RESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the death of motor neurons in the spinal cord and the brain. Although this disease is characterized by motoneuron degeneration, non-neuronal cells such as oligodendrocytes play an important role in the disease onset and progression. The aim of our study was to examine functional properties of oligodendrocytes in the SOD1G93A rat model of ALS with a particular focus on the inwardly rectifying potassium channel Kir4.1 that is abundantly expressed in these glial cells and plays a role in the regulation of extracellular K+ . First, we demonstrate that the expression of Kir4.1 is diminished in the spinal cord oligodendrocytes of the SOD1G93A rat. Moreover, our data show an elevated number of dysmorphic oligodendrocytes in the ALS spinal cord that is indicative of a degenerative phenotype. In order to assess physiological properties of oligodendrocytes, we prepared cell cultures from the rat spinal cord. Oligodendrocytes isolated from the SOD1G93A spinal cord display similar ramification of the processes as the control but express a lower level of Kir4.1. We further demonstrate an impairment of oligodendrocyte functional properties in ALS. Remarkably, whole-cell patch-clamp recordings revealed compromised membrane biophysical properties and diminished inward currents in the SOD1G93A oligodendrocytes. In addition, the Ba2+ -sensitive Kir currents were decreased in ALS oligodendrocytes. Altogether, our findings provide the evidence of impaired Kir4.1 expression and function in oligodendrocytes of the SOD1G93A spinal cord, suggesting oligodendrocyte Kir4.1 channel as a potential contributor to the ALS pathophysiology.
Assuntos
Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Canais de Potássio Corretores do Fluxo de Internalização , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Neurônios Motores , Oligodendroglia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Ratos , Medula EspinalRESUMO
Electrical activity is important for brain development. In brain slices, human subplate neurons exhibit spontaneous electrical activity that is highly sensitive to lanthanum. Based on the results of pharmacological experiments in human fetal tissue, we hypothesized that hemichannel-forming connexin (Cx) isoforms 26, 36, and 45 would be expressed on neurons in the subplate (SP) zone. RNA sequencing of dissected human cortical mantles at ages of 17-23 gestational weeks revealed that Cx45 has the highest expression, followed by Cx36 and Cx26. The levels of Cx and pannexin expression between male and female fetal cortices were not significantly different. Immunohistochemical analysis detected Cx45- and Cx26-expressing neurons in the upper segment of the SP zone. Cx45 was present on the cell bodies of human SP neurons, while Cx26 was found on both cell bodies and dendrites. Cx45, Cx36, and Cx26 were strongly expressed in the cortical plate, where newborn migrating neurons line up to form cortical layers. New information about the expression of 3 "neuronal" Cx isoforms in each cortical layer/zone (e.g., SP, cortical plate) and pharmacological data with cadmium and lanthanum may improve our understanding of the cellular mechanisms underlying neuronal development in human fetuses and potential vulnerabilities.
