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