RESUMO
Amyotrophic Lateral Sclerosis (ALS) is considered the prototype of motor neuron disease, characterized by motor neuron loss and muscle waste. A well-established pathogenic hallmark of ALS is mitochondrial failure, leading to bioenergetic deficits. So far, pharmacological interventions for the disease have proven ineffective. Trimetazidine (TMZ) is described as a metabolic modulator acting on different cellular pathways. Its efficacy in enhancing muscular and cardiovascular performance has been widely described, although its molecular target remains elusive. We addressed the molecular mechanisms underlying TMZ action on neuronal experimental paradigms. To this aim, we treated murine SOD1G93A-model-derived primary cultures of cortical and spinal enriched motor neurons, as well as a murine motor-neuron-like cell line overexpressing SOD1G93A, with TMZ. We first characterized the bioenergetic profile of the cell cultures, demonstrating significant mitochondrial dysfunction that is reversed by acute TMZ treatments. We then investigated the effect of TMZ in promoting autophagy processes and its impact on mitochondrial morphology. Finally, we demonstrated the effectiveness of TMZ in terms of the mitochondrial functionality of ALS-rpatient-derived peripheral blood mononuclear cells (PBMCs). In summary, our results emphasize the concept that targeting mitochondrial dysfunction may represent an effective therapeutic strategy for ALS. The findings demonstrate that TMZ enhances mitochondrial performance in motor neuron cells by activating autophagy processes, particularly mitophagy. Although further investigations are needed to elucidate the precise molecular pathways involved, these results hold critical implications for the development of more effective and specific derivatives of TMZ for ALS treatment.
Assuntos
Esclerose Lateral Amiotrófica , Doenças Mitocondriais , Trimetazidina , Camundongos , Animais , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Superóxido Dismutase-1/metabolismo , Trimetazidina/farmacologia , Trimetazidina/uso terapêutico , Camundongos Transgênicos , Leucócitos Mononucleares/metabolismo , Superóxido Dismutase/metabolismo , Autofagia , Modelos Animais de DoençasRESUMO
Since its initial involvement in numerous neurodegenerative pathologies in 2006, either as a principal actor or as a cofactor, new pathologies implicating transactive response (TAR) DNA-binding protein 43 (TDP-43) are regularly emerging also beyond the neuronal system. This reflects the fact that TDP-43 functions are particularly complex and broad in a great variety of human cells. In neurodegenerative diseases, this protein is often pathologically delocalized to the cytoplasm, where it irreversibly aggregates and is subjected to various post-translational modifications such as phosphorylation, polyubiquitination, and cleavage. Until a few years ago, the research emphasis has been focused particularly on the impacts of this aggregation and/or on its widely described role in complex RNA splicing, whether related to loss- or gain-of-function mechanisms. Interestingly, recent studies have strengthened the knowledge of TDP-43 activity at the chromatin level and its implication in the regulation of DNA transcription and stability. These discoveries have highlighted new features regarding its own transcriptional regulation and suggested additional mechanistic and disease models for the effects of TPD-43. In this review, we aim to give a comprehensive view of the potential epigenetic (de)regulations driven by (and driving) this multitask DNA/RNA-binding protein.
Assuntos
Cromatina , Proteínas de Ligação a DNA , Humanos , Citoplasma , Proteínas de Ligação a DNA/genética , Epigênese Genética , EpigenômicaRESUMO
Proteins oxidation by reactive species is implicated in the aetiology or progression of a panoply of disorders and diseases such as neurodegenerative disorders. It is becoming increasingly evident that redox imbalance in the brain mediates neurodegeneration. Free radicals, as reactive species of oxygen (ROS) but also reactive nitrogen species (RNS) and reactive sulfur species (RSS), are generated in vivo from several sources. Within the cell the mitochondria represent the main source of ROS and mitochondrial dysfunction is both the major contributor to oxidative stress (OS) as well its major consequence.To date there are no doubts that a condition of OS added to other factors as mitochondrial damage in mtDNA or mitochondrial respiratory chain, may contribute to trigger or amplify mechanisms leading to neurodegenerative disorders.In this chapter, we aim at illustrate the molecular interplay occurring between mitochondria and OS focusing on Amyotrophic Lateral Sclerosis, describing a phenotypic reprogramming mechanism of mitochondria in complex neurological disorder.
