2-phenyl-4-4-5-5-tetramethylimidazoline-1-oxyl-3-oxide and Neuroblastoma

Sporadic Alzheimer's disease (sAD) is associated with energy metabolism deficiency and impairment of insulin receptor (IR) signaling in the brain. In this context, low doses of intracerebroventricular (icv) injection of streptozotocin (STZ) in rodents has been used as an experimental model of sAD which leads to an insulin-resistant brain state and neurodegeneration. However, the STZ effects on brain insulin signaling-related proteins it is not appropriately elucidated. The aim of this study was to evaluate the beginning and progression of alterations in the brain IR pathway of rats after 1, 3, 5, and 7 days of STZ injection and investigate intracellular signaling involved on STZ induced insulin resistance. We observed that STZ injection causes cognitive impairment in the animals, a temporal variation of the insulin signaling-related proteins and apoptosis cell death in the hippocampus. We also have shown that STZ causes insulin resistance and impairment on phosphoinositide 3-kinase (PI3K) activity in the Neuro-2a cells through protein kinase B (Akt) inactivation by S-nitrosylation, which could upregulate GSK3-β activity. STZ ability to cause an insulin-resistant neuron state involves NO production and ROS production which may play an important role in the mechanism linked to STZ-induced neurotoxicity. The icv injection of STZ model and STZ exposed Neuro-2a cells may be potential experimental models for assessing molecules related to the pathogenesis of sAD.

Topics: Animals; Antibiotics, Antineoplastic; Cells, Cultured; Cyclic N-Oxides; Free Radical Scavengers; Gene Expression Regulation; Hippocampus; Imidazoles; Injections, Intraventricular; Insulin Resistance; Male; Memory Disorders; Neuroblastoma; Neurons; Nitric Oxide; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Reactive Oxygen Species; Recognition, Psychology; Signal Transduction; Streptozocin

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is thought to be caused in part by the age-related accumulation of amyloid-β (Aβ) in the brain. Recent findings have revealed that nitric oxide (NO) modulates the processing of amyloid-β precursor protein (APP) and alters Aβ production; however, the previously presented data are contradictory and the underlying molecular mechanisms are still incomplete. Here, using human SH-SY5Y neuroblastoma cells stably transfected with wild-type APPwt695, we found that NO, derived from NO donor sodium nitroprusside (SNP), bi-directionally modulates APP processing in vitro. The data from ELISA and Western blot (WB) tests indicated that SNP at lower concentrations (0.01 and 0.1 μM) inhibits BACE1 expression, thus consequently suppresses APP β-cleavage and decreases Aβ production. In contrast, SNP at higher concentrations (10 and 20 μM) biases the APP processing toward the amyloidogenic pathway as evidenced by an increased BACE1 but a decreased ADAM10 expression, together with an elevated Aβ secretion. This bi-directional modulating activity of SNP on APP processing was completely blocked by specific NO scavenger c-PTIO, indicating NO-dependent mechanisms. Moreover, the anti-amyloidogenic activity of SNP is sGC/cGMP/PKG-dependent as evidenced by its reversal by sGC/PKG inhibitions, whereas the amyloidogenic activity of SNP is peroxynitrite-related and can be reversed by peroxynitrite scavenger uric acid. In summary, these present findings predict a double-edged role of NO in APP processing in vitro. Low (physiological) levels of NO inhibit the amyloidogenic processing of APP, whereas extra-high (pathological) concentrations of NO favor the amyloidogenic pathway of APP processing. This preliminary study may provide further evidence to clarify the molecular roles of NO and NO-related signaling in AD and supply potential molecular targets for AD treatment.

Topics: ADAM Proteins; ADAM10 Protein; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Aspartic Acid Endopeptidases; Carbazoles; Cell Line, Tumor; Cyclic GMP; Cyclic N-Oxides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation; Humans; Imidazoles; Membrane Proteins; Neuroblastoma; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Peroxynitrous Acid; Signal Transduction; Superoxides; Transfection

Activated microglial cells are an important pathological component in brains of patients with neurodegenerative diseases. The purpose of this study was to investigate the effect of He-Ne (632.8 nm, 64.6 mW/cm2) low-level laser therapy (LLLT), a non-damaging physical therapy, on activated microglia, and the subsequent signaling events of LLLT-induced neuroprotective effects and phagocytic responses.. To model microglial activation, we treated the microglial BV2 cells with lipopolysaccharide (LPS). For the LLLT-induced neuroprotective study, neuronal cells with activated microglial cells in a Transwell™ cell-culture system were used. For the phagocytosis study, fluorescence-labeled microspheres were added into the treated microglial cells to confirm the role of LLLT.. Our results showed that LLLT (20 J/cm2) could attenuate toll-like receptor (TLR)-mediated proinflammatory responses in microglia, characterized by down-regulation of proinflammatory cytokine expression and nitric oxide (NO) production. LLLT-triggered TLR signaling inhibition was achieved by activating tyrosine kinases Src and Syk, which led to MyD88 tyrosine phosphorylation, thus impairing MyD88-dependent proinflammatory signaling cascade. In addition, we found that Src activation could enhance Rac1 activity and F-actin accumulation that typify microglial phagocytic activity. We also found that Src/PI3K/Akt inhibitors prevented LLLT-stimulated Akt (Ser473 and Thr308) phosphorylation and blocked Rac1 activity and actin-based microglial phagocytosis, indicating the activation of Src/PI3K/Akt/Rac1 signaling pathway.. The present study underlines the importance of Src in suppressing inflammation and enhancing microglial phagocytic function in activated microglia during LLLT stimulation. We have identified a new and important neuroprotective signaling pathway that consists of regulation of microglial phagocytosis and inflammation under LLLT treatment. Our research may provide a feasible therapeutic approach to control the progression of neurodegenerative diseases.

Topics: Actins; Analysis of Variance; Animals; Animals, Newborn; Brain; Cells, Cultured; Chromones; Cyclic N-Oxides; Cytokines; Cytotoxicity Tests, Immunologic; Enzyme Inhibitors; Free Radical Scavengers; Gene Expression Regulation; Humans; Imidazoles; Lipopolysaccharides; Low-Level Light Therapy; Mice; Mice, Inbred C57BL; Microglia; Microscopy, Confocal; Morpholines; Myeloid Differentiation Factor 88; Neuroblastoma; Nitric Oxide; Phagocytosis; Phalloidine; Proto-Oncogene Proteins pp60(c-src); rac1 GTP-Binding Protein; Signal Transduction; Statistics as Topic; Time Factors; Transfection; Tyrosine