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The Role of EphA7 in Synapse Formation during Cortical Development
Marie-Sophie van der GoesGeorgetown University Senior, Neurobiology
Sigma Xi Student Virtual Research Showcase March 13 - 23 2013
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Background
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Johnston et al, 2009
The formation of synapses is mediated by signaling molecules
Signaling molecules in the post-synaptic density
Feng & Zhang, 2009
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Signaling molecules in the post-synaptic density
Feng and Zhang, 2009
Eph-Ephrins are a receptor-ligand family of signaling molecules
between neurons
Alexandra Russo, 2011
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Steps in the shaping of post-synaptic protrusions in layers IV-V of the cortex
Alexandra Russo, 2011
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Noren And Pasquale 2004
Egea and Klein 2007
Structure and Function of EphA-EphrinA
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Miller et al 2006
Complementary expression pattern of EphA7 and EphrinA5 in the mouse
cortexE 15.5 P 0
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Question: What role plays EphA7 receptor in the formation of the synapses in layers IV-V of the cortex?Hypothesis:EphA7 mediates signaling early in development to regulate protrusion of potential post-synaptic sites
EphA7 signaling ?
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3) DIV 14 - 18 - 21: Single whole-cell recordings
1) Ex Utero Electroporation (Mouse )
2) Primary Cortical Cell Culture
Experimental Procedure
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Experimental Procedure (Continued)
5) AMPA mediated mEPSCs with acute treatment with TTX+BMR
4) Selection for Regular Spiking Neurons
What is a mEPSC ?
Pharmacological isolation of AMPA (excitatory) receptors
Anderen & Soleng, 1999
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Minis allow to measure Synaptic Strength
Parameters of Measurement:
-Frequency (# synaptic sites)
-Amplitude (# receptors)
Slide credit: Bridget Queenan
Results (a): In vitro Recordings show a slowed maturation of the synapses of A7-/- cortical
neurons
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Results(b): Spine analysis shows similar deficiencies in A7-/- neurons at
DIV 18
WT
EphA7 -/-
PSD 95 MergeGFP
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Next Research Step: More Background
Carrie Leonard (2012)
The FL form is expressed early in development, the TR later
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Structure of two alternative splice forms of EphA7 Receptor
Miller et al, 2006
FL: Full Length sequence
TR: the protein lacks the intracellular catalytic domain
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Holmberg et al., 2000
Red: EphA7-expressing cellsGreen: Bed of EphrinA5-expressing cells
FL A7 TR A7
EphrinA5-EphA7-FL mediates repulsive signaling between the cells, EphrinA5-
EphA7-TR adhesive
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Question: What is the specific role of each form of EphA7 receptor in the
formation of the appropriate synapses in Layers IV-V of the cortex?
A7FL signaling ? A7TR signaling ?
Hypothesized mechanism of action of the two alternative splice forms of
EphA7 receptorEphA7FL
EphA7TR
1) In Utero Electroporation: E 15.5
Genetic Manipulation in A7-/-:a) dsRed+A7FLb) dsRed+A7TR
Experimental Procedure
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2) Slice recording
4) Analysis of AMPA-mediated mEPSCs
3) Biocytin injection of untransfected neurons
Experimental Procedure (Continued)
WT
A7 -/-
A7 -/- + TRA7 rescue
Significance across age
Significance within age
Preliminary Results from In vivo Experiments *
*Note: Technical difficulties have prevented data collection for FL-A7 rescue experiments and increase in the N. The lack of normal distribution and the low N prevent test with Two-Way ANOVA. Preliminary T-tests are shown.
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Preliminary conclusions from in vivo experiments
- In WT synapses get stronger after P14 in terms of active sites, but not in terms of post-synaptic receptors
- The average number of active sites is larger in the TR rescue than in WT and in KO at P14-16
- The average strength of the post-synaptic sites is larger in the TR rescue than in WT at P12-13 and P14-16 and also than in KO at P14-16
-The strength of post-synaptic sites increases in KO from P14-16 to P17-19
P13 P16
Future Directions-Add experimental group/ increase N (KO, A7FL and A7TR
Rescues)
-Perform Immunohistochemical Analysis
-Correlate “cell function” with “cell morphology” (Confocal Microscopy)
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References-Ciossek, T., Lerch, M. M. and Ullrich, A. (1995). Cloning, characterization, and differential expression of MDK2 and MDK5, two novel receptor tyrosine kinases of the eck/eph family. Oncogene 11, 2085-2095.
-Ciossek, T., Lerch, M. M. and Ullrich, A. (1999) Identification of alternatively spliced mRNAs encoding variants of MDK1, a novel receptor tyrosine kinase expressed in the murine nervous system. Oncogene 10(1): 97-108
-Noren and Pasquale, E. (2003) Eph receptor-ephrin bidirectional signals that target Ras and Rhoproteins. J. Cellular Signaling,16 (2004) 655–666
-Goel A., Lee H. K. (2007). Persistence of experience-induced homeostatic synaptic plasticity through adulthood in superficial layers of mouse visual cortex. J. Neurosci. 27, 6692–6700. doi: 10.1523/JNEUROSCI.5038-06.2007.
-Holmberg J, Clarke DL, Frisen J (2000) Regulation of repulsion versus adhesion by different splice forms of an Eph receptor. Nature 408:203–206.
-Miller K, Kolk SM, Donoghue MJ (2006) EphA7-ephrin-A5 signaling in mouse somatosensory cortex: developmental restriction of molecular domains and postnatal maintenance of functional compartments. J Comp Neurol 496:627–642.
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Acknowledgements
- Stefano Vicini, PhD-Maria Donoghue, PhD-John Partridge, PhD-Rupa Lalchandani-Bridget Queenan-Meredith Clifford, PhD-Xiumei Zhao-Carrie Leonard-Wardah Athar-Chao Chen-Ruixi Luo