Microglial activation induces nitric oxide signalling and alters protein S‐nitrosylation patterns in extracellular vesicles

Abstract 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.