RESUMEN
Neuroinflammation is initiated through microglial activation and cytokine release which can be induced through lipopolysaccharide treatment (LPS) leading to a transcriptional cascade culminating in the differential expression of target proteins. These differentially expressed proteins can then be packaged into extracellular vesicles (EVs), a form of cellular communication, further propagating the neuroinflammatory response over long distances. Despite this, the EV proteome in the brain, following LPS treatment, has not been investigated. Brain tissue and brain derived EVs (BDEVs) isolated from the cortex of LPS-treated mice underwent thorough characterisation to meet the minimal information for studies of extracellular vesicles guidelines before undergoing mass spectrometry analysis to identify the differentially expressed proteins. Fourteen differentially expressed proteins were identified in the LPS brain tissue samples compared to the controls and 57 were identified in the BDEVs isolated from the LPS treated mice compared to the controls. This included proteins associated with the initiation of the inflammatory response, epigenetic regulation, and metabolism. These results allude to a potential link between small EV cargo and early inflammatory signalling.
RESUMEN
Background: Accumulating evidence underscores the pivotal role of heightened inflammation in the pathophysiology of stress-related diseases, but the underlying mechanisms remain elusive. The complement system, a key effector of the innate immune system, produces the C5-cleaved activation product C5a upon activation, initiating inflammatory responses through the canonical C5a receptor 1 (C5aR1). While C5aR1 is expressed in stress-responsive brain regions, its role in stress responsiveness remains unknown. Methods: To investigate C5a-C5aR1 signaling in stress responses, mice underwent acute and chronic stress paradigms. Circulating C5a levels and messenger RNA expression of C5aR1 in the hippocampus and adrenal gland were measured. C5aR1-deficient mice were used to elucidate the effects of disrupted C5a-C5aR1 signaling across behavioral, hormonal, metabolic, and inflammation parameters. Results: Chronic restraint stress elevated circulating C5a levels while reducing C5aR1 messenger RNA expression in the hippocampus and adrenal gland. Notably, the absence of C5aR1 signaling enhanced adrenal sensitivity to adrenocorticotropic hormone, concurrently reducing pituitary adrenocorticotropic hormone production and enhancing the response to acute stress. C5aR1-deficient mice exhibited attenuated reductions in locomotor activity and body weight under chronic stress. Additionally, these mice displayed increased glucocorticoid receptor sensitivity and disrupted glucose and insulin homeostasis. Chronic stress induced an increase in C5aR1-expressing microglia in the hippocampus, a response mitigated in C5aR1-deficient mice. Conclusions: C5a-C5aR1 signaling emerges as a key metabolic regulator during stress, suggesting that complement activation and dysfunctional C5aR1 signaling may contribute to neuroinflammatory phenotypes in stress-related disorders. The results advocate for further exploration of complement C5aR1 as a potential therapeutic target for stress-related conditions.
How the immune system, particularly the complement system, influences responses to stress has not been fully clear. In this study, we focus on C5a-C5aR1 signaling, a part of the immune system, and found that it significantly affects stress-related reactions in mice. In chronic stress, we observed increased inflammation, altered hormonal responses, and disrupted metabolic regulation. Mice lacking C5aR1 showed reduced stress-induced behavioral changes, indicating that this receptor may play a vital role in modulating the stress response. Understanding these immune mechanisms sheds light on stress-related disorders and may open avenues for therapeutic interventions.