Assuntos
Cádmio/administração & dosagem , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiologia , Conexinas/metabolismo , Lantânio/administração & dosagem , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Conexina 26/metabolismo , Feminino , Feto , Humanos , Masculino , Potenciais da Membrana , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Proteína delta-2 de Junções ComunicantesRESUMO
BACKGROUND: Microglia are essential to maintain cell homeostasis in the healthy brain and are activated after brain injury. Upon activation, microglia polarize towards different phenotypes. The course of microglia activation is complex and depends on signals in the surrounding milieu. Recently, it has been suggested that microglia respond to ion currents, as a way of regulating their activity and function. METHODS AND RESULTS: Under the hypothesis that HCN and KCNQ/Kv7 channels impact on microglia, we studied primary rat microglia in the presence or absence of specific pharmacological blockade or RNA silencing. Primary microglia expressed the subunits HCN1-4, Kv7.2, Kv7.3, and Kv7.5. The expression of HCN2, as well as Kv7.2 and Kv7.3, varied among different microglia phenotypes. The pharmacological blockade of HCN channels by ZD7288 resulted in cell depolarization with slowly rising intracellular calcium levels, leading to enhanced survival and reduced proliferation rates of resting microglia. Furthermore, ZD7288 treatment, as well as knockdown of HCN2 RNA by small interfering RNA, resulted in an attenuation of later microglia activation-both towards the anti- and pro-inflammatory phenotype. However, HCN channel inhibition enhanced the phagocytic capacity of IL4-stimulated microglia. Blockade of Kv7/KCNQ channel by XE-991 exclusively inhibited the migratory capacity of resting microglia. CONCLUSION: These observations suggest that the HCN current contributes to various microglia functions and impacts on the course of microglia activation, while the Kv7/KCNQ channels affect microglia migration. Characterizing the role of HCN channels in microglial functioning may offer new therapeutic approaches for targeted modulation of neuroinflammation as a hallmark of various neurological disorders.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Microglia/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Microglia/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Pirimidinas/farmacologia , Interferência de RNA , Ratos , Ratos WistarRESUMO
Interaction between autoreactive immune cells and astroglia is an important part of the pathologic processes that fuel neurodegeneration in multiple sclerosis. In this inflammatory disease, immune cells enter into the central nervous system (CNS) and they spread through CNS parenchyma, but the impact of these autoreactive immune cells on the activity pattern of astrocytes has not been defined. By exploiting naïve astrocytes in culture and CNS-infiltrated immune cells (CNS IICs) isolated from rat with experimental autoimmune encephalomyelitis (EAE), here we demonstrate previously unrecognized properties of immune cell-astrocyte interaction. We show that CNS IICs but not the peripheral immune cell application, evokes a rapid and vigorous intracellular Ca2+ increase in astrocytes by promoting glial release of ATP. ATP propagated Ca2+ elevation through glial purinergic P2X7 receptor activation by the hemichannel-dependent nucleotide release mechanism. Astrocyte Ca2+ increase is specifically triggered by the autoreactive CD4+ T-cell application and these two cell types exhibit close spatial interaction in EAE. Therefore, Ca2+ signals may mediate a rapid astroglial response to the autoreactive immune cells in their local environment. This property of immune cell-astrocyte interaction may be important to consider in studies interrogating CNS autoimmune disease.
Assuntos
Astrócitos/metabolismo , Sinalização do Cálcio , Imunidade Celular , Receptores Purinérgicos/imunologia , Trifosfato de Adenosina/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Neuroglia/metabolismo , Ratos , Receptores Purinérgicos P2X7/imunologia , Receptores Purinérgicos P2X7/metabolismo , Transdução de Sinais , Medula Espinal/citologia , Medula Espinal/imunologiaRESUMO
Extracellular vesicles (EVs) have recently attracted a great deal of interest as they may represent a new biosignaling paradigm. According to the mode of biogenesis, size and composition, two broad categories of EVs have been described, exosomes and microvesicles. EVs have been shown to carry cargoes of signaling proteins, RNA species, DNA and lipids. Once released, their content is selectively taken up by near or distant target cells, influencing their behavior. Exosomes are involved in cell-cell communication in a wide range of embryonic developmental processes and in fetal-maternal communication. In the present review, an outline of the role of EVs in neural development, regeneration and diseases is presented. EVs can act as regulators of normal homeostasis, but they can also promote either neuroinflammation/degeneration or tissue repair in pathological conditions, depending on their content. Since EV molecular cargo constitutes a representation of the origin cell status, EVs can be exploited in the diagnosis of several diseases. Due to their capability to cross the blood-brain barrier (BBB), EVs not only have been suggested for the diagnosis of central nervous system disorders by means of minimally invasive procedures, i.e., "liquid biopsies", but they are also considered attractive tools for targeted drug delivery across the BBB. From the therapeutic perspective, mesenchymal stem cells (MSCs) represent one of the most promising sources of EVs. In particular, the neuroprotective properties of MSCs derived from the dental pulp are here discussed.
