RESUMEN

Motor neuron (MN) loss is the primary pathological hallmark of amyotrophic lateral sclerosis (ALS). Histone deacetylase 4 (HDAC4) is one of several factors involved in nerve-muscle communication during MN loss, hindering muscle reinnervation, as shown in humans and in animal models of ALS, and may explain the differential progression observed in patients with ALS - rapid versus slow progression. In this work, we inhibited HDAC4 activity through the administration of a pan-histone deacetylase inhibitor, sodium butyrate, in an in vivo model of chronic spinal MN death induced by AMPA-mediated excitotoxicity. We infused AMPA into the spinal cord at low and high doses, which mimic the rapid and slow progression observed in humans, respectively. We found that muscle HDAC4 expression was increased by high-dose infusion of AMPA. Treatment of animals with sodium butyrate further decreased expression of muscle HDAC4, although non-significantly, and did not prevent the paralysis or the MN loss induced by AMPA infusion. These results inform on the role of muscle HDAC4 in MN degeneration in vivo and provide insights for the search for more suitable therapeutic strategies.

Asunto(s)

Esclerosis Amiotrófica Lateral , Humanos , Animales , Esclerosis Amiotrófica Lateral/patología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismo , Ácido Butírico/farmacología , Ácido Butírico/metabolismo , Neuronas Motoras/patología , Médula Espinal/patología

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that mainly affects the motor system. It is a very heterogeneous disorder, so far more than 40 genes have been described as responsible for ALS. The cause of motor neuron degeneration is not yet fully understood, but there is consensus in the literature that it is the result of a complex interplay of several pathogenic processes, which include alterations in nucleocytoplasmic transport, defects in transcription and splicing, altered formation and/or disassembly of stress granules and impaired proteostasis. These defects result in protein aggregation, impaired DNA repair, mitochondrial dysfunction and oxidative stress, neuroinflammation, impaired axonal transport, impaired vesicular transport, excitotoxicity, as well as impaired calcium influx. We argue here that all the above functions ultimately lead to defects in protein synthesis. Fused in Sarcoma (FUS) is one of the genes associated with ALS. It causes ALS type 6 when mutated and is found mislocalized to the cytoplasm in the motor neurons of sporadic ALS patients (without FUS mutations). In addition, FUS plays a role in all cellular functions that are impaired in degenerating motor neurons. Moreover, ALS patients with FUS mutations present the first symptoms significantly earlier than in other forms of the disease. Therefore, the aim of this review is to further discuss ALS6, detail the cellular functions of FUS, and suggest that the localization of FUS, as well as protein synthesis rates, could be hallmarks of the ALS phenotype and thus good therapeutic targets.

Asunto(s)

Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Humanos , Esclerosis Amiotrófica Lateral/patología , Neuronas Motoras/patología , Mutación , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/patología , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive death of motor neurons and muscle atrophy, with defective neuron-glia interplay and emergence of aberrant glial phenotypes having a role in disease pathology. Here, we have studied if the pigment violacein with several reported protective/antiproliferative properties may control highly neurotoxic astrocytes (AbAs) obtained from spinal cord cultures of symptomatic hSOD1G93A rats, and if it could be neuroprotective in this ALS experimental model. At concentrations lower than those reported as protective, violacein selectively killed aberrant astrocytes. Treatment of hSOD1G93A rats with doses equivalent to the concentrations that killed AbAs caused a marginally significant delay in survival, partially preserved the body weight and soleus muscle mass and improved the integrity of the neuromuscular junction. Reduced motor neuron death and glial reactivity was also found and likely related to decreased inflammation and matrix metalloproteinase-2 and -9. Thus, in spite that new experimental designs aimed at extending the lifespan of hSOD1G93A rats are needed, improvements observed upon violacein treatment suggest a significant therapeutic potential that deserves further studies.