Assuntos
Esclerose Lateral Amiotrófica , Mitocôndrias , Estresse Oxidativo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/fisiopatologia , DNA Mitocondrial/genética , Humanos , Mitocôndrias/patologia , Espécies Reativas de Nitrogênio , Espécies Reativas de Oxigênio/metabolismoRESUMO
A number of factors can trigger amyotrophic lateral sclerosis (ALS), although its precise pathogenesis is still uncertain. In a previous study done by us, poisonous liquoral levels of hydrogen sulphide (H2S) in sporadic ALS patients were reported. In the same study very high concentrations of H2S in the cerebral tissues of the familial ALS (fALS) model of the SOD1G93A mouse, were measured. The objective of this study was to test whether decreasing the levels of H2S in the fALS mouse could be beneficial. Amino-oxyacetic acid (AOA)-a systemic dual inhibitor of cystathionine-ß-synthase and cystathionine-γ lyase (two key enzymes in the production of H2S)-was administered to fALS mice. AOA treatment decreased the content of H2S in the cerebral tissues, and the lifespan of female mice increased by approximately ten days, while disease progression in male mice was not affected. The histological evaluation of the spinal cord of the females revealed a significant increase in GFAP positivity and a significant decrease in IBA1 positivity. In conclusion, the results of the study indicate that, in the animal model, the inhibition of H2S production is more effective in females. The findings reinforce the need to adequately consider sex as a relevant factor in ALS.
Assuntos
Ácido Amino-Oxiacético/farmacologia , Esclerose Lateral Amiotrófica/metabolismo , Cistationina beta-Sintase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Sulfeto de Hidrogênio/metabolismo , Ácido Amino-Oxiacético/uso terapêutico , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Inibidores Enzimáticos/uso terapêutico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/efeitos dos fármacos , Fatores Sexuais , Superóxido Dismutase-1/genéticaRESUMO
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.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Modelos Animais de Doenças , Córtex Motor/metabolismo , Fosfotirosina/metabolismo , Medula Espinal/metabolismo , Superóxido Dismutase/biossíntese , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Córtex Motor/patologia , Transdução de Sinais/fisiologia , Medula Espinal/patologia , Superóxido Dismutase/genéticaRESUMO
OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a motor neuron disease whose pathophysiological deficits, causing impairment in motor function, are largely unknown. Here we propose that hydrogen sulfide (H2 S), as a glial-released inflammatory factor, contributes to ALS-mediated motor neuron death. METHODS: H2 S concentrations were analyzed in the cerebrospinal fluid of 37 sporadic ALS patients and 14 age- and gender-matched controls, in tissues of a familial ALS (fALS) mouse model, and in spinal cord culture media by means of a specific and innovative high-performance liquid chromatography method. The effects of H2 S on motor neurons cultures was analyzed immunohistochemically and by patch clamp recordings and microfluorometry. RESULTS: We found a significantly high level of H2 S in the spinal fluid of the ALS patients. Consistently, we found increased levels of H2 S in the tissues and in the media from mice spinal cord cultures bearing the fALS mutation SOD1G93A. In addition, NaHS, an H2 S donor, added to spinal culture, obtained from control C57BL/6J mice, is toxic for motor neurons, and induces an intracellular Ca(2+) increase, attenuated by the intracytoplasmatic application of adenosine triphosphate. We further show that H2 S is mainly released by astrocytes and microglia. INTERPRETATION: This study unravels H2 S as an astroglial mediator of motor neuron damage possibly involved in the cellular death characterizing ALS.
