RESUMEN
Myelination is essential for neuronal function and health. In peripheral nerves, >100 causative mutations have been identified that cause Charcot-Marie-Tooth disease, a disorder that can affect myelin sheaths. Among these, a number of mutations are related to essential targets of the posttranslational modification neddylation, although how these lead to myelin defects is unclear. Here, we demonstrate that inhibiting neddylation leads to a notable absence of peripheral myelin and axonal loss both in developing and regenerating mouse nerves. Our data indicate that neddylation exerts a global influence on the complex transcriptional and posttranscriptional program by simultaneously regulating the expression and function of multiple essential myelination signals, including the master transcription factor EGR2 and the negative regulators c-Jun and Sox2, and inducing global secondary changes in downstream pathways, including the mTOR and YAP/TAZ signaling pathways. This places neddylation as a critical regulator of myelination and delineates the potential pathogenic mechanisms involved in CMT mutations related to neddylation.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Células de Schwann , Animales , Ratones , Vaina de Mielina/genética , Enfermedad de Charcot-Marie-Tooth/genética , Mutación , Procesamiento Proteico-PostraduccionalRESUMEN
Oligodendrocytes are the myelin forming cells of the central nervous system, and their vulnerability to excitotoxicity induced by glutamate contributes to the pathogenesis of neurological disorders including brain ischemia and neurodegenerative diseases, such as multiple sclerosis. In addition to glutamate receptors, oligodendrocytes express GABA receptors (GABAR) that are involved in their survival and differentiation. The interactions between glutamate and GABAergic systems are well documented in neurons, under both physiological and pathological conditions, but this potential crosstalk in oligodendrocytes has not been studied in depth. Here, we evaluated the protective effect of GABAR agonists, baclofen (GABAB) and muscimol (GABAA), against AMPA-induced excitotoxicity in cultured rat oligodendrocytes. First, we observed that both baclofen and muscimol reduced cell death and caspase-3 activation after AMPA insult, proving their oligoprotective potential. Interestingly, analysis of the cell-surface expression of calcium-impermeable GluR2 subunits in oligodendrocytes revealed that GABAergic agonists significantly reverted GluR2 internalization induced by AMPA. We determined that baclofen and muscimol also impaired AMPA-induced intracellular calcium increase and subsequent mitochondrial membrane potential alteration, ROS generation, and calpain activation. However, AMPA-triggered activation of Src, Akt, JNK and CREB was not affected by baclofen or muscimol. Overall, our results suggest that GABAR activation initiates alternative molecular mechanisms that attenuate AMPA-mediated apoptotic excitotoxicity in oligodendrocytes by interfering with expression of GluR subunits in membranes and with calcium-dependent intracellular signaling pathways. Together, these findings provide evidence of GABAR agonists as potential oligodendroglial protectants in central nervous system disorders.
RESUMEN
Promoting remyelination is considered as a potential neurorepair strategy to prevent/limit the development of permanent neurological disability in patients with multiple sclerosis (MS). To this end, a number of clinical trials are investigating the potential of existing drugs to enhance oligodendrocyte progenitor cell (OPC) differentiation, a process that fails in chronic MS lesions. We previously reported that oligodendroglia express GABAB receptors (GABAB Rs) both in vitro and in vivo, and that GABAB R-mediated signaling enhances OPC differentiation and myelin protein expression in vitro. Our goal here was to evaluate the pro-remyelinating potential of GABAB R agonist baclofen (Bac), a clinically approved drug to treat spasticity in patients with MS. We first demonstrated that Bac increases myelin protein production in lysolecithin (LPC)-treated cerebellar slices. Importantly, Bac administration to adult mice following induction of demyelination by LPC injection in the spinal cord resulted in enhanced OPC differentiation and remyelination. Thus, our results suggest that Bac repurposing should be considered as a potential therapeutic strategy to stimulate remyelination in patients with MS.
