1. MATERIALS AND METHODS Primary Neuron Culture The experiments described in this report conformed to guidelines established by the National Institutes of Health and University of Wisconsin Eau Claire for the humane treatment of animals. Primary cultures of cortical neurons were prepared from Sprague Dawley rat pups on embryonic day 18 (E18) as described in Brewer et al. (1993) and Brewer & Price (1996) Briefly, cortices were sorted by biological sex and dissected in Hibernate-E media (Brain Bits Inc, Springfield, Il) containing B27 supplement. Sexual Differences in pERK/ERK due to 17-Beta-Estradiol Treatments Student: Daniel F. Ferrise Jr.; Faculty: Dr. Damani Bryant Biology Department │ University of Wisconsin - Eau Claire │ 2013-2014 ABSTRACT Alzheimer's disease is an important disease, affecting both women and men. One area of study is the effect of 17-β-Estradiol (E2) on neuro- protection. Females have a sharp increase in Alzheimer's onset following menopause. The purpose of this study is to determine where the cell signaling differences inlay. Our point of interest is whether phosphorylated ERK concentrations are altered in response to E2 treatments with male and female rat pup neurons. Rat pup neurons will be collected and plated, treated with E2 or an ethanol control, and collected so western blots can be run to determine basal and E2-induced levels of pERK. Based on preliminary data, we expect to see a decrease in ERK phosphorylation in males. Furthermore, we do not expect to see a decrease in ERK phosphorylation in females. ACKNOWLEDGEMENTS: This project was funded by the National Science Foundation. I would like to thank Dr. Damani Bryant for everything I’ve learned, being a great Professor, and giving me the opportunity to do research with him. Also, Dr. Daniel Herman for allowing me to use his EpiChemi Dark Room, as well as Luke Mike, Seyeon Kim, and Ben Ziebart for helping process the samples for this study. Figure 1. Western Blots and Graph of pERK and Total ERK. Neurons were starved for 5hrs and then treated with (10 nanomolar) Ethanol or Estrogen for 15 minutes. Densitometry of the western blots were graphed to show the relationship between sexes and treatment. 2-way ANOVA showed that there is no statistically significant effect of E2 on ERK phosphorylation/activation between the sexes (p = 0.3692). CONCLUSIONS AND FUTURE STUDIES Our tests show a significant difference in ERK phosphorylation between the sexes due to 20 min E2 treatments (p=0.0427). This difference was due to the ERK phosphorylation/activation between E2 treated male and E2 treated female neurons; however, there was no significant difference in the 15 min E2 treatments. The differences may be due to length of treatment or the concentration of plated cells. Possible future studies could look at time tables of treatment to determine if there is a point where E2 has a significant effect on female neurons. BACKGROUND Estrogen is a hormone that is found most prominently in females. There is a sharp increase in Alzheimer’s disease in females following menopause due to the decrease in hormone production. Estrogen is neuro-protective through binding to the Estrogen Receptors (Giddabasappa et al, 2010). It has been shown that the ERK signaling pathway can protect cells from apoptotic proteins. Knocking out ERK can lead to cell death (Tasyriq et al and Liu et al). When Estrogen is studied in more detail, it appears that the binding of Estrogen to ERs can initiate the ERK signaling pathway to protect cells against apoptosis. So how are the effects of Estrogen on neuro-protection through the ERK signaling pathway different in females and males? RESULTS REFERENCES 1) Bryant, Damani N.; Dorsa, Daniel M. CREB-CBP Interactions are Important for Sexually Dimorphic Estradiol Neuroprotection. Neuroscience 170, 1261 (2010) 2) Giddabasappa, Anand; Bauler, Matthew; Yepuru, Muralimohan; Chaum, Edward; Dalton, James T.; Eswaraka, Jeetendra. 17-β Estradiol Protects ARPE-19 Cells from Oxidative Stress through Estrogen Receptor-β. Investigative Ophthalmology and Visual Science. May 12, 2010. 3) Liu, Ling-juan; Liu, Li-qun; Bo, Tau; Li, Shi-jun; Zhu, Zhen; Cui, Rong-rong; Mao, Ding-an. Peurarin Suppress Apoptosis of Human Osteoblasts via ERK Signaling Pathway. International Journal of Endocrinology. June 12, 2013. 4) Tasyriq, Mohammad; Najmuldeen, Ibrahim A.; In, Lionel L. A.; Mohamad, Khalit; Awang, Khalijah; Hasima, Noor. 7α-Hydroxy-β-Sitosterol from Chisocheton tomentosus Induces Apoptosis via Dysregulation of Cellular Bax/Bcl-2 Ratio and Cell Cycle Arrest by Down-regulating ERK1/2 Activation. Evidence-Based Complementary and Alternative Medicine. September 11, 2012. pERK ERK Vehicle V 1 V 2 V 3 Estrogen E 1 E 2 E 3 Figure 2. Western Blots and Graph of pERK and Total ERK. Neurons were starved for 5hrs and then treated with (10 nanomolar) Ethanol or Estrogen for 20 minutes. Densitometry of the western blots were graphed to show the relationship between sexes and treatment. 2-way ANOVA showed that there is a statistically significant effect of E2 on ERK phosphorylation/activation between the sexes (p = 0.0427). Female Male pERK ERK Magic Mark Dissected cortices were incubated in Papain (20units/ml) in Hibernate-E minus calcium for 30 minutes at room temperature. Neurons were subsequently dissociated by manual trituration in Hibernate/B27. Neurons were counted and plated on poly-d-lysine coated plates in Neurobasal media containing 2% B27, 1% Glutamax and penicillin- streptomycin (Invitrogen, Carlsbad California). Cultures were maintained in a 5% CO 2 atmosphere for 10 days in vitro (DIV) in a humidified incubator. Neurons cultured as Described in (Bryant & Dorsa, 2010). Vehicle V 1 V 2 V 3 Estrogen E 1 E 2 E 3 Female pERK ERK pERK ERK Male Blue Magic 15 min E2 treatment 20 min E2 treatment