Impact of Sustained Exposure to β-Amyloid on Calcium Homeostasis and Neuronal Integrity in Model Nerve Cell System Expressing α4β2 Nicotinic Acetylcholine Receptors

Nicotinic Acetylcholine Receptors Sensitize a MAPK-linked Toxicity Pathway on Prolonged Exposure to β-Amyloid

“…Cell Culture and Transfection-Hybrid neuroblastoma NG108 -15 cells were used as a model nerve cell system, as they are normally devoid of functional nAChRs (22)(23)(24). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 15% FBS and hypoxanthine/aminopterin/ thymidine (HAT selection).…”

“…Immunocytochemistry-Cells were differentiated on CellTak-coated coverslips and transfected as described (22)(23)(24). After various treatments, cells were fixed using freshly prepared 4% paraformaldehyde for 40min and rinsed with PBS for 30 min.…”

“…Reactive Oxygen Species (ROS)/Hoechst Staining-Relative oxidative stress was assessed in live cultures of differentiated cells, transfected as described (22), as changes in the levels of reactive molecular species. Following various treatments, the Image-iT Live Reactive Oxygen Species Detection assay (Life Technologies) was performed in accordance with the manufacturer's protocol.…”

“…The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 15% FBS and hypoxanthine/aminopterin/ thymidine (HAT selection). Cells were differentiated on poly-L-lysine (plates) or Cell-Tak (coverslips) with 1 mM dibutyryl cyclic AMP in DMEM in the presence of reduced serum (1% FBS) and penicillin-streptomycin-glutamine for 72 h, as described previously (22)(23)(24). pcDNA3.1 expression vectors harboring mouse sequences for ␣4 and ␤2 nAChR subunits (1:4 ratio, respectively) were transfected into the differentiated cells using FuGENE HD, a lipid-based transfection reagent, for 48 h. Mock-transfected NG108 -15 cells, exposed only to FuGENE HD and not plasmid DNA, were used as controls (22)(23)(24).…”

“…As we have previously found that the presence of a relatively high-affinity target receptor for A␤ sensitizes nerve cells to A␤ toxicity (22), potential roles for select intracellular pathways involved in this sensitization were evaluated. The study focused on the MAPK pathway, assessing the relationship between receptor-linked, A␤-induced alterations in signaling and oxidative stress.…”