Epigenetic regulation of Cpt1a and Cpt1c and link to multiple sclerosis
Translated title
Epigenetisk regulering af Cpt1a og Cpt1c og link til multipel sklerose
Author
Jønsson, Katrine
Term
4. term
Publication year
2018
Submitted on
2018-05-30
Pages
61
Abstract
Multipel sklerose (MS) er en invaliderende sygdom, der rammer over to millioner mennesker på verdensplan. Carnitinpalmitoyltransferase 1A (CPT1A) er et enzym, der hjælper celler med at bruge fedt som energi. Varianter, der nedsætter CPT1A-aktiviteten, er sat i forbindelse med lavere forekomst af MS. Epigenetik – kemiske ændringer på DNA og histonproteiner, som påvirker genaktivitet uden at ændre DNA-sekvensen – kan bidrage til at forklare sådanne sammenhænge. Der vides dog lidt om epigenetisk regulering af de beslægtede gener Cpt1a og Cpt1c i blod-hjerne-barriere (BBB)-celler eller i dyremodeller for MS. Vi undersøgte, hvor og hvor meget Cpt1a og Cpt1c udtrykkes i primære rotte-BBB-celler (endotelceller, astrocytter og pericytter) i kultur og i hjernevæv fra mus med eksperimentel autoimmun encefalomyelitis (EAE), en udbredt MS-model. Vi kortlagde også epigenetiske markeringer på disse gener og testede effekten af histondeacetylase (HDAC)-hæmmere, stoffer der påvirker histonacetylering. Vi målte genaktivitet (mRNA) med RT-qPCR og proteinmængder med immunocytokemi. Vi vurderede DNA-methylering – andelen af cytosiner med methylgrupper – før og efter HDAC-hæmmerbehandling in vitro og i EAE- versus kontrolmus in vivo. In vivo blev histonmodifikationer analyseret med kromatin-immunpræcipitation. Vi viser for første gang, at begge gener udtrykkes i BBB-associerede celler. Cpt1a var højest i endotelceller og lavest i pericytter, mens Cpt1c var højest i astrocytter. Efter HDAC-hæmmerbehandling steg udtrykket af begge gener i endotelceller og pericytter, men faldt i astrocytter; immunocytokemi understøttede disse mønstre. DNA-methylering steg efter behandling i endotelceller og astrocytter og faldt i pericytter sammenlignet med ubehandlede kontroller. In vivo havde EAE-mus lavere Cpt1c-udtryk end kontroller, ledsaget af en tendens til højere DNA-methylering og reduceret histon H4-acetylering ved Cpt1c. Konklusion: Cpt1a og Cpt1c er epigenetisk regulerede i dyrkede BBB-celler, og Cpt1c-udtrykket er nedsat og epigenetisk ændret i EAE-modellen. Resultaterne udvider forståelsen af, hvordan fedtstofskiftegener kan reguleres i MS-relevante celler og tilstande.
Multiple sclerosis (MS) is a disabling disease affecting over two million people worldwide. Carnitine palmitoyl transferase 1A (CPT1A) is an enzyme that helps cells use fat for energy. Variants that reduce CPT1A activity have been linked to lower MS incidence. Epigenetics—chemical changes to DNA and histone proteins that influence gene activity without altering the DNA sequence—may help explain such links. However, little is known about the epigenetic regulation of the related genes Cpt1a and Cpt1c in blood-brain barrier (BBB) cells or in animal models of MS. We examined where and how much Cpt1a and Cpt1c are expressed in primary rat BBB cells (endothelial cells, astrocytes, and pericytes) grown in culture and in brain tissue from mice with experimental autoimmune encephalomyelitis (EAE), a widely used MS model. We also mapped epigenetic marks on these genes and tested the effect of histone deacetylase (HDAC) inhibitors, compounds that modify histone acetylation. We measured gene activity (mRNA) by RT-qPCR and protein levels by immunocytochemistry. We assessed DNA methylation—the fraction of cytosines carrying methyl groups—before and after HDAC inhibitor treatment in vitro and in EAE versus control mice in vivo. In vivo histone modifications were assessed by chromatin immunoprecipitation. We show for the first time that both genes are expressed in BBB-associated cells. Cpt1a was highest in endothelial cells and lowest in pericytes, while Cpt1c was highest in astrocytes. After HDAC inhibitor treatment, Cpt1a and Cpt1c expression increased in endothelial cells and pericytes but decreased in astrocytes; protein staining supported these patterns. DNA methylation increased after treatment in endothelial cells and astrocytes and decreased in pericytes compared with untreated controls. In vivo, EAE mice had lower Cpt1c expression than controls, accompanied by a tendency toward higher DNA methylation and reduced histone H4 acetylation at Cpt1c. Conclusion: Cpt1a and Cpt1c are epigenetically regulated in cultured BBB cells, and Cpt1c expression is reduced and epigenetically altered in the EAE model. These findings broaden our understanding of how fat-metabolism genes may be regulated in MS-relevant cells and conditions.
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