Asunto(s)

Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Fármacos Neuroprotectores , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/genética , Animales , Modelos Animales de Enfermedad , Indoles , Metaloproteinasa 2 de la Matriz , Ratones , Ratones Transgénicos , Neuronas Motoras/patología , Enfermedades Neurodegenerativas/patología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Ratas , Médula Espinal/patología

RESUMEN

Degeneration of motor neurons, glial cell reactivity, and vascular alterations in the CNS are important neuropathological features of amyotrophic lateral sclerosis (ALS). Immune cells trafficking from the blood also infiltrate the affected CNS parenchyma and contribute to neuroinflammation. Mast cells (MCs) are hematopoietic-derived immune cells whose precursors differentiate upon migration into tissues. Upon activation, MCs undergo degranulation with the ability to increase vascular permeability, orchestrate neuroinflammation and modulate the neuroimmune response. However, the prevalence, pathological significance, and pharmacology of MCs in the CNS of ALS patients remain largely unknown. In autopsy ALS spinal cords, we identified for the first time that MCs express c-Kit together with chymase, tryptase, and Cox-2 and display granular or degranulating morphology, as compared with scarce MCs in control cords. In ALS, MCs were mainly found in the niche between spinal motor neuron somas and nearby microvascular elements, and they displayed remarkable pathological abnormalities. Similarly, MCs accumulated in the motor neuron-vascular niche of ALS murine models, in the vicinity of astrocytes and motor neurons expressing the c-Kit ligand stem cell factor (SCF), suggesting an SCF/c-Kit-dependent mechanism of MC differentiation from precursors. Mechanistically, we provide evidence that fully differentiated MCs in cell cultures can be generated from the murine ALS spinal cord tissue, further supporting the presence of c-Kit+ MC precursors. Moreover, intravenous administration of bone marrow-derived c-Kit+ MC precursors infiltrated the spinal cord in ALS mice but not in controls, consistent with aberrant trafficking through a defective microvasculature. Pharmacological inhibition of c-Kit with masitinib in ALS mice reduced the MC number and the influx of MC precursors from the periphery. Our results suggest a previously unknown pathogenic mechanism triggered by MCs in the ALS motor neuron-vascular niche that might be targeted pharmacologically.

Asunto(s)

Esclerosis Amiotrófica Lateral/inmunología , Mastocitos/inmunología , Microvasos/patología , Neuronas Motoras/patología , Enfermedades Neuroinflamatorias/inmunología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Médula Espinal/inmunología , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Astrocitos/metabolismo , Astrocitos/patología , Benzamidas/farmacología , Estudios de Casos y Controles , Quimasas/metabolismo , Ciclooxigenasa 2/metabolismo , Femenino , Humanos , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , Microvasos/metabolismo , Persona de Mediana Edad , Neuronas Motoras/metabolismo , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/patología , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Piridinas/farmacología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patología , Factor de Células Madre/metabolismo , Tiazoles/farmacología , Triptasas/metabolismo

RESUMEN

Correct operation of neuronal networks depends on the interplay between synaptic excitation and inhibition processes leading to a dynamic state termed balanced network. In the spinal cord, balanced network activity is fundamental for the expression of locomotor patterns necessary for rhythmic activation of limb extensor and flexor muscles. After spinal cord lesion, paralysis ensues often followed by spasticity. These conditions imply that, below the damaged site, the state of balanced networks has been disrupted and that restoration might be attempted by modulating the excitability of sublesional spinal neurons. Because of the widespread expression of inhibitory GABAergic neurons in the spinal cord, their role in the early and late phases of spinal cord injury deserves full attention. Thus, an early surge in extracellular GABA might be involved in the onset of spinal shock while a relative deficit of GABAergic mechanisms may be a contributor to spasticity. We discuss the role of GABA A receptors at synaptic and extrasynaptic level to modulate network excitability and to offer a pharmacological target for symptom control. In particular, it is proposed that activation of GABA A receptors with synthetic GABA agonists may downregulate motoneuron hyperexcitability (due to enhanced persistent ionic currents) and, therefore, diminish spasticity. This approach might constitute a complementary strategy to regulate network excitability after injury so that reconstruction of damaged spinal networks with new materials or cell transplants might proceed more successfully.

Asunto(s)

Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Inhibidores/fisiología , Red Nerviosa/metabolismo , Receptores de GABA-A/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Médula Espinal/metabolismo , Animales , Neuronas GABAérgicas/fisiología , Humanos , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Red Nerviosa/patología , Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología

RESUMEN

Patients suffering of amyotrophic lateral sclerosis (ALS) present motoneuron degeneration leading to muscle atrophy, dysphagia, and dysarthria. The Wobbler mouse, an animal model of ALS, shows a selective loss of motoneurons, astrocytosis, and microgliosis in the spinal cord. The incidence of ALS is greater in men; however, it increases in women after menopause, suggesting a role of sex steroids in ALS. Testosterone is a complex steroid that exerts its effects directly via androgen (AR) or Sigma-1 receptors and indirectly via estrogen receptors (ER) after aromatization into estradiol. Its reduced-metabolite 5α-dihydrotestosterone acts via AR. This study analyzed the effects of testosterone in male symptomatic Wobblers. Controls or Wobblers received empty or testosterone-filled silastic tubes for 2 months. The cervical spinal cord from testosterone-treated Wobblers showed (1) similar androgen levels to untreated control and (2) increased levels of testosterone, and its 5α-reduced metabolites, 5α- dihydrotestosterone, and 3ß-androstanediol, but (3) undetectable levels of estradiol compared to untreated Wobblers. Testosterone-treated controls showed comparable steroid concentrations to its untreated counterpart. In testosterone- treated Wobblers a reduction of AR, ERα, and aromatase and high levels of Sigma-1 receptor mRNAs was demonstrated. Testosterone treatment increased ChAT immunoreactivity and the antiinflammatory mediator TGFß, while it lessened vacuolated motoneurons, GFAP+ astrogliosis, the density of IBA1+ microgliosis, proinflammatory mediators, and oxidative/nitrosative stress. Clinically, testosterone treatment in Wobblers slowed the progression of paw atrophy and improved rotarod performance. Collectively, our findings indicate an antiinflammatory and protective effect of testosterone in the degenerating spinal cord. These results coincided with a high concentration of androgen-reduced derivatives after testosterone treatment suggesting that the steroid profile may have a beneficial role on disease progression.

Asunto(s)

Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Neuronas Motoras/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Testosterona/uso terapéutico , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Aromatasa/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Modelos Animales de Enfermedad , Receptor alfa de Estrógeno/metabolismo , Masculino , Ratones , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Fármacos Neuroprotectores/farmacología , Receptores Androgénicos/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patología , Testosterona/metabolismo , Testosterona/farmacología , Resultado del Tratamiento

RESUMEN

Motor neuron degeneration and neuroinflammation are the most striking pathological features of amyotrophic lateral sclerosis (ALS). ALS currently has no cure and approved drugs have only a modest clinically therapeutic effect in patients. Drugs targeting different deleterious inflammatory pathways in ALS appear as promising therapeutic alternatives. Here, we have assessed the potential therapeutic effect of an electrophilic nitroalkene benzoic acid derivative, (E)-4-(2-nitrovinyl) benzoic acid (BANA), to slow down paralysis progression when administered after overt disease onset in SOD1G93A rats. BANA exerted a significant inhibition of NF-κB activation in NF-κB reporter transgenic mice and microglial cell cultures. Systemic daily oral administration of BANA to SOD1G93A rats after paralysis onset significantly decreased microgliosis and astrocytosis, and significantly reduced the number of NF-κB-p65-positive microglial nuclei surrounding spinal motor neurons. Numerous microglia bearing nuclear NF-κB-p65 were observed in the surrounding of motor neurons in autopsy spinal cords from ALS patients but not in controls, suggesting ALS-associated microglia could be targeted by BANA. In addition, BANA-treated SOD1G93A rats after paralysis onset showed significantly ameliorated spinal motor neuron pathology as well as conserved neuromuscular junction innervation in the skeletal muscle, as compared to controls. Notably, BANA prolonged post-paralysis survival by ~30%, compared to vehicle-treated littermates. These data provide a rationale to therapeutically slow paralysis progression in ALS using small electrophilic compounds such as BANA, through a mechanism involving microglial NF-κB inhibition.

Asunto(s)

Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Nitrobenzoatos/uso terapéutico , Esclerosis Amiotrófica Lateral/mortalidad , Esclerosis Amiotrófica Lateral/patología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Células HT29/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Ratas , Médula Espinal/efectos de los fármacos , Médula Espinal/patología

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder that is characterized by progressive loss of the upper and lower motor neurons at the spinal or bulbar level. Oxidative stress (OS) associated with mitochondrial dysfunction and the deterioration of the electron transport chain are factors that contribute to neurodegeneration and perform a potential role in the pathogenesis of ALS. Natural antioxidant molecules have been proposed as an alternative form of treatment for the prevention of age-related neurological diseases, in which ALS is included. Researches support that regulations in cellular reduction/oxidation (redox) processes are being increasingly implicated in this disease, and antioxidant drugs are aimed at a promising pathway to treatment. Among the strategies used for obtaining new drugs, we can highlight the isolation of secondary metabolite compounds from natural sources that, along with semisynthetic derivatives, correspond to approximately 40% of the drugs found on the market. Among these compounds, we emphasize oxygenated and nitrogenous compounds, such as flavonoids, coumarins, and alkaloids, in addition to the fatty acids, that already stand out in the literature for their antioxidant properties, consisting in a part of the diets of millions of people worldwide. Therefore, this review is aimed at presenting and summarizing the main articles published within the last years, which represent the therapeutic potential of antioxidant compounds of natural origin for the treatment of ALS.

