Transcript FerriseSpr14x

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
Male
pERK
Male
pERK
Ratio of pERK/Total ERK
2.0
1.0
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Magic
Mark
2.5
20 min E2
treatment
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ERK
1.5
Vehicle
Estrogen
15 min E2
treatment
Vehicle
Estrogen
1.5
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ERK
Sex
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).
CONCLUSIONS AND FUTURE STUDIES
1.0
0.5
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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.
Estrogen
E1
E2
E3
pERK
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MATERIALS AND METHODS
ERK
M
V1
Blue Magic
Estrogen
E1 E2 E3
pERK
Female
Vehicle
V2
V3
Female
ERK
RESULTS
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?
Vehicle
V1 V2 V3
Ratio of pERK/Total ERK
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 neuroprotection. 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.
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 penicillinstreptomycin (Invitrogen, Carlsbad California). Cultures
were maintained in a 5% CO2 atmosphere for 10 days
in vitro (DIV) in a humidified incubator. Neurons cultured
as Described in (Bryant & Dorsa, 2010).
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ABSTRACT
Sex
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).
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.
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.
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.
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.