RESUMEN
Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease characterised by the deposition of aggregated proteins including TAR DNA-binding protein 43 (TDP-43) in vulnerable motor neurons and the brain. Extracellular vesicles (EVs) facilitate the spread of neurodegenerative diseases and can be easily accessed in the bloodstream. This study aimed to identify a panel of EV miRNAs that can capture the pathology occurring in the brain and peripheral circulation. EVs were isolated from the cortex (BDEVs) and serum (serum EVs) of 3 month-old and 6-month-old TDP-43*Q331K and TDP-43*WT mice. Following characterisation and miRNA isolation, the EVs underwent next-generation sequencing where 24 differentially packaged miRNAs were identified in the TDP-43*Q331K BDEVs and 7 in the TDP-43*Q331K serum EVs. Several miRNAs, including miR-183-5p, were linked to ALS. Additionally, miR-122-5p and miR-486b-5p were identified in both panels, demonstrating the ability of the serum EVs to capture the dysregulation occurring in the brain. This is the first study to identify miRNAs common to both the serum EVs and BDEVs in a mouse model of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Biomarcadores , Encéfalo , Modelos Animales de Enfermedad , Vesículas Extracelulares , MicroARNs , Animales , Esclerosis Amiotrófica Lateral/sangre , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/metabolismo , Vesículas Extracelulares/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , MicroARNs/sangre , Biomarcadores/sangre , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Ratones , Ratones Transgénicos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Masculino , Ratones Endogámicos C57BLRESUMEN
Neuroinflammation is an underlying feature of neurodegenerative conditions, often appearing early in the aetiology of a disease. Microglial activation, a prominent initiator of neuroinflammation, can be induced through lipopolysaccharide (LPS) treatment resulting in expression of the inducible form of nitric oxide synthase (iNOS), which produces nitric oxide (NO). NO post-translationally modifies cysteine thiols through S-nitrosylation, which can alter function of the target protein. Furthermore, packaging of these NO-modified proteins into extracellular vesicles (EVs) allows for the exertion of NO signalling in distant locations, resulting in further propagation of the neuroinflammatory phenotype. Despite this, the NO-modified proteome of activated microglial EVs has not been investigated. This study aimed to identify the protein post-translational modifications NO signalling induces in neuroinflammation. EVs isolated from LPS-treated microglia underwent mass spectral surface imaging using time of flight-secondary ion mass spectrometry (ToF-SIMS), in addition to iodolabelling and comparative proteomic analysis to identify post-translation S-nitrosylation modifications. ToF-SIMS imaging successfully identified cysteine thiol side chains modified through NO signalling in the LPS treated microglial-derived EV proteins. In addition, the iodolabelling proteomic analysis revealed that the EVs from LPS-treated microglia carried S-nitrosylated proteins indicative of neuroinflammation. These included known NO-modified proteins and those associated with LPS-induced microglial activation that may play an essential role in neuroinflammatory communication. Together, these results show activated microglia can exert broad NO signalling changes through the selective packaging of EVs during neuroinflammation.
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Vesículas Extracelulares , Lipopolisacáridos , Microglía , Óxido Nítrico , Transducción de Señal , Microglía/metabolismo , Vesículas Extracelulares/metabolismo , Óxido Nítrico/metabolismo , Animales , Lipopolisacáridos/farmacología , Ratones , Proteómica/métodos , Procesamiento Proteico-Postraduccional , Cisteína/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismoRESUMEN
Extracellular vesicles (EVs) are potentially useful biomarkers for disease detection and monitoring. Development of a label-free technique for imaging and distinguishing small volumes of EVs from different cell types and cell states would be of great value. Here, we have designed a method to explore the chemical changes in EVs associated with neuroinflammation using Time-of-Flight Secondary Ion Mass spectrometry (ToF-SIMS) and machine learning (ML). Mass spectral imaging was able to identify and differentiate EVs released by microglia following lipopolysaccharide (LPS) stimulation compared to a control group. This process requires a much smaller sample size (1 µL) than other molecular analysis methods (up to 50 µL). Conspicuously, we saw a reduction in free cysteine thiols (a marker of cellular oxidative stress associated with neuroinflammation) in EVs from microglial cells treated with LPS, consistent with the reduced cellular free thiol levels measured experimentally. This validates the synergistic combination of ToF-SIMS and ML as a sensitive and valuable technique for collecting and analysing molecular data from EVs at high resolution.