Assuntos
Axônios/metabolismo , Micropartículas Derivadas de Células/metabolismo , Exossomos/metabolismo , Doenças do Sistema Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Barreira Hematoencefálica/metabolismo , Comunicação Celular , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Feminino , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/prevenção & controle , Células-Tronco Neurais/citologia , Placenta/metabolismo , Gravidez , Regeneração/genéticaRESUMO
Amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, is the most common adult onset neurodegenerative disorder affecting motor neurons. Disruptions in metal ion homeostasis have been described in association with ALS, but the pathological mechanisms are still poorly understood. One of the familial ALS cases is caused by mutations in the metallo-enzyme copper-zinc superoxide dismutase (SOD1). In this study, we employed orthogonal cellular synchrotron radiation based spectro-microscopies to investigate the astrocytes of an ALS animal model: the rat hSOD1 G93A that overexpresses human mutated SOD1, which is known to increase the susceptibility of the SOD1 protein to form insoluble intracellular aggregates. Specifically, we applied soft X-ray transmission tomography and hard X-ray fluorescence microscopy in situ, Fourier transform infrared spectro-microscopy to detect and analyze aggregates, as well as to determine the alterations in the cellular ultrastructure and the elemental and the organic composition of ALS model astrocytes with respect to the control astrocytes isolated from nontransgenic littermates (NTg). The present study demonstrates that large aggregates in the form of multivesicular inclusions form exclusively in the ALS model astrocytes and not in the NTg counterpart. Furthermore, the number of mitochondria, the cellular copper concentration, and the amount of antiparallel ß-sheet structures were significantly changed within the cells of the ALS model as well as the lipid localization and composition. Also, our data indicate that choline was decreased in the ALS model astrocytes, which could explain their higher sensitivity to oxidative stress that we observed. These results show that the hG93A SOD1 mutation causes metabolic and ultrastructural cellular changes and point to a link between an increased copper concentration and aggregation: the most probable that the aggregation of G93A hSOD1 may perturb its binding to Cu, thus directly or indirectly affecting Cu homeostasis.
Assuntos
Esclerose Lateral Amiotrófica/diagnóstico por imagem , Esclerose Lateral Amiotrófica/patologia , Astrócitos/patologia , Microscopia/instrumentação , Mutação , Superóxido Dismutase-1/genética , Síncrotrons , Esclerose Lateral Amiotrófica/genética , Animais , Humanos , RatosRESUMO
Pathological mechanisms in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, are still poorly understood. One subset of familial ALS cases is caused by mutations in the metallo-enzyme copper-zinc superoxide dismutase (SOD1), increasing the susceptibility of the SOD1 protein to form insoluble intracellular aggregates. Here, we employed synchrotron radiation-based Fourier transform infrared spectroscopy and microscopy to investigate brainstem cross-sections from the transgenic hSOD1 G93A rat model of ALS that overexpresses human-mutated SOD1. We compared the biomacromolecular organic composition in brainstem tissue cross-sections of ALS rats and their non-transgenic littermates (NTg). We demonstrate that the proteins and especially their antiparallel ß-sheet structure significantly differed in all three regions: the facial nucleus (FN), the gigantocellular reticular nucleus (GRN) and the trigeminal motor nucleus (TMN) in the brainstem tissue of ALS rats. The protein levels varied between different brainstem areas, with the highest concentration observed in the region of the FN in the brainstem tissue of NTg animals. Furthermore, the concentration of lipids and esters was significantly decreased in the TMN and FN of ALS animals. A similar pattern was detected for choline and phosphate assigned to nucleic acids with the highest concentrations in the FN of NTg animals. The spectroscopic analysis showed significant differences in phosphates, amide and lipid structure in the FN of NTg animals in comparison with the same area of ALS rats. These results show that the hG93A SOD1 mutation causes metabolic cellular changes and point to a link between bioorganic composition and hallmarks of protein aggregation.
