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Anti-inflammatory effect of carbon monoxide on the neuron-microglia bidirectional communication
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RESUMO: Doenças neurológicas refere-se a qualquer tipo de patologia que afecta o cérebro, espinal medula e
sistema nervoso periférico. Entre outras, podemos identificar doenças como o acidente vascular cerebral
(AVC), epilepsia, doenças psiquiátricas, demência, infecções neurológicas e tumores. A Organização Mundial
da Saúde (OMS) estima que, em 2030, quase 8 mil milhões de pessoas irão morrer devido a este tipo de
doenças.
A fisiopatologia e progressão dos distúrbios do Sistema Nervoso Central (SNC) são, na maior parte
das vezes, multifatoriais e mal compreendidos, tornando um tratamento eficiente particularmente
complicado. Porém, existem vários mecanismos característicos de disfunção e dano. Neuroinflamação
descontrolada é uma das causas principais de propagação de dano e desequilíbrios homeostáticos.
Desregulação de neurogénese adulta e morte de populações neuronais especificas são outra característica
patológica. Descobertas recentes têm ajudado a esclarecer mecanismos moleculares de doença, mas muitas
questões permanecem sem resposta. Desta forma, existe um esforço contínuo para melhor entender estas
patologias e encontrar novas alternativas terapêuticas.
O monóxido de carbono (CO) é uma molécula gasosa endógena, que é protetora em vários tecidos,
incluindo no SNC. O principal objectivo desta tese de Doutoramento foi entender de melhor forma os
mecanismos de neuroprotecção do CO. O efeito modulatório do CO foi estudado em dois processos
biológicos diferentes: neurogénese e neuroinflamação. Para isto, vários modelos experimentais foram
implementados.
O Capítulo I introduz conceitos teóricos relativos ao cérebro, ao neurónio e às células da glia. É
também feita uma descrição do estado-da-arte relativo às vias moleculares de diferenciação, função da
microglia na imunidade e homeostasia, comunicação microglia neurónio, fagocitose e morte celular. Existe
um foco nas propriedades biológicas do CO.
No Capítulo II, é reportado que o CO reforça a produção de neurónios num modelo de diferenciação
neuronal usando a linha celular de neuroblastoma SH-SY5Y. O efeito estimulatório do CO na diferenciação
neuronal havia sido relacionado com modulação de morte celular e melhoramento do metabolismo
mitocondrial em outros modelos. Aqui, o CO melhorou a neurogénese na linha celular SH-SY5Y, ao actuar
sobre o enzima Glucose-6-fosfato desidrogenase (G6PD) e aumentar o fluxo da via das pentoses fosfato. O
efeito aqui reportado foi independente da síntese de novo de glutatião. Knockdown da expressão do enzima
limitante da via, G6PD, bloqueou a estimulação da diferenciação neuronal induzido pelo CO. Sumariamente,
o CO promove uma reprogramação metabólica que tem um papel importante no destino da célula e em
mecanismos de diferenciação neuronal O Capítulo III foca-se no CO como mediador da comunicação remota neurónio-microglia. Utilizando
um protocolo de meio condicionado, o CO protegeu os neurónios incubados com meio pro-inflamatório de
microglia. O CO inibiu a elevada secreção de TNF-α e NO em microglia reactiva. Além disso, o envolvimento
do CO na comunicação neurónio-microglia foi também avaliado na ausência de um estímulo inflamatório. O
meio condicionado de microglia tratada com CO protegeu parcialmente os neurónios contra morte celular
induzida via stress oxidativo. Adicionalmente, o sobrenadante de microglia tratada com CO promoveu um
aumento na complexidade morfológica neuronal. Este efeito neurotrófico gerado pela microglia tratada com
CO parece ser dependente de um aumento na secreção microglial de IL-10. Este capítulo indica que o CO tem
um papel anti-inflamatório celular não-autónomo, promovendo neuroprotecção através da regulação da
função microglial e comunicação unidirecional com os neurónios. Aqui, foi demonstrado pela primeira vez
que o CO estimula a função neurotrófica da microglia.
Um sistema de cultura de células em microfluídicas é usado no Capítulo IV para explorar o
interactoma directo neurónio-microglia no contexto neuroinflamatório, focando também na regulação da
fagocitose. Administração de CO inibiu a secreção de mediadores inflamatórios por parte da microglia e
limitou apoptose neuronal e atrofia morfológica. Sob condições inflamatórias, a remoção de conteúdo
sináptico por parte da microglia decresceu. Porém, o CO reverteu este efeito, indicando que esta molécula
melhora a fagocitose de sinapses sob condições de stress. Em conjunto com a poda sináptica, o CO também
regulou a fagocitose microglial de neurónios durante a inflamação. Na verdade, o CO teve um efeito de
controlo de qualidade na fagocitose de neurónios: bloqueou a remoção disfuncional de células viáveis, mas
promoveu a fagocitose de resíduos neuronais e material apoptótico. Este mecanismo aparentemente
homeostático, promovido pelo CO, não havia sido descrito e experiências estão a decorrer de modo a
entender as vias moleculares que o regulam.
