Graduation Year
2024
Document Type
Master's Thesis
Degree
Master of Science
Program
Biological Science
Program Director
Meredith Protas, PhD
First Reader
Lisa Ellerby
Second Reader
Danilo Medinas
Abstract
The protein disulfide isomerases (PDIs) are a family of enzymes within the endoplasmic reticulum (ER) that catalyze disulfide bond formation in proteins. This posttranslational modification— a process termed redox protein folding— regulates protein function and structural stability. The genetic loss-of-function mutation c.170G>A in PDIA3 causes a severe intellectual disability (ID) during childhood. To understand how PDIA3 c.170G>A causes this disease, we engineered a knockin mouse model, Pdia3 c.170G>A, using CRISPR/Cas9 technology to model the endogenous disease pathology more accurately. We hypothesized that the ID pathology and behavioral deficits would be present in the Pdia3 c.170G>A knockin mice. In congruence with our hypothesis, assessment of recognition memory, long-term spatial memory, and contextual memory showed a deficit in the Pdia3 c.170G>A heterozygous mice, constituting abnormal behavior reminiscent of the human condition. The spontaneous working memory, exploration/anxiety behavior, general mobility/activity, and weight of these mice, however, were not statistically significantly altered by the mutation. Additionally, we performed proteomics analysis to understand the molecular mechanisms for the impaired learning of the Pdia3 c.170G>A knockin mice. We found pathways enriched to respiratory electron transport, ATP biosynthesis, Alzheimer’s disease, tricarboxylic acid cycle, Huntington’s, and Parkinson’s disease. Overall, our results demonstrated we could model aspects of severe ID with the Pdia3 c.170G>A knockin mouse model. Consequently, the first part of this thesis provides foundational research for the analysis of redox protein folding and the result of stunting PDIA3 function, which is a pivotal part of the redox protein folding machinery.
Cellular senescence is a post-mitotic state in which cells secrete proinflammatory factors, may lose cell-type-specific functions, and become apoptosis-resistant. Although senescence has useful biological functions in development and cancer immune response, an accumulation of senescent cells is associated with most age-related diseases including dementia. The first stage in cellular senescence is cell-cycle arrest, induced by either the p16/ retinoblastoma (Rb) or the p21/p53 pathway. Although a great deal of research has been done to understand the p16/Rb pathway, much less is known about the p21/p53 pathway. A recently generated p21-clearance mouse model suggests the removal of p21-positive cells is beneficial in bone aging models. However, how removal of p21 cells impacts cognition is unknown. We therefore hypothesize that the chemical induction of cellular senescence in this mouse model will result in cognitive deficits that may subsequently be ameliorated by the removal of p21-positive cells in the brain. In this pilot project, we attempted to induce brain cell senescence in mice using doxorubicin to study its cognitive impacts. We then measured recognition and spatial memory, general mobility and activity, and weights after doxorubicin treatment. We found no statistically significant trends of decline in long-term spatial memory as a result of doxorubicin treatment and no noticeable deficits in recognition memory, weights, or general mobility and activity. This pilot study concludes that doxorubicin does not have a statistically significant impact on cognitive function in this particular mouse system. Since the trend for our study was toward decreased cognitive function in doxorubicin-treated mice, we may need to use a larger number of mice to make a final conclusion.
Comments
This submission includes 2 chapters within the same document. There is one title page, acknowledgments, table of contents, list of figures, list of abbreviations, and references sections. There is, however, two introductions, materials and methods, results, and discussion sections.