"Ependymin (EPN) is a goldfish neurotrophic factor (NTF) that is one of the most abundant secreted glycoprotein components of brain extracellular fluid (ECF) and cerebrospinal fluid. This protein was first discovered due to its enhanced turnover following learning events, but has since been found to function in other important cellular events such as long-term memory formation and optic nerve elongation (Shashoua, 1976; Shashoua, 1977; Shashoua, 1985). Goldfish EPN has several demonstrated effects on mammalian cells, and immuno-reactive EPN-like proteins have been observed in a variety of organisms ranging from invertebrates (Limulus) to mice. Some NTFs have been shown to alleviate oxidative stress, one of the primary mediators of cell damage in neurodegenerative conditions. One mechanism by which they accomplish this is to increase cellular levels of anti-oxidative enzyme superoxide dismutase (SOD). In fact, our lab recently showed that a synthetic EPN fragment (CMX-8933) increases SOD mRNA and protein levels in rat primary cortical cultures (Parikh, 2003). Transgenic mice and rabbits that overexpress SOD are resistant to ischemia, while mice that lack SOD present with worse ischemic damage. Thus, due to this important SOD activating NTF-like feature of EPN may have potential therapeutic applications for treating neurodegenerative conditions such as Alzheimer’s disease, Parkinson’s or stroke. Because full-length NTFs do not efficiently cross the blood brain barrier (BBB) when administered intravenously, our lab, in collaboration with Ceremedix, Inc. (Boston, MA), is interested in designing short peptides that mimic the action of full-length NTFs, especially EPN. Due to proteases that naturally exist in ECF, EPN is partially cleaved to release the 8 aa peptide KKETLQFR. Other brain proteins, like encephalins and endorphins, also release similar short active components, so we hypothesized that this 8 aa peptide may represent an active component of EPN. Indeed, our lab demonstrated that administration of this sequence in synthetic form (termed CMX-8933) to rat primary cortical cells increases cellular titers of SOD (Parkih, 2003). This thesis was divided into three parts. The first part investigated which signal transduction pathway is responsible for CMX-8933’s ability to upregulate SOD. Because our lab also showed that CMX-8933 activates the MAPK pathway (Hasson, 1998; El-Khishin, 1999; Adams et al., 2003) we hypothesized that CMX-8933 may use this pathway to upregulate SOD. Inhibition experiments were performed to test three known components of the MAPK pathway, and a member of an unrelated pathway. Six independent SOD immunoblot experiments demonstrated that pre-treatment of rat primary cortical cultures with specific inhibitors for the protein kinase-C family (PKC), protein tyrosine kinases (PTKs), or MEK protein kinases (MEKK), completely blocked (p = 0.0001) CMX-8933’s average 15-fold upregulation of SOD. Thus, these three critical components of the MAPK pathway appear to be involved in the CMX-8933-induced upregulation of SOD. An inhibitor of transcription factor NF-êB in an unrelated pathway had no significant effect (p = 0.901). The second part of this thesis tested whether treatment of rat primary cortical cultures with CMX-8933 