Assuntos
Esclerose Lateral Amiotrófica/líquido cefalorraquidiano , Esclerose Lateral Amiotrófica/diagnóstico , Sulfeto de Hidrogênio/líquido cefalorraquidiano , Idoso , Animais , Biomarcadores/líquido cefalorraquidiano , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Medula Espinal/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease pathologically characterized by the massive loss of motor neurons in the spinal cord, brain stem and cerebral cortex. There is a consensus in the field that ALS is a multifactorial pathology and a number of possible mechanisms have been suggested. Among the proposed hypothesis, glutamate toxicity has been one of the most investigated. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor mediated cell death and impairment of the glutamate-transport system have been suggested to play a central role in the glutamate-mediated motor neuron degeneration. In this context, the role played by the N-methyl-d-aspartate (NMDA) receptor has received considerable less attention notwithstanding its high Ca(2+) permeability, expression in motor neurons and its importance in excitotoxicity. This review overviews the critical role of NMDA-mediated toxicity in ALS, with a particular emphasis on the endogenous modulators of the NMDAR.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Esclerose Lateral Amiotrófica/patologia , Morte Celular , Humanos , Neurônios Motores/patologia , Receptores de N-Metil-D-Aspartato/metabolismo , Medula Espinal/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive loss of motor neurons (MNs) and astrogliosis. Recent evidence suggests that factors secreted by activated astrocytes might contribute to degeneration of MNs. We focused on endothelin-1 (ET-1), a peptide which is strongly up-regulated in reactive astrocytes under different pathological conditions. We show that ET-1 is abundantly expressed by reactive astrocytes in the spinal cord of the SOD1-G93A mouse model and sporadic ALS patients. To test if ET-1 might play a role in degeneration of MNs, we investigated its effect on MN survival in an in vitro model of mixed rat spinal cord cultures (MSCs) enriched of astrocytes exhibiting a reactive phenotype. ET-1 exerted a toxic effect on MNs in a time- and concentration-dependent manner, with an exposure to 100-200nM ET-1 for 48h resulting in 40-50% MN cell death. Importantly, ET-1 did not induce MN degeneration when administered on cultures treated with AraC (5µM) or grown in a serum-free medium that did not favor astrocyte proliferation and reactivity. We found that both ETA and ETB receptors are enriched in astrocytes in MSCs. The ET-1 toxic effect was mimicked by ET-3 (100nM) and sarafotoxin S6c (10nM), two selective agonists of endothelin-B receptors, and was not additive with that of ET-3 suggesting the involvement of ETB receptors. Surprisingly, however, the ET-1 effect persisted in the presence of the ETB receptor antagonist BQ-788 (200nM-2µM) and was slightly reversed by the ETA receptor antagonist BQ-123 (2µM), suggesting an atypical pharmacological profile of the astrocytic receptors responsible for ET-1 toxicity. The ET-1 effect was not undone by the ionotropic glutamate receptor AMPA antagonist GYKI 52466 (20µM), indicating that it is not caused by an increased glutamate release. Conversely, a 48-hour ET-1 treatment increased MN cell death induced by acute exposure to AMPA (50µM), which is indicative of two distinct pathways leading to neuronal death. Altogether these results indicate that ET-1 exerts a toxic effect on cultured MNs through mechanisms mediated by reactive astrocytes and suggest that ET-1 may contribute to MN degeneration in ALS. Thus, a treatment aimed at lowering ET-1 levels or antagonizing its effect might be envisaged as a potential therapeutic strategy to slow down MN degeneration in this devastating disease.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Endotelina-1/farmacologia , Neurônios Motores/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/genética , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Gravidez , Ratos , Ratos Wistar , Medula Espinal/citologia , Superóxido Dismutase/genética , Fatores de TempoRESUMO
The modulation of synaptic strength associated with learning is post-synaptically regulated by changes in density and shape of dendritic spines. The transcription factor CREB (cAMP response element binding protein) is required for memory formation and in vitro dendritic spine rearrangements, but its role in learning-induced remodeling of neurons remains elusive. Using transgenic mice conditionally expressing a dominant-negative CREB (CREBS133A: mCREB) mutant, we found that inhibiting CREB function does not alter spine density, spine morphology, and levels of polymerized actin in naive CA1 neurons. CREB inhibition, however, impaired contextual fear conditioning and produced a learning-induced collapse of spines associated with a blockade of learning-dependent increase in actin polymerization. Blocking mCREB expression with doxycycline rescued memory and restored a normal pattern of learning-induced spines, demonstrating that CREB controls structural adaptations of neurons selectively involved in memory formation.
