RESUMO
The development of neuroprotective therapies is a sought-after goal. By screening combinatorial chemical libraries using in vitro assays, we identified the small molecule BN201 that promotes the survival of cultured neural cells when subjected to oxidative stress or when deprived of trophic factors. Moreover, BN201 promotes neuronal differentiation, the differentiation of precursor cells to mature oligodendrocytes in vitro, and the myelination of new axons. BN201 modulates several kinases participating in the insulin growth factor 1 pathway including serum-glucocorticoid kinase and midkine, inducing the phosphorylation of NDRG1 and the translocation of the transcription factor Foxo3 to the cytoplasm. In vivo, BN201 prevents axonal and neuronal loss, and it promotes remyelination in models of multiple sclerosis, chemically induced demyelination, and glaucoma. In summary, we provide a new promising strategy to promote neuroaxonal survival and remyelination, potentially preventing disability in brain diseases.
Assuntos
Amidas/uso terapêutico , Axônios/efeitos dos fármacos , Encefalite/tratamento farmacológico , Bainha de Mielina/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Peptoides/uso terapêutico , Pirrolidinonas/uso terapêutico , Animais , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Feminino , Imunofluorescência , Glaucoma/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nervo Óptico/efeitos dos fármacos , Proguanil , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , TriazinasRESUMO
BACKGROUND: Newly proliferated oligodendrocyte precursor cells (OPCs) migrate and surround lesions of patients with multiple sclerosis (MS) and other demyelinating diseases, but fail to differentiate into oligodendrocytes (OLs) and remyelinate remaining viable axons. The abundance of secreted inflammatory factors within and surrounding these lesions likely plays a major inhibitory role, promoting cell death and preventing OL differentiation and axon remyelination. To identify clinical candidate compounds that may protect existing and differentiating OLs in patients, we have developed a high throughput screening (HTS) assay that utilizes purified rat OPCs. RESULTS: Using a fluorescent indicator of cell viability coupled with image quantification, we developed an assay to allow the identification of compounds that promote OL viability and differentiation in the presence of the synergistic inflammatory cytokines, tumor necrosis factor α and interferon-γ. We have utilized this assay to screen the NIH clinical collection library and identify compounds that protect OLs and promote OL differentiation in the presence of these inflammatory cytokines. CONCLUSION: This primary OL-based cytokine protection assay is adaptable for HTS and may be easily modified for profiling of compounds in the presence of other potentially inhibitory molecules found in MS lesions. This assay should be of use to those interested in identifying drugs for the treatment of MS and other demyelinating diseases.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Oligodendroglia/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Inflamação , Interferon gama/metabolismo , Masculino , Esclerose Múltipla/patologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Ratos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Multiple sclerosis is caused by an autoimmune response resulting in demyelination and neural degeneration. The adult central nervous system has the capacity to remyelinate axons in part through the generation of new oligodendrocytes (OLs). To identify clinical candidate compounds that may promote remyelination, we have developed a high throughput screening (HTS) assay to identify compounds that promote the differentiation of oligodendrocyte precursor cells (OPCs) into OLs. RESULTS: Using acutely dissociated and purified rat OPCs coupled with immunofluorescent image quantification, we have developed an OL differentiation assay. We have validated this assay with a known promoter of differentiation, thyroid hormone, and subsequently used the assay to screen the NIH clinical collection library. We have identified twenty-seven hit compounds which were validated by dose response analysis and the generation of half maximal effective concentration (EC50) values allowed for the ranking of efficacy. The assay identified novel promoters of OL differentiation which we attribute to (1) the incorporation of an OL toxicity pre-screen to allow lowering the concentrations of toxic compounds and (2) the utilization of freshly purified, non-passaged OPCs. These features set our assay apart from other OL differentiation assays used for drug discovery efforts. CONCLUSIONS: This acute primary OL-based differentiation assay should be of use to those interested in screening large compound libraries for the identification of drugs for the treatment of MS and other demyelinating diseases.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Animais , Técnicas In Vitro , Esclerose Múltipla/tratamento farmacológico , Ratos , Células-Tronco/citologiaRESUMO
BACKGROUND: Regeneration of new myelin is impaired in persistent multiple sclerosis (MS) lesions, leaving neurons unable to function properly and subject to further degeneration. Current MS therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system (CNS). Development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid CNS culture system. RESULTS: We established a high throughput cell-based assay to identify compounds that promote myelination. Culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. Using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a 96 well plate format. We have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. We have validated the assay scalability and consistency by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination. Half maximal effective concentration (EC50) values for these compounds were determined to rank them according to potency. CONCLUSIONS: We have designed the first high capacity in vitro assay that assesses myelination of live axons. This assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. Identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for MS patients.
