RESUMO
Amyotrophic lateral sclerosis (ALS) is a late-onset fatal neurodegenerative disease reflecting degeneration of upper and lower motoneurons (MNs). The cause of ALS and the mechanisms of neuronal death are still largely obscure, thus impairing the establishment of efficacious therapies. Glutamate (Glu)-mediated excitotoxicity plays a major role in MN degeneration in ALS. We recently demonstrated that the activation of Group I metabotropic Glu autoreceptors, belonging to both type 1 and type 5 receptors (mGluR1 and mGluR5), at glutamatergic spinal cord nerve terminals, produces excessive Glu release in mice over-expressing human superoxide-dismutase carrying the G93A point mutation (SOD1(G93A)), a widely used animal model of human ALS. To establish whether these receptors are implicated in ALS, we generated mice expressing half dosage of mGluR1 in the SOD1(G93A) background (SOD1(G93A)Grm1(crv4/+)), by crossing the SOD1(G93A) mutant mouse with the Grm1(crv4/+) mouse, lacking mGluR1 because of a spontaneous recessive mutation. SOD1(G93A)Grm1(crv4/+) mice showed prolonged survival probability, delayed pathology onset, slower disease progression and improved motor performances compared to SOD1(G93A) mice. These effects were associated to reduction of mGluR5 expression, enhanced number of MNs, decreased astrocyte and microglia activation, normalization of metallothionein and catalase mRNA expression, reduced mitochondrial damage, and decrease of abnormal Glu release in spinal cord of SOD1(G93A)Grm1(crv4/+)compared to SOD1(G93A) mice. These results demonstrate that a lower constitutive level of mGluR1 has a significant positive impact on mice with experimental ALS, thus providing the rationale for future pharmacological approaches to ALS by selectively blocking Group I metabotropic Glu receptors.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Superóxido Dismutase/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Catalase/metabolismo , Progressão da Doença , Transportador 2 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Metalotioneína/metabolismo , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Atividade Motora , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação Puntual , RNA Mensageiro/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores de Glutamato Metabotrópico/genética , Índice de Gravidade de Doença , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Análise de SobrevidaRESUMO
Axonal transport ensures long-range delivery of essential components and signals between proximal and distal areas of the neuron, and it is crucial for neuronal homeostasis and survival. Several pathogens and virulence factors use this route to gain access to the central nervous system, exploiting the complex and still poorly understood trafficking mechanisms that regulate the dynamics of their cellular receptors. Studying the intracellular transport of neurotropic pathogens is therefore instrumental to glean new insights into these important molecular events. Botulinum (BoNT) and tetanus (TeNT) neurotoxins bind with high affinity to a variety of neurons and are internalised by specialised endocytic pathways leading to specific intracellular fates. Whereas BoNT trafficking is largely confined to the neuromuscular junction, TeNT is internalised in signalling endosomes shared with neurotrophins and their receptors, which are recruited to the fast axonal retrograde transport pathway. Recently, important paradigms regarding the mechanisms by which BoNT and TeNT interact with their cellular targets and are transported in neurons have been challenged. In this review, we summarise new findings concerning the uptake and intracellular trafficking of these neurotoxins, and discuss their implications in terms of the physiological effects of BoNT and TeNT in the central nervous system.
Assuntos
Transporte Axonal , Clostridium tetani/metabolismo , Toxina Tetânica/metabolismo , Animais , Membrana Celular/metabolismo , Endocitose , Gangliosídeos/metabolismo , Neurônios Motores/metabolismo , Fatores de Crescimento Neural/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Receptores de Superfície Celular/metabolismo , Vesículas Sinápticas/metabolismoRESUMO
Glutamate-mediated excitotoxicity plays a major role in ALS and reduced astrocytic glutamate transport was suggested as a cause. Based on previous work we have proposed that abnormal release may represent another source of excessive glutamate. In this line, here we studied the modulation of glutamate release in ALS by Group I metabotropic glutamate (mGlu) receptors, that comprise mGlu1 and mGlu5 members. Synaptosomes from the lumbar spinal cord of SOD1/G93A mice, a widely used murine model for human ALS, and controls were used in release, confocal or electron microscopy and Western blot experiments. Concentrations of the mGlu1/5 receptor agonist 3,5-DHPG >0.3 µM stimulated the release of [(3)H]d- aspartate, used to label the releasing pools of glutamate, both in control and SOD1/G93A mice. At variance, ≤0.3 µM 3,5-DHPG increased [(3)H]d-aspartate release in SOD1/G93A mice only. Experiments with selective antagonists indicated the involvement of both mGlu1 and mGlu5 receptors, mGlu5 being preferentially involved in the high potency effects of 3,5-DHPG. High 3,5-DHPG concentrations increased IP3 formation in both mouse strains, whereas low 3,5-DHPG did it in SOD1/G93A mice only. Release experiments confirmed that 3,5-DHPG elicited [(3)H]d-aspartate exocytosis involving intra-terminal Ca(2+) release through IP3-sensitive channels. Confocal microscopy indicated the co-existence of both receptors presynaptically in the same glutamatergic nerve terminal in SOD1/G93A mice. To conclude, activation of mGlu1/5 receptors produced abnormal glutamate release in SOD1/G93A mice, suggesting that these receptors are implicated in ALS and that selective antagonists may be predicted for new therapeutic approaches. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.
Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Autorreceptores/fisiologia , Exocitose/fisiologia , Ácido Glutâmico/metabolismo , Receptores de Glutamato Metabotrópico/fisiologia , Esclerose Lateral Amiotrófica/metabolismo , Animais , Ácido Aspártico/metabolismo , Autorreceptores/antagonistas & inibidores , Autorreceptores/biossíntese , Cálcio/metabolismo , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/farmacologia , Exocitose/efeitos dos fármacos , Feminino , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Fosfatos de Inositol/metabolismo , Vértebras Lombares , Masculino , Camundongos , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Receptor de Glutamato Metabotrópico 5 , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/biossíntese , Resorcinóis/farmacologia , Medula Espinal , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/fisiologia , Sinaptossomos/ultraestruturaRESUMO
Amblyopia is one of the most common forms of visual impairment, arising from an early functional imbalance between the two eyes. It is currently accepted that, due to a lack of neural plasticity,amblyopia is an untreatable pathology in adults. Environmental enrichment (EE) emerged as a strategy highly effective in restoring plasticity in adult animals, eliciting recovery from amblyopia through a reduction of intracortical inhibition. It is unknown whether single EE components are able to promote plasticity in the adult brain, crucial information for designing new protocols of environmental stimulation suitable for amblyopic human subjects. Here, we assessed the effects of enhanced physical exercise,increased social interaction, visual enrichment or perceptual learning on visual function recovery in adult amblyopic rats. We report a complete rescue of both visual acuity and ocular dominance in exercised rats, in animals exposed to visual enrichment and in animals engaged in perceptual learning.These effects were accompanied by a reduced inhibition/excitation balance in the visual cortex. In contrast, we did not detect any sign of recovery in socially enriched rats or in animals practicing a purely associative visual task. These findings could have a bearing in orienting clinical research in the field of amblyopia therapy.
Assuntos
Ambliopia/reabilitação , Meio Ambiente , Relações Interpessoais , Atividade Motora/fisiologia , Condicionamento Físico Animal/fisiologia , Recuperação de Função Fisiológica/fisiologia , Fatores Etários , Ambliopia/fisiopatologia , Ambliopia/psicologia , Animais , Aprendizagem/fisiologia , Inibição Neural/fisiologia , Estimulação Luminosa/métodos , Condicionamento Físico Animal/métodos , Ratos , Ratos Long-Evans , Privação Sensorial/fisiologia , Percepção Visual/fisiologiaRESUMO
Despite some advances in the understanding of amyotrophic lateral sclerosis (ALS) pathogenesis, significant achievements in treating this disease are still lacking. Mesenchymal stromal (stem) cells (MSCs) have been shown to be effective in several models of neurological disease. To determine the effects of the intravenous injection of MSCs in an ALS mouse model during the symptomatic stage of disease, MSCs (1 × 106) were intravenously injected in mice expressing human superoxide dismutase 1 (SOD1) carrying the G93A mutation (SOD1/G93A) presenting with experimental ALS. Survival, motor abilities, histology, oxidative stress markers and [³H]D-aspartate release in the spinal cord were investigated. MSC injection in SOD1/G93A mice improved survival and motor functions compared with saline-injected controls. Injected MSCs scantly home to the central nervous system and poorly engraft. We observed a reduced accumulation of ubiquitin agglomerates and of activated astrocytes and microglia in the spinal cord of MSC-treated SOD1/G93A mice, with no changes in the number of choline acetyltransferase- and glutamate transporter type 1-positive cells. MSC administration turned around the upregulation of metallothionein mRNA expression and of the activity of the antioxidant enzyme glutathione S-transferase, both associated with disease progression. Last, we observed that MSCs reverted both spontaneous and stimulus-evoked neuronal release of [³H]D-aspartate, a marker of endogenous glutamate, which is upregulated in SOD1/G93A mice. These findings suggest that intravenous administration of MSCs significantly improves the clinical outcome and pathological scores of mutant SOD1/G93A mice, thus providing the rationale for their exploitation for the treatment of ALS.