Assuntos
Proteína de Ligação a CREB/fisiologia , Condicionamento Psicológico/fisiologia , Neurônios/metabolismo , Alanina/genética , Análise de Variância , Animais , Animais Recém-Nascidos , Proteína de Ligação a CREB/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Doxiciclina/farmacologia , Eletrochoque/efeitos adversos , Medo/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Hipocampo/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Fosforilação , Serina/genética , Coloração pela PrataRESUMO
Ever since its presence was reported in the brain, the nature and role of hydrogen sulfide (H2S) in the Central Nervous System (CNS) have changed. Consequently, H2S has been elected as the third gas transmitter, along with carbon monoxide and nitric oxide, and a number of studies have focused on its neuromodulatory and protectant functions in physiological conditions. The research on H2S has highlighted its many facets in the periphery and in the CNS, and its role as a double-faced compound, switching from protective to toxic depending on its concentration. In this review, we will focus on the bell-shaped nature of H2S as an angiogenic factor and as a molecule released by glial cells (mainly astrocytes) and non-neuronal cells acting on the surrounding environment (paracrine) or on the releasing cells themselves (autocrine). Finally, we will discuss its role in Amyotrophic Lateral Sclerosis, a paradigm of a neurodegenerative disease.
Assuntos
Esclerose Lateral Amiotrófica , Sulfeto de Hidrogênio , Doenças Neurodegenerativas , Humanos , Sistema Nervoso Central , Óxido NítricoRESUMO
Amyotrophic lateral sclerosis (ALS) is an adult-onset disease characterized by the progressive degeneration of motoneurons (MNs). Altered electrical properties have been described in familial and sporadic ALS patients. Cortical and spinal neurons cultured from the mutant Cu,Zn superoxide dismutase 1 (SOD1G93A) mouse, a murine model of ALS, exhibit a marked increase in the persistent Na+ currents. Here, we investigated the effects of the SOD1G93A mutation on the expression of the voltage-gated Na+ channel alpha subunit SCN8A (Nav1.6) and the beta subunits SCN1B (beta1), SCN2B (beta2), and SCN3B (beta3) in MNs of the spinal cord in presymptomatic (P75) and symptomatic (P120) mice. We observed a significant increase, within lamina IX, of the beta3 transcript and protein expression. On the other hand, the beta1 transcript was significantly decreased, in the same area, at the symptomatic stage, while the beta2 transcript levels were unaltered. The SCN8A transcript was significantly decreased at P120 in the whole spinal cord. These data suggest that the SOD1G93A mutation alters voltage-gated Na+ channel subunit expression. Moreover, the increased expression of the beta3 subunit support the hypothesis that altered persistent Na+ currents contribute to the hyperexcitability observed in the ALS-affected MNs.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Subunidades Proteicas/metabolismo , Canais de Sódio/metabolismo , Medula Espinal/fisiologia , Superóxido Dismutase/metabolismo , Adulto , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Humanos , Masculino , Camundongos , Neurônios Motores/citologia , Neurônios Motores/patologia , Neurônios Motores/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.6 , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Subunidades Proteicas/genética , Canais de Sódio/genética , Medula Espinal/citologia , Medula Espinal/patologia , Superóxido Dismutase/genética , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem , Subunidade beta-2 do Canal de Sódio Disparado por Voltagem , Subunidade beta-3 do Canal de Sódio Disparado por VoltagemRESUMO
