ABSTRACT This thesis was divided into two parts. The purpose of part I was to clone and sequence the full-length ependymin gene from the invertebrate Limulus polyphemus, or portions of the gene, and to use RT-PCR to determine whether expression of this gene increases during leg regeneration. PCR was chosen as the method for obtaining the gene due to the success our lab had previously characterizing several ependymin genes using this approach. Three sets of primers were designed based on the conserved domains between teleost fish and three invertebrate ependymin sequences. “Sea primers" were designed based on the nucleotide sequence of the sea cucumber H. glaberrima for each conserved domain, and these primers produced all four of the expected size amplicons with Limulus DNA, but surprisingly only one such band with the sea cucumber Sclerodactyla briareus. The consensus primers (con-primers) were designed based on the most conserved nucleotide among all known ependymin species at each particular position in the conserved domains. Primers designated“5-11 primers" were designed based on the absolutely conserved domains among the three known invertebrate ependymins. Neither con-primers nor 5-11 primers produced any bands of the expected size; this was true for both species of DNA. One very strong band was produced using“5-11" primer pair 6/10 with both species. One of the bands from this reaction from Limulus was cloned and sequenced, and showed a very strong homology (88% over 292 bp) with mouse FGF-14, a neurotrophic factor involved in mouse neurogenesis. The expression of this gene during leg regeneration will be tested in future experiments. Limulus GAPDH was also cloned and sequenced, and a genomic intron was identified for the first time in this study. This Limulus housekeeping gene will be used in future studies for gene expression comparisons. The purpose of part two of this thesis was to study the up-regulation of growth-related genes induced by treatment of a human neuroblastoma SH-SY5Y cell line with a human ependymin peptide mimetic (hEPN-1), in an attempt to help provide a basis for using human EPN mimetics as therapeutics in stroke and neurodegenerative diseases. The sequence of this mimetic is derived from an area of human MERP-1 analogous to goldfish mimetic CMX-8933. The human mimetic was previously found to up-regulate growth related genes L-19, EF-2 and ATP Synthase in the mouse neuroblastoma cell line Nb2a (Saif, 2004). The expression levels of genes encoding ribosomal proteins and ribosomal RNA were studied using RT-PCR as hallmarks of proliferating cells. hEPN-1 was found to increase the expression of the nuclear-encoded ribosomal proteins S-19 and S-12, an average of 2.76 fold and 1.74 fold, with statistically significant p-values of 0.031 and 0.015 (<0.05), respectively. The expression levels of nuclear-encoded 5.8S ribosomal RNA (p = 0.018) and the mitochondrial-encoded 16S RNA (p = 0.046) were found to be increased an average of 14.04 fold and 3.91 fold, respectively. Thus, human ependymin mimetic hEPN-1 appears to stimulate growth-related genes, a property which can be useful to regenerate neuronal tissue after injury.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in the world. The most accepted hypothesis for the cause of this disease is the amyloid cascade hypothesis, which postulates that the formation of extracellular neurotoxic amyloid-beta binds specific receptors on the surface of neuronal and glial cells to increase cell stress leading to cell death. Our laboratory previously showed that treatment of cultured human SHSY neuronal cells with amyloid beta increases the cellular levels of two key components (caspases-2 and -3) of the extrinsic apoptotic pathway, leading to cell death. The amyloid beta induced caspase elevation was blocked by simultaneously treating the cells with a short mimetic of human ependymin neurotrophic factor, hEPN-1, and the hEPN-1 treatment also blocked cell death. This thesis extends the AD investigation to show that treatment of SHSY cells with amyloid beta may also activate an intrinsic apoptotic mitochondrial stress pathway (assaying caspase-9 as a marker enzyme), and that hEPN-1 treatment significantly lowers this activation. In addition, our laboratory previously showed that treating SHSY cells with amyloid beta increases TUNEL staining, an assay for DNA fragmentation (a hallmark of end stage of apoptosis, and a different apoptotic marker than caspase activation). Treatment with hEPN-1 simultaneously with the amyloid beta, or 6 hrs post amyloid beta, significantly lowered the amyloid beta induced TUNEL signal. This thesis extended the earlier TUNEL experiments to show that hEPN-1 treatment can significantly lower the amyloid beta induced TUNEL staining even when added 18 hrs post amyloid beta. With respect to caspase-8, an initiator caspase in the extrinsic pathway, immunoblot assays of brain lysates from 8 month old transgenic AD mice showed that a 2 week oral delivery of hEPN-1 (conjugated to a carrier to deliver it across the blood brain barrier) significantly lowered caspase-8 levels. Finally, an assay of cellular inhibitors of apoptosis (cIAP) showed a significant increase in their cellular levels in SHSY cells, and in transgenic AD mice treated with hEPN-1, showing for the first time that hEPN-1 may aid cell survival by upregulating proteins known to directly bind specific caspases to block their activity leading to their degradation. The cIAP upregulation occurred in the presence or absence of amyloid beta, indicating that hEPN-1 likely does not block cell death by directly interfering with the interaction of amyloid beta with its cell surface receptors, but instead hEPN-1 may activate an independent cell survival signal transduction pathway in neuronal cells.

"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."