Ependymin (EPN) is a goldfish brain neurotrophic factor (NTF) previously shown to function in a variety of cellular events related to long-term memory formation and neuronal regeneration. Because of these functions, EPN and other NTFs have potential applications for treating neuro-degenerative conditions, including stroke. In previous experiments, our lab in collaboration with Victor Shashoua of Ceremedix Inc (Boston, MA), designed short synthetic peptide CMX-8933 (a proteolytic cleavage product of EPN) and CMX-9236 (an EPN-Calmodulin combination peptide) that mimic the action of full-length EPN. In a rat stroke model, administration of these peptides i.v. significantly lowered brain ischemic volume (Shashoua et al., 2003). Because oxidative stress is one of the primary mediators of cell damage following a stroke, we hypothesized that NTFs, and in particular our therapeutic peptides, may act in part by reducing neuronal oxidative stress. Thus, the purpose of this thesis was to determine whether CMX-8933 and CMX-9236 increase the cellular titers of anti-oxidative enzymes. A hybridization array was used as a“hypothesis generator" to obtain candidates for further analysis. This approach applied to rat primary brain cortical cells treated with CMX-8933 identified superoxide dismutase (SOD) as strongly upregulated. SOD immunoblots on whole cell lysates, and RT-PCR on total cellular RNA, were used to confirm this observation. In time-course and dose-response experiments, treatment of rat primary cortical cultures with either peptide showed an optimal 8.5 fold (N = 5, p < 0.001) increase in SOD protein, while administration of CMX-8933 to murine neuroblastoma cells caused a 6.5 fold (N = 3, p = 0.001) increase in SOD mRNA levels. Previous work in other laboratories indicated that systemic (i.v.) administration of full-length NTFs allows only an inefficient delivery across the blood brain barrier (BBB). We hypothesized that our short synthetic peptides may cross the BBB more efficiently. Immunoblot analysis of brains and hearts excised from mice treated i.v. with various doses of CMX-8933 confirmed the elevated SOD titers (10 fold in brain, and 8 fold in heart, at a 6 mg/kg dose for 5 hr; N = 5, p < 0.001). Furthermore, we hypothesized that conjugation of CMX-8933 to BBB carrier DHA, a natural neuronal membrane fatty acid shown previously to enhance the delivery of dopamine to the brain (Shashoua and Hesse, 1996), might further enhance the NTF therapy. Delivery of DHA-8933 increased SOD expression by 3 fold (N = 4, p < 0.001) relative to non-conjugated CMX-8933. Recently, the use of special incubators that allow the culture of cells under low oxygen conditions (anoxia) has been used as an in vitro model for stroke. When we tested our peptides in this new in vitro model, surprisingly SOD was upregulated 3 fold (N = 3, p = 0.003) in rat primary cortical cells cultured for 24 hr under oxygen deprivation, compared to normoxic conditions. This implies that these rat cultures may have an endogenous cellular system for responding to oxygen stress, a finding worthy of further investigation. Treatment of anoxic cells with CMX-8933 increased SOD levels another 2.8 fold (N = 3, p < 0.001) compared to the levels for anoxia alone (for a total of 8.5 fold relative to normoxic cells). Altogether, the data from this thesis illustrate that small NTF EPN peptide mimetics increase the cellular titers of the mRNA and protein for the anti-oxidative enzyme SOD, which may be an important step in their known therapeutic benefits.

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.