Many lines of evidence have highlighted the role played by heterogeneous nuclear ribonucleoproteins in amyotrophic lateral sclerosis. In this study, we have aimed to identify transcripts co-regulated by TAR DNA-binding protein 43â kDa and highly conserved heterogeneous nuclear ribonucleoproteins which have been previously shown to regulate TAR DNA-binding protein 43â kDa toxicity (deleted in azoospermia-associated protein 1, heterogeneous nuclear ribonucleoprotein -Q, -D, -K and -U). Using the transcriptome analyses, we have uncovered that Nitric Oxide Synthase 1 Adaptor Protein mRNA is a direct TAR DNA-binding protein 43â kDa target, and in flies, its modulation alone can rescue TAR DNA-binding protein 43â kDa pathology. In primary mouse cortical neurons, we show that TAR DNA-binding protein 43â kDa mediated downregulation of Nitric Oxide Synthase 1 Adaptor Protein expression strongly affects the NMDA-receptor signalling pathway. In human patients, the downregulation of Nitric Oxide Synthase 1 Adaptor Protein mRNA strongly correlates with TAR DNA-binding protein 43â kDa proteinopathy as measured by cryptic Stathmin-2 and Unc-13 homolog A cryptic exon inclusion. Overall, our results demonstrate that Nitric Oxide Synthase 1 Adaptor Protein may represent a novel disease-relevant gene, potentially suitable for the development of new therapeutic strategies.
RESUMO
ALS is a devastating neurodegenerative disorder with no effective treatment. In the present study, we found that daily doses of lithium, leading to plasma levels ranging from 0.4 to 0.8 mEq/liter, delay disease progression in human patients affected by ALS. None of the patients treated with lithium died during the 15 months of the follow-up, and disease progression was markedly attenuated when compared with age-, disease duration-, and sex-matched control patients treated with riluzole for the same amount of time. In a parallel study on a genetic ALS animal model, the G93A mouse, we found a marked neuroprotection by lithium, which delayed disease onset and duration and augmented the life span. These effects were concomitant with activation of autophagy and an increase in the number of the mitochondria in motor neurons and suppressed reactive astrogliosis. Again, lithium reduced the slow necrosis characterized by mitochondrial vacuolization and increased the number of neurons counted in lamina VII that were severely affected in saline-treated G93A mice. After lithium administration in G93A mice, the number of these neurons was higher even when compared with saline-treated WT. All these mechanisms may contribute to the effects of lithium, and these results offer a promising perspective for the treatment of human patients affected by ALS.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Compostos de Lítio/uso terapêutico , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Humanos , Compostos de Lítio/farmacologia , Camundongos , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Medula Espinal/efeitos dos fármacosRESUMO
Over the past 30 years a considerable amount of data has accumulated on the multifaceted role of hydrogen sulfide (H2S) in the central nervous system. Depending on its concentrations, H2S has opposite actions, ranging from neuromodulator to neurotoxic. Nowadays, accurate determination of H2S is still an important challenge to understand its biochemistry and functions. In this perspective, this study aims to explore H2S levels in cerebrospinal fluid (CSF), key biofluid for neurological studies, and to assess alleged correlations with neuroinflammatory and neurodegenerative mechanisms. A validated analytical determination combining selective electrochemical detection with ion chromatography was developed to measure free and bound sulfur forms of H2S. A first cohort of CSF samples (n = 134) was analyzed from patients with inflammatory and demyelinating disorders (acute disseminated encephalomyelitis; multiple sclerosis), chronic neurodegenerative diseases (Alzheimer disease; Parkinson disease), and motor neuron disease (Amyotrophic lateral sclerosis). Given its analytical features, the chromatographic method resulted sensitive, reproducible and robust. We also explored low molecular weight-proteome linked to sulphydration by proteomics analysis on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). This study is a first clinical report on CSF H2S concentrations from neurological diseases and opens up new perspectives on the potential clinical relevance of H2S and its potential therapeutic application.