Por último, a discussão final é apresentada no Capítulo V, integrando a informação de todos os
capítulos. Aqui, a inovação e impacto geral dos resultados são contextualizados tendo em conta a literatura
existente. Melhorias experimentais e próximos passos são discutidos.
Em suma, esta tese de Doutoramento oferece uma forte contribuição para o entendido de
mecanismos chave no funcionamento do sistema nervoso. Adicionalmente, contribui para a melhor
compreensão do CO como molécula citoprotectora e homeostática no SNC.
ABSTRACT: Neurological disorders refer to any pathology that affects the brain, spinal cord, and peripheral nerves. These include stroke, epilepsy, psychiatric disorders, dementia, neuroinfections and tumours. The World Health Organization (WHO) estimates that, in 2030, almost 8 billion people will die because of neurological complications. Moreover, it reveals that formal care costs related to neuropathology morbidities will continue skyrocketing in the future. The pathophysiology and progression of central nervous system (CNS) disorders are often multifactorial and poorly understood, which makes efficient treatment particularly difficult. There are several hallmark mechanisms of dysfunction: Uncontrolled neuroinflammation is a key driving force for damage propagation and homeostatic imbalances. Dysregulation of adult neurogenesis and loss of specific neuronal population are also neurological hallmarks of ageing and disease. Current day findings have helped to illuminate the underlying mechanisms of disease, but several questions remain unanswered. Therefore, there is an ongoing struggle to further disclose neurological disease aetiology and find new therapeutic alternatives in brain pathologies. Carbon monoxide (CO) is an endogenous gaseous molecule, which confers protection in different tissues, including the CNS. The main aim of this PhD thesis was to better understand the mechanisms of COinduced neuroprotection. The modulatory role of CO was addressed on two distinct processes: neurogenesis and neuroinflammation. Different models were employed. CO-releasing molecules (CORMs) were used to deliver CO in vitro. Chapter I introduces concepts regarding the brain, neuron, and glial cells. Additionally, state-of-the art knowledge about molecular pathways of neurodifferentiation, microglia function in immunity and homeostasis, as well as microglia-neuron communication, phagocytosis, and cell death. There is a great focus on the biological role of CO. In Chapter II, it is reported that CO enhances neuronal production yield in an SH-SY5Y neuroblastoma cell line differentiation model. The CO’s stimulatory effect on neuronal differentiation had previously been linked to modulation of cell death and improvement of mitochondrial metabolism in other models. In here, CO improved SH-SY5Y neuroblastoma cell line neurogenesis by acting on the Glucose-6-phosphate dehydrogenase (G6PD) and enhancing Pentose Phosphate Pathway (PPP) flux. The reported effect was independent of de novo Glutathione (GSH) synthesis. Knocking down the expression of the rate-limiting enzyme G6PD blocked the CO-induced improvement of SH-SY5Y differentiation. In summary, CO-driven metabolic reprogramming has a central role in cell fate and neuronal differentiation processes. Chapter III focuses on CO as a mediator of neuron-microglia remote communication. Neuronmicroglia conditioned media protocol revealed that CO provides cytoprotection to neurons exposed to microglia pro-inflammatory media. In fact, CO suppressed exacerbated microglia reactivity by limiting Tumour necrosis factor α (TNF-α) and nitric oxide (NO) secretion. Furthermore, the effect of CO on neuronmicroglia communication was also assessed in the absence of an inflammatory context. CO-treated microglia conditioned media partially protected neurons against cell death induced by oxidative stress. Moreover, supernatant from CO-treated microglia improved neuronal morphological complexity. This neurotrophic effect generated by CO-treated microglia might be dependent on microglia increased secretion of Interleukin-10 (IL-10). This chapter demonstrates that CO has an anti-inflammatory non-cell autonomous role, providing neuroprotection via regulation of microglia function and unidirectional communication with neurons. It was demonstrated for the first time that CO enhances microglial neurotrophic functions. After exploring CO’s mediation of remote communication (secretome) between neurons and microglia, a microfluidic cell system is used in Chapter IV, to explore neuron-microglia direct interactome during inflammation, while also focusing on the phagocytic process. CO’s administration inhibited microglia secretion of inflammatory mediators and limited neuronal apoptosis and morphological atrophy. Focusing on the direct cell contact, microglial clearance of synaptic material is decreased under inflammatory conditions, while CO reverted phagocytosis to control levels, indicating that CO improves microglial removal of synaptic content under stress conditions. Along with synaptic pruning, CO also regulates microglial phagocytosis in an inflammatory context. In fact, CO had a quality control effect on microglia engulfment of neuronal cells under inflammation by blocking removal of viable neurons but enhancing the phagocytosis of neuronal debris and apoptotic cells. This apparent homeostatic role of CO had never been described, and the underlying molecular pathways are under study. Lastly, a final discussion is presented in Chapter V, which integrates information from all chapters. The novelty and overall impact of the thesis generated data are contextualized in the scope of the existing literature. Experimental shortcomings and future steps are discussed. Altogether, this PhD thesis provides a strong contribution to comprehending key CNS mechanisms. Additionally, it contributes for the better understanding of CO as a potentially therapeutic cytoprotective and homeostatic molecule.
