Epigenetic regulation of Cpt1a and Cpt1c and link to multiple sclerosis

Student thesis: Master thesis (including HD thesis)

  • Katrine Jønsson
Introduction: Multiple sclerosis (MS) is a disabling disease that affects more than 2 million people worldwide. Carnitine palmitoyl transferase 1A (CPT1A) is an enzyme involved in fatty acid oxidation and mutations in the gene coding for this protein decreases the activity of the enzyme, which has shown to decrease the incidence of multiple sclerosis. Epigenetics of multiple sclerosis is a relatively new field and only a few studies has to date investigated the role of epigenetics of Cpt1a and Cpt1c, whereas there are no studies describing the expression and regulation of both genes in blood brain barrier (BBB)-associated cells and an experimental autoimmune encephalomyelitis (EAE) model. Aim: The aim of this study was to investigate the expression of Cpt1a and Cpt1c in vitro in rat primary BBB cells and in vivo in EAE mouse model of MS and investigate the epigenetic landscape of the two genes. Methods: This study investigates the relative mRNA expression of Cpt1a and Cpt1c in vitro in primary rat endothelial cells, astrocytes, and pericytes before and after treatment with histone deacetylase (HDAC) inhibitors and in vivo in brain tissue of a murine EAE model by reverse transcriptase-quantitative polymerase chain reaction. Immunocytochemistry was applied to investigate the amount of protein in primary cells before and after treatment with HDAC inhibitors. DNA methylation analysis was performed to investigate the fraction of DNA methylation in vitro before and after treatment and in vivo in controls and EAE animals. This study further investigated histone modifications in vivo by chromatin immunoprecipitation. Results: The results showed that both genes were expressed in BBB-associated cells, which was never shown before. The endothelial cells presented with the highest expression of Cpt1a, whereas pericytes presented with the lowest. Furthermore, astrocytes presented with the highest expression of Cpt1c. After treatment with HDAC inhibitors an increased expression of Cpt1a and Cpt1c is seen in endothelial cells and pericytes, whereas astrocytes presented with a decreased expression of both genes after treatment. Immunocytochemistry supported these results. DNA methylation analysis showed increased fraction of DNA methylation in endothelial cells and astrocytes after treatment compared to controls and decreased DNA methylation after treatment in pericytes compared to controls. In vivo, EAE animals presented with a decreased expression of Cpt1c compared to controls. DNA methylation analysis demonstrated a tendency of increased DNA methylation in EAE animals compared to controls. Chromatin immunoprecipitation demonstrated a decreased acetylation of histone 4 of Cpt1c in EAE mice compared to controls. Conclusion: Cpt1a and Cpt1c are epigenetically regulated in vitro. Furthermore, the expression of Cpt1c decreases in vivo in EAE animals and is epigenetically regulated.
Publication date30 May 2018
Number of pages61
ID: 280106930