Assuntos
Esclerose Lateral Amiotrófica/diagnóstico por imagem , Tronco Encefálico/diagnóstico por imagem , Microscopia/instrumentação , Mutação , Espectroscopia de Infravermelho com Transformada de Fourier , Superóxido Dismutase-1/genética , Síncrotrons , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Tronco Encefálico/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos Transgênicos , RatosRESUMO
Neuroinflammation is one of the major players in amyotrophic lateral sclerosis (ALS) pathogenesis, and astrocytes are significantly involved in this process. The astrocytic protein S100B can be released in pathological states activating the receptor for advanced glycation end products (RAGE). Different indications point to an aberrant expression of S100B and RAGE in ALS. In this work, we observed that S100B and RAGE are progressively and selectively upregulated in astrocytes of diseased rats with a tissue-specific timing pattern, correlated to the level of neurodegeneration. The expression of the full-length and soluble RAGE isoforms could also be linked to the degree of tissue damage. The mere presence of mutant SOD1 is able to increase the intracellular levels and release S100B from astrocytes, suggesting the possibility that an increased astrocytic S100B expression might be an early occurring event in the disease. Finally, our findings indicate that the protein may exert a proinflammatory role in ALS, since its inhibition in astrocytes derived from SOD1G93A mice limits the expression of reactivity-linked/proinflammatory genes. Thus, our results propose the S100B-RAGE axis as an effective contributor to the pathogenesis of the disease, suggesting its blockade as a rational target for a therapeutic intervention in ALS.
Assuntos
Receptor para Produtos Finais de Glicação Avançada/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Superóxido Dismutase-1/metabolismo , Animais , Animais Geneticamente Modificados , Astrócitos/metabolismo , Western Blotting , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Masculino , Microscopia de Fluorescência , Ratos , Receptor para Produtos Finais de Glicação Avançada/genética , Subunidade beta da Proteína Ligante de Cálcio S100/genética , Superóxido Dismutase-1/genéticaRESUMO
Alzheimer's disease (AD) involves selective loss of basal forebrain cholinergic neurons, particularly in the nucleus basalis (NB). Similarly, Parkinson's disease (PD) might involve the selective loss of pedunculopontine tegmental nucleus (PPT) cholinergic neurons. Therefore, lesions of these functionally distinct cholinergic centers in rats might serve as models of AD and PD cholinergic neuropathologies. Our previous articles described dissimilar sleep/wake-state disorders in rat models of AD and PD cholinergic neuropathologies. This study further examines astroglial and microglial responses as underlying pathologies in these distinct sleep disorders. Unilateral lesions of the NB or the PPT were induced with rats under ketamine/diazepam anesthesia (50 mg/kg i.p.) by using stereotaxically guided microinfusion of the excitotoxin ibotenic acid (IBO). Twenty-one days after the lesion, loss of cholinergic neurons was quantified by nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry, and the astroglial and microglial responses were quantified by glia fibrillary acidic protein/OX42 immunohistochemistry. This study demonstrates, for the first time, the anatomofunctionally related astroglial response following unilateral excitotoxic PPT cholinergic neuronal lesion. Whereas IBO NB and PPT lesions similarly enhanced local astroglial and microglial responses, astrogliosis in the PPT was followed by a remote astrogliosis within the ipslilateral NB. Conversely, there was no microglial response within the NB after PPT lesions. Our results reveal the rostrorostral PPT-NB astrogliosis after denervation of cholinergic neurons in the PPT. This hierarchically and anatomofunctionally guided PPT-NB astrogliosis emerged following cholinergic neuronal loss greater than 17% throughout the overall rostrocaudal PPT dimension.