Asunto(s)

Esclerosis Amiotrófica Lateral/metabolismo , Antioxidantes/metabolismo , Neuronas Motoras/patología , Estrés Oxidativo/fisiología , Metabolismo Secundario/fisiología , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Humanos , Neuronas Motoras/metabolismo

RESUMEN

Motor behavior alterations are a shared hallmark of neurodegenerative diseases affecting motor circuits, such as amyotrophic lateral sclerosis (ALS), Parkinson's, and Huntington's diseases. In patients and transgenic animal models of amyotrophic lateral sclerosis fine movements controlled by distal muscles are the first to be affected, but its study and knowledge remain poorly understood, mainly because most of the tests used for describing the motor alterations are focused on the function of proximal muscles and gross movements. In this study we demonstrate that alterations of phalangeal fine movements can be quantitatively evaluated using a novel procedure designed by us, phalangeal tension recording test, which showed high sensitivity to detect such alterations. The evaluation was carried out during the motor neuron (MN) degenerative process induced by the acute and chronic overactivation of AMPA receptors in the lumbar rat spinal cord, using previously described models. The new method allowed the quantification of significant alterations of the fine movements of the hindpaws phalanges when AMPA was infused in the lumbar segment controlling the distal muscles, but not when a more rostral spinal segment was infused, and these alterations were not detected by the rotarod or the stride tests. These changes occurred before the paralysis of the hindlimbs. Studying the early distal motor alterations before the total paralysis at late stages is essential for understanding the initial consequences of MN degeneration and therefore for designing new strategies for the control, treatment and prevention of MN diseases.

Asunto(s)

Neuronas Motoras/patología , Movimiento/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/administración & dosificación , Animales , Modelos Animales de Enfermedad , Fuerza de la Mano , Masculino , Ratas Wistar , Receptores AMPA/agonistas , Prueba de Desempeño de Rotación con Aceleración Constante

RESUMEN

Amyotrophic lateral sclerosis type 8 (ALS8) is an autosomal dominant form of ALS, which is caused by pathogenic variants in the VAPB gene. Here we investigated five ALS8 patients, classified as 'severe' and 'mild' from a gigantic Brazilian kindred, carrying the same VAPB mutation but displaying different clinical courses. Copy number variation and whole exome sequencing analyses in such individuals ruled out previously described genetic modifiers of pathogenicity. After deriving induced pluripotent stem cells (iPSCs) for each patient (N = 5) and controls (N = 3), motor neurons were differentiated, and high-throughput RNA-Seq gene expression measurements were performed. Functional cell death and oxidative metabolism assays were also carried out in patients' iPSC-derived motor neurons. The degree of cell death and mitochondrial oxidative metabolism were similar in iPSC-derived motor neurons from mild patients and controls and were distinct from those of severe patients. Similar findings were obtained when RNA-Seq from such cells was performed. Overall, 43 genes were upregulated and 66 downregulated in the two mild ALS8 patients when compared with severe ALS8 individuals and controls. Interestingly, significantly enriched pathways found among differentially expressed genes, such as protein translation and protein targeting to the endoplasmic reticulum (ER), are known to be associated with neurodegenerative processes. Taken together, the mitigating mechanisms here presented appear to maintain motor neuron survival by keeping translational activity and protein targeting to the ER in such cells. As ALS8 physiopathology has been associated with proteostasis mechanisms in ER-mitochondria contact sites, such differentially expressed genes appear to relate to the bypass of VAPB deficiency.

Asunto(s)

Esclerosis Amiotrófica Lateral/genética , Mitocondrias/genética , Degeneración Nerviosa/genética , Proteínas de Transporte Vesicular/genética , Anciano , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Diferenciación Celular/genética , Retículo Endoplásmico/genética , Femenino , Regulación de la Expresión Génica/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Degeneración Nerviosa/patología , Estrés Oxidativo/genética , RNA-Seq , Proteínas de Transporte Vesicular/deficiencia

