An explorative study of iron homeostasis and the contribution of ferroptosis to disease pathogenesis in NPC2(Gt(LST105)BygNya) mice
Translated title
Et eksplorativt studie af jern homeostasen og ferroptoses bidrag til sygdomspatogenesen i NPC2(Gt(LST105)BygNya) mus
Author
Zinn, Mikela Reinholdt
Term
4. term
Publication year
2024
Submitted on
2024-05-31
Pages
115
Abstract
Dette eksplorative studie undersøgte, om jern akkumulerer i hjernen hos NPC2Gt(LST105)BygNya (NPC2-/-) mus, om sådanne ændringer er forbundet med ferroptose, og om blod-hjerne-barrieren (BBB) er intakt. NPC2-/- mus i alderen 6, 8 og 12 uger samt aldersmatchede kontrolmus blev analyseret. Jern, kobber og zink i cerebrum og cerebellum blev målt med ICP-OES, mens RT-qPCR vurderede mRNA-ekspression af gener for jernhåndtering (tfrc, ferritin, dmt1, ferroportin, metallothionein III), ferroptosemarkører (gpx4, SEC24b, acsl4) og BBB-integritet (claudin 5, occludin, zo-1). Immunohistokemi visualiserede GPX4 i neuroner (NeuN eller Calbindin) og TFRC, ELISA målte GPX4-protein, og Western blot vurderede TFRC og ferritin. Resultaterne pegede på jernophobning særligt i cerebellum hos NPC2-/- mus og ledsagende transskriptionelle ændringer i jerntransport og -lagring (øget ferritin og ferroportin samt nedsat TFRC og DMT1 mRNA). Gpx4 mRNA var nedsat i cerebellum, men dette afspejlede sig ikke i GPX4-proteinniveauer, og SEC24b mRNA var reduceret i cerebrum; samlet gav dette ikke klare beviser for ferroptose. BBB-markører viste nedsat occludin mRNA uden ændringer i claudin 5, hvilket tyder på ændringer uden tydelig kompromittering af barriereintegriteten. Samlet tyder fundene på en jern-dyshomeostase i NPC2-deficiente mus, især i cerebellum, uden entydig indikation på ferroptose; oprindelsen af det akkumulerede jern forbliver ukendt.
This exploratory study examined whether iron accumulates in the brains of NPC2Gt(LST105)BygNya (NPC2-/-) mice, whether such changes are associated with ferroptosis, and whether the blood–brain barrier (BBB) remains intact. NPC2-/- mice aged 6, 8, and 12 weeks and age-matched controls were included. Iron, copper, and zinc in cerebrum and cerebellum were quantified by ICP-OES, while RT-qPCR assessed mRNA expression of iron-handling genes (tfrc, ferritin, dmt1, ferroportin, metallothionein III), ferroptosis markers (gpx4, SEC24b, acsl4), and BBB integrity markers (claudin 5, occludin, zo-1). Immunohistochemistry visualized GPX4 in neurons (NeuN or Calbindin) and TFRC, ELISA measured GPX4 protein, and Western blots assessed TFRC and ferritin. The results indicated iron accumulation particularly in the cerebellum of NPC2-/- mice, accompanied by transcriptional changes in iron transport and storage (increased ferritin and ferroportin and decreased TFRC and DMT1 mRNA). Although gpx4 mRNA was reduced in the cerebellum, this did not translate into lower GPX4 protein, and SEC24b mRNA was decreased in the cerebrum; overall, there was no clear evidence for ferroptosis. BBB markers showed reduced occludin mRNA without changes in claudin 5, suggesting alterations without overt loss of barrier integrity. Together, the findings indicate iron dyshomeostasis in NPC2-deficient mice, particularly in the cerebellum, without definitive support for ferroptosis; the source of the accumulated iron remains unknown.
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