Asunto(s)
Esclerosis Múltiple , Remielinización , Animales , Baclofeno/metabolismo , Baclofeno/farmacología , Baclofeno/uso terapéutico , Diferenciación Celular , Sistema Nervioso Central/metabolismo , Agonistas de Receptores GABA-B/metabolismo , Agonistas de Receptores GABA-B/farmacología , Agonistas de Receptores GABA-B/uso terapéutico , Lisofosfatidilcolinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/patología , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismoRESUMEN
Oligodendrocytes (OLs) express functional GABAA receptors (GABAARs) that are activated by GABA released at synaptic contacts with axons or by ambient GABA in extrasynaptic domains. In both instances, the receptors' molecular identity has not been fully defined. Furthermore, data on their structural diversity in different brain regions and information on age-dependent changes in their molecular composition are scant. This lack of knowledge has delayed access to a better understanding of the role of GABAergic signaling between neurons and OLs. Here, we used functional, and pharmacological analyses, as well as gene and protein expression of GABAAR subunits, to explore the subunit combination that could explain the receptor functional profile expressed in OLs from the neonate rat. We found that GABAAR composed of α3ß2γ1 subunits mimicked the characteristics of the endogenous receptor when expressed heterologously in Xenopus laevis oocytes. Either α3 or γ1 subunit silencing by small interfering RNA transfection changed the GABA-response characteristics in oligodendrocyte precursor cells, indicating their participation in the endogenous receptor conformation. Thus, α3 subunit silencing shifted the mean EC50 for GABA from 75.1 to 46.6 µM, whereas γ1 silencing reduced the current amplitude response by 55%. We also observed that ß-carbolines differentially enhance GABA responses in oligodendroglia as compared with those in neurons. These results contribute to defining the molecular and pharmacological properties of GABAARs in OLs. Additionally, the identification of ß-carbolines as selective enhancers of GABAARs in OLs may help to study the role of GABAergic signaling during myelination. SIGNIFICANCE STATEMENT: GABAergic signaling through GABAA receptors (GABAARs) expressed in the oligodendroglial lineage contributes to the myelination control. Determining the molecular identity and the pharmacology of these receptors is essential to define their specific roles in myelination. Using GABAAR subunit expression and silencing, we identified that the GABAAR subunit combination α3ß2γ1 conforms the bulk of GABAARs in oligodendrocytes from rat neonates. Furthermore, we found that these receptors have differential pharmacological properties that allow specific positive modulation by ß-carbolines.
Asunto(s)
Encéfalo/citología , Neuronas/citología , Oligodendroglía/citología , Receptores de GABA-A/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Carbolinas/farmacología , Células Cultivadas , Femenino , Silenciador del Gen , Ratones , Neuronas/metabolismo , Oligodendroglía/metabolismo , Ratas , Receptores de GABA-A/genética , Xenopus laevisRESUMEN
Myelin facilitates the fast transmission of nerve impulses and provides metabolic support to axons. Differentiation of oligodendrocyte progenitor cells (OPCs) and Schwann cell (SC) precursors is critical for myelination during development and myelin repair in demyelinating disorders. Myelination is tightly controlled by neuron-glia communication and requires the participation of a wide repertoire of signals, including neurotransmitters such as glutamate, ATP, adenosine, or γ-aminobutyric acid (GABA). GABA is the main inhibitory neurotransmitter in the central nervous system (CNS) and it is also present in the peripheral nervous system (PNS). The composition and function of GABA receptors (GABARs) are well studied in neurons, while their nature and role in glial cells are still incipient. Recent studies demonstrate that GABA-mediated signaling mechanisms play relevant roles in OPC and SC precursor development and function, and stand out the implication of GABARs in oligodendrocyte (OL) and SC maturation and myelination. In this review, we highlight the evidence supporting the novel role of GABA with an emphasis on the molecular identity of the receptors expressed in these glial cells and the possible signaling pathways involved in their actions. GABAergic signaling in myelinating cells may have potential implications for developing novel reparative therapies in demyelinating diseases.
RESUMEN
OBJECTIVE: Antibodies against neuronal N-methyl-D-aspartate receptors (NMDARs) in patients with anti-NMDAR encephalitis alter neuronal synaptic function and plasticity, but the effects on other cells of the nervous system are unknown. METHODS: Cerebrospinal fluid (CSF) of patients with anti-NMDAR encephalitis (preabsorbed or not with GluN1) and a human NMDAR-specific monoclonal antibody (SSM5) derived from plasma cells of a patient, along the corresponding controls, were used in the studies. To evaluate the activity of oligodendrocyte NMDARs and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in vitro after exposure to patients' CSF antibodies or SSM5, we used a functional assay based on cytosolic Ca2+ imaging. Expression of the glucose transporter (GLUT1) in oligodendrocytes was assessed by immunocytochemistry. RESULTS: NMDAR agonist responses were robustly reduced after preincubation of oligodendrocytes with patients' CSF or SSM5 but remained largely unaltered with the corresponding controls. These effects were NMDAR specific, as patients' CSF did not alter responses to AMPA receptor agonists and was abrogated by preabsorption of CSF with HEK cells expressing GluN1 subunit. Patients' CSF also reduced oligodendrocyte expression of glucose transporter GLUT1 induced by NMDAR activity. INTERPRETATION: Antibodies from patients with anti-NMDAR encephalitis specifically alter the function of NMDARs in oligodendrocytes, causing a decrease of expression of GLUT1. Considering that normal GLUT1 expression in oligodendrocytes and myelin is needed to metabolically support axonal function, the findings suggest a link between antibody-mediated dysfunction of NMDARs in oligodendrocytes and the white matter alterations reported in patients with this disorder. ANN NEUROL 2020;87:670-676.
