This document describes a study aiming to induce axonal regeneration in retinal ganglion cells through epithelial-mesenchymal transition. The researcher hypothesized that overexpressing epithelial-mesenchymal transition master regulators in retinal ganglion cells through AAV viruses would cause them to regain the ability to grow axons after injury. The experimental design involved cloning master regulators into AAV2 plasmids, transfecting retinal ganglion cells, performing optic nerve crush surgery, and observing axon regeneration. Preliminary results found that master regulators like Twist induced more axonal regeneration compared to controls, indicating the ability to initiate regeneration in senescent tissue.

1. The Induction of Axonal Regeneration
by Epithelial-MesenchymalTransition
in Retinal Ganglion Cells
Allison Belette
Bascom Palmer Eye Institute, University of Miami

2. 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.oregoneye
center.com/images/ey
es_glaucoma.jpg

3. 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

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

5. Long-Term Goal
 Reprogram the cells back to their embryonic state
where they were able to grow axons

6. What is Epithelial to
MesenchymalTransition?
 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 cells
http://jcs.biologists.org/content/118/19/4325/F1.small.gif

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

8. Experimental Design
Zeb1
AAV2
Slug
AAV2
Snai1
AAV2
Twist
AAV2
e47
AAV2
Clone EMT Master
Regulators Into
AAV2 Plasmid
Insert Plasmid with
Gene of Interest
intoVirus
Perform Optic
Nerve Crush &
Observe Axon
Regeneration

9. 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

10. Checking Insert Size & Excision
Slug Twist1
3 kb
3 kb
2 kb
1.5 kb
2 kb
1.5 kb
1 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
Figure1
Lane1: 1kb ladder, Lane2: SlugAAV2
plasmid digested with SmaI, HindIII,
Lane3: Undigested SlugAAV2 plasmid
Figure 2
Lane1: 1kb ladder, Lane2:Twist1AAV2
plasmid digested with BamHI, Xho1,
Lane3: UndigestedTwist1AAV2 plasmid

11. 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

12. 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 andTwist 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.

13. 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

14. VirusTiterValues
 Zeb had lowest titer value; not large enough to transfect the RGCs
 YFP and E47 had highest concentration in comparison toTwist, Slug, and Snai

15. Transfection Efficiency in RGCs
YFP  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

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

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

18. Optic NerveVisual 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 containingYFP AAV2, the control that did not
contain any MR sequence
 Second optic nerve was infected was infected with virus containing AAV2 plasmid
withTwist 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 representsTwist optic nerve that was as an outlier as it was 1 of 20
Twist optic nerves that demonstrated immense axonal regeneration

19. QuantificationNumberofaxons

20. Discussion
Large standard deviation inTwist 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

21. 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

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