RESUMEN

Amyotrophic Lateral Sclerosis (ALS) is a progressive, neurodegenerative disease characterized by loss of upper motor neurons (UMN) and lower motor neurons (LMN). Disease affects people all over the world and is more prevalent in men. Patients with ALS develop extensive muscle wasting, paralysis and ultimately death, with a median survival of usually fewer than five years after disease onset. ALS may be sporadic (sALS, 90%) or familial (fALS, 10%). The large majority of fALS cases are associated with genetic alterations, which are mainly related to the genes SOD1, TDP-43, FUS, and C9ORF72. In vitro and in vivo models have helped elucidate ALS etiology and pathogenesis, as well as its molecular, cellular, and physiological mechanisms. Many studies in cell cultures and animal models, such as Caenorhabditis elegans, Drosophila melanogaster, zebrafish, rodents, and non-human primates have been performed to clarify the relationship of these genes to ALS disease. However, there are inherent limitations to consider when using experimental models. In this review, we provide an updated overview of the most used in vitro and in vivo studies that have contributed to a better understanding of the different ALS pathogenic mechanisms.

Asunto(s)

Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Modelos Animales de Enfermedad , Neuronas Motoras/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Humanos , Neuronas Motoras/patología , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo

RESUMEN

The Wobbler mouse spinal cord shows vacuolated motoneurons, glial reaction, inflammation and abnormal glutamatergic parameters. Wobblers also show deficits of motor performance. These conditions resemble amyotrophic lateral sclerosis (ALS). Wobbler mice also show high levels of corticosterone in blood, adrenals and brain plus adrenal hypertrophy, suggesting that chronically elevated glucocorticoids prime spinal cord neuroinflammation. Therefore, we analyzed if treatment of Wobbler mice with the glucocorticoid receptor (GR) antagonist CORT113176 mitigated the mentioned abnormalities. 30 mg/kg CORT113176 given daily for 3 weeks reduced motoneuron vacuolation, decreased astro and microgliosis, lowered the inflammatory mediators high mobility group box 1 protein (HMGB1), toll-like receptor 4, myeloid differentiation primary response 88 (MyD88), p50 subunit of nuclear factor kappa B (NFκB), tumor necrosis factor (TNF) receptor, and interleukin 18 (IL18) compared to untreated Wobblers. CORT113176 increased the survival signal pAKT (serine-threonine kinase) and decreased the death signal phosphorylated Junk-N-terminal kinase (pJNK), symptomatic of antiapoptosis. There was a moderate positive effect on glutamine synthase and astrocyte glutamate transporters, suggesting decreased glutamate excitotoxicity. In this pre-clinical study, Wobblers receiving CORT113176 showed enhanced resistance to fatigue in the rota rod test and lower forelimb atrophy at weeks 2-3. Therefore, long-term treatment with CORT113176 attenuated degeneration and inflammation, increased motor performance and decreased paw deformity. Antagonism of the GR may be of potential therapeutic value for neurodegenerative diseases.

Asunto(s)

Isoquinolinas/administración & dosificación , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Pirazoles/administración & dosificación , Receptores de Glucocorticoides/antagonistas & inhibidores , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/patología , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Encefalitis/patología , Femenino , Ácido Glutámico/toxicidad , Masculino , Ratones , Microglía/efectos de los fármacos , Microglía/patología

RESUMEN

Distal axonopathy is a recognized pathological feature of amyotrophic lateral sclerosis (ALS). In the peripheral nerves of ALS patients, motor axon loss elicits a Wallerian-like degeneration characterized by denervated Schwann cells (SCs) together with immune cell infiltration. However, the pathogenic significance of denervated SCs accumulating following impaired axonal growth in ALS remains unclear. Here, we analyze SC phenotypes in sciatic nerves of ALS patients and paralytic SOD1G93A rats, and identify remarkably similar and specific reactive SC phenotypes based on the pattern of S100ß, GFAP, isolectin and/or p75NTR immunoreactivity. Different subsets of reactive SCs expressed colony-stimulating factor-1 (CSF1) and Interleukin-34 (IL-34) and closely interacted with numerous endoneurial CSF-1R-expressing monocyte/macrophages, suggesting a paracrine mechanism of myeloid cell expansion and activation. SCs bearing phagocytic phenotypes as well as endoneurial macrophages expressed stem cell factor (SCF), a trophic factor that attracts and activates mast cells through the c-Kit receptor. Notably, a subpopulation of Ki67+ SCs expressed c-Kit in the sciatic nerves of SOD1G93A rats, suggesting a signaling pathway that fuels SC proliferation in ALS. c-Kit+ mast cells were also abundant in the sciatic nerve from ALS donors but not in controls. Pharmacological inhibition of CSF-1R and c-Kit with masitinib in SOD1G93A rats potently reduced SC reactivity and immune cell infiltration in the sciatic nerve and ventral roots, suggesting a mechanism by which the drug ameliorates peripheral nerve pathology. These findings provide strong evidence for a previously unknown inflammatory mechanism triggered by SCs in ALS peripheral nerves that has broad application in developing novel therapies.

