Using Tet1 and Lipofection to catalyze memory extinction in PTSD BY ATREYI MITRA Post Traumatic Stress Disorder Constant vivid recollection of traumatic memory Hippocampus becomes smaller memory recall is too fast + inaccurate Ventromedial prefrontal cortex becomes smaller cant control amygdala Hypothalamus secretes hormones adrenal glands become hyperactive high levels of cortisol Increased chance of other neurological disorders Ventromedial prefrontal cortex DNA methylation and the role of Tet1 - DNMTs add a methyl group to CpG islands to form a 5-methylcytosine - MBDs (methyl CpG binding proteins) bind and attract proteins to turn off the gene w/ 5mC - Suppresses other genes and becomes tightly coiled - Cant bind to the protein no mRNA produced - Tet1 plays an important role in DNA demethylation - Important role in conversion to 5hmC through passive demethylation - 5hmC will eventually form cytosine through active demethylation DNMTs 5hmC Using Tet1KO mice to show memory extinction Memory extinction is the decline of intensity and frequency of a memory A control and Tet1KO mice both went through contextual fear conditioning in cage 24 hours later, both had similar freezing levels since conditioning 24 more hours later, Tet1KO had much higher freezing levels than one of control Tet1KO mouse had impaired memory extinction in comparison to control What was going on in the brain? Tet1KO was shown to have a down regulation of important neuronal activity-related genes Hypermethylation of Npas4 C-Fos important for memory extinction Arc, Egr2, Egr4 Decrease in 5hmC and increase in 5mC in hippocampus and cortex Tet1KO tended to experience LTD (long term depression) Could still form new memories because fear stimulus is strong enough to lower methylation of Npas4 and then activate Tet2 and Tet3 Levels of 5mC and 5hmC in Control and Tet1 KO Can increasing Tet1 expression in transgenic mice aid memory extinction in Post Traumatic Stress Disorder? Researchable Question The Experiment 1)Inject plasmids into mice 2)Fear conditioning with hot plates 3)Test for freezing levels of control + transgenic mice 4)Determining the levels of 5mC and 5hmC 5)Assess important neuronal activated-regulated genes 6)Sacrifice mice and use their hippocampal slices to check for traces of LTD Lipofection Tet1 CRISPR/Cas9 KO plasmid Tet1 CRISPR activation plasmid Liposome w/ genetically engineered pY antibodies on cell membrane Scalpel Stereotaxic machine Anesthesia Microinjector Fear Conditioning + Freezing Levels Hot plate cages w/ bell + fear stimulus Alternate cages w/bell Image FZ Microarray Analysis Buffer solution Columns with Oligo-dT Beads Solvent solution Labelling mixes (3 different colors) Microarray analysis solution Microarray Scanner and workstation Pipetts + pipetmen + tips Microcentrifuge Vortex Sample tubes 5hmC + 5mC Analysis Master Mix PCR thermal cycler machine EpiMark 5-hmC and 5-mC Analysis Kit Water Incubator Hippocampal slices Low Calcium Cerebral Fluid Vibrotome Folded paper towel Beebee scissors Bone rongeurs Hippocampus tool Surgical scissors Thine/wide spatula 100 mm glass petri dish Plastic spoon *More than listed Using Pre-engineered Plasmids Tet1 CRISPR/Cas9 KO plasmid Inhibits Tet1 gene expression through a disruption or replacement of artificial DNA 3 plasmids w/ Cas9 nuclease + gRNA of Tet1 Tet1 CRISPR activation plasmid Synergistic Activation Mediator (SAM) transcription activation system used to overexpress upstream expression of Tet1 2-3 plasmids Deactivated Cas9 restriction enzyme + VP64 (transactivation domain) + blasticidan resistance gene MS2-p65-HSF1 chimeric protein + hygromycin resistance gene Courtesy to Life Technologies Using a liposome to act as a vector for the plasmid Place the plasmids into liposomes with genetically engineered pY antibodies pY antibodies will only bind to the pY antigens which will ensure accuracy Lipid bilayer on both the liposome and neuron will allow for easy pass through of the liposome into nucleus The plasmid will then incorporate itself to the genome Using a liposome to act as a vector for the plasmid pY antibodies Liposome Tet1 KO/Overexpression Plasmid Neuron pY antigen Plasmid incorporated in genome Using Stereotaxic Technique to Transfect Mice In vivo, postnatal surgery taken place when a mouse is just a baby Microinjector with glass