RESUMO
Frontotemporal Lobar Degeneration (FTD) is a neurodegenerative disease characterized by a progressive deterioration of cognitive functions. Currently, no effective treatment exists. We have studied cytotoxicity and neuronal functionality in cortical and spinal cord cultures upon exposure to cerebrospinal fluid (CSF) from 39 FTD patients. FTD-CSF alters the miniature excitatory postsynaptic currents in the cortical cultures and it is toxic to spinal cord cultures, particularly to GABAergic+ and calbindin-D28k + neurons.
Assuntos
Biomarcadores/líquido cefalorraquidiano , Líquido Cefalorraquidiano , Demência Frontotemporal/patologia , Neurônios/patologia , Idoso , Estudos de Casos e Controles , Feminino , Demência Frontotemporal/líquido cefalorraquidiano , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
[This corrects the article DOI: 10.3389/fimmu.2020.575792.].
RESUMO
One of the most challenging frontiers in biological systems understanding is fluorescent label-free imaging. We present here the NeuriTES platform that revisits the standard paradigms of video analysis to detect unlabeled objects and adapt to the dynamic evolution of the phenomenon under observation. Object segmentation is reformulated using robust algorithms to assure regular cell detection and transfer entropy measures are used to study the inter-relationship among the parameters related to the evolving system. We applied the NeuriTES platform to the automatic analysis of neurites degeneration in presence of amyotrophic lateral sclerosis (ALS) and to the study of the effects of a chemotherapy drug on living prostate cancer cells (PC3) cultures. Control cells have been considered in both the two cases study. Accuracy values of 93% and of 92% are achieved, respectively. NeuriTES not only represents a tool for investigation in fluorescent label-free images but demonstrates to be adaptable to individual needs.
RESUMO
Transgenic mice expressing the human superoxide dismutase 1 (SOD-1) mutant at position 93 (G93A) develop a phenotype resembling amyotrophic lateral sclerosis (ALS). In fact, G93A mice develop progressive motor deficits which finally lead to motor palsy and death. This is due to the progressive degeneration of motor neurons in the ventral horn of the spinal cord. Although a similar loss is reported for specific cranial motor nuclei, only a few studies so far investigated degeneration in a few brainstem nuclei. We recently reported that chronic lithium administration delays onset and duration of the disease, while reducing degeneration of spinal motor neuron. In the present study, we extended this investigation to all somatic motor nuclei of the brain stem in the G93A mice and we evaluated whether analogous protective effects induced by lithium in the spinal cord were present at the brain stem level. We found that all motor but the oculomotor nuclei were markedly degenerated in G93A mice, and chronic treatment with lithium significantly attenuated neurodegeneration in the trigeminal, facial, ambiguus, and hypoglossal nuclei. Moreover, in the hypoglossal nucleus, we found that recurrent collaterals were markedly lost in G93A mice while they were rescued by chronic lithium administration.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Tronco Encefálico/efeitos dos fármacos , Nervos Cranianos/efeitos dos fármacos , Lítio/farmacologia , Neurônios Motores/metabolismo , Fármacos Neuroprotetores/farmacologia , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Biomarcadores/análise , Biomarcadores/metabolismo , Mapeamento Encefálico/métodos , Tronco Encefálico/patologia , Tronco Encefálico/fisiopatologia , Colina O-Acetiltransferase/análise , Colina O-Acetiltransferase/metabolismo , Nervos Cranianos/patologia , Nervos Cranianos/fisiopatologia , Citoproteção/efeitos dos fármacos , Citoproteção/fisiologia , Modelos Animais de Doenças , Esquema de Medicação , Nervo Facial/efeitos dos fármacos , Nervo Facial/patologia , Nervo Facial/fisiopatologia , Humanos , Nervo Hipoglosso/efeitos dos fármacos , Nervo Hipoglosso/patologia , Nervo Hipoglosso/fisiopatologia , Lítio/uso terapêutico , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Degeneração Neural/tratamento farmacológico , Degeneração Neural/fisiopatologia , Degeneração Neural/prevenção & controle , Fármacos Neuroprotetores/uso terapêutico , Resultado do Tratamento , Nervo Trigêmeo/efeitos dos fármacos , Nervo Trigêmeo/patologia , Nervo Trigêmeo/fisiopatologia , Nervo Vago/efeitos dos fármacos , Nervo Vago/patologia , Nervo Vago/fisiopatologiaRESUMO