Asunto(s)

Esclerosis Amiotrófica Lateral/patología , Inflamación/metabolismo , Interleucinas/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Células de Schwann/metabolismo , Factor de Células Madre/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Axones/patología , Modelos Animales de Enfermedad , Humanos , Masculino , Neuronas Motoras/patología , Neuroglía/metabolismo , Ratas Transgénicas

Asunto(s)

Enfermedad de Huntington/patología , Neuronas Motoras/patología , Fibras Musculares de Contracción Rápida/patología , Unión Neuromuscular/patología , Animales , Extremidad Inferior , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/inervación , Músculo Esquelético/patología , Transmisión Sináptica/fisiología

RESUMEN

BACKGROUND: The development of new therapeutic strategies to treat amyotrophic lateral sclerosis (ALS) is of utmost importance. The use of cyclic nitroxides such as tempol may provide neuroprotection and improve lifespan. We investigated whether tempol (50 mg/kg) presents therapeutic potential in SOD1G93A transgenic mice. METHODS: Tempol treatment began at the asymptomatic phase of the disease (10th week) and was administered every other day until week 14, after which it was administered twice a week until the final stage of the disease. The animals were sacrificed at week 14 (initial stage of symptoms-ISS) and at the end stage (ES) of the disease. The lumbar spinal cord of the animals was dissected and processed for use in the following techniques: Nissl staining to evaluate neuronal survival; immunohistochemistry to evaluate astrogliosis and microgliosis (ISS and ES); qRT-PCR to evaluate the expression of neurotrophic factors and pro-inflammatory cytokines (ISS); and transmission electron microscopy to evaluate the alpha-motoneurons (ES). Behavioral analyses considering the survival of animals, bodyweight loss, and Rotarod motor performance test started on week 10 and were performed every 3 days until the end-stage of the disease. RESULTS: The results revealed that treatment with tempol promoted greater neuronal survival (23%) at ISS compared to untreated animals, which was maintained until ES. The intense reactivity of astrocytes and microglia observed in vehicle animals was reduced in the lumbar spinal cords of the animals treated with tempol. In addition, the groups treated with tempol showed reduced expression of proinflammatory cytokines (IL1ß and TNFα) and a three-fold decrease in the expression of TGFß1 at ISS compared with the group treated with vehicle. CONCLUSIONS: Altogether, our results indicate that treatment with tempol has beneficial effects, delaying the onset of the disease by enhancing neuronal survival and decreasing glial cell reactivity during ALS progression in SOD1G93A mice.

Asunto(s)

Esclerosis Amiotrófica Lateral/fisiopatología , Óxidos N-Cíclicos/uso terapéutico , Inflamación/tratamiento farmacológico , Destreza Motora/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Médula Espinal/efectos de los fármacos , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Supervivencia Celular/efectos de los fármacos , Óxidos N-Cíclicos/farmacología , Modelos Animales de Enfermedad , Femenino , Inflamación/metabolismo , Inflamación/patología , Interleucina-1beta/metabolismo , Masculino , Ratones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Destreza Motora/fisiología , Fármacos Neuroprotectores/farmacología , Prueba de Desempeño de Rotación con Aceleración Constante , Marcadores de Spin , Médula Espinal/metabolismo , Médula Espinal/patología , Superóxido Dismutasa-1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of upper and lower motor neurons accompanied by proliferation of reactive microglia in affected regions. However, it is unknown whether the hematopoietic marker CD34 can identify a subpopulation of proliferating microglial cells in the ALS degenerating spinal cord. Immunohistochemistry for CD34 and microglia markers was performed in lumbar spinal cords of ALS rats bearing the SOD1G93A mutation and autopsied ALS and control human subjects. Characterization of CD34-positive cells was also performed in primary cell cultures of the rat spinal cords. CD34 was expressed in a large number of cells that closely interacted with degenerating lumbar spinal cord motor neurons in symptomatic SOD1G93A rats, but not in controls. Most CD34+ cells co-expressed the myeloid marker CD11b, while only a subpopulation was stained for Iba1 or CD68. Notably, CD34+ cells actively proliferated and formed clusters adjacent to damaged motor neurons bearing misfolded SOD1. CD34+ cells were identified in the proximity of motor neurons in autopsied spinal cord from sporadic ALS subjects but not in controls. Cell culture of symptomatic SOD1G93A rat spinal cords yielded a large number of CD34+ cells exclusively in the non-adherent phase, which generated microglia after successive passaging. A yet unrecognized CD34+ cells, expressing or not the microglial marker Iba1, proliferate and accumulate adjacent to degenerating spinal motor neurons, representing an intriguing cell target for approaching ALS pathogenesis and therapeutics.

