"Alzheimer’s disease (AD) is a neurodegenerative disorder that currently affects an estimated 4.2 million to 5.8 million Americans. Although the cause of AD is not fully known, the current working model proposes that amyloid precursor protein (APP) is unnaturally cleaved by beta and gamma secretases to form the highly neurotoxic peptide beta-amyloid (Aâ) which engages cell surface receptors to cause cell death through a series of events involving oxidative stress and apoptosis. An in vitro model for AD uses cultured human SHSY-5Y (commonly abbreviated SHSY) neuroblastoma cells treated with Yankner peptide, an 11 amino acid peptide representing Aâ residues 25-35 that strongly binds receptor. Treatment of SHSY cells with 20 µM Yankner peptide strongly induces cellular apoptosis. Synthetic peptide human ependymin-1 (hEPN-1) is a derivative of a naturally occurring protein within the human brain, previously shown by our laboratory to upregulate antioxidative enzymes in SHSY cells, and AP-1 transcription factor associated with long-term memory formation. Since hEPN-1 has anti-oxidative potential as a therapeutic, we hypothesized that hEPN-1 can reverse the neurotoxic effects of Yankner peptide treatment of cultured human SHSY neuronal cells. Microtiter dishes were plated with SHSY cells under control conditions (no Yankner peptide), in the presence of 20 µM Yankner peptide, or in the presence of Yankner peptide plus various concentrations of hEPN-1 therapeutic, then cultured for 3 days to 80% confluency. Unattached dying cells were gently washed away, then the residual cells were monitored by measuring cell number, cell viability (Trypan blue exclusion), LDH activity per mg protein (an indirect measure of cell viability), and nuclear blebbing (a measure of apoptosis). Statistical significance was determined using a One Way ANOVA under the LSD stringency, using SPSS. In three independent trials, average cell numbers per microtiter well decreased 44.7% (from 3.11 x 105 to 1.72 x 105) in the presence of 20 µM Yankner peptide (p < 0.05 compared to control), were 2.73 x 105 when 75 µM hEPN-1 was added simultaneously with Yankner (p < 0.05 compared to Yankner), and were 2.96 x 105 when 75 µM hEPN-1 was added 24 hrs post-Yankner (p < 0.05 relative to Yankner alone). The control mean was not statistically distinguishable from either of the hEPN-1-treated samples (p = 0.220 and p = 0.671, respectively). With respect to the trypan blue data, in three independent trials, the mean percent viable cells (excluding trypan blue) decreased 41.0% (from 68.7% to 40.5%) in the presence of 20 µM Yankner peptide (p < 0.001 relative to control), was 60.7% when 75 µM hEPN-1 was added simultaneously with Yankner (p < 0.001 relative to Yankner alone), and was 61.4% when 75 µM hEPN-1 was added 24 hrs post-Yankner (p < 0.001 relative to Yankner alone). The control mean was not statistically distinguishable from either of the hEPN-1-treated samples (p = 0.013 and 0.03, respectively). In the LDH activity experiments, in four independent trials, the average LDH OD decreased 80.8% (from 0.47 to 0.09) in the presence of 20 µM Yankner peptide (p < 0.001 relative to control), was 0.47 when 75 µM hEPN-1 was added simultaneously with Yankner (p < 0.001 relative to Yankner alone), and was 0.48 when 75 µM hEPN-1 was added 24 hrs post-Yankner (p < 0.001 relative to Yankner alone). The control mean was not statistically distinguishable from either of the hEPN-1-treated samples (p = 0.174 and 0.479, respectively). Although previous reports in the literature indicated LDH expression is constitutive in SHSY cells (thus its activity is an indirect measure of cell numbers or viability), it was possible the hEPN-1 treatments upregulated LDH activity. So to ensure our observed changes in LDH activity levels did not represent changes per unit protein, the LDH activity values were divided by the mg of protein present in the sample, and all four experimental samples were statistically indistinguishable (p values = 0.184, 0.995, 0.872, respectively, relative to control). In the nuclear blebbing experiments, in five independent trials, the mean percent blebbed nuclei (a measure of apoptosis) doubled from 7.5% to 16.0% in the presence of 20 µM Yankner peptide (p < 0.001 relative to control), was 6.7% when 75 µM hEPN-1 was added simultaneously with Yankner (p < 0.001 relative to Yankner alone), and was 6.5% when 75 µM hEPN-1 was added 24 hrs post-Yankner (p < 0.001 relative to Yankner alone). The decreased apoptosis observed in the hEPN-1-treated samples was however, not statistically significant (p = 0.381 and 0.279, respectively). Overall, the data suggest that hEPN-1 can protect human neuronal cells from Yankner-induced cell death, whether added simultaneous to the insult, or 24 hrs post. Because the therapeutic can act 24 hrs post-insult, it may interfere with a late-stage apoptotic event. As there is currently no known drug that blocks Yankner-induced toxicity, the hEPN-1 therapeutic shows potential in combating the underlying apoptosis of Alzheimer’s disease."
| Identifer | oai:union.ndltd.org:wpi.edu/oai:digitalcommons.wpi.edu:etd-theses-1943 |
| Date | 21 August 2006 |
| Creators | Stovall, Kirk Hiatt |
| Contributors | Eric W. Overström, Committee Member, Daniel G. Gibson III, Committee Member, David S. Adams, Advisor |
| Publisher | Digital WPI |
| Source Sets | Worcester Polytechnic Institute |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses (All Theses, All Years) |
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