The link between the size of soluble amyloid β (Aβ) oligomers and their toxicity to rat cerebellar granule cells (CGC) was investigated. Variation in conditions during in vitro oligomerization of Aβ 1-42 resulted in peptide assemblies with different particle size as measured by atomic force microscopy and confirmed by the dynamic light scattering and fluorescence correlation spectroscopy. Small oligomers of Aβ 1-42 with a mean particle z-height of 1-2 nm exhibited propensity to bind to the phospholipid vesicles and they were the most toxic species that induced rapid neuronal necrosis at submicromolar concentrations whereas the bigger aggregates (z-height above 4-5 nm) did not bind vesicles and did not cause detectable neuronal death. Similar neurotoxic pattern was also observed in primary cultures of cortex neurons whereas Aβ 1-42 oligomers, monomers and fibrils were nontoxic to glial cells in CGC cultures or macrophage J774 cells. However, both oligomeric forms of Aβ 1-42 induced reduction of neuronal cell densities in the CGC cultures.

Beta amyloid (Ab) oligomers are thought to contribute to the pathogenesis of Alzheimer's disease. However, clinical trials using Ab 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 Ab would prevent the neurotoxicity of Ab oligomers in primary neuronal-glial cultures. However, surprisingly, the antibodies dramatically increased the neurotoxicity of Ab. Antibodies bound to monomeric Ab 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 antibodyoligomeric 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 Ab 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 Ab or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation.

Damage to cerebral mitochondria, particularly opening of mitochondrial permeability transition pore (MPTP), is a key mechanism of ischemic brain injury, therefore, modulation of MPTP may be a potential target for a neuroprotective strategy in ischemic brain pathologies. The aim of this study was to investigate whether biguanides—metformin and phenformin as well as other inhibitors of Complex I of the mitochondrial electron transfer system may protect against ischemia-induced cell death in brain slice cultures by suppressing MPTP, and whether the effects of these inhibitors depend on the age of animals. Experiments were performed on brain slice cultures prepared from 5–7-day (premature) and 2–3-month old (adult) rat brains. In premature brain slice cultures, simulated ischemia (hypoxia plus deoxyglucose) induced necrosis whereas in adult rat brain slice cultures necrosis was induced by hypoxia alone and was suppressed by deoxyglucose. Phenformin prevented necrosis induced by simulated ischemia in premature and hypoxia-induced—in adult brain slices, whereas metformin was protective in adult brain slices cultures. In premature brain slices, necrosis was also prevented by Complex I inhibitors rotenone and amobarbital and by MPTP inhibitor cyclosporine A. The latter two inhibitors were protective in adult brain slices as well. Short-term exposure of cultured neurons to phenformin, metformin and rotenone prevented ionomycin-induced MPTP opening in intact cells. The data suggest that, depending on the age, phenformin and metformin may protect the brain against ischemic damage possibly by suppressing MPTP via inhibition of mitochondrial Complex I.

Although it is well documented that soluble beta amyloid (Aβ) oligomers are critical factors in the pathogenesis of Alzheimer's disease (AD) by causing synaptic dysfunction and neuronal death, the primary mechanisms by which Aβ oligomers trigger neurodegeneration are not entirely understood. We sought to investigate whether toxic small Aβ(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 Aβ(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 Aβ-induced neuronal depolarization. In contrast, Aβ1(-42) oligomer-induced decrease in plasma membrane potential of microglia was prevented by MK801. Small Aβ(1-42) oligomers significantly elevated extracellular glutamate and caused neuronal necrosis, and both were prevented by MK801. Also, small Aβ(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 Aβ oligomers on neurons is rapid, NMDA-R-independent plasma membrane depolarization, which leads to neuronal death. Aβ oligomers-induced depolarization of microglial cells is NMDA-R dependent.

BackgroundThe aim of this study is to evaluate the role of NADPH oxidase in primary intestinal epithelial cells during the active phase of UC.MethodsThe primary human colonic epithelial cells were isolated from 19 patients with mild to moderate inflammatory activity of UC and 14 controls using chelation method. The cells were cultivated under the effect of mediators. Viability of cells was assessed by fluorescent microscopy. Production of reactive oxygen species (ROS) by the cells was measured fluorimetrically using Amplex Red. Production of TNF-α cytokine by the colonic epithelial cells was analysed by ELISA.ResultsThe results of our study showed that unstimulated cells of UC patients had a decreased viability, increased ROS production, but similar TNF-α level when compared to the controls. Stimulation with LPS increased hydrogen peroxide and TNF-α level in the UC group. Treatment of colonic epithelial cells with NADPH oxidase inhibitor increased cell viability decreased the levels of ROS and TNF-α in the LPS-treated cells isolated from UC patients.ConclusionsOur study showed that bacterial endotoxins induced NADPH oxidase activation in the colonic epithelial cells. Moreover, we revealed that treatment with NADPH oxidase inhibitors had a protective effect against pro-inflammatory action of LPS in human colonic epithelium cells during inflammation.