Asunto(s)

Esclerosis Amiotrófica Lateral/patología , Antígenos CD34/análisis , Microglía/patología , Neuronas Motoras/patología , Esclerosis Amiotrófica Lateral/genética , Animales , Proliferación Celular , Células Cultivadas , Humanos , Masculino , Microglía/citología , Mutación Puntual , Pliegue de Proteína , Ratas , Médula Espinal/patología , Superóxido Dismutasa-1/análisis , Superóxido Dismutasa-1/genética

RESUMEN

The Wobbler mouse has been proposed as an experimental model of the sporadic form of amyotrophic lateral sclerosis (ALS). The administration of natural progesterone (PROG) to Wobbler mice attenuates neuropathology, inhibits oxidative stress, enhances the expression of genes involved in motoneuron function, increases survival and restores axonal transport. However, current pharmacological treatments for ALS patients are still partially effective. This encouraged us to investigate if the synthetic progestin norethindrone (NOR), showing higher potency than PROG and used for birth control and hormone therapy might also afford neuroprotection. Two-month-old Wobbler mice (wr/wr) were left untreated or received either a 20 mg pellet of PROG or a 1 mg pellet of NOR for 18 days. Untreated control NFR/NFR mice (background strain for Wobbler) were also employed. Wobblers showed typical clinical and spinal cord abnormalities, while these abnormalities were normalized with PROG treatment. Surprisingly, we found that NOR did not increase immunoreactivity and gene expression for choline-acetyltransferase, drastically decreased GFAP + astrogliosis, favored proinflammatory mediators, promoted the inflammatory phenotype of IBA1+ microglia, increased the receptor for advanced glycation end products (RAGE) mRNA and protein expression and the activity of nitric oxide synthase (NOS)/NADPH diaphorase in the cervical spinal cord. Additionally, NOR treatment produced atrophy of the thymus. The combined negative effects of NOR on clinical assessments (forelimb atrophy and rotarod performance) suggest a detrimental effect on muscle trophism and motor function. These findings reinforce the evidence that the type of progestin used for contraception, endometriosis or replacement therapy, may condition the outcome of preclinical and clinical studies targeting neurodegenerative diseases.

Asunto(s)

Modelos Animales de Enfermedad , Neuronas Motoras/efectos de los fármacos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Neuroprotección/efectos de los fármacos , Noretindrona/farmacología , Progesterona/farmacología , Progestinas/farmacología , Animales , Anticonceptivos Sintéticos Orales/farmacología , Ratones , Neuronas Motoras/patología

RESUMEN

Astrogliosis and microglial reactions are correlated with the formation of scar tissue and synapse loss. 4-hydroxy-tempo (TEMPOL) is a reactive oxygen species scavenger with proven neuroprotective efficacy in experimental models of traumatic injury and cerebral ischemia. TEMPOL has not, however, been applied following ventral root lesions, which are particularly correlated with the degeneration of spinal motoneurons following brachial plexus injuries. Thus, the present study investigated the effects of TEMPOL on motoneurons and adjacent glial reactions, with a particular focus on the preservation of excitatory and inhibitory spinal circuits. Adult female Sprague Dawley rats were subjected to ventral root crush (VRC) at the lumbar intumescence. Animals were divided into the following experimental groups: (a) VRC-saline treatment; (b) VRC-TEMPOL treatment (12 mg/kg, n = 5), and (c) VRC-TEMPOL treatment (250 mg/kg, n = 5). The spinal cord tissue located contralateral to the lesion was used as the control. Fourteen days after lesioning, the rats were euthanized and the spinal cords were removed for motoneuron counting and immunolabeling with glial (GFAP and Iba-1) and synapse markers (synaptophysin, VGLUT-1, and GAD65). Although TEMPOL did not exert neuroprotective effects at the studied concentrations, the modulation of glial reactions was significant at higher doses. Thus, synaptophysin staining was preserved and, in particular, VGLUT-1-positive inputs were maintained, thereby indicating that TEMPOL preserved proprioceptive glutamatergic inputs without exacerbating the rate of motoneuron degeneration. Consequently, its administration with other efficient neuroprotective substances may significantly improve the outcomes following spinal cord lesioning.