Recent preclinical and clinical evidence suggest that immune system has a role in the progression and prognosis of Amyotrophic Lateral Sclerosis (ALS), but the identification of a clear mechanism and immune players remains to be elucidated. Here, we have investigated, in 30 and 60 days (presymptomatic) and 120 days (symptomatic) old SOD1-G93A mice, systemic, peripheral, and central innate and adaptive immune and inflammatory response, correlating it with the progression of the neurodegeneration in neuromuscular junction, sciatic nerves, and spinal cord. Surprisingly, we found a very initial (45-60 days) presence of IgG in sciatic nerves together with a gradual enhancement of A20/TNFAIP3 (protein controlling NF-κB signalling) and a concomitantly significant increase and activation of circulating mast cells (MCs) as well as MCs and macrophages in sciatic nerve and an enhancement of IL-6 and IL-10. This immunological frame coincided with a myelin aggregation. The 30-60 days old SOD1-G93A mice didn't show real elements of neuroinflammation and neurodegeneration in spinal cord. In 120 days old mice macrophages and monocytes are widely diffused in sciatic nerves, peripheral neurodegeneration reaches the tip, high circulating levels of TNFα and IL-2 were found and spinal cord exhibits clear signs of neural damage and infiltrating immune cells. Our results underpin a clear immunological disorder at the origin of ALS axonopathy, in which MCs are involved in the initiation and sustaining of inflammatory events. These data cannot be considered a mere epiphenomenon of motor neuron degeneration and reveal new potential selective immune targets in ALS therapy.
Assuntos
Esclerose Lateral Amiotrófica/imunologia , Imunidade Inata , Neuroimunomodulação , Junção Neuromuscular/imunologia , Nervo Isquiático/imunologia , Medula Espinal/imunologia , Superóxido Dismutase-1/metabolismo , Degeneração Walleriana , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Progressão da Doença , Predisposição Genética para Doença , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , NF-kappa B/metabolismo , Junção Neuromuscular/enzimologia , Junção Neuromuscular/patologia , Fenótipo , Nervo Isquiático/enzimologia , Nervo Isquiático/patologia , Transdução de Sinais , Medula Espinal/enzimologia , Medula Espinal/patologia , Superóxido Dismutase-1/genética , Fatores de TempoRESUMO
Hydrogen sulfide (H2S) is an endogenous gasotransmitter recognized as an essential body product with a dual, biphasic action. It can function as an antioxidant and a cytoprotective, but also as a poison with a high probability of causing brain damage when present at noxious levels. In a previous study, we measured toxic liquoral levels of H2S in sporadic amyotrophic lateral sclerosis (ALS) patients and in the familial ALS (fALS) mouse model, SOD1G93A. In addition, we experimentally demonstrated that H2S is extremely and selectively toxic to motor neurons, and that it is released by glial cells and increases Ca2+ concentration in motor neurons due to a lack of ATP. The presented study further examines the effect of toxic concentrations of H2S on embryonic mouse spinal-cord cultures. We performed a proteomic analysis that revealed a significant H2S-mediated activation of pathways related to oxidative stress and cell death, particularly the Nrf-2-mediated oxidative stress response and peroxiredoxins. Furthermore, we report that Na2S (a stable precursor of H2S) toxicity is, at least in part, reverted by the Bax inhibitor V5 and by necrostatin, a potent necroptosis inhibitor.