S100 calcium-binding protein A9 (S100A9, also known as calgranulin B) is expressed and secreted by myeloid cells under inflammatory conditions, and S100A9 can amplify inflammation. There is a large increase in S100A9 expression in the brains of patients with neurodegenerative diseases, such as Alzheimer's disease, and S100A9 has been suggested to contribute to neurodegeneration, but the mechanisms are unclear. Here we investigated the effects of extracellular recombinant S100A9 protein on microglia, neurons and synapses in primary rat brain neuronal-glial cell cultures. Incubation of cell cultures with 250-500 nM S100A9 caused neuronal loss without signs of apoptosis or necrosis, but accompanied by exposure of the "eat-me" signal - phosphatidylserine on neurons. S100A9 caused activation of microglial inflammation as evidenced by an increase in the microglial number, morphological changes, release of pro-inflammatory cytokines, and increased phagocytic activity. At lower concentrations, 10-100 nM S100A9 induced synaptic loss in the cultures. Depletion of microglia from the cultures prevented S100A9-induced neuronal and synaptic loss, indicating that neuronal and synaptic loss was mediated by microglia. These results suggest that extracellular S100A9 may contribute to neurodegeneration by activating microglial inflammation and phagocytosis, resulting in loss of synapses and neurons. This further suggests the possibility that neurodegeneration may be reduced by targeting S100A9 or microglia.

Alzheimer’s disease (AD) is the most common form of dementia worldwide, and it contributes up to 70% of cases. AD pathology involves abnormal amyloid beta (Aβ) accumulation, and the link between the Aβ_1-42 structure and toxicity is of major interest. NMDA receptors (NMDAR) are thought to be essential in Aβ-affected neurons, but the role of this receptor in glial impairment is still unclear. In addition, there is insufficient knowledge about the role of Aβ species regarding mitochondrial redox states in neurons and glial cells, which may be critical in developing Aβ-caused neurotoxicity. In this study, we investigated whether different Aβ_1-42 species—small oligomers, large oligomers, insoluble fibrils, and monomers—were capable of producing neurotoxic effects via microglial NMDAR activation and changes in mitochondrial redox states in primary rat brain cell cultures. Small Aβ_1-42 oligomers induced a concentration- and time-dependent increase in intracellular Ca²⁺ and necrotic microglial death. These changes were partially prevented by the NMDAR inhibitors MK801, memantine, and D-2-amino-5-phosphopentanoic acid (DAP5). Neither microglial intracellular Ca²⁺ nor viability was significantly affected by larger Aβ_1-42 species or monomers. In addition, the small Aβ_1-42 oligomers caused mitochondrial reactive oxygen species (mtROS)-mediated mitochondrial depolarization, glutamate release, and neuronal cell death. In microglia, the Aβ_1-42-induced mtROS overproduction was mediated by intracellular calcium ions and Aβ-binding alcohol dehydrogenase (ABAD). The data suggest that the pharmacological targeting of microglial NMDAR and mtROS may be a promising strategy for AD therapy.

Microglial functioning depends on Ca²⁺ signaling. By using Ca²⁺ sensitive fluorescence dye, we studied how inhibition of mitochondrial respiration changed spontaneous Ca²⁺ signals in soma of microglial cells from 5–7-day-old rats grown under normoxic and mild-hypoxic conditions. In microglia under normoxic conditions, metformin or rotenone elevated the rate and the amplitude of Ca²⁺ signals 10–15 min after drug application. Addition of cyclosporin A, a blocker of mitochondrial permeability transition pore (mPTP), antioxidant trolox, or inositol 1,4,5-trisphosphate receptor (IP3R) blocker caffeine in the presence of rotenone reduced the elevated rate and the amplitude of the signals implying sensitivity to reactive oxygen species (ROS), and involvement of mitochondrial mPTP together with IP3R. Microglial cells exposed to mild hypoxic conditions for 24 h showed elevated rate and increased amplitude of Ca²⁺ signals. Application of metformin or rotenone but not phenformin before mild hypoxia reduced this elevated rate. Thus, metformin and rotenone had the opposing fast action in normoxia after 10–15 min and the slow action during 24 h mild-hypoxia implying activation of different signaling pathways. The slow action of metformin through inhibition of complex I could stabilize Ca²⁺ homeostasis after mild hypoxia and could be important for reduction of ischemia-induced microglial activation.