Sigma Xi Powerpoint 2014
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Transcript of Sigma Xi Powerpoint 2014
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
References1. Properzi, Francesca, Daniela Carulli, Richard A. Asher, Elizabeth Muir, Luiz M. Camargo, Toin H. Van Kuppevelt, Gerdy B.
Ten Dam, Yoko Furukawa, Tadishima Mikami, Kazuyuki Sugahara, Toshihiko Toida, Herbert M. Geller, and James W. Fawcett. "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, 20 Jan. 2005. Web. 24 Sept. 2013. http://www.ncbi.nlm.nih.gov/pubmed/15673437.
2. Goldberg, Jeffrey L., Mauricio E. Vargas, Jack T. Wang, Wim Mandemakers, Stephen F. Oster, David W. Sretavan, and Ben A. Barres. "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, 26 May 2004. Web. 24 Sept. 2013. http://www.ncbi.nlm.nih.gov/pubmed/15163691.
3. Moore, Darcie L. "KLF Family Members Regulate Intrinsic Axon Regeneration Ability." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 25 Oct. 2012. http://www.ncbi.nlm.nih.gov/pubmed/19815778.
4. De Lima, Silmara. "Full-length Axon Regeneration in the Adult Mouse Optic Nerve and Partial Recovery of Simple Visual Behaviors." Full-length Axon Regeneration in the Adult Mouse Optic Nerve and Partial Recovery of Simple Visual Behaviors. PNAS, 21 May 2012. Web. 25 Oct. 2012. http://www.pnas.org/content/early/2012/05/15/1119449109.abstract.
5. Park, Kevin K. "Promoting Axon Regeneration in the Adult CNS by Modulation of the PTEN/mTOR Pathway." Promoting Axon Regeneration in the Adult CNS by Modulation of the PTEN/mTOR Pathway. Science Mag, 2008. Web. 25 Oct. 2012. http://www.sciencemag.org/content/322/5903/963.short.
6. Sun, Fang. "Sustained axon regeneration induced by co-deletion of PTEN and SOCS3." National Center for Biotechnology Information. U.S. National Library of Medicine, 2011. Web. 25 Oct. 2012. http://www.ncbi.nlm.nih.gov/pubmed/22056987.
7. Lee, Jonathan M. " Molecular requirements for epithelial-mesenchymal transition during tumor progression." National Center for Biotechnology Information. U.S. National Library of Medicine, 2005. Web. 25 Oct. 2012. http://www.ncbi.nlm.nih.gov/pubmed/16098727.
8. Casas, Esmeralda. "Snail2 Is an Essential Mediator of Twist1-Induced Epithelial Mesenchymal Transition and Metastasis." Cancer Research. Cancer Research, 01 Jan. 2011. Web. 25 Oct. 2012. http://cancerres.aacrjournals.org/content/71/1/245.short.
9. Hung, J. J. " Prognostic significance of hypoxia-inducible factor-1α, TWIST1 and Snail expression in resectable non-small cell lung cancer." National Center for Biotechnology Information. U.S. National Library of Medicine, 23 Sept. 2009. Web. 25 Oct. 2012. <http://www.ncbi.nlm.nih.gov/pubmed/19778933>.
10. Blackmore, Murray G., Zimei Wang, Jessica K. Lerch, Dario Motti, Yi P. Zhang, Christopher B. Shields, Jae K. Lee, Jeffrey L. Goldberg, Vance P. Lemmon, and John L. Bixby. "Krüppel-like Factor 7 Engineered for Transcriptional Activation Promotes Axon Regeneration in the Adult Corticospinal Tract." PNAS (2012): n. pag. Web. 3 Sept. 2013.
11. Yin, Yuqin, Michael T. Henzl, Barbara Lorber, Toru Nakazawa, Tommy T. Thomas, Fan Jiang, Robert Langer, and Larry I. Benowitz. "Oncomodulin Is a Macrophage-derived Signal for Axon Regeneration in Retinal Ganglion Cells." Nature Neuroscience 9.6 (2006): 843-52. Print.
12. Unpublished research performed by Jeffrey L. Goldberg in Bascom Palmer Eye Institue in the University of Miami