Specific Aims
Down syndrome is caused by a trisomy of chromosome 21 (having three copies instead of two copies) [1]. This extra copy of chromosome 21 is a result of abnormal cell division during development. Several neuronal defects are observed in affected individuals in their developmental years [2]. Down syndrome cannot be cured, and symptoms will stay with individuals for the rest of their lives. The current support and treatment can only help affected individuals manage their symptoms. Although several genes may be involved, the DYRK1A is one gene that has been associated with cognitive defects in Down syndrome [2]. DYRK1A encodes a kinase that plays a role in neural development and neuronal differentiation [3]. Interestingly, DYRK1A also mediates cell proliferation [4], and it is unclear if the levels of DYRK1A protein may mediate cell division events during neural development.
My primary goal is to determine how DYRK1A modulates the cell cycle during neural development. I hypothesize that the DYRK1A kinase downregulates cell cycle factors leading to reduced cell proliferation during critical neural development periods. I will use wild-type and DYRK1A mutant zebrafish (Danio rerio) as a model because the DYRK1A domains are well-conserved, and their transparent nervous systems allow for the visualization of neural proliferation. My long-term goal is to analyze how neural development is impacted by cell cycle modulation.
Aim 1: Identify the specific DYRK1A domains that are necessary for cell cycle processes in the brain.
Approach: First, NCBI BLAST will be used to determine the homologs of DYRK1A with diverse nervous systems. The homolog sequences will be aligned using ClustalOMEGA, and highly conserved domains will be determined. I will then create DYRK1A mutants by using CRISPR/Cas9 to knockout the identified domains one at a time. The neural proliferation in the mutant zebrafish will be compared with the neural proliferation in the wild-type zebrafish.
Rationale: DYRK1A is known to reduce neuronal development and is also known to have phosphorylating capabilities through its P-kinase regions. Removing regions of the gene that may play a role in mediating the cell cycle in neurons is a priority as these will phenocopy the human disease.
Hypothesis: Knockout of the P-kinase region in species with diverse nervous systems will be necessary for mediating cell division in neurons.
Aim 2: Identify genes in the brain that are expressed differently within the DYRK1A mutants
Approach: Brain tissue samples will be taken from both wild-type and the DYRK1A mutant zebrafish from Aim #1. The gene expression profiles of wild-type and P-kinase knockout mutants will be determined by conducting RNA sequencing on these tissue samples. Through GO analysis, the differentially expressed genes will be sorted into categories such as neurogenesis, neural cell differentiation, and neural cell proliferation.
Rationale: Identifying genes that are differentially expressed in DYRK1A mutants will elucidate the interactions of DYRK1A that play a role in neural cell proliferation.
Hypothesis: Wild-type and DYRK1A mutant zebrafish will differ in gene expression profiles related to neural cell proliferation. Specifically, the DYRK1A mutants without the P-kinase domain will have increased expression of neural cell proliferative genes.
Aim 3: Determine DYRK1A protein interactions that regulate the cell cycle in neurons in the brain
Approach: I will determine the difference in protein interactions between DYRK1A protein with and without the P-kinase domain. By using BioID, proteins that interact with DYRK1A will be tagged in brain samples of wild-type and mutant zebrafish without a P-kinase domain. Using affinity chromatography, the proteins that are tagged from both samples will be purified and analyzed using mass spectrometry. Next, we can identify the proteins and group them by gene ontology groups such as neurogenesis, neural cell differentiation, and neural cell proliferation to understand the biological and functional relationships with DYRK1A.
Rationale: Identifying DYRK1A protein interaction changes in the brain between wild-type and DYRK1A mutants will further elucidate the biological processes that mediate neural cell proliferation.
Hypothesis: DYRK1A interacts with proteins that are implicated in mediating neural cell proliferation through its P-kinase domain.
Through these specific aims, I expect to elucidate how the specific domains, gene expression profiles, and protein interactions of DYRK1A are involved in mediating cell division and neural proliferation in the brain. This research is especially crucial because it can lead to a better understanding of the biological mechanisms that lead to the pathology of neurodegenerative diseases such as Down syndrome.
My primary goal is to determine how DYRK1A modulates the cell cycle during neural development. I hypothesize that the DYRK1A kinase downregulates cell cycle factors leading to reduced cell proliferation during critical neural development periods. I will use wild-type and DYRK1A mutant zebrafish (Danio rerio) as a model because the DYRK1A domains are well-conserved, and their transparent nervous systems allow for the visualization of neural proliferation. My long-term goal is to analyze how neural development is impacted by cell cycle modulation.
Aim 1: Identify the specific DYRK1A domains that are necessary for cell cycle processes in the brain.