Asunto(s)
Encefalitis Antirreceptor N-Metil-D-Aspartato/metabolismo , Autoanticuerpos/inmunología , Oligodendroglía/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Adolescente , Adulto , Animales , Encefalitis Antirreceptor N-Metil-D-Aspartato/inmunología , Autoanticuerpos/líquido cefalorraquídeo , Autoanticuerpos/farmacología , Autoantígenos/inmunología , Células Cultivadas , Niño , Femenino , Transportador de Glucosa de Tipo 1/biosíntesis , Humanos , Masculino , Oligodendroglía/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/inmunología , Adulto JovenRESUMEN
Differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs) is a key event for axonal myelination in the central nervous system (CNS). Several growth factors and neurotransmitters like GABA are postulated as important regulators of that process, and different protein kinases may also participate in OL differentiation and myelination. However, the molecular mechanisms underlying the regulation of myelination by neurotransmitters are only partially known. In the present study, we provide evidence showing that GABA receptors (GABARs) play an important role in OL differentiation. First, we observed that OPCs and OLs synthesize GABA and expressed GABAR and transporters, both in vitro and in vivo and, in contrast to GABAARs, the subunits GABAB1R and GABAB2R are expressed in OLs over time. Then, we found that exogenous GABA increases the number of myelin segments and MBP expression in DRG-OPC cocultures, indicating that GABA regulates myelination when OLs are in contact with axons. Notably, in purified rat OPC cultures, chronic treatment with GABA and baclofen, specific GABABR agonist, accelerates OPC differentiation by enhancing the processes branching and myelin protein expression, effects that are reverted in presence of GABABR specific antagonist CGP55845. Exposure of OPCs to baclofen promotes the Src-phosphorylation, and the baclofen-induced maturation is attenuated in presence of the Src-family kinases inhibitor PP2. None of these effects are mediated by the GABAAR agonist muscimol. Together, these results highlight the relevance of the GABAergic system in OL differentiation, and indicate that this functional role is mediated through GABABR involving the participation of Src-family kinases. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.
Asunto(s)
Oligodendroglía , Receptores de GABA-B , Animales , Diferenciación Celular , Células Cultivadas , Vaina de Mielina , Ratas , Ácido gamma-AminobutíricoRESUMEN
Alzheimer´s disease (AD) is characterized by a progressive cognitive decline that correlates with the levels of amyloid ß-peptide (Aß) oligomers. Strong evidences connect changes of oligodendrocyte function with the onset of neurodegeneration in AD. However, the mechanisms controlling oligodendrocyte responses to Aß are still elusive. Here, we tested the role of Aß in oligodendrocyte differentiation, maturation, and survival in isolated oligodendrocytes and in organotypic cerebellar slices. We found that Aß peptides specifically induced local translation of 18.5-kDa myelin basic protein (MBP) isoform in distal cell processes concomitant with an increase of process complexity of MBP-expressing oligodendrocytes. Aß oligomers required integrin ß1 receptor, Src-family kinase Fyn and Ca2+/CaMKII as effectors to modulate MBP protein expression. The pharmacological inhibition of Fyn kinase also attenuated oligodendrocyte differentiation and survival induced by Aß oligomers. Similarly, using ex vivo organotypic cerebellar slices Aß promoted MBP upregulation through Fyn kinase, and modulated oligodendrocyte population dynamics by inducing cell proliferation and differentiation. Importantly, application of Aß to cerebellar organotypic slices enhanced remyelination and oligodendrocyte lineage recovery in lysolecithin (LPC)-induced demyelination. These data reveal an important role of Aß in oligodendrocyte lineage function and maturation, which may be relevant to AD pathogenesis.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Integrina beta1/metabolismo , Oligodendroglía/metabolismo , Organoides/crecimiento & desarrollo , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Animales , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Enfermedades Desmielinizantes/metabolismo , Proteína Básica de Mielina/metabolismo , Oligodendroglía/citología , Oligodendroglía/enzimología , Organoides/citología , Organoides/enzimología , Organoides/metabolismo , Proteínas Proto-Oncogénicas c-fyn/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fyn/genética , Ratas , Ratas Sprague-Dawley , Transducción de Señal/genéticaRESUMEN
