Cytochrome c can be released into extracellular space and modulate functions of human astrocytes in a toll-like receptor 4-dependent manner

Background: Chronic activation of glial cells contributes to neurodegenerative diseases. Cytochrome c (CytC) is a soluble mitochondrial protein that can act as a damage-associated molecular pattern (DAMP) when released into the extracellular space from damaged cells. CytC causes immune activation of microglia in a toll-like receptor (TLR) 4-dependent manner. The effects of extracellular CytC on astrocytes are unknown. Astrocytes, which are the most abundant glial cell type in the brain, express TLR 4 and secrete inflammatory mediators; therefore, we hypothesized that extracellular CytC can interact with the TLR 4 of astrocytes inducing their release of inflammatory molecules and cytotoxins. Method: Experiments were conducted using primary human astrocytes, U118 MG human astrocytic cells, BV-2 murine microglia, and SH-SY5Y human neuronal cells. Results: Extracellularly applied CytC increased the secretion of interleukin (IL)-1 beta, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-12 p70 by cultured primary human astrocytes. Anti-TLR 4 antibodies blocked the CytC-induced secretion of IL-1 beta and GM-CSF by astrocytes. Supernatants from CytC-activated astrocytes were toxic to human SH-SY5Y neuronal cells. We also demonstrated CytC release from damaged glial cells by measuring CytC in the supernatants of BV-2 microglia after their exposure to cytotoxic concentrations of staurosporine, amyloid-beta peptides (A beta 42) and tumor necrosis factor-alpha. Conclusion: CytC can be released into the extracellular space from damaged glial cells causing immune activation of astrocytes in a TLR 4-dependent manner. General significance: Astrocyte activation by CytC may contribute to neuroinflammation and neuronal death in neurodegenerative diseases. Astrocyte TLR 4 could be a potential therapeutic target in these diseases.