increases the cellular titers of mRNAs related to translation. Previous observations indicated that treatment of these cells with with CMX-8933 induces neurite sprouting (Shashoua, unpublished), and that EPN plays a role in optic nerve elongation (Schmidt and Shashoua, 1988), two processes related to growth. So we hypothesized that EPN, or CMX-8933, may stimulate the transcriptioin of mRNAs related to growth. We tested mRNAs for translation factor EF-2, and ribosomal proteins S12 and L19 based on previous observations in our lab with hybridization arrays (Parikh, 2003). RT-PCR experiments indicated that treatment of rat primary cortical cultures with 10 ng/ml CMX-8933 for 5 hrs increased the mRNAs for S12 an average of 12-fold relative to untreated cultures (N = 3, p = 0.02), L19 an average of 9-fold (N = 3, p = 0.048), and EF-2 an average of 11-fold (N = 3, p = 0.045). Levels of housekeeper polyubiquitin remained unchanged. Thus 3 gene products related to growth are indeed upregulated by CMX-8933. The third part of this thesis investigated the SOD stimulatory effects of full-length EPN versus its cleavage product CMX-8933. Previous studies showed that full-length NTFs BDNF and NGF upregulate SOD in neuronal cells. Because CMX-8933 upregulates SOD, maybe full-length EPN does too. We hypothesized that if CMX-8933 represents the receptor-binding domain of full-length EPN, that full-length EPN may show the same stimulatory effects as CMX-8933, and may upregulate SOD. Extracellular fluid (ECF) was prepared from goldfish brains, the traditional source for isolating EPN. Analysis of the ECF on protein gels demonstrated the presence of a complex protein pattern dominated by two bands at 37,000 and 31,000 daltons, the known sizes of EPN-â (glycosylated) and EPN-ã (non-glycosylated), respectively. Immunoblots performed with EPN antibody “Sheila” (directed against the C-terminal end of EPN, Shashoua and Moore, 1978) confirmed the identity of these two ECF bands as EPN. Cultured mouse Nb2a neuronal cells were treated in six independent experiments with 12 ìg/ml ECF protein for 5 hrs, and whole cell lysates were tested for levels of SOD by immunoblots. This ECF treatment of neuronal cells produced a mean 4-fold increase in SOD levels (p = 0.007), supporting our hypothesis. However, since ECF is a complex mixture, this data did not show which ECF component was resonsible for the SOD signal. Since ECF is known to contain CMX-8933 EPN cleavage product, which by itself can upregulate SOD, it is possible CMX-8933 was responsible for the signal, not full-length EPN. To address this issue, microdialysis was performed using an 8,000 dalton MWCO membrane to remove low MW components from the ECF (including CMX-8933, MW = 1149), leaving EPN-â (MW 37,000) and EPN-ã (MW 31,000) present in the dialyzed ECF. Four independent experiments indicated no significant difference (p = 0.116) between dialyzed versus non-dialyzed ECF for activating SOD. Thus, CMX-8933 does not appear to be responsible for ECF’s ability to increase SOD, but instead, high MW molecules (including full-length EPN) appear to be the active components. Altogether, the data from this thesis extends our knowledge of the mechanism of action of both full-length EPN and its cleavage product CMX-8933."

