Poster Presentation Australia and New Zealand Society for Extracellular Vesicles Conference 2023

Microglial activation induces nitric oxide signalling and alters protein nitrosylation patterns in extracellular vesicles (#60)

Natasha Vassileff 1 2 3 , Jereme G Spiers 1 2 3 , Sarah E Bamford 4 , Rohan G.T Lowe 5 , Keshava K Datta 5 , Paul Pigram 4 , Andrew F Hill 1 6
  1. Department of Biochemistry and Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia
  2. The Australian National University, Acton, ACT, Australia
  3. Clear Vision Research, Eccles Institute of Neuroscience, John Curtin School of Medical Research, College of Health and Medicine, The Australian National University, Acton, ACT, Australia
  4. Centre for Materials and Surface Science, Department of Mathematical and Physical Sciences, La Trobe University, Melbourne, Victoria, Australia
  5. Proteomics Metabolomics Platform, La Trobe University , Melborune, Victoria, Australia
  6. Institute for Health and Sport, Victoria University, Melbourne, Victoria, Australia

Neuroinflammation is a common feature of neurodegenerative conditions, often appearing very early in disease pathogenesis. Microglial activation is known to be a prominent initiator of neuroinflammation and can be induced through lipopolysaccharide treatment (LPS). This activation leads to the expression of the inducible form of nitric oxide synthase (iNOS), resulting in the production of nitric oxide (NO). NO targets cysteine thiols resulting post-translational S-nitrosylation, which can alter the target protein’s function. Furthermore, packaging of these NO-modified proteins into Extracellular Vesicles (EVs) further propagates the neuroinflammatory phenotype by allowing the exertion of NO signalling in distant locations. Despite this, the NO-modified proteome of activated microglial EVs has not been investigated. Therefore, this study aimed to identify the effect NO signalling exerts through protein post translational modifications in neuroinflammation. Extracellular Vesicles (EVs) isolated from LPS-treated microglia underwent novel advanced surface imaging using time of flight-secondary ion mass spectrometry (ToF-SIMS) in addition to, iodolabelling and comparative proteomic analysis to identify post-translation nitrosylation changes. ToF-SIMS imaging successfully identified NO modified cysteine thiol side chains in the EV proteins isolated from LPS treated microglia. Additionally, the iodolabelling proteomic analysis revealed the EVs from LPS treated microglia carried nitrosylated proteins indicative of neuroinflammation. These included known NO-modified proteins and those associated with LPS-induced microglial activation that may play an important role in neuroinflammatory communication. Together, these results show activated microglia are capable of exerting broad signalling changes through the selective packaging of EVs during neuroinflammation.