In both cases, left panels illustrate the dot blot images and right panels the Western blot detection Validation of Nrf2 nuclear translocation and concomitant increase in the expression of the antioxidant proteins SOD1, and HO-1 was performed by immunocytochemistry in SH-SY5Y cells incubated with MTZ, MEL, and Trolox as well as with the electrophilic compound SFN, in the presence/absence of PI3K and GSK-3 inhibitors

In both cases, left panels illustrate the dot blot images and right panels the Western blot detection Validation of Nrf2 nuclear translocation and concomitant increase in the expression of the antioxidant proteins SOD1, and HO-1 was performed by immunocytochemistry in SH-SY5Y cells incubated with MTZ, MEL, and Trolox as well as with the electrophilic compound SFN, in the presence/absence of PI3K and GSK-3 inhibitors. nuclear fluorescence signal from images depicted in Physique ?Physique13.13. Fluorescence of at least 400 cells was quantitated utilizing ImageJ software and expressed in fold of control cells. Data is usually represented as mean SD; **** indicates em p /em 0.0001. 13195_2019_578_MOESM2_ESM.pdf (95K) GUID:?2AFC327E-3B52-4CB8-B98F-0F2E86BCBED7 Additional file 3: Physique S3. Quantitation of cytoplasmic SOD-1 and HO-1 fluorescence induced by Methazolamide, Melatonin, and Trolox in the presence of PI3K- and GSK-3 inhibitors. Graphs in Panels A and B depict the quantitation of the cytoplasmic SOD-1 and HO-1fluorescence transmission, respectively, of at least 400 cells utilizing ImageJ software and expressed in fold of control. Data is usually represented as mean SD; **** indicates em p /em 0.0001. 13195_2019_578_MOESM3_ESM.pdf (127K) GUID:?1463E6BF-BF93-44FA-AA32-8F3F3789643C Data Availability StatementThe datasets used and/or analyzed during the current study are available from your corresponding authors on affordable request. Abstract Background Mounting evidence points to a crucial role of amyloid- (A) in the pathophysiology of Alzheimers disease (AD), a disorder in which brain glucose hypometabolism, downregulation of central elements of phosphorylation pathways, reduced ATP levels, and enhanced oxidative damage coexist, and sometimes precede, synaptic alterations and clinical manifestations. Since the brain has limited energy storage capacity, mitochondria play essential roles in maintaining the high levels of energy demand, but, as major consumers of oxygen, these organelles are also the most important generators of reactive oxygen species (ROS). Thus, it is not amazing that mitochondrial dysfunction is usually tightly linked to synaptic loss and AD pathophysiology. In spite of their relevance, the mechanistic links among ROS homeostasis, metabolic alterations, and cell bioenergetics, particularly in relation to A, still remain elusive. Methods We have used classic biochemical and immunocytochemical methods together with the evaluation of real-time changes in global energy metabolism in a Seahorse Metabolic Analyzer to provide insights into the detrimental role of oligA in SH-SY5Y and main neurons screening their pharmacologic protection by small molecules. Results Our findings indicate that oligomeric A induces a dramatic increase in ROS production and severely affects neuronal metabolism and bioenergetics. Assessment of global energy metabolism in real time demonstrated A-mediated reduction in oxygen consumption affecting basal and maximal respiration and causing decreased ATP production. Pharmacologic targeting of A-challenged neurons with a set of small molecules of known antioxidant and cytoprotective activity prevented the metabolic/bioenergetic changes induced by the peptide, fully restoring mitochondrial function while inducing an antioxidant response that counterbalanced the ROS production. Search for a mechanistic link among the protective small molecules tested recognized the transcription factor Nrf2compromised by age and downregulated in AD and transgenic modelsas their main target and the PI3K/GSK-3 axis as the central pathway through which the compounds elicit their A protective action. Conclusions Our study provides insights into the complex molecular mechanisms brought on by oligA which profoundly impact mitochondrial overall performance and argues for the inclusion of small molecules targeting the PI3K/GSK-3 6-(γ,γ-Dimethylallylamino)purine axis and Nrf2-mediated pathways as part of the current or future combinatorial therapies. strong class=”kwd-title” Keywords: Alzheimers disease, Amyloid-, Rabbit polyclonal to Cyclin B1.a member of the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance through the cell cycle.Cyclins function as regulators of CDK kinases. Mitochondria, Methazolamide, Melatonin, Trolox, Oxidative stress, Cell metabolism and bioenergetics, Oxygen consumption, Cellular respiration Background Alzheimers disease (AD), the most common type of dementia, is usually neuropathologically characterized by the presence of hyperphosphorylated tau in intraneuronal neurofibrillary tangles and the deposition of amyloid- (A) in the brain parenchyma and cerebral vasculature [1]. Although it remains unclear what primarily triggers and drives the progression of AD, strong evidence supports a pathogenic role for 6-(γ,γ-Dimethylallylamino)purine any oligomeric conformations [2, 3]. It is now considered that this transition from soluble monomeric species normally present in body fluids to the oligomeric, protofibrillar, and end-point fibrillar assemblies contributes significantly to disease pathogenesis. Intermediate oligomeric and protofibrillar forms seem to display the most potent effects in neuronal cells inducing synaptic disruption, neurotoxicity, and ultimately neurodegenerative cell death [3, 4]. The molecular mechanisms leading to AD pathophysiology are complex and not fully elucidated with mounting evidence highlighting a central role for mitochondrial dysfunction taking place at the early stages of the disease and supporting a causative role for these abnormalities in AD pathogenesis [5, 6]. Previous studies from our lab as well as the work of others show that 6-(γ,γ-Dimethylallylamino)purine A accumulation prospects to a cascade of events affecting mitochondrial function not only in neurons and glial cell populations but also in cells of the cerebral.