Approach: First, NCBI BLAST will be used to determine the homologs of DYRK1A with diverse nervous systems. The homolog sequences will be aligned using ClustalOMEGA, and highly conserved domains will be determined. I will then create DYRK1A mutants by using CRISPR/Cas9 to knockout the identified domains one at a time. The neural proliferation in the mutant zebrafish will be compared with the neural proliferation in the wild-type zebrafish.
Rationale: DYRK1A is known to reduce neuronal development and is also known to have phosphorylating capabilities through its P-kinase regions. Removing regions of the gene that may play a role in mediating the cell cycle in neurons is a priority as these will phenocopy the human disease.
Hypothesis: Knockout of the P-kinase region in species with diverse nervous systems will be necessary for mediating cell division in neurons.
Aim 2: Identify genes in the brain that are expressed differently within the DYRK1A mutants
Approach: Brain tissue samples will be taken from both wild-type and the DYRK1A mutant zebrafish from Aim #1. The gene expression profiles of wild-type and P-kinase knockout mutants will be determined by conducting RNA sequencing on these tissue samples. Through GO analysis, the differentially expressed genes will be sorted into categories such as neurogenesis, neural cell differentiation, and neural cell proliferation.
Rationale: Identifying genes that are differentially expressed in DYRK1A mutants will elucidate the interactions of DYRK1A that play a role in neural cell proliferation.
Hypothesis: Wild-type and DYRK1A mutant zebrafish will differ in gene expression profiles related to neural cell proliferation. Specifically, the DYRK1A mutants without the P-kinase domain will have increased expression of neural cell proliferative genes.
Aim 3: Determine DYRK1A protein interactions that regulate the cell cycle in neurons in the brain
Approach: I will determine the difference in protein interactions between DYRK1A protein with and without the P-kinase domain. By using BioID, proteins that interact with DYRK1A will be tagged in brain samples of wild-type and mutant zebrafish without a P-kinase domain. Using affinity chromatography, the proteins that are tagged from both samples will be purified and analyzed using mass spectrometry. Next, we can identify the proteins and group them by gene ontology groups such as neurogenesis, neural cell differentiation, and neural cell proliferation to understand the biological and functional relationships with DYRK1A.
Rationale: Identifying DYRK1A protein interaction changes in the brain between wild-type and DYRK1A mutants will further elucidate the biological processes that mediate neural cell proliferation.
Hypothesis: DYRK1A interacts with proteins that are implicated in mediating neural cell proliferation through its P-kinase domain.
Through these specific aims, I expect to elucidate how the specific domains, gene expression profiles, and protein interactions of DYRK1A are involved in mediating cell division and neural proliferation in the brain. This research is especially crucial because it can lead to a better understanding of the biological mechanisms that lead to the pathology of neurodegenerative diseases such as Down syndrome.
Specific Aims Drafts:
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References:
1. Feki, A., & Hibaoui, Y. (2018). DYRK1A Protein, A Promising Therapeutic Target to Improve Cognitive Deficits in Down Syndrome. Brain sciences, 8(10), 187. Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210095/
2. Nguyen, Thu Lan et al. “Correction of cognitive deficits in mouse models of Down syndrome by a pharmacological inhibitor of DYRK1A.” Disease models & mechanisms vol. 11,9 dmm035634. 27 Sep. 2018. Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5125364/?report=classic
3. Liu, Y., Lin, Z., Liu, M., Wang, H., & Sun, H. (2017). Overexpression of DYRK1A, a Down Syndrome Candidate gene, Impairs Primordial Germ Cells Maintenance and Migration in zebrafish. Scientific reports, 7(1), 15313. Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681638
4. Thompson, B. J., Bhansali, R., Diebold, L., Cook, D. E., Stolzenburg, L., Casagrande, A. S., Besson, T., Leblond, B., Désiré, L., Malinge, S., & Crispino, J. D. (2015). DYRK1A controls the transition from proliferation to quiescence during lymphoid development by destabilizing Cyclin D3. The Journal of experimental medicine, 212(6), 953–970. https://doi.org/10.1084/jem.20150002
5. Olson L.E., et al. Down syndrome mouse models Ts65Dn, Ts1Cje, and Ms1Cje/Ts65Dn exhibit variable severity of cerebellar phenotypes. Dev. Dyn. 2004;230:581–589.
6. Liu, X, et al An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and sub-cellular localizations. Nat Commun. 2018 Mar 22;9(1):1188. doi: 10.1038/s41467-018-03523-2.
7. Liu, X., Salokas, K., Tamene, F. et al. An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations. Nat Commun 9, 1188 (2018). https://doi.org/10.1038/s41467-018-03523-2
This web page was produced as an assignment for Genetics 564, an undergraduate capstone course at UW-Madison.