Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jun;13(6):e12455.
doi: 10.1002/jev2.12455.

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

Natasha Vassileff  1 , Jereme G Spiers  1   2   3 , Sarah E Bamford  4 , Rohan G T Lowe  5 , Keshava K Datta  5 , Paul J Pigram  4 , Andrew F Hill  1   6
Affiliations

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

Natasha Vassileff et al. J Extracell Vesicles. 2024 Jun.

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.

Keywords: Microglia; Neuroinflammation; Nitric Oxide; Nitrosylation; Post‐translational modification; Proteomics; ToF‐SIMS; extracellular vesicles; mass Spectral Imaging.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Characterisation of the LPS treatment performed on SIM‐A9 microglial cells. 1 µg/µL of lipopolysaccharide (LPS) treatment elicited a strong inflammatory response in treated microglial cells. (a) Cells treated with LPS exhibited a significant increase in their gene expression of cytokines: Il6, Il1β, Tnf, and Ccl2, in addition to Nfkbia and the inducible form of nitric oxide synthase (Nos2) compared to untreated control cells. (b) NO metabolites (nitrate and nitrite) levels were also found to be significantly increased in cells treated with LPS compared to controls, confirming the presence of a neuroinflammatory phenotype. Data was compared using unpaired t‐test and expressed as mean ± SEM, *p < 0.05.
FIGURE 2
FIGURE 2
Characterisation of extracellular vesicles (EVs) from microglia. EVs isolated from microglial cells with/without LPS treatment appear to exhibit characteristics consistent with that of small EVs. (a) The isolated vesicles were positive for small EV enriched markers tsg101, flotillin, and actin, and negative for small EV non‐enriched markers; calnexin and ApoB. (b) Nanoparticle tracking analysis, performed on the ZetaView© Quatt PMX‐420, demonstrates the vesicles appear to be between 100 and 150 nm in diameter, consistent with small EVs. This result is representative of n = 6. (c) Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images exhibit a population of vesicles with depressed cup‐like structures and a diameter of 100–200 nm, consistent with that of small EVs. These images are of a representative sample.
FIGURE 3
FIGURE 3
Physiological target analysis from ToF‐SIMS imaging data. (a) Nitrotyrosine(C6H4NO3 −), Cysteine (C3H7SNO2 −) and sulphur‐bound hydrogen thiol (HS−) imaging data were pseudo‐coloured green. Each image was overlaid with NO2 − (red), and colocalised pixels were recoloured white. After background removal co‐located pixels were re‐coloured yellow. All mass spectral images were captured using Fast–Imaging mode. Each image has a 20 µm × 20 µm field of view. (b) The NO2 peak collected in Spectrometry mode correlated with the 3‐nitrotyrosine levels which were found to be significantly elevated in EVs isolated from LPS‐treated microglial cells compared to controls. Data were compared using unpaired t‐test and expressed as mean ± SEM, *p < 0.05.
FIGURE 4
FIGURE 4
Significant proteins in the total proteome and those with altered S‐nitrosylation in the LPS treated microglial EVs compared to controls. (a) Fifteen proteins were found to be significantly differentially expressed in the LPS treated microglial EVs compared to the control microglial EVs with many being key players in the inflammatory response. (b) The detection of proteins with altered S‐ntirosylation encompasses those with either increased or decreased S‐nitrosylation when compared to their total proteome.
FIGURE 5
FIGURE 5
Gene ontology (GO) analysis and search tool for the retrieval of interacting genes/proteins (STRING) analysis of proteins with altered S‐nitrosylation in the LPS treated microglial EVs compared to controls. (a) GO analysis based on fold enrichment revealed the proteins exhibit molecular functions including complement and interleukin receptor activity; cellular component locations including integrin complexes; biological processes including regulation of post‐synaptic density; protein domains including enolase and integrins; and reactome analysis uncovered the proteins to be involved in the inflammatory responses including those associated with LPS. (b) The majority of proteins with either increased or decreased S‐nitrosylation when compared to their total proteome, in the treated microglial EVs, appear to exhibit network relationships/connectivity. The graph was created using STRING.
FIGURE 6
FIGURE 6
Neuroinflammatory signalling alters microglia through S‐nitrosylation which is represented in their released extracellular vesicles (EVs). A (Left) Lipopolysaccharide (LPS) binds to Toll like receptor 4 (TLR4) leading to a cascade of signals culminating in nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) activation and expression of pro‐inflammatory cytokines and chemokines in addition to inducible nitric oxide synthase (iNOS) which generates nitric oxide (NO), a molecule that leads to nitrothiol modifications on proteins in a process known as S‐nitrosylation. These modified proteins (Right) are then packaged into EVs which when taken up by recipient cells are capable of potentiating the neuroinflammatory signal. B Upon LPS stimulation, both glycolytic proteins; α‐enolase and pyruvate kinase are nitrosylated leading to altered glycolysis in the microglia, a process that is required for microglia to maintain their activated state. These proteins undergo increased nitrosylation in the cells where they appear to be retained. C LPS treatment leads to nitrosylation of both Cathepsin B and the 26S proteasomal subunits resulting in impairment of the autophagy and proteasomal degradation pathways. The increased EV packaging of these nitrosylated proteins may be an attempt by the cells to restore protein degradation pathways through removal of these impaired proteins. D Increased nitrosylation of Cofilin‐1, as a result of LPS stimulation, enables the protein to sever actin filaments releasing actin, which also undergoes nitrosylation, for cytoskeletal remodelling. This allows microglia to undergo the structural changes required to achieve their activated state. The nitrosylated form of integrin α‐5 (ITGA5), a protein known to interact with actin and undergo nitrosylation, was found to have reduced EV packaging implying it may be retained in the cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Bamford, S. E. , Vassileff, N. , Spiers, J. G. , Gardner, W. , Winkler, D. A. , Muir, B. W. , Hill, A. F. , & Pigram, P. J. (2023). High resolution imaging and analysis of extracellular vesicles using mass spectral imaging and machine learning. Journal of Extracellular Biology, 2, e110.
    1. Bell‐Temin, H. , Culver‐Cochran, A. E. , Chaput, D. , Carlson, C. M. , Kuehl, M. , Burkhardt, B. R. , Bickford, P. C. , Liu, B. , & Stevens, S. M. Jr. (2015). Novel molecular insights into classical and alternative activation states of microglia as revealed by stable isotope labeling by amino acids in cell culture (SILAC)‐based proteomics. Molecular & Cellular Proteomics, 14, 3173–3184. - PMC - PubMed
    1. Bochmann, I. , Ebstein, F. , Lehmann, A. , Wohlschlaeger, J. , Sixt, S. U. , Kloetzel, P. M. , & Dahlmann, B. (2014). T lymphocytes export proteasomes by way of microparticles: A possible mechanism for generation of extracellular proteasomes. Journal of Cellular and Molecular Medicine, 18, 59–68. - PMC - PubMed
    1. Bolte, S. , & CordeliÈRes, F. P. (2006). A guided tour into subcellular colocalization analysis in light microscopy. Journal of Microscopy, 224, 213–232. - PubMed
    1. Bourgognon, J. M. , Spiers, J. G. , Robinson, S. W. , Scheiblich, H. , Glynn, P. , Ortori, C. , Bradley, S. J. , Tobin, A. B. , & Steinert, J. R. (2021). Inhibition of neuroinflammatory nitric oxide signaling suppresses glycation and prevents neuronal dysfunction in mouse prion disease. PNAS, 118, e2009579118. - PMC - PubMed

LinkOut - more resources

-