Assuntos
Lesões Encefálicas/patologia , Neurônios Colinérgicos/patologia , Neuroglia/metabolismo , Análise de Variância , Animais , Lesões Encefálicas/induzido quimicamente , Lesões Encefálicas/complicações , Antígeno CD11b/metabolismo , Denervação/métodos , Modelos Animais de Doenças , Agonistas de Aminoácidos Excitatórios/toxicidade , Lateralidade Funcional , Proteína Glial Fibrilar Ácida/metabolismo , Ácido Ibotênico/toxicidade , Masculino , Neuroglia/efeitos dos fármacos , Núcleo Tegmental Pedunculopontino/lesões , Núcleo Tegmental Pedunculopontino/patologia , Ratos , Ratos WistarRESUMO
GL2011 is a naturally occurring thiol compound and a series of thiol compounds have been proposed as radioprotectors. Radioprotective efficacy of a triple intraperitoneal dose of GL2011 of 100 mg/kg body weight of Wistar rats, 30 min prior to and 3 and 6 h following irradiation (6.7 Gy) was evaluated. Four groups of animals were used, vehicle-treated non-irradiated (VN), GL2011-treated and irradiated (GI), GL2011-treated and non-irradiated (GN) and vehicle-treated and irradiated (VI) (n = 30 per group). The radioprotective efficacy of GL2011 was determined by measuring 28-day survival and intestinal crypt cell survival. Neuroprotection in terms of behaviour was evaluated using the behavioural observational battery, open field test and elevated plus maze paradigm. An RNA microarray was carried out in order to show differences at the RNA level between VI and VN groups. Brain protein changes were identified using a gel-based proteomics method and major brain receptor complex levels were determined by blue-native gels followed by immunoblotting. 28-Day survival rate in VI was 30 %, in GI survival was 93 %, survival of VN and GN was 100 %. Jejunal crypt cell survival was significantly enhanced in GI. Protein-level changes of peroxiredoxin-5, Mn-superoxide dismutase 2, voltage-dependent anion-selective channel protein 1, septin 5 and dopamine D2 receptor complex levels were paralleling radiation damage and protection. Taken together, the findings demonstrate that GL2011 improves survival rates and jejunal crypt survival, provides partial neuroprotection at the behavioural level and modulates proteins known to be involved in protection against oxidative stress-mediated cell damage.
Assuntos
Aminas/farmacologia , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/efeitos da radiação , Raios gama/efeitos adversos , Proteínas/metabolismo , Protetores contra Radiação/farmacologia , Compostos de Sulfidrila/farmacologia , Irradiação Corporal Total/efeitos adversos , Animais , Comportamento Animal/efeitos da radiação , Encéfalo/metabolismo , Eletroforese em Gel Bidimensional , Injeções Intraperitoneais , Jejuno/citologia , Jejuno/efeitos dos fármacos , Jejuno/efeitos da radiação , Masculino , Aprendizagem em Labirinto , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas/genética , RNA/análise , Protetores contra Radiação/administração & dosagem , Ratos Wistar , Reprodutibilidade dos Testes , Taxa de SobrevidaRESUMO
AIM: To investigate the survival of laboratory rats after irradiation and to study the cellularity of their bone marrow and the multipotential mesenchymal stem cells (BM-MSCs) in groups treated with or without a new thiol-based radioprotector (GM2011). METHODS: Animals were irradiated by a Cobalt gamma source at 6.7 Gy. Treated animals were given i.p. GM2011 30 minutes before and 3 and 7 hours after irradiation. Controls consisted of sham irradiated animals without treatment and animals treated without irradiation. After 30 days post-irradiation, animals were sacrificed and bone marrow cells were prepared from isolated femurs. A colony forming unit-fibroblast (CFU-F) assay was performed to obtain the number of BM-MSCs. RESULTS: In the treated group, 87% of animals survived, compared to only 30% in the non-treated irradiated group. Irradiation induced significant changes in the bone marrow of the treated rats (total bone marrow cellularity was reduced by~60%--from 63 to 28 cells × 10(6)/femur and the frequency of the CFU-F per femur by~70% - from 357 to 97), however GL2011 almost completely prevented the suppressive effect observed on day 30 post-irradiation (71 cells × 10(6)/femur and 230 CFU-F/femur). CONCLUSION: Although the irradiation dosage was relatively high, GL2011 acted as a very effective new radioprotector. The recovery of the BN-MSCs and their counts support the effectiveness of the studied radioprotector.