Assuntos
Axônios/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Esclerose Múltipla/tratamento farmacológico , Bainha de Mielina/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Secretases da Proteína Precursora do Amiloide/farmacologia , Animais , Axônios/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Córtex Cerebral/fisiologia , Meios de Cultivo Condicionados/farmacologia , Imunofluorescência/métodos , Esclerose Múltipla/fisiopatologia , Bainha de Mielina/fisiologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/fisiologia , RatosRESUMO
Reactive gliosis is an aspect of neural plasticity and growth factor (GF) stimulation of astrocytes in vitro is widely regarded as a model system to study astrocyte plasticity. Astrocytes express receptors for several ligands including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), agonists for the G-protein-coupled lysophospholipid receptors (lpRs). Activation of lpRs by LPA or S1P leads to multiple pharmacological effects including the influx of calcium, phosphoinositide (PI) hydrolysis, phosphorylation of extracellular receptor regulated kinase (ERK), release of arachidonic acid, and induces mitogenesis. Treatment of astrocytes in vitro with a growth factor cocktail (containing epidermal growth factor [EGF], basic fibroblast growth factor [bFGF] and insulin) led to a marked attenuation of lpR-induced PI hydrolysis. In contrast, under identical conditions, GF treatment led to marked potentiation of PI hydrolysis downstream of activation of another abundantly expressed G-protein coupled receptor, mGluR5. Quantitative gene expression analysis of GF-treated or control astrocytes by TaqMan RT-PCR indicated that GF treatment did not change gene expression of lpa1 and s1p1, but increased gene expression of s1p5 which is expressed at very low levels in basal conditions. These results suggest that GF differentially affected PLC activation downstream of mGluR5 versus lpR activation and that the changes in mRNA levels of lpRs do not account for marked attenuation of agonist-induced phosphoinositide turnover.
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Substâncias de Crescimento/farmacologia , Fosfatidilinositóis/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Fosfatos de Inositol/metabolismo , Insulina/farmacologia , Fosforilação , Ratos , Ratos Sprague-Dawley , Receptor de Glutamato Metabotrópico 5 , Receptores de Lisofosfolipídeos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have been proposed to play a key role in oligodendrocyte maturation and myelinogenesis. In this study, we examined lysophospholipid receptor gene expression in differentiated rat oligodendrocyte cultures and signaling downstream of lysophospholipid receptor activation by LPA and S1P. Differentiated oligodendrocytes express mRNAs encoding lysophospholipid receptors with the relative abundance of lpa1>s1p5>s1p1=s1p2=lpa3>s1p3. LPA and S1P transiently increased phosphorylation of extracellular signal-regulated kinase (ERK) with EC50 values of 956 and 168 nM, respectively. LPA- and S1P-induced ERK phosphorylation was dependent on the activation of mitogen-activated protein kinase (MAPK), phospholipase C (PLC), and protein kinase C (PKC), but was insensitive to pertussis toxin (PTX). LPA increased intracellular calcium levels in oligodendrocytes and these increases were partially blocked by a PLC inhibitor but not by PTX. In contrast, S1P was not found to induce measurable changes of intracellular calcium. These results taken together suggest that lysophospholipid receptor activation involves receptor coupling to heterotrimeric Gq subunits with consequent activation of PLC, PKC, and MAPK pathways leading to ERK phosphorylation.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Oligodendroglia/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Animais , Córtex Cerebral/metabolismo , Relação Dose-Resposta a Droga , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oligodendroglia/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologiaRESUMO
