Sigma Xi Powerpoint 2014

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  • 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/l 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 1. Properzi, Francesca, Daniela Carulli, Richard A. Asher, Elizabeth Muir, Luiz M. Camargo, Toin H. Van Kuppe