Oligodendrocytes are highly vulnerable to glutamate excitotoxicity, a central mechanism involved in tissue damage in Multiple Sclerosis (MS). Sustained activation of AMPA receptors in rat oligodendrocytes induces cytosolic calcium overload, mitochondrial depolarization, increase of reactive oxygen species, and activation of intracelular pathways resulting in apoptotic cell death. Although many signals driven by excitotoxicity have been identified, some of the key players are still under investigation. Casein kinase 2 (CK2) is a serine/threonine kinase, constitutively expressed in all eukaryotic tissues, involved in cell proliferation, malignant transformation and apoptosis. In this study, we identify CK2 as a critical regulator of oligodendrocytic death pathways and elucidate its role as a signal inductor following excitotoxic insults. We provide evidence that CK2 activity is up-regulated in AMPA-treated oligodendrocytes and CK2 inhibition significantly diminished AMPA receptor-induced oligodendroglial death. In addition, we analyzed mitogen-activated protein kinase (MAPK) signaling after excitotoxic insult. We observed that AMPA receptor activation induced a rapid increase in c-Jun N-terminal kinase (JNK) and p38 phosphorylation that was reduced after CK2 inhibition. Moreover, blocking their phosphorylation, we enhanced oligodendrocyte survival after excitotoxic insult. Finally, we observed that the tumor suppressor p53 is activated during AMPA receptor-induced cell death and, interestingly, down-regulated by JNK or CK2 inhibition. Together, these data indicate that the increase in CK2 activity induced by excitotoxic insults regulates MAPKs, triggers p53 activation and mediates subsequent oligodendroglial loss. Therefore, targeting CK2 may be a useful strategy to prevent oligodendrocyte death in MS and other diseases involving central nervous system (CNS) white matter.
RESUMEN
Multiple sclerosis (MS) is a chronic demyelinating disease of unknown etiology in which tissue pathology suggests both immune-dependent attacks to oligodendroglia and primary oligodendrocyte demise. The endocannabinoid system has been crucially involved in the control of autoimmune demyelination and cannabinoid-based therapies exhibit therapeutic potential, but also limitations, in MS patients. In this context, growing evidence suggests that targeting the hydrolysis of the main endocannabinoid 2-arachidonoylglycerol (2-AG) may offer a more favorable benefit-to-risk balance in MS than existing cannabinoid medicines. Here we evaluated the modulation of endocannabinoid signaling and the therapeutic potential of targeting the 2-AG hydrolytic enzyme alpha/beta-hydrolase domain-containing 6 (ABHD6) in the cuprizone model of non-immune dependent demyelination. The concentrations of N-arachidonoylethanolamine (anandamide, AEA) and its congener N-palmitoylethanolamine (PEA) were reduced following 6â¯weeks of cuprizone feeding. Deregulation of AEA and PEA levels was not due to differences in the expression of the hydrolytic and biosynthetic enzymes fatty acid amide hydrolase and N-acylphosphatidylethanolamine-phospholipase D, respectively. Conversely, we measured elevated transcript levels of 2-AG hydrolytic enzymes monoacylglycerol lipase, ABHD6 and ABHD12 without changes in bulk 2-AG concentration. Upregulated CB1 and CB2 receptors expression, ascribed in part to microglia, was also detected in the brain of cuprizone-treated mice. Administration of an ABHD6 inhibitor partially attenuated myelin damage, astrogliosis and microglia/macrophage reactivity associated to cuprizone feeding. However, ABHD6 blockade was ineffective at engaging protective or differentiation promoting effects in oligodendrocyte cultures. These results show specific alterations of the endocannabinoid system and modest beneficial effects resulting from ABHD6 inactivation in a relevant model of primary demyelination.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Endocannabinoides/metabolismo , Glicéridos/metabolismo , Monoacilglicerol Lipasas/antagonistas & inhibidores , Vaina de Mielina/efectos de los fármacos , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Masculino , Ratones Endogámicos C57BL , Esclerosis Múltiple/inducido químicamente , Esclerosis Múltiple/tratamiento farmacológico , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismoRESUMEN
Oligodendrocytes are the myelin-forming cells in the central nervous system (CNS) and their loss or dysfunction is a hallmark of CNS demyelinating diseases, such as multiple sclerosis (MS), hypoxic-ischemic demyelination, or spinal cord injury. In the rodent CNS, oligodendrocyte progenitor cells (OPCs) arise in multiple ventral and dorsal locations of the forebrain during late embryogenesis and early postnatal periods. OPCs migrate out from these germinal zones and disperse throughout the CNS, to populate the developing white and gray matter. There, OPCs can begin to mature through a series of intermediate states characterized by the expression of stage-specific proteins until completely differentiated into postmitotic myelinating oligodendrocytes. Elucidating the cellular and molecular mechanisms that control oligodendrocyte maturation requires isolating OPCs and premyelinating oligodendrocytes by rapid and reliable methods that provide high yield, pure and viable culture, being a powerful tool to characterize their differentiation and potential capacity for myelin repair after injury. This chapter describes in detail two simple and efficient protocols for the preparation of highly enriched rat OPC populations and immature oligodendrocytes derived from mixed glial cultures and optic nerves, respectively. Functional oligodendrocytes obtained with these protocols can be cocultured with primary neurons to study myelination.
