The effects of increased camp levels on neuronal differentiation in murine embryonic stem cells, and the creation of a CRISPR-induced c.1252c>t point mutation in the ADCY5 gene

Author

Elizabeth Zepeda, Humboldt State UniversityFollow

Graduation Date

Fall 2018

Document Type

Thesis

Program

Master of Science degree with a major in Biology

Committee Chair Name

Amy Sprowles

Committee Chair Affiliation

HSU Faculty or Staff

Second Committee Member Name

Bruce O'Gara

Second Committee Member Affiliation

HSU Faculty or Staff

Third Committee Member Name

John Steele

Third Committee Member Affiliation

HSU Faculty or Staff

Fourth Committee Member Name

Christina Waters

Fourth Committee Member Affiliation

Community Member or Outside Professional

Keywords

ADCY5, dyskinesia, movement disorders

Subject Categories

Biology

Abstract

ADCY5-related dyskinesia is a rare movement disorder with early onset in childhood and adolescence. Previous studies linked this disease to various point mutations in the ADCY5 gene. Recent studies show that two of the point mutations cause an increase in cyclic adenosine monophosphate (cAMP) levels. However, it remains unknown how increased levels of cAMP result in the phenotypes associated with this disease. My study examines the effects of increased cAMP levels on neuronal differentiation of mouse embryonic stem cells (mESCs). My experiments demonstrated successful differentiation of mESCs into the dopaminergic neuronal lineage, indicated by the presence of Tuj 1 (a class III beta-tubulin neuronal cell marker), and tyrosine hydroxylase (TH) (an enzyme found in dopaminergic neurons), as detected by immunocytochemistry (ICC). To determine the effect of cAMP on this process, murine embryonic stem cells were differentiated in the presence of a range of db-cAMP concentrations. Cells treated with 700 μM of db-cAMP during the differentiation step of the protocol showed increased percentage of the neuronal cell-type, followed by a decrease in percent neuronal-cell type treated with 1400 μM and 2800 μM db-cAMP. There was no significant effect of db-cAMP on percent dopamine-containing neurons. In addition, there were lower levels of tyrosine hydroxylase (TH) protein present in the membrane fraction, in cells treated with 1400 μM db-cAMP compared to no treatment, and no significant change in cytosolic TH. My results also revealed an increase in dopamine receptor D2 in both the membrane and cytosol. In order to characterize the effects of the point mutation p.R418W on cellular cAMP levels in HEK293T cells, I attempted to create a mutation in a HEK293T cell line using CRISPR. Once the mutant cell line was generated, the plan was to treat with pharmacological agonists to beta-adrenergic receptors to stimulate cAMP production in mutated cells as well as control cells. The amount of cAMP produced in mutated cells versus wild-type would then be quantified using enzyme-linked immunosorbent assay (ELISA). After the first round, CRISPR failed to produce the desired mutation, the guide RNA was modified, and the process repeated three times. Although we successfully created the desired edit in the third round, other mutations directly flanking upstream and downstream predicted that ADCY5 protein would be non-functional, so the ELISA was not performed. Overall our results provide a model of the molecular basis of ADCY5 related-dyskinesia, such that high levels of cAMP during early development decreases neuronal cell-type production, as well as exerting its downstream effects on D2 (dopamine 2) receptor and tyrosine hydroxylase protein expression, thereby disrupting the dopamine pathway. Understanding the pathophysiological mechanisms underlying this disease will help differentiate ADCY5 related-dyskinesia from other motor disorders, in order for clinicians to make informed decisions about treatment. Future steps will include the use of patient iPSCs to individually diagnose mutation type, quantify cAMP production level, and customize drug treatment.

Citation Style

CSE

Recommended Citation

Zepeda, Elizabeth, "The effects of increased camp levels on neuronal differentiation in murine embryonic stem cells, and the creation of a CRISPR-induced c.1252c>t point mutation in the ADCY5 gene" (2018). Cal Poly Humboldt theses and projects. 221.
https://digitalcommons.humboldt.edu/etd/221