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Elucidating the Molecular Etiology of Levodopa Responsive Dystonia

Title: Elucidating the Molecular Etiology of Levodopa Responsive Dystonia.
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Name(s): Jones, Lataisia C. (Cherie'), author
Bhide, Pradeep, professor directing dissertation
Fadool, Debra Ann, university representative
Nowakowski, Richard S., committee member
Flynn, Heather A., committee member
Gunjan, Akash, committee member
Florida State University, degree granting institution
College of Medicine, degree granting college
Department of Biomedical Sciences, degree granting department
Type of Resource: text
Genre: Text
Doctoral Thesis
Issuance: monographic
Date Issued: 2017
Publisher: Florida State University
Place of Publication: Tallahassee, Florida
Physical Form: computer
online resource
Extent: 1 online resource (103 pages)
Language(s): English
Abstract/Description: My research is designed to test the hypothesis that a single nucleotide polymorphism (SNPs) in the non-coding region of the GCH1 gene suppresses GCH1 mRNA translation and generates a novel peptide, and that both of these phenomena contribute to downregulation of the dopamine synthesis pathways. The GCH1 gene codes for the protein guanosine triphosphate (GTP) cyclohydrolase 1 (GCH1), a key regulator of dopamine synthesis. I focused on a SNP resulting from substitution of a cytosine (C) with thymine (T) at location +142 (position from the transcription start site) in the 5’ untranslated region (UTR) of the GCH1 gene (+142C>T SNP), because it is associated with a heritable movement disorder and subtype of dystonia called DYT5. Thus, my research is designed to investigate molecular mechanisms associated with the pathogenesis of DYT5. To test my hypothesis, I used an in vitro model in which human embryonic kidney (HEK) 293T cells were transfected with cDNA for wild type GCH1, GCH1 containing the +142C>T SNP sequence, or control sequences. Firefly luciferase (Fluc) reporter assays showed that the +142C>T SNP introduces an upstream open reading frame (uORF), which suppresses translation of GCH1. The reduced GCH1 translation fits well with the reduction in GCH1 protein and dopamine reported in DYT5. The uORF introduced by the +142C>T SNP is predicted to synthesize a 73 amino acid novel peptide, which I have named DRDp73. As a first step toward understanding the role of DRDp73 cellular function, I performed a bioinformatics analysis. This analysis predicted existence of a nuclear localization signal, a large “disordered” region and a nucleic acid binding alpha-helical domain in DRDp73 revealing interesting clues to DRDp73’s potential biological function. To test predictions provided by the bioinformatics analysis, I characterized the expression, subcellular localization and the impact of DRDp73 on cell viability. I used a custom-made DRDp73 antibody, and a commercially available GCH1 antibody to detect both of the proteins. I detected DRDp73 and GCH1 in HEK293T cells overexpressing the +142T GCH1 (GCH1 in DYT5) plasmid. However, GCH1 expression in these cells was significantly lower than that in HEK293T cells transfected with wildtype GCH1 plasmid. Since HEK293T cells divide rapidly, I predicted that DRDp73 is degraded and cleared during cellular division. Therefore, in additional studies, I inhibited the proteasome and autophagy pathways, key regulators of protein degradation. Under these circumstances, DRDp73 was localized to the nucleus, consistent with the predicted nuclear localization signal revealed by bioinformatics, and GCH1 was localized to the cytoplasm. In addition, viability of the HEK293T cells expressing DRDp73 was compromised. These findings confirmed that the +142C>T SNP is associated with generation of DRDp73, downregulation of GCH1 expression, and provided evidence that DRDp73 accumulation may adversely affect cell viability. The HEK293T cells do not synthesize dopamine, the neurotransmitter involved in motor function. Moreover, since the HEK293T cells divide rapidly, analysis of the long term effects of protein accumulation becomes difficult. Therefore, I used human origin SK-N-BE(2)-M17 (a twice sub-cloned cell line from the SK-N-BE (2) neuroblastoma cell line) and SH-SY5Y cells (third sub-clone of SK-N-SH neuroblastoma cell line). Both cell lines differentiate into non-dividing cells, express GCH1 and neuronal markers upon differentiation, and are excellent models of dopaminergic cells, such as the midbrain dopaminergic neurons affected in DYT5 dystonia. My data from SK-N-BE(2)-M17 and SH-SY5Y cells show that endogenous GCH1 protein is localized to the nucleus, cytoplasm and processes. In both types of cells transfected with the +142T GCH1 plasmid, DRDp73 also localizes to the nucleus, cytoplasm and processes. GCH1 expression is reduced in the nucleus in both neuroblastoma cells expressing DRDp73, and is found predominantly in the cytoplasm and processes. The dopamine biosynthetic pathway is compromised in DRDp73 expressing SK-N-BE(2)-M17 cells. Thus, the +142C>T SNP downregulated GCH1 expression and compromised dopamine biosynthesis in the neuronal-like cells. In the final set of studies, I analyzed GCH1 expression in lymphoblastoid cells from DYT5 patients. GCH1 expression was reduced in the patient-derived cells compared to that in cells from non-DYT5 individuals. In summary, my work identified dual consequences of DYT5-associated +142C>T SNP in the non-coding region of the GCH1 gene: Suppression of GCH1 translation, and production of the novel DRDp73 peptide. DRDp73 is a novel biomarker for DYT5, and the DRDp73 antibody is a potential novel diagnostic tool. My results expand current understanding of the SNP-uORF encoded proteins, and advances the field of SNP-uORF associated diseases by offering novel mechanistic and functional insights into SNP-uORF encoded peptides. My work also identifies future studies, for example using DYT5 patient derived cells, primary neuronal cultures and animal models that could significantly advance this emerging biomedical field.
Identifier: FSU_SUMMER2017_Jones_fsu_0071E_14035 (IID)
Submitted Note: A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the Doctor of Philosophy.
Degree Awarded: Summer Semester 2017.
Date of Defense: July 19, 2017.
Keywords: dopamine, dystonia, gene, regulation, SNP, uORF
Bibliography Note: Includes bibliographical references.
Advisory Committee: Pradeep G. Bhide, Professor Directing Dissertation; Debra Ann Fadool, University Representative; Richard S. Nowakowski, Committee Member; Heather Flynn, Committee Member; Akash Gunjan, Committee Member.
Subject(s): Molecular biology
Neurosciences
Persistent Link to This Record: http://purl.flvc.org/fsu/fd/FSU_SUMMER2017_Jones_fsu_0071E_14035
Owner Institution: FSU

Choose the citation style.
Jones, L. C. (C. '). (2017). Elucidating the Molecular Etiology of Levodopa Responsive Dystonia. Retrieved from http://purl.flvc.org/fsu/fd/FSU_SUMMER2017_Jones_fsu_0071E_14035