"This web page was produced as an assignment for Genetics 564, an undergraduate capstone course at UW-Madison."
Introduction
Lewy body dementia is a progressive neurodegenerative disease characterized by small protein aggregates, called Lewy bodies, in the neurons in the brain. Lewy bodies consist primarily of misfolded alpha synuclein proteins. Normally, misfolded proteins are not a problem, as the cell degrades them through various mechanisms. However, in Lewy body dementia, these protein aggregates form through the improper degradation of these misfolded alpha synuclein proteins. Lewy bodies form in the presynaptic terminals of neuron cells, ultimately affecting proper neuron function or causing neuron cell death (2).
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The SNCA gene is associated with lewy body dementia. SNCA encodes the alpha synuclein protein. The molecular function of the SNCA gene involves copper binding (3). Interestingly, increased levels of copper has been found in patients with Parkinson's disease through Cu(II) binding to alpha synuclein (3). Furthermore, SNCA's biological process involves regulating dopamine neurotransmission, likely through the maintenance of synaptic vesicles (2). Lastly, SNCA localizes in the cytoplasm of the neuron cells.
SNCA consists of one synuclein domain, and the N terminus of the protein has been shown to be important for proper protein folding (5). SNCA is highly conserved throughout vertebrate model organisms, making it an easy gene to study. Interestingly, human SNCA shares a high percent identity with the SNCA protein of an ancient mammalian relative, the Coelacanth. This conservation shows that SNCA has been highly conserved throughout a large part of evolution.
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Primary goal
It has been shown that patients with Lewy body dementia over express this SNCA protein, leading to a disruption in dopamine signaling. However, it is not known how SNCA regulates this dopamine signaling and brain function. Therefore, my primary goal of this project is to determine how SNCA regulates dopamine expression. To do this, I will use zebrafish to assay dopaminergic neurons and behavioral changes in dopamine deficient zebrafish versus wild type fish. Zebrafish are a great model organism to study Lewy body dementia because they share similar brain structure to humans, their neurons are easily observed, and they have behavioral phenotypes that are clear and easy to assay, such as highly irregular swimming patters in dopamine deficient fish (4).
Specific aim 1
Aim 1 is to identify conserved amino acids in alpha synuclein that are important for regulating dopamine release. To do this, I used Clustal Omega to align the SNCA protein sequences of nine different species, including the model organisms and other closely related species. Then, paying close attention to the less characterized C terminus, I found two regions, region A and region B, that were highly conserved, but differed in the zebrafish.
I will then use CRISPR/Cas9 to edit these two regions, generating two mutants, mutant A and mutant B. In mutant A, I will use CRISPR/Cas9 to change the glutamate residue (E) to a glycine (G) residue. In mutant B, I am doing a similar editing, but this time changing the glutamine residue (Q) to a glutamate (E). In doing this, I am changing the differing amino acid in the zebrafish, to the amino acid found in humans, and many of the other species.
I will then assay for differences in dopaminergic neuron expression and behavioral changes. For these results, it would be expected that mutant A, a strain with near wild type dopamine expression levels, would show much more regular swimming patters and a higher expression of dopaminergic neurons compared to mutant B, a strain with decreased dopamine expression levels.
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Specific aim 2
In aim 2, I will identify small molecules that rescue dopamine expression. I will screen wild type, mutant A and mutant B zebrafish embryos against a focused chemical library. I will then assay the fish's dopaminergic neuron expression and swimming behavior. I would the expect molecules that interact with and rescue the dopamine expression of mutant B to revert the zebrafish's swimming behavior and dopaminergic neuron expression levels to near wild type.
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Specific aim 3
Aim 3 is to use Co-IP and mass spectrometry to identify proteins that interact with alpha synuclein. I will use Co-immunoprecipitation on wild type, mutant A and mutant B SNCA proteins to isolate any interacting proteins. Then, I will use mass spectrometry to identify and characterize those interacting proteins. I expect that the proteins interacting with wild type and mutant A will be different than the proteins interacting with the dopamine deficient mutant B. Identifying those proteins that interact with wild type and mutant A, but not mutant B, may be important for proper dopamine release and regulation.
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Conclusions and future directions
Future directions involve characterizing the function of the unknown proteins in the zebrafish protein interaction network. These proteins may work to regulate dopamine release directly, or maintain the machinery necessary for dopamine release. Characterization of these interacting proteins can lead to a better understanding of dopamine expression and help develop novel drugs for this debilitating disease.
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References
2. SNCA Gene-Genetics Home Reference. (2017). Retrieved from https://ghr.nlm.nih.gov/gene/SNCA#conditions
3. Neurodegenerative Diseases-German Research School for Simulation Sciences. Retrieved from http://www.grs-sim.de/research/computational-biophysics/research-activity/neurodegenerative-diseases.html
4. 1.Huang, J., Zhong, Z., Wang, M., Chen, X., Tan, Y., Zhang, S., He, W., He, X., Huang, G., Lu, H., Wu, P., Che, Y., Yan, Y., John, P. H., Chen, W., Wang, H. (February, 2015). Circadian Modulation of Dopamine Levels and Dopaminergic Neuron Development Contributes to Attention Deficiency and Hyperactive Behavior. The Journal of Neuroscience, 35 (6); 2572- 2587. DOI: https://doi.org/10.1523/JNEUROSCI.2551-14.2015
5. Siddiqui, I. J., Pervaiz, N., & Abbasi, A. A. (2016). The Parkinson's Disease Gene SNCA: Evolutionary and structural insights with pathological implication. Scientific Reports, 6, 1-11. DOI: 10.1038/srep24475
3. Neurodegenerative Diseases-German Research School for Simulation Sciences. Retrieved from http://www.grs-sim.de/research/computational-biophysics/research-activity/neurodegenerative-diseases.html
4. 1.Huang, J., Zhong, Z., Wang, M., Chen, X., Tan, Y., Zhang, S., He, W., He, X., Huang, G., Lu, H., Wu, P., Che, Y., Yan, Y., John, P. H., Chen, W., Wang, H. (February, 2015). Circadian Modulation of Dopamine Levels and Dopaminergic Neuron Development Contributes to Attention Deficiency and Hyperactive Behavior. The Journal of Neuroscience, 35 (6); 2572- 2587. DOI: https://doi.org/10.1523/JNEUROSCI.2551-14.2015
5. Siddiqui, I. J., Pervaiz, N., & Abbasi, A. A. (2016). The Parkinson's Disease Gene SNCA: Evolutionary and structural insights with pathological implication. Scientific Reports, 6, 1-11. DOI: 10.1038/srep24475