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Intra- versus extracellular effects of microglia-derived cysteine proteases in a conditioned medium transfer model
Wiebke Wendt1, Roxane Schulten, Christine C Stichel
1Department of Animal Physiology, Ruhr-University of Bochum, Bochum, Germany. wiebke.wendt@rub.de
Abstract:
Activated microglia release inflammatory mediators that display either beneficial or harmful effects on neuronal survival and signaling. In the present study we demonstrate that exposure to lipopolysaccharide leads to an increase in the lysosomal cysteine proteases, cathepsin B, K, S, and X, in culture supernatants of the microglia cell line BV-2. In addition, we observed an up-regulation of cathepsins in the cytoplasmic fraction in response to stimulation with lipopolysaccharide. Conditioned medium from these cells was toxic to the neuroblastoma cell line Neuro2a. Experiments with membrane-permeable and membrane-impermeable cysteine protease inhibitors suggested that blocking extracellular cathepsins had no effect on microglia-mediated neuron death in this medium transfer model. However, intracellular cathepsins seem to trigger the release of neurotoxic factors. In lipopolysaccharide-stimulated BV-2 cells, inhibition of intracellular cathepsins significantly diminished microglial activation characterized by reduced expression of different proinflammatory cytokines, thereby reducing the neurotoxic effects of the medium. This hitherto unknown intracellular effect of cysteine proteases in activated microglia might connect chronic neuroinflammation with neurodegeneration.
Insights
Lipopolysaccharide stimulation increases cysteine proteases in microglia. Inhibiting intracellular cathepsins reduces neuroinflammation and neurotoxicity, suggesting a link between neuroinflammation and neurodegeneration.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Activated microglia release mediators affecting neuronal survival.
- The role of specific microglial enzymes in neuroinflammation is not fully understood.
Purpose of the Study:
- To investigate the role of lysosomal cysteine proteases in lipopolysaccharide-activated microglia.
- To determine the impact of these proteases on neurotoxicity and neuroinflammation.
Main Methods:
- Utilized the BV-2 microglia cell line stimulated with lipopolysaccharide.
- Measured cathepsin levels in culture supernatants and cytoplasmic fractions.
- Assessed neurotoxicity using the Neuro2a cell line and conditioned medium.
- Employed cysteine protease inhibitors to differentiate extracellular and intracellular effects.
Main Results:
- Lipopolysaccharide increased cathepsins B, K, S, and X in microglia supernatants and cytoplasm.
- Conditioned medium from stimulated microglia was neurotoxic.
- Inhibiting extracellular cathepsins did not affect neurotoxicity.
- Inhibiting intracellular cathepsins reduced microglial activation, pro-inflammatory cytokine expression, and neurotoxicity.
Conclusions:
- Intracellular cysteine proteases in activated microglia contribute to neuroinflammation and neurotoxicity.
- This finding suggests a novel mechanism linking chronic neuroinflammation to neurodegeneration.
