The Induction of Axonal Regenerationby Epithelial-Mesenchymal Transition

in Retinal Ganglion Cells

Allison BeletteBascom Palmer Eye Institute, University of Miami

Glaucoma

Affects over 60 million individuals worldwide

Pressure accumulates in retina causing damage to optic nerve

Optic nerve contains cell structures called axons that take signals from retina to brain

Damage to axons causes lack of function (blindness) which is commonly nonreversible

http://www.oregoneyecenter.com/images/eyes_glaucoma.jpg

Current Problem

Regeneration of axons in the Central Nervous System does not occur in adult mammals

This is partially due to the fact that, naturally during development, axons lose the ability to grow

Introduction

If you think of an axon as a straw, if you extend the straw to its maximum length and cut the straw, no matter how hard you try to pull the straw, it will never reach its original maximum length

Neurons extend their axons until they reach their adult stage where they reach their maximum length

If an axon is injured, it will never be able to regenerate and reach that same maximum length

http://www.nature.com/nrn/journal/v2/n11/images/nrn1101-831a-f1.gif

http://www.polyvore.com/cgi/img-thing?.out=jpg&size=l&tid=75883183

Long-Term Goal

Reprogram the cells back to their embryonic state where they were able to grow axons

What is Epithelial to Mesenchymal Transition?

EMT Process where cells in

epithelial state gain mobility, lose adhesion proteins, and are able to move about freely

Master Regulators (transcription factors) known to cause this process that I use in my project: Zeb1 E47 Slug Snai1 Twist1

Occurs in cancerous cells, not in retinal ganglion cellshttp://jcs.biologists.org/content/118/19/4325/F1.small.gif

Hypothesis

If overexpressed in Retinal Ganglion Cells (RGCs), these master regulators (MRs) will induce a similar process to Epithelial-Mesenchymal Transition (EMT) in RGCs and cause the RGCs to regain their ability to grow axons after injury

Experimental Design

Zeb1

AAV2 Slug

AAV2

Snai1

AAV2 Twis

t AAV

2e47 AAV

2

Clone EMT Master

Regulators Into AAV2 Plasmid

Insert Plasmid with Gene of Interest into

Virus

Perform Optic Nerve Crush & Observe Axon Regeneration

Original Plasmid

AAV2 Plasmid

RESTRICTION ENZYME DIGESTION

Master Regulator

Restriction Enzymes

Zeb1 Xba I & EcoR I

E47 Hind III & Xho I

Slug Sma I & Hind III

Snai1 Xba I & Xho I

Twist1 BamH I & Xho I

Performed restriction enzyme digestion to remove each master regulator from its original plasmid and place it in AAV2 plasmid

With MRs, I also removed 3XFlag sequence that would be used to ensure presence and proper cloning of MR sequence in the AAV2 plasmid

Checking Insert Size & Excision

Slug Twist1

3 kb3 kb

2 kb1.5 kb

2 kb

1.5 kb1 kb

1 kb

0.5 kb 0.5

kb

1 2 3 321

Performed gel extraction to remove MR insert from original plasmid vector

Master regulator and 3XFlag from digested plasmid

Figure1Lane1: 1kb ladder, Lane2: Slug AAV2 plasmid digested with SmaI, HindIII, Lane3: Undigested Slug AAV2 plasmid

Figure 2Lane1: 1kb ladder, Lane2: Twist1 AAV2 plasmid digested with BamHI, Xho1, Lane3: Undigested Twist1 AAV2 plasmid

Checking Sequencing: Twist

Plasmid DNA was sequenced to ensure proper cloning of 3XFlag sequence and MR sequence into AAV2 plasmid

Verification of presence of correct master regulator and 3XFlag sequences

Immunostaining Human Embryonic Kidney (HEK) cells with Slug AAV2, Twist AAV2, and Control with

DAPI and FLAG stains

Immunostaining of HEK cells performed to view presence of 3XFlag sequence and therefore assure presence and proper cloning of MR sequence in plasmids

Blue coloring depicts DAPI which labels cell nuclei, while green coloring depicts 3XFlag expression

Stained with DAPI to demonstrate that 3XFlag and DAPI are colocalized in nuclei of the cells

In third column, when compared to the control, Slug and Twist both displayed presence of 3XFlag sequence and therefore presence of the MR sequence

DAPI / 3XFLAG DAPI 3XFLAG

Figure 6 Immunostaining HEK cells with Slug master regulator gene, Twist master regulator gene, and Control, respectively, with DAPI and FLAG stains.

Maxipreps

Performed Maxipreps to amplify the amount of plasmid DNA

>1000 ng/μl concentration, >1.80 ration of DNA to protein, and >2.00 ratio of DNA to solvent needed to make viruses

Virus Titer Values

Zeb had lowest titer value; not large enough to transfect the RGCs

YFP and E47 had highest concentration in comparison to Twist, Slug, and Snai

Transfection Efficiency in RGCsYFP RGCs were

transfected with the viruses

Green coloring depicts 3XFlag expression

YFP and e47, which had the highest titer values also had the largest transfection efficiencies

Surgery Timeline

• Ctxb-594 is stain used to label axonal regeneration in red

Axonal Regeneration

YFP

Figure 7. Visual Representation of Twist optic nerve section axonal regeneration compared to that of YFP optic nerve section axonal regeneration.

*

Optic Nerve Visual Representation continued

Representation of 3 specific optic nerves in which axonal regeneration was quantified

Axonal regeneration was quantified from site of ONC to where axonal regeneration ended (represented by colored bands)

Number of axons quantified at specific length intervals from ONC site

First optic nerve was infected with virus containing YFP AAV2, the control that did not contain any MR sequence

Second optic nerve was infected was infected with virus containing AAV2 plasmid with Twist MR sequence

Twist optic nerves demonstrated more axonal regeneration than the control optic nerves

Similar results were seen in all MR optic nerves

Third optic nerve represents Twist optic nerve that was as an outlier as it was 1 of 20 Twist optic nerves that demonstrated immense axonal regeneration

QuantificationN

umbe

r of

axo

ns

Discussion

Large standard deviation in Twist was caused by the one optic nerve outlier

The presence of the master regulator sequences caused more axonal regeneration than in the control

EMT-like transition in RGCs indicating the ability of these cells to initiate regeneration in senescent tissue

Research Applications

Project is not only applicable in the retina

Axons extend from the Central Nervous System to several parts of the body

Individuals who suffer from spinal cord injuries, strokes, or traumatic brain injuries suffer from loss of function throughout the body

For example, there are 1/50 Americans (approximately 5.6 million individuals) who suffer from paralysis

http://medicalterms.info/img/uploads/anatomy/spinal-cord.gif

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12. Unpublished research performed by Jeffrey L. Goldberg in Bascom Palmer Eye Institue in the University of Miami