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Investigating the role of innate immunity and inflammation in the nervous system
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Resumo: A inflamação tem sido progressivamente associada como um importante fator na progressão de inúmeras patologias, incluindo doenças do sistema nervoso. No sistema nervoso, a ativação crónica de resposta pro-inflamatórias, como a via mediada pelo fator de transcrição nuclear kappa-B (NF-κB), particularmente em células da glia, tem demonstrado capacidade de comprometer a função neuronal, perturbando a comunicação sináptica e induzindo morte celular, contribuindo, ao longo do tempo, para défices na funcionalidade motora e cognitiva. Apesar do papel central da inflamação na progressão deste tipo de patologias, os mecanismos de ativação de respostas inflamatórias, como os tipos de celulares com a capacidade de ativar diretamente a NF-κB, assim como o seu impacto em mecanismos que influenciam a morfologia neuronal e a organização dos circuitos, permanecem ainda pouco estudadas.
Neste trabalho, apresentamos um método in vivo inovador que utiliza a Drosophila para avaliar a ativação do NF-κB em distintos subtipos gliais do sistema nervoso periférico. A nossa abordagem baseou-se na utilização de sistemas com repórteres fluorescentes para monitorizar, em tempo real, a sinalização do NF-κB. Através deste sistema, verificámos que as células glia periféricas exibem diferentes perfis de resposta após exposição ao peptidoglicano (PGN), um péptido bacteriano reconhecido por vias pro-inflamatórias. Especificamente, dois subtipos gliais da periferia revelaram ter capacidade de desencadear respostas através da imunidade inata, enquanto que células do terceiro subtipo, maioritariamente, não demonstraram qualquer ativação da via NF-κB nas mesmas condições. Adicionalmente, avaliamos a presença do recetor canónico de reconhecimento do PGN, PGRP-LC, em subtipos celulares. Surpreendentemente, este recetor essencial para a deteção de PGN encontrava-se ausente em todos os subtipos gliais periféricos e nas células musculares. Para além disto, o modelo aqui descrito permitiu investigar o impacto da ativação continuada de vias pro-inflamatórias na morfologia neuronal dos terminais pre-sinápticos e no cérebro, oferecendo uma plataforma única para o estudo de processos neuroinflamatórios e sua ligação com doenças neurodegenerativas. Demonstrámos que a exposição contínua a agentes pró-inflamatórios induz alterações significativas no cordão nervoso central e no citoesqueleto dos terminais pré-sinápticos de neurónios motores, ao nível da junção neuromuscular (JNM). Adicionalmente, mostrámos que ao PGN desde a embriogénese resulta numa diminuição acentuada da proteína estabilizadora de microtúbulos, Futsch, na JNM. Além disso, a exposição prolongada ao PGN. Em síntese, este método constitui uma ferramenta útil para o estudo, em tempo real, dos mecanismos associados a respostas pro-inflamatórias agudas e crónicas, com elevado potencial para impulsionar o desenvolvimento e teste de estratégias terapêuticas direcionadas à mitigação da neurodegeneração associada à inflamação, preservando simultaneamente funções imunitárias essenciais.
