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Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
Microglial activation induces nitric oxide signalling and alters protein S-nitrosylation patterns in extracellular
Natasha Vassileff1, Jereme G Spiers1,2,3, Sarah E Bamford4
1The Department of Biochemistry and Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.
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.
Insights
Neuroinflammation involves nitric oxide (NO) modifying proteins in extracellular vesicles (EVs) released by activated microglia. This study identified these S-nitrosylated proteins, revealing how microglia propagate neuroinflammation.
Area of Science:
- Neuroscience
- Immunology
- Biochemistry
Background:
- Neuroinflammation is a key factor in neurodegenerative diseases.
- Microglial activation by lipopolysaccharide (LPS) leads to nitric oxide (NO) production.
- NO can modify proteins via S-nitrosylation, affecting their function and intercellular signaling through extracellular vesicles (EVs).
Purpose of the Study:
- To investigate the S-nitrosylated proteome of extracellular vesicles (EVs) from activated microglia.
- To identify specific protein modifications induced by NO signaling in the context of neuroinflammation.
- To understand the role of NO-modified proteins in EVs in propagating neuroinflammatory signals.
Main Methods:
- Isolation of EVs from lipopolysaccharide (LPS)-treated microglia.
- Time-of-flight secondary ion mass spectrometry (ToF-SIMS) for surface imaging.
- Iodine labeling and comparative proteomic analysis to identify S-nitrosylated proteins.
Main Results:
- ToF-SIMS confirmed S-nitrosylation of cysteine thiols in microglial EVs.
- Proteomic analysis identified S-nitrosylated proteins within EVs from LPS-treated microglia.
- These proteins are associated with microglial activation and neuroinflammation, indicating their role in intercellular communication.
Conclusions:
- Activated microglia selectively package S-nitrosylated proteins into EVs.
- These EVs contribute to the propagation of neuroinflammation through NO signaling.
- The study provides insights into molecular mechanisms of neuroinflammation mediated by microglial EVs.