Telomerase is an enzyme that helps maintain the telomeric ends of chromosomes during DNA replication. Telomere lengths represent a balance between telomerase activity attempting to elongate their ends, and cell division that causes telomere shortening. As cells age, diminished telomerase activity allows a shortening of telomere lengths until they reach a target length that stimlulates apoptosis. Identifying a drug capable of upregulating telomerase activity may help increase cell (and even organismal) lifespan. The purpose of this thesis was to determine whether treatment of human primary foreskin fibroblast cultures with a 14 amino acid (aa) ependymin peptide mimetic upregulates (or at least maintains) telomerase activity and telomere lengths during cellular ageing. The 14aa peptide was previously shown to significantly increase the murine lifespan by 25%, so its activity was a logical candidate to test in this thesis. In a preliminary set of experiments, the human primary fibroblast cells were shown to respond to the 14aa drug by upregulating the antioxidative enzyme superoxide dismutase (SOD), thus human fibroblast cells likely contain the appropriate receptor for binding this drug. This same dose proved optimal for upregulating telomerase activity in the fibroblast cells an average of 57% relative to untreated cells (p value = 0.003). The upregulation appears to be specific for the sequence of aa in the 14aa drug since a“scrambled" peptide containing the same aa but in a different order showed no upregulation, even at doses 10-fold higher. Treatment of mice once per day or twice per day with the 14aa peptide was also found to upregulate telomerase activity in vivo in brain and heart. The activity was optimal at a 3.3 mg/kg dose for each aged organ, and was generally high in young organs. The activity observed in heart was a total surprise since heart cells are generally thought to be quiescent, and telomerase is usually associated with cell division, so perhaps telomerase has a function other than in cell division. The second part of the hypothesis tested whether treatment of fibroblast cells with the 14aa drug elongated (or prevented from shortening) telomere lengths in aged cells. A telomere length assay (TLA) based on a Southern hybridization approach using a telomere probe appeared to work well, since marker DNAs showed appropriate differences in their“telomere smears", and aged fibroblast cells showed shorter smears than young cells. However, no difference was observed between drug-treated versus vehicle-treated cells, even at the 10 ng/ml dose previously shown to strongly upregulate telomerase activity. So perhaps the upregulation of telomerase activity was not sufficient to provide a measurable increase in telomere lengths. Telomerase has been shown to extend the lifespan of virus-transformed human cells without showing any visible telomere lengthening (Blackburn et al, 1999), so perhaps telomerase can increase cell lifespan without increasing telomere lengths. To our knowledge, this is the only drug demonstrated to upregulate telomerase activity. Transforming cells with the viral T-antigen can upregulate telomerase, but T-antigen is not a therapeutic drug since it also causes cancer. Telomerase upregulation is known to occur during oncogenesis, but telomerase itself is not an oncogene since oncogenesis also requires the upregulation of oncogenes. Our lab previously showed this peptide does not upregulate the potent oncogene myc. If this proves to be the case for other oncogenes, using this 14aa drug to upregulate telomerase activity without activating oncogenes could prove extremely useful for helping prove telomerase is not an oncogene, and for extending cell lifespan.