Asunto(s)

Óxidos N-Cíclicos/farmacología , Hidroxilamina/farmacología , Neuroglía/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Raíces Nerviosas Espinales/lesiones , Raíces Nerviosas Espinales/metabolismo , Sinapsis/efectos de los fármacos , Animales , Antioxidantes , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis , Neuronas Motoras/patología , Ratas , Ratas Sprague-Dawley , Asta Lateral de la Médula Espinal/metabolismo , Raíces Nerviosas Espinales/patología , Sinaptofisina/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo

RESUMEN

Neuroinflammation is a recognized pathogenic mechanism underlying motor neuron degeneration in amyotrophic lateral sclerosis (ALS), but the inflammatory mechanisms influencing peripheral motor axon degeneration remain largely unknown. A recent report showed a pathogenic role for c-Kit-expressing mast cells mediating inflammation and neuromuscular junction denervation in muscles from SOD1G93A rats. Here, we have explored whether mast cells infiltrate skeletal muscles in autopsied muscles from ALS patients. We report that degranulating mast cells were abundant in the quadriceps muscles from ALS subjects but not in controls. Mast cells were associated with myofibers and motor endplates and, remarkably, interacted with neutrophils forming large extracellular traps. Mast cells and neutrophils were also abundant around motor axons in the extensor digitorum longus muscle, sciatic nerve, and ventral roots of symptomatic SOD1G93A rats, indicating that immune cell infiltration extends along the entire peripheral motor pathway. Postparalysis treatment of SOD1G93A rats with the tyrosine kinase inhibitor drug masitinib prevented mast cell and neutrophil infiltration, axonal pathology, secondary demyelination, and the loss of type 2B myofibers, compared with vehicle-treated rats. These findings provide further evidence for a yet unrecognized contribution of immune cells in peripheral motor pathway degeneration that can be therapeutically targeted by tyrosine kinase inhibitors.

Asunto(s)

Esclerosis Amiotrófica Lateral/inmunología , Mastocitos/inmunología , Neuronas Motoras/patología , Unión Neuromuscular/patología , Neutrófilos/inmunología , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/patología , Animales , Axones/efectos de los fármacos , Axones/inmunología , Axones/patología , Benzamidas , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/inmunología , Modelos Animales de Enfermedad , Humanos , Masculino , Mastocitos/efectos de los fármacos , Neuronas Motoras/citología , Neuronas Motoras/inmunología , Músculo Esquelético/citología , Músculo Esquelético/inervación , Músculo Esquelético/patología , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/inmunología , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Piperidinas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas , Ratas , Ratas Transgénicas , Superóxido Dismutasa/genética , Superóxido Dismutasa-1/genética , Tiazoles/farmacología , Tiazoles/uso terapéutico , Resultado del Tratamiento

RESUMEN

In recent years, the role of autophagy in the pathogenesis of most neurodegenerative diseases has transitioned into a limbo of protective or detrimental effects. Genetic evidence indicates that mutations in autophagy-regulatory genes can result in the occurrence of amyotrophic lateral sclerosis (ALS), suggesting a physiological role of the pathway to motoneuron function. However, experimental manipulation of autophagy in ALS models led to conflicting results depending on the intervention strategy and the disease model used. A recent work by the Maniatis group systematically explored the role of cell-specific autophagy in motoneurons at different disease stages, revealing surprising and unexpected findings. Autophagy activity at early stages may contribute to maintaining the structure and function of neuromuscular junctions, whereas at later steps of the disease it has a pathogenic activity possibly involving cell-nonautonomous mechanisms related to glial activation. This new study adds a new layer of complexity in the field, suggesting an intricate interplay between proteostasis alterations, the time-differential function of autophagy in neurons, and muscle innervation in ALS.

Asunto(s)

Esclerosis Amiotrófica Lateral/metabolismo , Autofagia/fisiología , Neuronas Motoras/patología , Unión Neuromuscular/metabolismo , Animales , Autofagia/genética , Modelos Animales de Enfermedad , Humanos , Neuronas Motoras/metabolismo , Superóxido Dismutasa/metabolismo