Assuntos
Esclerose Lateral Amiotrófica/terapia , Transplante de Células-Tronco Mesenquimais , Atividade Motora , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/mortalidade , Animais , Ácido Aspártico/metabolismo , Movimento Celular , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Progressão da Doença , Feminino , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Transgênicos , Estresse Oxidativo , Medula Espinal/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1RESUMO
The striking differences between the clinical symptoms of tetanus and botulism have been ascribed to the different fate of the parental neurotoxins once internalised in motor neurons. Tetanus toxin (TeNT) is known to undergo transcytosis into inhibitory interneurons and block the release of inhibitory neurotransmitters in the spinal cord, causing a spastic paralysis. In contrast, botulinum neurotoxins (BoNTs) block acetylcholine release at the neuromuscular junction, therefore inducing a flaccid paralysis. Whilst overt experimental evidence supports the sorting of TeNT to the axonal retrograde transport pathway, recent findings challenge the established view that BoNT trafficking is restricted to the neuromuscular junction by highlighting central effects caused by these neurotoxins. These results suggest a more complex scenario whereby BoNTs also engage long-range trafficking mechanisms. However, the intracellular pathways underlying this process remain unclear. We sought to fill this gap by using primary motor neurons either in mass culture or differentiated in microfluidic devices to directly monitor the endocytosis and axonal transport of full length BoNT/A and BoNT/E and their recombinant binding fragments. We show that BoNT/A and BoNT/E are internalised by spinal cord motor neurons and undergo fast axonal retrograde transport. BoNT/A and BoNT/E are internalised in non-acidic axonal carriers that partially overlap with those containing TeNT, following a process that is largely independent of stimulated synaptic vesicle endo-exocytosis. Following intramuscular injection in vivo, BoNT/A and TeNT displayed central effects with a similar time course. Central actions paralleled the peripheral spastic paralysis for TeNT, but lagged behind the onset of flaccid paralysis for BoNT/A. These results suggest that the fast axonal retrograde transport compartment is composed of multifunctional trafficking organelles orchestrating the simultaneous transfer of diverse cargoes from nerve terminals to the soma, and represents a general gateway for the delivery of virulence factors and pathogens to the central nervous system.
Assuntos
Transporte Axonal/efeitos dos fármacos , Toxinas Botulínicas Tipo A/farmacologia , Toxinas Botulínicas/farmacologia , Neurônios Motores/efeitos dos fármacos , Neurotransmissores/antagonistas & inibidores , Acetilcolina/metabolismo , Animais , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas Tipo A/metabolismo , Células Cultivadas , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Endocitose/efeitos dos fármacos , Camundongos , Neurônios Motores/metabolismo , Junção Neuromuscular/metabolismo , Paralisia/metabolismo , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Toxina Tetânica/metabolismo , Toxina Tetânica/farmacologiaRESUMO
The endocannabinoid system and endocannabinoid receptor-driven modulation of glutamate release were studied in rat brain cortex astroglial gliosomes. These preparations contained the endocannabinoids N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol, as well their major biosynthetic (N-acyl-phosphatidylethanolamines-hydrolyzing-phospholipase D and diacylglycerol-lipase) and catabolic (fatty acid amide-hydrolase and monoacylglycerol-lipase) enzymes. Gliosomes expressed type-1 (CB1R), type-2 (CB2R) cannabinoid, and type-1 vanilloid (TRPV1) receptors, as ascertained by Western blotting and confocal microscopy. Methanandamide, a stable analogue of anandamide acting as CB1R, CB2R, and TRPV1 agonist, stimulated or inhibited the depolarization-evoked gliosomal [(3)H]D: -aspartate release, at lower and higher concentrations, respectively. Experiments with ACEA (arachidonyl-2'-chloroethylamide), JWH133 ((6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]-pyran) and capsaicin, selective agonists at CB1R, CB2R and TRPV1, respectively, demonstrated that potentiation of [(3)H]D: -aspartate release was due to CB1R while inhibition to CB2R and TRPV1 engagement. These findings were confirmed by using selective receptor antagonists. Furthermore, CB1R activation caused increase of intracellular IP3 and Ca(2+) concentration, suggesting an involvement of phospholipase C.