Thesis (M.S.) -- Worcester Polytechnic Institute. / Keywords: ependymin; SOD. Includes bibliographical references (p. 61-67).

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: ependymin; telomerase; telomere Includes bibliographical references. (p.46-49)

Thesis (M.S.) -- Worcester Polytechnic Institute. / Keywords: Alzheimer's; Ependymins; Caspases; SOD. Includes bibliographical references (p. 55-59).

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: SHSY, LDH, Ependymin, Alzheimer's. Includes bibliographical references (leaves 34-38 ).

Thesis (M.S.) -- Worcester Polytechnic Institute. / Keywords: AP-1; ependymin; neurotrophic factor. Includes bibliographical references (p.63-69).

Ependymin (EPN) is a neurotrophic factor (NTF) that functions in goldfish long-term memory formation and optic nerve elongation (Shashoua, 1976; Shashoua, 1977; Shashoua, 1985). Goldfish EPN, or CMX-8933 (a short goldfish EPN mimetic studied by our lab), surprisingly have several demonstrated effects on mammalian cells, including neuroregenerative effects in a rat stroke model (Shashoua et al, 2003), and the activation of therapeutic superoxide dismutase (SOD) (Parikh, 2003) and transcription factor AP-1 (Adams et al, 2003) in mouse neuroblastoma cells or rat primary cortical neuronal cultures. Among its various functions, AP-1 can function as a master switch in long-term memory consolidation (Sanyal et al, 2002), so it may be a key event in EPN's mechanism of action. AP-1 activation is also a characteristic associated with other full-sized neurotrophic factors, including nerve growth factor and brain-derived nerve growth factor. This thesis was divided into three parts. The purpose of part I was to determine whether our previously observed upregulation of SOD by CMX-8933 is dependent upon (or merely concurrent with) AP-1 activation. Four independent SOD immunoblot experiments demonstrated that pre-treatment of rat primary cortical cultures with trifluoromethyl pyrimidine carboxylate (TFPC), a specific inhibitor of AP-1, significantly (p = 0.0004) decreased cellular levels of SOD by 67% at its IC50 concentration of 1 ìM, and completely inhibited the upregulation at 10 and 100 ìM concentrations. Thus, the CMX-8933-induced upregulation of SOD appears to depend (directly or indirectly) on AP-1 activation. Part II of this thesis included the use of bioinformatics to re-verify exciting recent observations that EPN-like proteins exist in mammals, termed mammalian-ependymin-related proteins or MERPs (Apostolopoulos et al, 2001). If our analyses were convincing, human EPN mimetics would then be designed and tested for AP-1 activation. Computer alignments and hydropathy plots performed with EPN amino acid sequences deduced from gene entries in GenBank verified the existence of mammalian homologs containing highly conserved domains with fish EPN's, suggesting the possibilities of similar protein conformation and function. Two human EPN mimetics were designed, hEPN-1 (8 aa long, corresponding to the same region as CMX-8933) and hEPN-2 (14 aa long, containing CMX-8933 and 6 upstream aa). Several mimetic doses were tested on mouse Neuro-2a cultures for nuclear translocation of c-Jun and c-Fos proteins (comprising the AP-1 particle upregulated by fish CMX-8933). Seven independent c-Jun immunoblot experiments, and five c-Fos experiments, demonstrated a strong (as high as 25-fold) dose-dependent increase in the nuclear titers of the AP-1 proteins. Both peptides had statistically equivalent effects. Thus, human EPN appears to exist, and two mimetics derived from its sequence appear to be biologically active against mouse neuroblastoma cells. Since hEPN-1 and -2 have only a few residues in common with CMX-8933, we hypothesize that the mimetic shape rather than sequence may be important for biological activity. In part III of this thesis, the biological effects of hEPN-1 and hEPN-2 on mouse Neuro-2a cells were studied further using RT-PCR to analyze potential increases in specific mRNAs. mRNAs related to growth, energy production, and protein translation were tested since previous data in our lab (Kaska, 2003) indicated mRNAs for translational elongation factor-2 (EF-2), and ribosomal proteins L19 and S12 were upregulated in rat primary cortical cultures by fish mimetic CMX-8933 (Kaska, 2003). Treatment of Neuro-2a cells with 1.0 ìg/ml hEPN-1 (the highest dose tested for the AP-1 translocation experiments) for 24 hrs appeared to increase (N = 1) mRNAs for ATP Synthase-C, ribosomal protein L19, and translational EF-2, relative to the levels of housekeeper polyubiquitin. Thus hEPN-1 may be involved in processes related to growth. Altogether, the data from this thesis extends our knowledge of fish EPN mimetic CMX-8933 (showing that its induction of SOD requires AP-1), demonstrates that human EPN may exist (bioinformatics), shows that two human EPN mimetics are biologically active (induce AP-1 translocation), and shows that one mimetic hEPN-1 may activate several mRNAs related to growth in mouse Neuro-2a 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."

"Alzheimer’s disease (AD) is a neurodegenerative disorder that leads to dementia in adults. The mechanism of neurodegeneration is thought to involve the extracellular production of a highly toxic A-beta peptide that engages cell surface receptors to induce cellular oxidative stress and apoptosis, but the signal transduction pathways that lead to A-beta induced cell death are unknown. We previously showed that a human ependymin neurotrophic peptide mimetic (hEPN-1) can promote cell survival in an in vitro AD model system. This initial observation was extended in this thesis by investigating the mechanism of A-beta induced apoptosis and hEPN-1 induced survival. Immunoblots were used to assay the total cellular levels of specific caspase proteins. The results show that A-beta induced apoptosis uses an extrinsic caspase pathway involving caspases-2 and -3, and that hEPN-1 treatment can reduce those caspase levels. A caspase activity assay showed that A-beta increased caspase-3/7 activity, while hEPN-1 treatment lowered it. Moreover, in vivo studies with AD transgenic mice showed that hEPN-1 treatment increased antioxidative superoxide dismutase levels in brain. Thus, hEPN-1 holds potential as a therapeutic to treat the underlying neurodegenerative cause of AD, not merely its symptoms as with other currently approved AD drugs."