Asunto(s)
Encéfalo/citología , Diferenciación Celular , Separación Celular/métodos , Oligodendroglía/citología , Nervio Óptico/citología , Animales , Animales Recién Nacidos , Biomarcadores , Técnicas de Cultivo de Célula , Linaje de la Célula , Técnica del Anticuerpo Fluorescente , Neuroglía/citología , Neuroglía/metabolismo , Oligodendroglía/metabolismo , Cultivo Primario de Células , RatasRESUMEN
Amyloid beta- (Aß-) mediated ROS overproduction disrupts intraneuronal redox balance and exacerbates mitochondrial dysfunction which leads to neuronal injury. Polyphenols have been investigated as therapeutic agents that promote neuroprotective effects in experimental models of brain injury and neurodegenerative diseases. The aim of this study was to identify the neuroprotective effects of morin and mangiferin against Aß oligomers in cultured cortical neurons and organotypic slices as well as their mechanisms of action. Cell death caused by Aß oligomers in neuronal cultures was decreased in the presence of micromolar concentrations of mangiferin or morin, which in turn attenuated oxidative stress. The neuroprotective effects of antioxidants against Aß were associated with the reduction of Aß-induced calcium load to mitochondria; mitochondrial membrane depolarization; and release of cytochrome c from mitochondria, a key trigger of apoptosis. Additionally, we observed that both polyphenols activated the endogenous enzymatic antioxidant system and restored oxidized protein levels. Finally, Aß induced an impairment of energy homeostasis due to a decreased respiratory capacity that was mitigated by morin and mangiferin. Overall, the beneficial effects of polyphenols in preventing mitochondrial dysfunction and neuronal injury in AD cell models suggest that morin and mangiferin hold promise for the treatment of this neurological disorder.
Asunto(s)
Flavonoides/farmacología , Xantonas/farmacología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citosol/metabolismo , Inmunohistoquímica , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The present study has been designed to explore the molecular mechanism and signaling pathway targets of chlorogenic acid (CGA) and its main hydrolysates, caffeic (CA) and quinic acid in the protective effect against glutamate-excitotoxicity. For this purpose 8-DIV cortical neurons in primary culture were exposed to 50 µM L-glutamic acid plus 10 µM glycine, with or without 10-100 µM tested compounds. Chlorogenic acid and caffeic acid via their antioxidant properties inhibited cell death induced by glutamate in dose depended manner. However, quinic acid slightly protects neurons at a higher dose. DCF, JC-1 and Ca2+sensitive fluorescent dye fura-2, were used to measure intracellular ROS accumulation, mitochondrial membrane potential integration and intracellular calcium concentration [Ca2+] i . Results indicate that similarly, CGA acts as a protective agent against glutamate-induced cortical neurons injury by suppressing the accumulation of endogenous ROS and restore the mitochondrial membrane potential, activate the enzymatic antioxidant system by the increase levels of SOD activity and modulate the rise of intracellular calcium levels by increasing the rise of intracellular concentrations of Ca2+caused by glutamate overstimulation. PKC signaling cascade appear to be engaged in this protective mechanism. Interseling, CGA and CA also exhibit antiapoptotic properties against glutamate-induced cleaved activation of pro-caspases; caspase 1,8 and 9 and calpain (PD 150606,Calpeptin and MDL 28170).These data suggest that neuroprotective activity of CGA ester may occurs throught its hydrolysate,the caffeic acid and its interaction with intracellular molecules suggesting that CGA exert its neuroprotection via its caffeoly acid group that might potentially be used as a therapeutic agent in neurodegeneratives disorders associated with glutamate excitotoxicity.