Lysophosphatidic acid (1-acyl-2-lyso-sn-glycero-3-phosphate; LPA) and sphingosine-1-phosphate (S1P) are bioactive phospholipids which respectively act as agonists for the G-protein-coupled lpA receptors (LPA1, LPA2, and LPA3) and s1p receptors (S1P1, S1P2, S1P3, S1P4, and S1P5), collectively referred to as lysophospholipid receptors (lpR). Since astrocytes are responsive to LPA and S1P, we examined mechanisms of lpR signaling in rat cortical secondary astrocytes. Rat cortical astrocyte mRNA expression by quantitative TaqMan polymerase chain reaction (PCR) analysis revealed the following order of relative expression of lpR mRNAs: s1p3>s1p1>lpa1>s1p2=lpa3>>s1p5. Activation of lpRs by LPA or S1P led to multiple pharmacological effects, including the influx of calcium, phosphoinositide (PI) hydrolysis, phosphorylation of extracellular receptor regulated kinase (ERK) and release of [3H]-arachidonic acid (AA). These signalling events downstream of lpR activation were inhibited to varying degrees by pertussis toxin (PTX) pretreatment or by the inhibition of sphingosine kinase (SK), a rate-limiting enzyme in the biosynthesis of S1P from sphingosine. These results suggest that astrocyte lpR signalling mechanisms likely involve both Gi- and Gq-coupled GPCRs and that receptor-mediated activation of SK leads to intracellular generation of S1P, which in turn amplifies the lpR signalling in a paracrine/autocrine manner.
Assuntos
Astrócitos/fisiologia , Córtex Cerebral/fisiologia , Lisofosfolipídeos/metabolismo , Receptores de Lipoproteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Actinas/biossíntese , Actinas/genética , Animais , Ácido Araquidônico/metabolismo , Astrócitos/efeitos dos fármacos , Western Blotting , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Clonagem Molecular , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Fosfatos de Inositol/metabolismo , Lisofosfolipídeos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Receptor Cross-Talk/efeitos dos fármacos , Receptores de Lipoproteínas/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingosina/farmacologiaRESUMO
Glia play a pivotal role in glutaminergic excitatory neurotransmission in the central nervous system by regulating synaptic levels of glutamate and by providing glutamine as the sole precursor for the neurotransmitter pool glutamate to neurons through the glutamate-glutamine cycle. In the present investigation, we examined the influence of glutamate application on glutamine, serine and aspartate release from rat cortical glial cultures. The glial glutamate transporters rapidly cleared exogenously applied glutamate and this was accompanied by rapid increases in aspartate and glutamine, and a delayed increase in serine levels in the glial-conditioned medium. While glutamate-induced increases in glutamine and serine were sustained for up to 24 h, increases in aspartate lasted only for up to 6 h. The glutamate-induced increases in aspartate and glutamine were dependent both on the concentration and the duration of glutamate stimulus, but were largely insensitive to the inhibition of non-N-methyl-D-aspartate receptors or the metabotropic glutamate receptor 5. Inhibition of the glutamate transporter function by L-trans-pyrrolidine 2,4-dicarboxylate decreased the rate of glutamate uptake but not completely abrogated the uptake process, and this resulted in the attenuation of rate of glutamate induced glutamine synthesis. Dexamethasone treatment increased serine and glutamine levels in conditioned medium and increased glutamate induced glutamine release suggesting an upregulation of glutamine synthase activity. These results further substantiate coupling between glutamate and glutamine, and shed light on glutamate-dependent release of serine and aspartate, which may further contribute to excitatory neurotransmission.