The Role of Intracellular Ca2+ and Mitochondrial ROS in Small Aß1-42 Oligomer-Induced Microglial Death.

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 (Aß) accumulation, and the link between the Aß1-42 structure and toxicity is of major interest. NMDA receptors (NMDAR) are thought to be essential in Aß-affected neurons, but the role of this receptor in glial impairment is still unclear. In addition, there is insufficient knowledge about the role of Aß species regarding mitochondrial redox states in neurons and glial cells, which may be critical in developing Aß-caused neurotoxicity. In this study, we investigated whether different Aß1-42 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 Aß1-42 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 Aß1-42 species or monomers. In addition, the small Aß1-42 oligomers caused mitochondrial reactive oxygen species (mtROS)-mediated mitochondrial depolarization, glutamate release, and neuronal cell death. In microglia, the Aß1-42-induced mtROS overproduction was mediated by intracellular calcium ions and Aß-binding alcohol dehydrogenase (ABAD). The data suggest that the pharmacological targeting of microglial NMDAR and mtROS may be a promising strategy for AD therapy.

Imeglimin Is Neuroprotective Against Ischemic Brain Injury in Rats-a Study Evaluating Neuroinflammation and Mitochondrial Functions.

Imeglimin is a novel oral antidiabetic drug modulating mitochondrial functions. However, neuroprotective effects of this drug have not been investigated. The aim of this study was to investigate effects of imeglimin against ischemia-induced brain damage and neurological deficits and whether it acted via inhibition of mitochondrial permeability transition pore (mPTP) and suppression of microglial activation. Ischemia in rats was induced by permanent middle cerebral artery occlusion (pMCAO) for 48 h. Imeglimin (135 µg/kg/day) was injected intraperitoneally immediately after pMCAO and repeated after 24 h. Immunohistochemical staining was used to evaluate total numbers of neurons, astrocytes, and microglia as well as interleukin-10 (IL-10) producing cells in brain slices. Respiration of isolated brain mitochondria was assessed using high-resolution respirometry. Assessment of ionomycin-induced mPTP opening in intact cultured primary rat neuronal, astrocytic, and microglial cells was performed using fluorescence microscopy. Treatment with imeglimin significantly decreased infarct size, brain edema, and neurological deficits after pMCAO. Moreover, imeglimin protected against pMCAO-induced neuronal loss as well as microglial proliferation and activation, and increased the number of astrocytes and the number of cells producing anti-inflammatory cytokine IL-10 in the ischemic hemisphere. Imeglimin in vitro acutely prevented mPTP opening in cultured neurons and astrocytes but not in microglial cells; however, treatment with imeglimin did not prevent ischemia-induced mitochondrial respiratory dysfunction after pMCAO. This study demonstrates that post-stroke treatment with imeglimin exerts neuroprotective effects by reducing infarct size and neuronal loss possibly via the resolution of neuroinflammation and partly via inhibition of mPTP opening in neurons and astrocytes.

Effects of Metformin on Spontaneous Ca2+ Signals in Cultured Microglia Cells under Normoxic and Hypoxic Conditions.

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.

Cerebrospinal fluids from Alzheimer's disease patients exhibit neurotoxic effects on neuronal cell cultures.

A growing number of studies suggest amyloid-ß and tau present in cerebrospinal fluid (CSF) and blood as putative biomarkers for Alzheimer's disease (AD). However, there is a question whether these compounds present in patients' bodily fluids can directly cause neurotoxic effects. We investigated effects of AD and other dementia (OD) patients' blood serum and CSF on viability of cells in primary cerebellar granule cell cultures. Overall, 59 individuals participated in the study from whom 55 samples of biological fluids were taken. Participants were classified into early (E-AD) and middle (M-AD) stages of AD, cognitively healthy control (HC) and OD groups. We found that concentrations of total and phosphorylated tau were higher in CSF from AD patients, while amyloid-ß42 and amyloid-ß40 in the serum was lower compared to HC. The most cytotoxic effects were induced by CSFs from M-AD patients which caused neuronal necrosis and suppressed microglial proliferation, whereas CSFs from the groups of other patients did not kill neurons. Serum and CSF from the E-AD group caused a reduction of neuronal numbers in cultures. There were no significant differences in levels of CSF biomarkers between the AD groups although both tau species in CSFs from M-AD patients were found to be significantly elevated compared to HC. Our data suggest that biological fluids from E-AD induce neuronal loss, whereas effects of CSF on the reduction in neuronal viability can serve as an indicator of M-AD and may be associated with extracellular tau.

Small Aß1-42 oligomer-induced membrane depolarization of neuronal and microglial cells: role of N-methyl-D-aspartate receptors.

Although it is well documented that soluble beta amyloid (Aß) oligomers are critical factors in the pathogenesis of Alzheimer's disease (AD) by causing synaptic dysfunction and neuronal death, the primary mechanisms by which Aß oligomers trigger neurodegeneration are not entirely understood. We sought to investigate whether toxic small Aß(1-42) oligomers induce changes in plasma membrane potential of cultured neurons and glial cells in rat cerebellar granule cell cultures leading to neuronal death and whether these effects are sensitive to the N-methyl-D-aspartate receptor (NMDA-R) antagonist MK801. We found that small Aß(1-42) oligomers induced rapid, protracted membrane depolarization of both neurons and microglia, whereas there was no change in membrane potential of astrocytes. MK801 did not modulate Aß-induced neuronal depolarization. In contrast, Aß1(-42) oligomer-induced decrease in plasma membrane potential of microglia was prevented by MK801. Small Aß(1-42) oligomers significantly elevated extracellular glutamate and caused neuronal necrosis, and both were prevented by MK801. Also, small Aß(1-42) oligomers decreased resistance of isolated brain mitochondria to calcium-induced opening of mitochondrial permeability transition pore. In conclusion, the results suggest that the primary effect of toxic small Aß oligomers on neurons is rapid, NMDA-R-independent plasma membrane depolarization, which leads to neuronal death. Aß oligomers-induced depolarization of microglial cells is NMDA-R dependent.

Antibodies bound to Aß oligomers potentiate the neurotoxicity of Aß by activating microglia.

Beta amyloid (Aß) oligomers are thought to contribute to the pathogenesis of Alzheimer's disease. However, clinical trials using Aß immunization were unsuccessful due to strong brain inflammation, the mechanisms of which are poorly understood. In this study we tested whether monoclonal antibodies to oligomeric Aß would prevent the neurotoxicity of Aß oligomers in primary neuronal-glial cultures. However, surprisingly,the antibodies dramatically increased the neurotoxicity of Aß. Antibodies bound to monomeric Aß fragments were non-toxic to cultured neurons, while antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein, human metapneumovirus nucleocapsid protein, and measles virus nucleocapsid protein, strongly potentiated the neurotoxicity of their antigens. The neurotoxicity of antibody-antibody oligomeric antigen complexes was abolished by removal of the Fc region from the antibodies or by removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. In conclusion, we find that immune complexes formed by Aß oligomers or other oligomeric/multimeric antigens and their specific antibodies can cause death and loss of neurons in primary neuronal-glial cultures via Fc-dependent microglial activation. The results suggest that therapies resulting in antibodies to oligomeric Aß or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation.