Synthesizing FingRs for a Greater Understanding of Parkinson’s Disease Mechanisms

Synthesizing FingRs for a Greater Understanding of Parkinson’s Disease Mechanisms Introduction Results Parkinson’s Disease (PD): Parkinson’s is a neurodegenerative disease characterized by a loss of dopamine-producing neurons, leading to motor deficits. However, there is currently little known about how the Figure 1. This indicates the death of dopaminergic neurons leads to difference in dopamine levels disrupted synaptic changes that affect the between a Parkinson’s patient 2 (right) and a control (left) ability to learn and use motor functions. FingRs: In order to study the synaptic changes after the onset of PD, molecular cloning was used to create fibronectin intrabodies generated with mRNA display (FingRs), a novel fluorescence-based genetic tool. FingRs bind to neuronal proteins to visualize excitatory and inhibitory synapses. In this case, the FingRs were designed to bind to the Gephyrin protein, a marker for inhibitory postsynaptic regions. Research objective: Using fluorescent visualization of synapses allows for a greater understanding of the way synapses are disrupted after the onset of PD and will hopefully lead to more effective treatments of the disease. Stbl3 Cell Transformation: The plasmid was successfully transformed into the Stbl3 cells as shown by the colonies grown on the bacterial plate that contains an antibiotic and kills off colonies without the plasmid. Methods mScarlet Construct Gibson Assembly Virus Production Plasmid DNA Purification Stbl3 Cell Transformation Gibson Assembly: DNA fragment ligations can be accomplished in a single reaction through Gibson assemblies. In this case, the red fluorescent tag (mRuby or mScarlet) was inserted into a plasmid backbone. Plasmid Backbone mRuby/ mScarlet insert Plasmid Stbl3 Cell Transformation: The plasmid DNA was transformed into Stbl3 E. coli cells. Transformation of plasmid into Stbl3 cells Plasmid Stbl3 Cell with mRuby/ mScarlet gene Place cells on LB agar bacterial plates Bacterial Plate Plasmid DNA Purification: Minipreps (50-100 ng/μL) and maxipreps (100500 ng/μL) were performed to purify and elute the DNA. Cell Harvest Cell Lysis Bind DNA to Matrix Wash DNA Matrix DNA Elution Virus Production: HEK293 cells were used to create an adeno-associated virus (AAV) that can be injected into the mice brains and label the targeted synapses with red fluorescence. DNA from Maxiprep Helper Vector Plate Hek293 Cells Replication Hek293 Cell Rep/Cap Vector AAV Transfection Viral Proteins AAV ssDNA AAV Particle Viral Packaging Figure 2. Bacterial colonies for mScarlet after transformation Figure 3. Bacterial colonies for mRuby after transformation Sequencing: Sequencing of the FingRs after the minipreps and maxipreps demonstrated high fidelity in the sequencing based on what was desired. mRuby Construct A B C D Gibson Forward mRuby GPHN.FingR A B C D WPRE Gibson Forward mScarlet GPHN.FingR WPRE A. Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element – when transcribed, it creates a tertiary structure that enhances gene expression B. Forward primer for the Gibson Assembly C. Fluorescent tags inserted into the backbone D. Allows the FingR to bind to the Gephyrin protein Conclusion The mRuby and mScarlet FingRs were successfully synthesized based on the high fidelity in sequencing. These FingRs will be helpful as a genetic tool for a greater understanding of why PD causes synaptic changes that affect motor function and hopefully help with treatment of the disease. Future Work After injecting the FingRs into the mice brain, a neurotoxin, 6-OHDA, that simulates PD by destroying dopaminergic neurons can be injected into half of the brain. After imaging, the red fluorescence from the injected FingRs can be used to compare the dopamine-producing inhibitory synapses in each half of the brain.