syringe of micrometers diameter Use anesthesia to ensure a painless surgery Identify area of interest (into the CA3 region of hippocampus using stereotaxic coordinates Inject approximately 50 nanometers of liposome solution onto surface Contextual Fear Conditioning -Both placed in a cage with a hot plate and are shocked after hearing a 30 second warning signal for 2 seconds -Mice will learn to associate sound with fear -This procedure should repeat sporadically each hour for one day to build fear Testing For Freezing Levels Mice tend to freeze in fear in response to a fear stimulus After the first week, the mice will return to another similar triangular cage The 30 second sound will play again to stimulate memory recall in both mice Can be measured by human observations or automated systems Image FZ uses a camera and specialized software to calculate the freezing time by analyzing the distance travelled Microarray Analysis on Neuronal Activated-Regulated Genes Microarray analysis is often used to express contrast in gene expression between different samples by analyzing mRNA Get a tissue sample from hippocampus CA3 region Extract mRNA and then create cDNAs with reporters using poly-T primers, reverse transcriptase, and dNTPs Spread cDNA onto the microarray and analyze the contrast in gene expression through different colors of each of the samples Tet1 Overexpression Red Tet1KO Blue Tet1+/+ - Green Determining Levels of 5mC and 5hmC with the Epimark 5-hmC and 5-mC Analysis Kit Analyzes a specific locus and then measures the levels of 5mhC and 5mC Use T4-BGT to add glucose to 5-hmC to form 5- ghmC to change Msp1 restriction site to become blocked Restriction enzymes cut the sites very specifically Perform PCR and results give the levels Courtesy to New England Biolabs Looking for traces of LTD: Preparation Decapitate mice Use scalpel to make incisions from the nasal to occipital bone Use rongeurs to cut through occipital + frontal + parietal plates Slide brain out and immediately immerse in calcium free ACSF Separate the hippocampi from the rest of brain using the scalpel Get a 400 micrometer slice of hippocampus using the microtone Transfer the hippocampal slice to recording chamber Analyzing the hippocampal slices Allow slice to acclimate on the recording chamber for 15 minutes Lower the stimulating electrodes down to the CA1 region till detection of slice is found With Clamp X, we can figure out the synaptic strength and plasticity by delivering multiple stimuli in varying intensity levels LTD by a decrease in synaptic strength LTP by an increase in synaptic strength Anticipated Results Tet1 Overexpression Tet1KOTet1+/+ Freeze Conditioning Relatively low60%20% Levels of 5mC + 5hmC More 5mhCMore 5mCEqual Neuronal Activated- Regulated Genes YesNoYes Hippocampus slices of LTD NoYesNo (if no other factors) Relevance of another treatment? 7-8 % of people had or will have PTSD Current medications are not completely reliable Correlation between current treatment and likelihood to get involved with drugs and self inflicted harm Many possible future implications Bibliography "DNA Microarray." DNA Microarray. Web. 30 July http://learn.genetics.utah.edu/content/labs/microarray/ Mathis, D. M., Furman, J. L., Norris, C. M. Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology. J. Vis. Exp. (49), e2330, doi: /2330 (2011) "Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology." Preparation of Acute Hippocampal Slices from Rats And... Web. 30 July "Related Products." Arraystar. Web. 30 July "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 30 July Rudenko, Andrii, Meelad Dawlaty, Jinsoo Seo, Albert Cheng, Jia Meng, Thuc Le, Kim Faull, Rudolph Jaenisch, and Li-Huei Tsai. "Tet1 Is Critical for Neuronal Activity-Regulated Gene Expression and Memory Extinction." 18 Sept Web. 30 July Schnyder, Anita, and Jrg Huwyler. "Drug Transport to Brain with Targeted Liposomes." NeuroRx. The American Society for Experimental NeuroTherapeutics, Inc. Web. 30 July Web. 30 July https://www.neb.com/products/e3317-epimark-5-hmc-and-5-mc-analysis-kit Bibliography (Images) https://classconnection.s3.amazonaws.com/475/flashcards/721475/jpg/brain_ jpg https://classconnection.s3.amazonaws.com/475/flashcards/721475/jpg/brain_ jpg monoclonal-antibody-production/antibody.pngmonoclonal-antibody-production/antibody.png