Asunto(s)
Ácido Clorogénico/farmacología , Ácido Glutámico/metabolismo , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Antioxidantes/farmacología , Ácidos Cafeicos/metabolismo , Muerte Celular/efectos de los fármacos , Células Cultivadas , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Neuronas/metabolismo , Neuroprotección/efectos de los fármacos , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Inwardly rectifying K+ (Kir) channel expression signals at an advanced stage of maturation during oligodendroglial differentiation. Knocking down their expression halts the generation of myelin and produces severe abnormalities in the central nervous system. Kir4.1 is the main subunit involved in the tetrameric structure of Kir channels in glial cells; however, the precise composition of Kir channels expressed in oligodendrocytes (OLs) remains partially unknown, as participation of other subunits has been proposed. Kir channels are sensitive to H+; thus, intracellular acidification produces Kir current inhibition. Since Kir subunits have differential sensitivity to H+, we studied the effect of intracellular acidification on Kir currents expressed in cultured OLs derived from optic nerves of 12-day-old rats. Unexpectedly, Kir currents in OLs (2-4 DIV) did not change within the pH range of 8.0-5.0, as observed when using standard whole-cell voltage-clamp recording or when preserving cytoplasmic components with the perforated patch-clamp technique. In contrast, low pH inhibited astrocyte Kir currents, which was consistent with the involvement of the Kir4.1 subunit. The H+-insensitivity expressed in OL Kir channels was not intrinsic because Kir cloning showed no difference in the sequence reported for the Kir4.1, Kir2.1, or Kir5.1 subunits. Moreover, when Kir channels were heterologously expressed in Xenopus oocytes they behaved as expected in their general properties and sensitivity to H+. It is therefore concluded that Kir channel H+-sensitivity in OLs is modulated through an extrinsic mechanism, probably by association with a modulatory component or by posttranslational modifications.
Asunto(s)
Oligodendroglía/fisiología , Nervio Óptico/fisiología , Canales de Potasio de Rectificación Interna/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Concentración de Iones de Hidrógeno , Potenciales de la Membrana/fisiología , Nervio Óptico/citología , Ratas , Ratas Sprague-Dawley , Xenopus laevisRESUMEN
Stimulated by the results of a recent paper on the effects of tiagabine, a selective inhibitor of the main GABA transporter GAT-1, on oligodendrogenesis, we verified the possibility that GAT-1 may be expressed in oligodendrocytes using immunocytochemical methods and functional assays. Light microscopic analysis of the subcortical white matter of all animals revealed the presence of numerous GAT-1+ cells of different size (from 3 to 29 µm) and morphology. An electron microscope analysis revealed that, besides fibrous astrocytes and interstitial neurons, GAT-1 immunoreactivity was present in immature and mature oligodendrocytes. Co-localization studies between GAT-1 and markers specific for oligodendrocytes (NG2 and RIP) showed that about 12% of GAT-1 positive cells in the white matter were immature oligodendrocytes, while about 15% were mature oligodendrocytes. In vitro functional assays showed that oligodendrocytes exhibit tiagabine-sensitive Na+ -dependent GABA uptake. Although relationships between GABA and oligodendrocytes have been known for many years, this is the first demonstration that GAT-1 is expressed in oligodendrocytes. The present results on the one hand definitely closes the era of "neuronal" and "glial" GABA transporters, on the other they suggest that oligodendrocytes may contribute to pathophysiology of the several diseases in which GAT-1 have been implicated to date. GLIA 2017;65:514-522.