ABSTRACT: Neurological disorders refer to any pathology that affects the brain, spinal cord, and peripheral nerves. These include stroke, epilepsy, psychiatric disorders, dementia, neuroinfections and tumours. The World Health Organization (WHO) estimates that, in 2030, almost 8 billion people will die because of neurological complications. Moreover, it reveals that formal care costs related to neuropathology morbidities will continue skyrocketing in the future. The pathophysiology and progression of central nervous system (CNS) disorders are often multifactorial and poorly understood, which makes efficient treatment particularly difficult. There are several hallmark mechanisms of dysfunction: Uncontrolled neuroinflammation is a key driving force for damage propagation and homeostatic imbalances. Dysregulation of adult neurogenesis and loss of specific neuronal population are also neurological hallmarks of ageing and disease. Current day findings have helped to illuminate the underlying mechanisms of disease, but several questions remain unanswered. Therefore, there is an ongoing struggle to further disclose neurological disease aetiology and find new therapeutic alternatives in brain pathologies. Carbon monoxide (CO) is an endogenous gaseous molecule, which confers protection in different tissues, including the CNS. The main aim of this PhD thesis was to better understand the mechanisms of COinduced neuroprotection. The modulatory role of CO was addressed on two distinct processes: neurogenesis and neuroinflammation. Different models were employed. CO-releasing molecules (CORMs) were used to deliver CO in vitro. Chapter I introduces concepts regarding the brain, neuron, and glial cells. Additionally, state-of-the art knowledge about molecular pathways of neurodifferentiation, microglia function in immunity and homeostasis, as well as microglia-neuron communication, phagocytosis, and cell death. There is a great focus on the biological role of CO. In Chapter II, it is reported that CO enhances neuronal production yield in an SH-SY5Y neuroblastoma cell line differentiation model. The CO’s stimulatory effect on neuronal differentiation had previously been linked to modulation of cell death and improvement of mitochondrial metabolism in other models. In here, CO improved SH-SY5Y neuroblastoma cell line neurogenesis by acting on the Glucose-6-phosphate dehydrogenase (G6PD) and enhancing Pentose Phosphate Pathway (PPP) flux. The reported effect was independent of de novo Glutathione (GSH) synthesis. Knocking down the expression of the rate-limiting enzyme G6PD blocked the CO-induced improvement of SH-SY5Y differentiation. In summary, CO-driven metabolic reprogramming has a central role in cell fate and neuronal differentiation processes. Chapter III focuses on CO as a mediator of neuron-microglia remote communication. Neuronmicroglia conditioned media protocol revealed that CO provides cytoprotection to neurons exposed to microglia pro-inflammatory media. In fact, CO suppressed exacerbated microglia reactivity by limiting Tumour necrosis factor α (TNF-α) and nitric oxide (NO) secretion. Furthermore, the effect of CO on neuronmicroglia communication was also assessed in the absence of an inflammatory context. CO-treated microglia conditioned media partially protected neurons against cell death induced by oxidative stress. Moreover, supernatant from CO-treated microglia improved neuronal morphological complexity. This neurotrophic effect generated by CO-treated microglia might be dependent on microglia increased secretion of Interleukin-10 (IL-10). This chapter demonstrates that CO has an anti-inflammatory non-cell autonomous role, providing neuroprotection via regulation of microglia function and unidirectional communication with neurons. It was demonstrated for the first time that CO enhances microglial neurotrophic functions. After exploring CO’s mediation of remote communication (secretome) between neurons and microglia, a microfluidic cell system is used in Chapter IV, to explore neuron-microglia direct interactome during inflammation, while also focusing on the phagocytic process. CO’s administration inhibited microglia secretion of inflammatory mediators and limited neuronal apoptosis and morphological atrophy. Focusing on the direct cell contact, microglial clearance of synaptic material is decreased under inflammatory conditions, while CO reverted phagocytosis to control levels, indicating that CO improves microglial removal of synaptic content under stress conditions. Along with synaptic pruning, CO also regulates microglial phagocytosis in an inflammatory context. In fact, CO had a quality control effect on microglia engulfment of neuronal cells under inflammation by blocking removal of viable neurons but enhancing the phagocytosis of neuronal debris and apoptotic cells. This apparent homeostatic role of CO had never been described, and the underlying molecular pathways are under study. Lastly, a final discussion is presented in Chapter V, which integrates information from all chapters. The novelty and overall impact of the thesis generated data are contextualized in the scope of the existing literature. Experimental shortcomings and future steps are discussed. Altogether, this PhD thesis provides a strong contribution to comprehending key CNS mechanisms. Additionally, it contributes for the better understanding of CO as a potentially therapeutic cytoprotective and homeostatic molecule.
Descrição
Palavras-chave
Neurological disorders Neurogenesis Mechanisms of disease Neuroinflammation CORMs