Epigenetic regulation of Cpt1a and Cpt1c and link to multiple sclerosis

Abstract

Multipel sklerose er en invaliderende sygdom, der påvirker mere end 2 millioner mennesker på verdensplan. Carnitin palmitoyl transferase 1A (CPT1A) er et enzym, der er involveret i fedtsyre metabolismen. Dens funktion er at omdanne acyl-CoA til acylcarnitin, så denne kan passere den impermeable mitokondriale membran for dernæst at kunne fortsætte i β-oxidation. I to populationer har det vist sig, at en mutation i genet, der koder for CPT1A, medfører en nedsat incidens af multipel sklerose. Epigenetik af multipel skerose er et relativt nyt felt, hvor kun få studier har undersøgt epigenetikken bag Cpt1a og Cpt1c. Der er på nuværende tidspunkt ingen der har undersøgt udtrykkelsen og reguleringen af disse to gener i blod hjerne barriere-associerede celler og i en eksperimentiel autoimmun encephalomyelitis (EAE) model. Dette studie undersøger den relative mRNA udtrykkelse af Cpt1a og Cpt1c in vitro i rotte primærceller, endothelceller, astrocytter og pericytter, både før og efter behandling med histon deacetylase (HDAC) inhibitorer og in vivo i hjernevæv fra en EAE model. Dette undersøges ved RT-qPCR. Desuden bliver immuncytokemi brugt for at undersøge udtrykkelsen af CPT1A proteinet i primærceller før og efter behandling med HDAC inhibitorer. Både in vitro før og efter behandling og in vivo i EAE modellen bliver DNA methylering undersøgt. Derudover bliver histon modifikationer undersøgt in vivo ved hjælp af kromatin immunprecipitation. Dette studie finder, at begge gener er udtrykt i blod hjerne barriere-associerede celler, hvilket ikke før er blevet vist. Endothelcellerne havde den højeste udtrykkelse af Cpt1a, mens pericytterne havde den laveste udtrykkelse af genet. Derudover havde astrocytterne den højeste udtrykkelse af Cpt1c sammenlignet med endothelceller og pericytter. Efter behandling med HDAC inhibitorer ses en øget udtrykkelse af Cpt1a og Cpt1c i endothelceller og pericytter, hvorimod der ses en nedsat udtrykkelse af Cpt1a og Cpt1c i astrocytter. Immuncytokemi bakkede op om disse resultater. DNA methyleringsanalysen viste en øget DNA methylering i endothelceller og astrocytter efter behandling sammenlignet med kontroller og nedsat DNA methylering efter behandling i pericytter sammenlignet med kontrol. EAE mus udviste nedsat udtrykkelse af Cpt1c sammenlignet med kontroller. Kromatin immunprecipitation viste en nedsat acetylering af histon 4 af Cpt1c i EAE mus sammenlignet med kontroller. Cpt1a og Cpt1c er dermed epigenetisk reguleret in vitro, hvorimod Cpt1c er epigenetisk reguleret in vivo.

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.

A master thesis from Aalborg University

Epigenetic regulation of Cpt1a and Cpt1c and link to multiple sclerosis

[Epigenetisk regulering af Cpt1a og Cpt1c og link til multipel sklerose]