Assuntos
Medula Óssea/efeitos da radiação , Células-Tronco Mesenquimais/fisiologia , Lesões Experimentais por Radiação/prevenção & controle , Protetores contra Radiação/uso terapêutico , Regeneração/fisiologia , Animais , Radioisótopos de Cobalto , Ensaio de Unidades Formadoras de Colônias , Masculino , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/mortalidade , Ratos , Ratos Wistar , Taxa de Sobrevida , Irradiação Corporal TotalRESUMO
Background: Zinc (Zn), an essential micronutrient, regulates and maintains neurological functions. However, both Zn deficiency and excess can cause oxidative stress and neurodegenerative diseases. As previously reported, immunoglobulin G (IgG) can modulate oxidative stress in various disorders. Aims: This study aimed to investigate whether IgG treatment can alleviate oxidative stress caused by Zn0 on microglia in vitro. Study Design: In vitro study. Methods: The feasibility of Zn0 treatment was evaluated using the MTS assay. Oxidative stress following treatment with Zn0, either alone or with IgG supplementation, was determined with dihydrorhodamine 123 staining. Flow cytometry was employed to ascertain the intracellular protein levels of TRIM21, PINK, PARKIN, MFN2, Beclin-1, and active LC3B. Results: In silico screening confirmed the association between Zn0 cytotoxicity and apoptosis. Furthermore, oxidative stress was identified as a critical mechanism that underlies Zn0 neurotoxicity. The in silico analysis revealed that Zn can interact with the constant region of the Ig heavy chain, suggesting a potential role for IgG in alleviating Zn0-induced cytotoxicity. Experimental findings supported this hypothesis, as IgG administration significantly reduced Zn0-induced mitochondrial stress in a dose-dependent manner. The upregulation of PINK1 levels by Zn0 exposure suggests that mitochondrial injury promotes mitophagy. Interestingly, Zn0 decreased TRIM21 levels, which is reversed by IgG administration. Conclusion: These findings elucidate the cellular responses to Zn0 and highlight the potential use of intravenous immunoglobulin in mitigating the adverse effects of acute Zn0 exposure.
RESUMO
Neuronal plasticity is a crucial mechanism for an adapting nervous system to change. It is shown to be regulated by perineuronal nets (PNNs), the condensed forms of the extracellular matrix (ECM) around neuronal bodies. By assessing the changes in the number, intensity, and structure of PNNs, the ultrastructure of the PNN mesh, and the expression of inhibitory and excitatory synaptic inputs on these neurons, we aimed to clarify the role of an ECM glycoprotein, tenascin-C (TnC), in the dorsal hippocampus. To enhance neuronal plasticity, TnC-deficient (TnC-/-) and wild-type (TnC+/+) young adult male mice were reared in an enriched environment (EE) for 8 weeks. Deletion of TnC in TnC-/- mice showed an ultrastructural reduction of the PNN mesh and an increased inhibitory input in the dentate gyrus (DG), and an increase in the number of PNNs with a rise in the inhibitory input in the CA2 region. EE induced an increased inhibitory input in the CA2, CA3, and DG regions; in DG, the change was also followed by an increased intensity of PNNs. No changes in PNNs or synaptic expression were found in the CA1 region. We conclude that the DG and CA2 regions emerged as focal points of alterations in PNNs and synaptogenesis with EE as mediated by TnC.