Asunto(s)
Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Oligodendroglía/metabolismo , Animales , Animales Recién Nacidos , Antígenos/metabolismo , Encéfalo/citología , Células Cultivadas , Masculino , Microscopía Confocal , Microscopía Inmunoelectrónica , Inhibidores de la Captación de Neurotransmisores/farmacología , Ácidos Nipecóticos/farmacología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/ultraestructura , Nervio Óptico/citología , Proteoglicanos/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Sodio/metabolismo , Tiagabina , Tritio/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Myelination requires oligodendrocyte-neuron communication, and both neurotransmitters and contact interactions are essential for this process. Oligodendrocytes are endowed with neurotransmitter receptors whose expression levels and properties may change during myelination. However, only scant information is available about the extent and timing of these changes or how they are regulated by oligodendrocyte-neuron interactions. Here, we used electrophysiology to study the expression of ionotropic GABA, glutamate, and ATP receptors in oligodendrocytes derived from the optic nerve and forebrain cultured either alone or in the presence of dorsal root ganglion neurons. We observed that oligodendrocytes from both regions responded to these transmitters at 1 day in culture. After the first day in culture, however, GABA sensitivity diminished drastically to less than 10%, while that of glutamate and ATP remained constant. In contrast, the GABA response amplitude was sustained and remained stable in oligodendrocytes cocultured with dorsal root ganglion neurons. Immunochemistry and pharmacological properties of the responses indicated that they were mediated by distinctive GABAA receptors and that in coculture with neurons, the oligodendrocytes bearing the receptors were those in direct contact with axons. These results reveal that GABAA receptor regulation in oligodendrocytes is driven by axonal cues and that GABA signaling may play a role in myelination and/or during axon-glia recognition.
Asunto(s)
Axones/metabolismo , Comunicación Celular/fisiología , Neuroglía/metabolismo , Oligodendroglía/metabolismo , Receptores de GABA-A/biosíntesis , Animales , Axones/efectos de los fármacos , Axones/ultraestructura , Comunicación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/ultraestructura , Regulación de la Expresión Génica , Neuroglía/efectos de los fármacos , Neuroglía/ultraestructura , Oligodendroglía/efectos de los fármacos , Oligodendroglía/ultraestructura , Prosencéfalo/efectos de los fármacos , Prosencéfalo/metabolismo , Prosencéfalo/ultraestructura , Ratas , Ratas Sprague-Dawley , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Pío del Río Hortega (1882-1945) discovered microglia and oligodendrocytes (OLGs), and after Ramón y Cajal, was the most prominent figure of the Spanish school of neurology. He began his scientific career with Nicolás Achúcarro from whom he learned the use of metallic impregnation techniques suitable to study non-neuronal cells. Later on, he joined Cajal's laboratory. and Subsequently, he created his own group, where he continued to develop other innovative modifications of silver staining methods that revolutionized the study of glial cells a century ago. He was also interested in neuropathology and became a leading authority on Central Nervous System (CNS) tumors. In parallel to this clinical activity, del Río Hortega rendered the first systematic description of a major polymorphism present in a subtype of macroglial cells that he named as oligodendroglia and later OLGs. He established their ectodermal origin and suggested that they built the myelin sheath of CNS axons, just as Schwann cells did in the periphery. Notably, he also suggested the trophic role of OLGs for neuronal functionality, an idea that has been substantiated in the last few years. Del Río Hortega became internationally recognized and established an important neurohistological school with outstanding pupils from Spain and abroad, which nearly disappeared after his exile due to the Spanish civil war. Yet, the difficulty of metal impregnation methods and their variability in results, delayed for some decades the confirmation of his great insights into oligodendrocyte biology until the development of electron microscopy and immunohistochemistry. This review aims at summarizing the pioneer and essential contributions of del Río Hortega to the current knowledge of oligodendrocyte structure and function, and to provide a hint of the scientific personality of this extraordinary and insufficiently recognized man.
RESUMEN
BACKGROUND: 5'-deoxy-5'-methylthioadenosine (MTA) is an endogenous compound produced through the metabolism of polyamines. The therapeutic potential of MTA has been assayed mainly in liver diseases and, more recently, in animal models of multiple sclerosis. The aim of this study was to determine the neuroprotective effect of this molecule in vitro and to assess whether MTA can cross the blood brain barrier (BBB) in order to also analyze its potential neuroprotective efficacy in vivo. METHODS: Neuroprotection was assessed in vitro using models of excitotoxicity in primary neurons, mixed astrocyte-neuron and primary oligodendrocyte cultures. The capacity of MTA to cross the BBB was measured in an artificial membrane assay and using an in vitro cell model. Finally, in vivo tests were performed in models of hypoxic brain damage, Parkinson's disease and epilepsy. RESULTS: MTA displays a wide array of neuroprotective activities against different insults in vitro. While the data from the two complementary approaches adopted indicate that MTA is likely to cross the BBB, the in vivo data showed that MTA may provide therapeutic benefits in specific circumstances. Whereas MTA reduced the neuronal cell death in pilocarpine-induced status epilepticus and the size of the lesion in global but not focal ischemic brain damage, it was ineffective in preserving dopaminergic neurons of the substantia nigra in the 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-mice model. However, in this model of Parkinson's disease the combined administration of MTA and an A2A adenosine receptor antagonist did produce significant neuroprotection in this brain region. CONCLUSION: MTA may potentially offer therapeutic neuroprotection.
