEPPs e GMEPPs não foram alteradas pelo CGS 21680 (p>0.05 Paired t-test). O aumento na frequência dos MEPPs resultante da ativação dos A2AR não foi significativamente diferente entre os dois grupos na presença de 3 e 10 nM do agonista dos A2AR (p>0.05 Unpaired t-test) mas foi estatisticamente superior em murganhos SOD1G93A quando perfundido a 5nM (p<0.05 Unpaired t-test). As NMJs dos animais SOD1G93A pré-sintomáticos apresentaram aumento na frequência dos GMEPPs na presença de CGS 21680 (5 nM) quando comparadas com as dos animais WT (p<0.05 Unpaired t-test). Na fase sintomática, o CGS 21680 não alterou a resposta evocada (amplitude dos EPP e QC) nem a atividade espontânea (frequência e amplitude dos MEPPs e GMEPPs) dos animais SOD1G93A (p>0.05 Paired t-test). Após estimulação de 0.5 Hz a amplitude dos EPPs, QC e frequência dos MEPPs aumentaram em murganhos WT na presença de 3, 5, 10 e 25 nM de CGS 21680 (p<0.05 Paired t-test). Estes parâmetros, juntamente com frequência dos GMEPPs foram significativamente mais elevados do que em murganhos sintomáticos (p<0,05 Unpaired t-test). A amplitude dos GMEPPs e MEPPs não foram alteradas pela aplicação de CGS 21680 em murganhos WT (p>0.05 Paired t-test). O antagonista dos A2AR SCH 58261 (50nM) não alterou a actividade espontânea e evocada (p>0.05 Paired t-test) mas bloqueou eficazmente o efeito facilitatório do CGS 21680 (a 5nM em animais de 4-6 semanas e a 10nM em murganhos WT com 12-14 semanas) (p<0.05 Unpaired t-test). Os resultados deste estudo apontam para a existência de uma disfunção da modulação adenosinérgica na libertação de ACh antes do aparecimento dos sintomas. Esta disfunção envolve uma sobrerregulação funcional dos A2AR nas NMJs de diafragma de murganhos SOD1G93A pré-sintomáticos. Quando os sintomas começam a aparecer, a função dos A2AR parece desaparecer em murganhos sintomáticos, mas permanece presente em animais WT de 12-14 semanas de idade. Além disso, o CGS 21680 promoveu uma facilitação A2AR superior em murganhos WT de 12-14 semanas do que de 4-6 semanas. Isto pode ser atribuído a uma característica normal de maturação da sinapse, contrastando com as observações em animais SOD1G93A. A passagem de uma sobrerregulação funcional dos A2AR antes da sintomatologia surgir para uma aparente perda da funcionalidade dos A2AR durante a fase sintomática em animais SOD1G93A, destaca o papel deste subtipo de recetores P1 no âmbito da ALS. Tendo isto em consideração, fármacos que atuem nos A2AR poderão eventualmente desempenhar um papel importante no atraso desta doença.