Assuntos
Matriz Extracelular , Hipocampo , Plasticidade Neuronal , Sinapses , Tenascina , Animais , Tenascina/metabolismo , Tenascina/genética , Masculino , Camundongos , Hipocampo/metabolismo , Matriz Extracelular/metabolismo , Sinapses/metabolismo , Camundongos Knockout , Neurônios/metabolismo , Camundongos Endogâmicos C57BL , Giro Denteado/metabolismoRESUMO
In vitro and in vivo studies on the role of tenascins have shown that the two paradigmatic glycoproteins of the tenascin family, tenascin-C (TnC) and tenascin-R (TnR) play important roles in cell proliferation and migration, fate determination, axonal pathfinding, myelination, and synaptic plasticity. As components of the extracellular matrix, both molecules show distinct, but also overlapping dual functions in inhibiting and promoting cell interactions depending on the cell type, developmental stage and molecular microenvironment. They are expressed by neurons and glia as well as, for TnC, by cells of the immune system. The functional relationship between neural and immune cells becomes relevant in acute and chronic nervous system disorders, in particular when the blood brain and blood peripheral nerve barriers are compromised. In this review, we will describe the functional parameters of the two molecules in cell interactions during development and, in the adult, in synaptic activity and plasticity, as well as regeneration after injury, with TnC being conducive for regeneration and TnR being inhibitory for functional recovery. Although not much is known about the role of tenascins in neuroinflammation, we will describe emerging knowledge on the interplay between neural and immune cells in autoimmune diseases, such as multiple sclerosis and polyneuropathies. We will attempt to point out the directions of experimental approaches that we envisage would help gaining insights into the complex interplay of TnC and TnR with the cells that express them in pathological conditions of nervous and immune systems.
Assuntos
Doenças do Sistema Nervoso/imunologia , Tenascina/imunologia , Animais , Humanos , Doenças do Sistema Nervoso/genética , Tenascina/genéticaRESUMO
Compelling evidence supports the use of a moderate static magnetic field (SMF) for therapeutic purposes. In order to provide insight into the mechanisms underlying SMF treatment, it is essential to examine the cellular responses elicited by therapeutically applied SMF, especially in the nervous system. The Na(+)/K(+) pump, by creating and maintaining the gradient of Na(+) and K(+) ions across the plasma membrane, regulates the physiological properties of neurons. In this study, we examined the expression of the Na(+)/K(+) pump in the isolated brain-subesophageal ganglion complex of the garden snail Helix pomatia, along with the immunoreactivity and current of the Na(+)/K(+) pump in isolated snail neurons after 15 min exposure to a moderate (10 mT) SMF. Western blot and immunofluorescence analysis revealed that 10 mT SMF did not significantly change the expression of the Na(+)/K(+) pump α-subunit in the snail brain and the neuronal cell body. However, our immunofluorescence data showed that SMF treatment induced a significant increase in the Na(+)/K(+) pump α-subunit expression in the neuronal plasma membrane area. This change in Na(+)/K(+) pump expression was reflected in pump activity as demonstrated by the pump current measurements. Whole-cell patch-clamp recordings from isolated snail neurons revealed that Na(+)/K(+) pump current density was significantly increased after the 10 mT SMF treatment. The SMF-induced increase was different in the two groups of control snail neurons, as defined by the pump current level. The results obtained could represent a physiologically important response of neurons to 10 mT SMF comparable in strength to therapeutic applications.