Asunto(s)
Desoxiadenosinas/farmacología , Fármacos Neuroprotectores/farmacología , Tionucleósidos/farmacología , Enfermedad Aguda , Antagonistas Adrenérgicos/farmacología , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/patología , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/patología , Permeabilidad de la Membrana Celular , Células Cultivadas , Enfermedad Crónica , Desoxiadenosinas/uso terapéutico , Modelos Animales de Enfermedad , Glucosa/deficiencia , Masculino , Ratones , N-Metilaspartato/toxicidad , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/patología , Fármacos Neuroprotectores/uso terapéutico , Neurotoxinas/toxicidad , Oxígeno , Pilocarpina , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Estado Epiléptico/tratamiento farmacológico , Estado Epiléptico/patología , Tionucleósidos/uso terapéutico , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/toxicidadRESUMEN
Adenosine receptor activation is involved in myelination and in apoptotic pathways linked to neurodegenerative diseases. In this study, we investigated the effects of adenosine receptor activation in the viability of oligodendrocytes of the rat optic nerve. Selective activation of A3 receptors in pure cultures of oligodendrocytes caused concentration-dependent apoptotic and necrotic death which was preceded by oxidative stress and mitochondrial membrane depolarization. Oligodendrocyte apoptosis induced by A3 receptor activation was caspase-dependent and caspase-independent. In addition to dissociated cultures, incubation of optic nerves ex vivo with adenosine and the A3 receptor agonist 2-CI-IB-MECA(1-[2-Chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide)-induced caspase-3 activation, oligodendrocyte damage, and myelin loss, effects which were prevented by the presence of caffeine and the A3 receptor antagonist MRS 1220 (N-[9-Chloro-2-(2-furanyl)[1,2,4]-triazolo [1,5-c]quinazolin-5-yl]benzene acetamide). Finally, ischemia-induced injury and functional loss to the optic nerve was attenuated by blocking A3 receptors. Together, these results indicate that adenosine may trigger oligodendrocyte death via activation of A3 receptors and suggest that this mechanism contributes to optic nerve and white matter ischemic damage.
Asunto(s)
Agonistas del Receptor de Adenosina A3/administración & dosificación , Agonistas del Receptor de Adenosina A3/farmacología , Apoptosis , Oligodendroglía/metabolismo , Nervio Óptico/metabolismo , Receptor de Adenosina A3/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacología , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Células Cultivadas , Oligodendroglía/citología , Ratas , Ratas Sprague-DawleyRESUMEN
Dyshomeostasis of cytosolic Zn(2+) is a critical mediator of neuronal damage during excitotoxicity. However, the role of this cation in oligodendrocyte pathophysiology is not well understood. The current study examined the contribution of Zn(2+) deregulation to oligodendrocyte injury mediated by AMPA receptors. Oligodendrocytes loaded with the Zn(2+)-selective indicator FluoZin-3 responded to mild stimulation of AMPA receptors with fast cytosolic Zn(2+) rises that resulted from intracellular release, as they were not blocked by the extracellular Zn(2+) chelator Ca-EDTA. Pharmacological experiments suggested that AMPA-induced Zn(2+) mobilization depends on cytosolic Ca(2+) accumulation, arises from mitochondria and protein-bound pools, and is triggered by mechanisms that do not involve the generation of reactive oxygen species. Moreover, intracellular Zn(2+) rises resulting from AMPA receptor activation seem to be promoted by Ca(2+)-dependent cytosolic acidification. Addition of the cell-permeable Zn(2+) chelator TPEN significantly reduced mitochondrial membrane depolarization, reactive oxygen species production, and cell death by sub-maximal activation of AMPA receptors both in vitro and in situ, suggesting that Zn(2+) deregulation is an important mediator of oligodendrocyte excitotoxicity. These data provide evidence that strategies aimed at maintaining Zn(2+) homeostasis may be useful for the treatment of disorders in which excitotoxicity is an important trigger of oligodendroglial death.