The neuronal microtubule-binding protein tau has been studied in the past for its suggested role in neurodegenerative diseases, including Alzheimer’s disease. Some studies suggest the hyperphosphorylation of tau can lead to apoptosis of neuronal cells through a number of different methods including destabilization of axons due to loss of microtubular support and tau tangles from the build up of hyperphosphorylated tau. Additionally, hyperphosphorylated tau may also play a crucial role in a number of intercellular signaling pathways including the activation of mitogen-activated protein kinase (MAPK)cite 3. Growth factors, cellular stressors and a number of other intracellular and extracellular stimuli lead to the activation of MAPK pathways including the JNK, p38, and ERK pathways cite 4.
Atypical activation or action in MAPK pathways can alter traditional functions of the pathway leading to apoptosis of neuronal cells. Such activation, specifically in the JNK and p38 signaling pathways, has been linked to neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Previous research has indicated that differing cellular stressors may The current study seeks to expand previous studies regarding the influence of cellular stressors on tau hyperphosphorylation and neuronal apoptosis by testing the effects of a high glucose condition in tau expressing and regular neuronal P12 cells. Method Cell Culture: RPMI media was used to maintain the clonal PC12 line cells. The media was supplemented using 10% equine serum, 5% HyClone fetal bovine serum, HEPES buffer, L-glutamine, and 50 ??g/mL penicillin/streptomycin. Cells were kept at 5% CO2 and at 37C via incubator throughout the study. DNA Transfection: Plasmid 3R-tau DNA was transfected into select PC12 cells thus allowing for tau expression in order to study differences between transfected and non transfected PC cells. Cells in a 6-well plate were transfected with 300 ??L OptiMEM, 7.5 ??L Lipofectamine 3000, 5 ??L P3000, and 12.5 ??L of 3R DNA per well. In the 96-well plate 10 ??L OptiMEM, 0.3 ??L Lipofectamine 3000, 0.2 ??L P3000, and 0.5 ??L of 3R DNA were used per treated well.
24 hours of incubation occurred following the transfection process before cells were treated with the high glucose condition. Induction of High Glucose Condition: Multiple trials of various glucose concentrations revealed a concentration of 7g/L of glucose to be the most effective concentration of high glucose media with nearly 50% cell death of non-transfected PC12 cells at 24 hr. Additional trials also supported the 24-hour time point, as opposed to a 19-hour option, to lead to optimal (50%) cell death. Cells in the 6-well plate for the high glucose condition were treated with Cells in the high glucose condition on the 96-well plate were treated with of Cell Viability Tyrpan Blue Stain: Cells from trials 1-7 were removed from the culture plate with trypsin/EDTA and a tyrpan blue exclusion assay was used to determine percent cell viability. Following treatment time (19hr or 24hr depending on the trial), cells were stained with 0.2 ??M filtered HyClone trypan blue solution. Four quadrants of each well were counted for live and dead cells using a hemocytometer. Cell viability was then calculated as a percentage of live or dead cells against the total number of cells counted. XTT Cell Viability Kit: Cell Viability for trials 8-9 was determined using an XTT assay kit. 50 ??L of XTT solution (containing a 1:50 ratio of reagent and electron coupling solution) was added to each well. Colored formazan dye is released by active cells in the assay allowing for quantification of live cells using a spectrophotometer. Absorbance was measured at a wavelength of 450nm.
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