Abstract: Inflammation has increasingly been implicated in the onset and progression of numerous neurological and systemic pathologies. In the nervous system, sustained activation of glial cells, particularly through nuclear factor kappa-B (NF-κB) signaling, has been shown to impair neuronal function, disrupt synaptic communication, and induce cell death, ultimately contributing to long-term neurological deficits and functional impairments. Despite its central role in neuroinflammation, the precise dynamics of glial NF-κB activation across different subcellular populations and its impact on neuronal morphology and circuitry remain poorly understood. Developing experimental strategies to study and modulate glial NF-κB activity in vivo is therefore critical for identifying potential therapeutic interventions that mitigate inflammation while preserving essential immune functions. Here, we present a novel in vivo method using Drosophila larvae to assess NF-κB activation across distinct glial subtypes in the peripheral nervous system. Our approach was based on the use of the genetic tractability of Drosophila and fluorescent reporter systems to monitor NF-κB signaling in real time. Using this system, we found that peripheral glia exhibit differential response profiles after exposure to peptidoglycan (PGN), a well-characterized bacterial peptide which is recognized by pro-inflammatory pathways. Specifically, subperineurial and wrapping glia were identified as immune-responsive, displaying robust NF-κB activation, whereas perineurial glia remained largely unresponsive under similar conditions. These findings highlight the functional and physiological heterogeneity of glial populations in their predisposition to sense inflammatory cues and mediating inflammatory signaling accordingly. Furthermore, we provided major insights in the presence of PGN recognition protein (PGRP), PGRP-LC, in glial subtypes and muscle cells. Surprisingly, this important PGN sensing receptor was absent in all peripheral glia subtypes and in muscle cells. Furthermore, our model enables systematic investigation of how prolonged or chronic activation of pro-inflammatory pathways impacts neuronal morphology, synaptic architecture, and functional output, offering a unique platform to study the progression of neuroinflammatory and neurodegenerative processes. By taking advantage of this method, we showed that continuous exposure to pro-inflammatory agents leads to major cytoskeleton alterations and bouton malformations in motor neurons, at the level of the neuromuscular junction (NMJ). Furthermore, we showed that when we exposed Drosophila larvae since embryogenesis to bacterial hazards (PGN), it lead to a significant decrease in the microtubule stabilizing protein, Futsch, at NMJ. Additionally, prolonged exposure to PGN resulted in striking morphological alterations in third-instar ventral nerve cord and opened a clear pathway to connect NF-κB sustained activation with neuronal death and with glial defects. Overall, this approach provides a powerful tool to study both acute and chronic neuroinflammatory responses in vivo and holds significant promise for advancing the development of therapeutic strategies aimed at mitigating inflammation-related neuronal dysfunction and promoting neuroprotection in diverse neurological disorders.
Abstract: Inflammation has increasingly been implicated in the onset and progression of numerous neurological and systemic pathologies. In the nervous system, sustained activation of glial cells, particularly through nuclear factor kappa-B (NF-κB) signaling, has been shown to impair neuronal function, disrupt synaptic communication, and induce cell death, ultimately contributing to long-term neurological deficits and functional impairments. Despite its central role in neuroinflammation, the precise dynamics of glial NF-κB activation across different subcellular populations and its impact on neuronal morphology and circuitry remain poorly understood. Developing experimental strategies to study and modulate glial NF-κB activity in vivo is therefore critical for identifying potential therapeutic interventions that mitigate inflammation while preserving essential immune functions. Here, we present a novel in vivo method using Drosophila larvae to assess NF-κB activation across distinct glial subtypes in the peripheral nervous system. Our approach was based on the use of the genetic tractability of Drosophila and fluorescent reporter systems to monitor NF-κB signaling in real time. Using this system, we found that peripheral glia exhibit differential response profiles after exposure to peptidoglycan (PGN), a well-characterized bacterial peptide which is recognized by pro-inflammatory pathways. Specifically, subperineurial and wrapping glia were identified as immune-responsive, displaying robust NF-κB activation, whereas perineurial glia remained largely unresponsive under similar conditions. These findings highlight the functional and physiological heterogeneity of glial populations in their predisposition to sense inflammatory cues and mediating inflammatory signaling accordingly. Furthermore, we provided major insights in the presence of PGN recognition protein (PGRP), PGRP-LC, in glial subtypes and muscle cells. Surprisingly, this important PGN sensing receptor was absent in all peripheral glia subtypes and in muscle cells. Furthermore, our model enables systematic investigation of how prolonged or chronic activation of pro-inflammatory pathways impacts neuronal morphology, synaptic architecture, and functional output, offering a unique platform to study the progression of neuroinflammatory and neurodegenerative processes. By taking advantage of this method, we showed that continuous exposure to pro-inflammatory agents leads to major cytoskeleton alterations and bouton malformations in motor neurons, at the level of the neuromuscular junction (NMJ). Furthermore, we showed that when we exposed Drosophila larvae since embryogenesis to bacterial hazards (PGN), it lead to a significant decrease in the microtubule stabilizing protein, Futsch, at NMJ. Additionally, prolonged exposure to PGN resulted in striking morphological alterations in third-instar ventral nerve cord and opened a clear pathway to connect NF-κB sustained activation with neuronal death and with glial defects. Overall, this approach provides a powerful tool to study both acute and chronic neuroinflammatory responses in vivo and holds significant promise for advancing the development of therapeutic strategies aimed at mitigating inflammation-related neuronal dysfunction and promoting neuroprotection in diverse neurological disorders.
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Palavras-chave
Innate Immunity Inflammation Nervous System