microRNA-146a modulates behavioural activity, neuroinflammation, and oxidative stress in adult mice

Small non-coding miRNA act as key regulators of several physiological processes due to their ability to interact with numerous target mRNA within a network. Whilst several miRNA can act in concert to regulate target mRNA expression, miR-146a has emerged as a critical modulator of inflammation by targeting key upstream signalling proteins of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and reductions in this miRNA have been observed in several neurological and neurodegenerative disorders. However, a targeted assessment of behaviour and neural tissues following the loss of miR-146a has not been documented. In this study, we examined the behavioural and neuroinflammatory phenotype of mice lacking miR-146a to determine the role of this miRNA in neurological function. Adult miR-146a−/− mice displayed no overt developmental phenotype with the exception of enlarged spleens. Behavioural testing revealed a mild but significant reduction in exploratory locomotor activity and increase in anxiety-like behaviour, with no changes in short-term spatial memory, fear conditioning, or sensorimotor gating. In the brain, the lack of miR-146a resulted in a significant compensatory miR-155 expression with no significant changes in expression of the target Interleukin 1 Receptor Associated Kinase (Irak) gene family. Despite these effects on upstream NF-κB mediators, downstream expression of cytokine and chemokine messengers was significantly elevated in miR-146a−/− mice compared to wild-type controls. Moreover, this increase in inflammatory cytokines was observed alongside an induction of oxidative stress, driven in part by nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, and included reduced thiol antioxidant concentrations and increased oxidised protein carbonyl concentrations. In female miR-146a mice, this increase in oxidative stress resulted in an increased expression of superoxide dismutase 1 (SOD1). Together, this suggests miR-146a plays a key role in regulating inflammation even in the absence of inflammatory stimuli and reduced levels of this miRNA have the capacity to induce limited behavioural effects whilst exacerbating both inflammation and oxidative stress in the brain

Neuroinflammatory modulation of extracellular vesicle biogenesis and cargo loading

Increasing evidence suggests neuroinflammation is a highly coordinated response involving multiple cell types and utilising several different forms of cellular communication. In addition to the well documented cytokine and chemokine messengers, extracellular vesicles (EVs) have emerged as key regulators of the inflammatory response. EVs act as vectors of intercellular communication, capable of travelling between different cells and tissues to deliver selectively packaged protein, miRNA, and lipids from the parent cell. During neuroinflammation, EVs transmit specific inflammatory mediators, particularly from microglia, to promote inflammatory resolution. This mini-review will highlight the novel neuroinflammatory mechanisms contributing to the biogenesis and selective packaging of EVs

Acute neuroinflammation promotes a metabolic shift that alters extracellular vesicle cargo in the mouse brain cortex

Abstract 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

High resolution imaging and analysis of extracellular vesicles using mass spectral imaging and machine learning

Abstract 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