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Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
Neuromelanin activates proinflammatory microglia through a caspase-8-dependent mechanism
Nikenza Viceconte1,2,3, Miguel A Burguillos4, Antonio J Herrera5,6
1Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Sevilla, 41012, Sevilla, Spain. nikenza.viceconte@yahoo.it.
Background:
We have uncovered a caspase-dependent (caspase-8/caspase-3/7) signaling governing microglia activation and associated neurotoxicity. Importantly, a profuse non-nuclear activation of cleaved caspases 8 and 3 was found in reactive microglia in the ventral mesencephalon from subjects with Parkinson's disease, thus supporting the existence of endogenous factors activating microglia through a caspase-dependent mechanism. One obvious candidate is neuromelanin, which is an efficient proinflammogen in vivo and in vitro and has been shown to have a role in the pathogenesis of Parkinson's disease. Consequently, the goal of this study is to test whether synthetic neuromelanin activates microglia in a caspase-dependent manner.
Results:
We found an in-vivo upregulation of CD16/32 (M1 marker) in Iba1-immunolabeled microglia in the ventral mesencephalon after neuromelanin injection. In vitro experiments using BV2 cells, a microglia-derived cell line, demonstrated that synthetic neuromelanin induced a significant chemotactic response to BV2 microglial cells, along with typical morphological features of microglia activation, increased oxidative stress and induction of pattern-recognition receptors including Toll-like receptor 2, NOD2, and CD14. Analysis of IETDase (caspase-8) and DEVDase (caspase-3/7) activities in BV2 cells demonstrated a modest but significant increase of both activities in response to neuromelanin treatment, in the absence of cell death.
Conclusions:
Caspase-8 inhibition prevented typical features of microglia activation, including morphological changes, a high rate of oxidative stress and expression of key proinflammatory cytokines and iNOS.
Insights
Neuromelanin activates microglia, a key immune cell in the brain, through a caspase-dependent pathway. This finding sheds light on Parkinson's disease pathogenesis and potential therapeutic targets.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia activation and neurotoxicity are governed by caspase-dependent signaling (caspase-8/caspase-3/7).
- Reactive microglia in Parkinson's disease (PD) exhibit non-nuclear activation of cleaved caspases 8 and 3.
- Neuromelanin, implicated in PD pathogenesis, is a potential endogenous activator of microglia.
Purpose of the Study:
- To investigate whether synthetic neuromelanin activates microglia in a caspase-dependent manner.
Main Methods:
- In vivo studies involved neuromelanin injection and analysis of microglia markers (Iba1, CD16/32) in the ventral mesencephalon.
- In vitro studies used BV2 cells to assess microglia chemotaxis, morphology, oxidative stress, and pattern-recognition receptor expression.
- Caspase-8 (IETDase) and caspase-3/7 (DEVDase) activities were measured in response to neuromelanin.
Main Results:
- Neuromelanin injection upregulated CD16/32 (M1 marker) on microglia in vivo.
- In vitro, synthetic neuromelanin induced microglia chemotaxis, morphological changes, oxidative stress, and upregulated Toll-like receptor 2, NOD2, and CD14.
- Neuromelanin treatment significantly increased caspase-8 and caspase-3/7 activities in BV2 cells without causing cell death.
Conclusions:
- Caspase-8 activation is crucial for neuromelanin-induced microglia activation.
- Inhibition of caspase-8 prevented microglia morphological changes, oxidative stress, and expression of proinflammatory cytokines and iNOS.
