The Role of G-Protein Coupled Estrogen Receptor (GPER) in Early Neurite Development. Kyle Pemberton
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1 The Role of G-Protein Coupled Estrogen Receptor (GPER) in Early Neurite Development Kyle Pemberton
2 Acknowledgement Dr. Xu Lab Members Brittany Mersman Nicki Patel Pallavi Mhaskar Jason Cocjin Committee Members Dr. Christopher Arnatt, Dr. Judith Ogilvie, Dr. Susan Spencer, Dr. Yuqi Wang
3 Outline Introduction Methods Results Future Direction
4 Outline Introduction Methods Results Future Direction
5 Introduction G-Protein-Coupled Receptors (GPCR) Membrane receptors Most diverse receptor type in eukaryotes Signals through secondary messenger Drug targets Li, J. et al. The Molecule Pages database. Nature 420, (2002).
6 Estrogen Primary female sex hormone Heim (1966) showed estrogen increases maturation rate of developing brain Estrogen studies on neuron development have been ongoing for decades without controlling for GPER PPT may activate GPER, not as selective for ERα as once thought (Srivastava, 2013) ER α and ER β may not play as many roles in estrogen signaling as previously believed
7 What is GPER G-Protein Coupled Estrogen Receptor (GPR30/GPER) Officially named Estrogen receptor in 2007 (Srivastava, 2013) Strongest response to E2 (17β-estradiol) G γ
8 GPER Research Has focused on classical estrogen receptors ER α and ER β Differs from traditional Estrogen Receptors GPER is Largely located in non-nuclear membranes GPER mediates Fast, non-genomic action GPER GPER Golgi ERα GPER Endoplasmic Reticulum Nucleus ERβ
9 Previous research about GPER functions in the nervous system Evidence of role in neuronal ion channel modulations: Intracellular Ca++ Mobilization from ER (Tran, 2015; Romano, 2008) Regulation of K+ channels (Broselid, 2014) Neuroprotective effects in PC-12 cells (Alyea, 2008) Increased post stroke recovery in sex specific manner(gibson, 2016) Research almost solely in mature neurons
10 Previous research: development 4 hours 36 hours 18 hours 48 hours 24 hours 72 hours Shi et al Biochem. Biophys. Res. Commun., 2013, 435, pp
11 Outline Introduction Methods Results Future Direction
12 Hypothesis Activation of GPER increases neurite outgrowth in early development
13 Methods: culture Culture rat embryonic day 18 neurons in estrogen free medium Agonists Antagonists Control Vehicle 10 nm G1 100 nm G1 1 um G1 10 nm G nm G15 1 um G15 Each treatment imaged at 8 hours, 20 hours, 48 hours, 72 hours, and 96 hours
14 Methods: quantifying neurite growth Initial Image
15 Methods: quantifying neurite growth Initial Image Adjust Image
16 Methods: quantifying neurite growth Initial Image Trace Growth Adjust Image
17 Outline Introduction Methods Results Future Direction
18 Results: cortical neurons Control G1 100 nm 1 um G nm 1 um
19 Results: cortical neurons
20 Control Vehicle G1 10nM G1 100nM G1 1uM G15 10nM G15 100nM G15 1uM Control Vehicle G1 10nM G1 100nM G1 1uM G15 10nM G15 100nM G15 1uM Control Vehicle G1 10nM G1 100nM G1 1uM G15 10nM G15 100nM G15 1uM Control Vehicle G1 10nM G1 100nM G1 1uM G15 10nM G15 100nM G15 1uM Control Vehicle G1 10nM G1 100nM G1 1uM G15 10nM G15 100nM G15 1uM Neurite Length (um) Results: cortical neurons *, p < 0.05 vs Control **, p < 0.01 vs Control #, p < 0.05 vs Vehicle ##, p < 0.01 vs vehicle Neurite outgrowth after 8 96 hours ## # ** ## ** ## ** ## # * ## ** ## ** ## ** ## ** ## 0 8 Hour 20 hour 48 hour 72 Hour 96 hour Time And Treatment
21 Results Hypothesis Activation of GPER increases neurite outgrowth in early development In cortical neurons-? Activation of GPER has no effect on neurite outgrowth, but blocking the receptor inhibited neurite outgrowth.
22 Results In cortical neurons-? Activation of GPER has no effect on neurite outgrowth, but blocking the receptor inhibited neurite outgrowth. High levels of endogenous E2 in early development may saturate GPER in cortex Cell specific co-localization may need activation of partner
23 Outline Introduction Methods Results Future Direction
24 Future directions Sub-cellular localization at different developmental times in cortex and hippocampus Co-localization of GPER with other growth promoting proteins Test other agonists of GPER and other estrogen receptors Ca++ mobilization in cortical neuron development Investigate second messenger pathways
25 Literature cited Gaudet, H. M., Cheng, S. B., Christensen, E. M., Filardo, E. J. (2015). "The G-protein coupled estrogen receptor, GPER: The inside and inside-out story." Mol Cell Endocrinol 418 Pt 3: LOUISE M. HEIM; Effect of Estradiol on Brain Maturation: Dose and Time Response Relationships, Endocrinology, Volume 78, Issue 6, 1 June 1966, Pages , Srivastava, D. P. and P. D. Evans (2013). "G-protein oestrogen receptor 1: trials and tribulations of a membrane oestrogen receptor." J Neuroendocrinol 25(11): Hara, Y., Waters, E. M., McEwen, B. S., & Morrison, J. H. (2015). Estrogen Effects on Cognitive and Synaptic Health Over the Lifecourse. Physiological Reviews, 95(3), Tran, Q. K., et al. (2015). "Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b." J Biol Chem 290(21): Romano, N., et al. (2008). "Nonclassical estrogen modulation of presynaptic GABA terminals modulates calcium dynamics in gonadotropin-releasing hormone neurons." Endocrinology 149(11): Broselid, S., et al. (2014). "G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits camp production." J Biol Chem 289(32): Alyea, R. A., et al. (2008). "The roles of membrane estrogen receptor subtypes in modulating dopamine transporters in PC-12 cells." J Neurochem 106(4): Gibson, C. L. and P. M. Bath (2016). "Feasibility of progesterone treatment for ischaemic stroke." J Cereb Blood Flow Metab 36(3): Shi, Y., Liu, X., Zhu, P., Li, J., Sham, K. W., Cheng, S. H., Li, S., Zhang, Y., Cheng, C. H., Lin, H.(2013) "G-protein-coupled estrogen receptor 1 is involved in brain development during zebrafish (Danio rerio) embryogenesis." Biochem Biophys Res Commun 435(1): Saunders, B., Lyon, S., Day, M., Riley, B., Chenette, E., & Subramaniam, S. (2008). The Molecule Pages database. Nucleic Acids Research, 36(Database issue), D700 D706.
26 Questions?
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