Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Physiology and Pharmacology

Collaborative Specialization

Musculoskeletal Health Research

Supervisor

Séguin, Cheryle A.

Abstract

This study used mice lacking equilibrative nucleoside transporter 1 (ENT1-/-) as a preclinical model to study pathogenesis of ectopic spine mineralization in diffuse idiopathic skeletal hyperostosis (DISH). We hypothesized that mineralization of the annulus fibrosus (AF), was driven by dysregulation of cellular processes and pathways associated with apoptosis, S100A9 proteins, the PI3K-Akt pathway, and lipid metabolism. Target pathways and processes were assessed using in situ localization and quantitative analyses. Metabolomic analysis of AF and plasma data was conducted to identify altered metabolites. Results demonstrate increased caspase-3 activity in the AF of ENT1-/- mice at both timepoints and decreased S100A9 in the AF of ENT1-/- mice at 2 months. PI3K and Akt proteins levels were decreased in male and female ENT1-/- mice at 6 months. Metabolomic analyses of AF and plasma suggested dysregulation in lipid metabolism and a decrease in betaine and choline levels, which may be linked to ectopic mineralization.

Summary for Lay Audience

Diffuse idiopathic skeletal hyperostosis (DISH) is a disease where soft connective tissues of the spine undergo mineralization. Symptoms of DISH include as pain and stiffness in the back, and in severe cases, difficulties in swallowing, decreased range of motion, or compression of the spinal cord. Recent studies have shown that 15-25% of people aged fifty and over suffer from DISH. Despite how common DISH is, the cause of the disease remains unknown and there are no disease-modifying treatments that can stop, or slow mineral formation. A gene-modified mouse model lacking the equilibrative nucleoside transporter 1 (ENT1-/-) has been established as a preclinical model of DISH since mice display progressive dystrophic calcification of fibrocartilaginous tissues of the spine, including the annulus fibrosus (AF) of the intervertebral disc with a spatial and temporal pattern that mimics DISH in humans. To understand the biology underlying ectopic spine mineralization, ENT1-/- mice were studied at 2 months (beginning of disease) and 6 months (established mineralization of the spine). Based on prior research in the Séguin lab, we hypothesized that mineralization of the spine is related to the disruption of processes and proteins linked to the breakdown of fat, cellular death, the caspase-3 protein, the S100A9 protein, and the PI3K-Akt pathway. To identify possible molecular mechanisms, we studied the localization or activation of targets of interest in AF and the difference of metabolite amounts in AF and blood of ENT1-/- mice compared to WT. We found cellular death was increased at 2 and 6 months and S100A9 protein amounts were decreased at 2 months in the AF of ENT1-/- mice. We then found that PI3K and Akt total protein amounts are decreased at 6 months in the AF of ENT1-/- mice, which may underlie the overall decrease in the cell survival in the AF and be associated with mineral formation. Finally, we found that multiple metabolites linked to the digestion of fats, betaine, and choline were dysregulated in the ENT1-/- mice. In conclusion, this work found several molecular changes that associate with calcification in the AF, which may further our understanding on the mechanism of DISH.

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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