Assuntos
Caracois Helix/enzimologia , Potenciais da Membrana/fisiologia , Sistema Nervoso/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Western Blotting , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Imunofluorescência , Caracois Helix/efeitos dos fármacos , Caracois Helix/fisiologia , Campos Magnéticos , Potenciais da Membrana/efeitos dos fármacos , Sistema Nervoso/efeitos dos fármacos , Neurônios/citologia , Neurônios/fisiologia , Ouabaína/farmacologia , Técnicas de Patch-Clamp , Subunidades Proteicas/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidoresRESUMO
In multiple sclerosis (MS), glial cells astrocytes interact with the autoreactive immune cells that attack the central nervous system (CNS), which causes and sustains neuroinflammation. However, little is known about the direct interaction between these cells when they are in close proximity in the inflamed CNS. By using an experimental autoimmune encephalomyelitis (EAE) model of MS, we previously found that in the proximity of autoreactive CNS-infiltrated immune cells (CNS-IICs), astrocytes respond with a rapid calcium increase that is mediated by the autocrine P2X7 receptor (P2X7R) activation. We now reveal that the mechanisms regulating this direct interaction of astrocytes and CNS-IICs involve the coupling between P2X7R, connexin-43, and ß3-integrin. We found that P2X7R and astroglial connexin-43 interact and concentrate in the immediate proximity of the CNS-IICs in EAE. P2X7R also interacts with ß3-integrin, and the block of astroglial αvß3-integrin reduces the P2X7R-dependent calcium response of astrocytes upon encountering CNS-IICs. This interaction was dependent on astroglial mitochondrial activity, which regulated the ATP-driven P2X7R activation and facilitated the termination of the astrocytic calcium response evoked by CNS-IICs. By further defining the interactions between the CNS and the immune system, our findings provide a novel perspective toward expanding integrin-targeting therapeutic approaches for MS treatment by controlling the cell-cell interactions between astrocytes and CNS-IICs.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Animais , Astrócitos , Receptores Purinérgicos P2X7 , Integrina beta3 , Cálcio , Comunicação CelularRESUMO
This review aims to summarize the latest evidence about the role of innate and adaptive immunity in Amyotrophic Lateral Sclerosis (ALS). ALS is a devastating neurodegenerative disease affecting upper and lower motor neurons, which involves essential cells of the immune system that play a basic role in innate or adaptive immunity, that can be neurotoxic or neuroprotective for neurons. However, distinguishing between the sole neurotoxic or neuroprotective function of certain cells such as astrocytes can be challenging due to intricate nature of these cells, the complexity of the microenvironment and the contextual factors. In this review, in regard to innate immunity we focus on the involvement of monocytes/macrophages, microglia, the complement, NK cells, neutrophils, mast cells, and astrocytes, while regarding adaptive immunity, in addition to humoral immunity the most important features and roles of T and B cells are highlighted, specifically different subsets of CD4+ as well as CD8+ T cells. The role of autoantibodies and cytokines is also discussed in distinct sections of this review.
RESUMO
Automated screening systems in conjunction with machine learning-based methods are becoming an essential part of the healthcare systems for assisting in disease diagnosis. Moreover, manually annotating data and hand-crafting features for training purposes are impractical and time-consuming. We propose a segmentation and classification-based approach for assembling an automated screening system for the analysis of calcium imaging. The method was developed and verified using the effects of disease IgGs (from Amyotrophic Lateral Sclerosis patients) on calcium (Ca2+) homeostasis. From 33 imaging videos we analyzed, 21 belonged to the disease and 12 to the control experimental groups. The method consists of three main steps: projection, segmentation, and classification. The entire Ca2+ time-lapse image recordings (videos) were projected into a single image using different projection methods. Segmentation was performed by using a multi-level thresholding (MLT) step and the Regions of Interest (ROIs) that encompassed cell somas were detected. A mean value of the pixels within these boundaries was collected at each time point to obtain the Ca2+ traces (time-series). Finally, a new matrix called feature image was generated from those traces and used for assessing the classification accuracy of various classifiers (control vs. disease). The mean value of the segmentation F-score for all the data was above 0.80 throughout the tested threshold levels for all projection methods, namely maximum intensity, standard deviation, and standard deviation with linear scaling projection. Although the classification accuracy reached up to 90.14%, interestingly, we observed that achieving better scores in segmentation results did not necessarily correspond to an increase in classification performance. Our method takes the advantage of the multi-level thresholding and of a classification procedure based on the feature images, thus it does not have to rely on hand-crafted training parameters of each event. It thus provides a semi-autonomous tool for assessing segmentation parameters which allows for the best classification accuracy.