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Bilirubin disrupts calcium homeostasis in neonatal hippocampal neurons: a new pathway of neurotoxicity

Rossana Rauti

  • Tel Aviv,
  • Israel
  • Rossana Rauti ¹
  • , Mohammed Qaisiya ⁴
  • , Claudio Tiribelli ³
  • , Laura Ballerini ¹
  • , Cristina Bellarosa ³
  • 1 International School for Advanced Studies (SISSA), Via Bonomea, 265, 34136 Trieste, Italy
  • 2 Department of Biomedical Engineering, Tel Aviv University, Tel Aviv, Israel
  • 3 Fondazione Italiana Fegato (FIF), Bldg Q-AREA Science Park Basovizza, SS14 Km 163,5, 34149 Trieste, Italy
  • 4 College of Pharmacy and Medical Sciences, Hebron University, West Bank, Hebron, Palestine

Severe hyperbilirubinemia leads to bilirubin encephalopathy in neonates, with irreversible neurological sequelae. We investigated the neuronal vulnerability to unconjugated bilirubin (UCB) toxicity. The calcium (Ca2+) homeostasis is crucial for neuron survival. Ca2+ release from endoplasmic reticulum (ER) during ER-stress can lead to apoptosis trough Caspase-12 activation. By live Ca2+ imaging we monitored Ca2+ signals in hippocampal neuroglia cells exposed to UCB doses, showing the ability of UCB to alter intracellular Ca2+ homeostasis. The contribution of intracellular Ca2+ stores and the activation of proteins involved in the apoptotic Ca2+ signaling were also assessed. Thapsigargin, specific inhibitor of Sarco/endoplasmic reticulum ATPase pumps, significantly reduced the duration of Ca2+ oscillation associated with UCB exposure indicating that UCB strongly interfered with the reticulum Ca2+ stores. Contrarily, in pure astrocyte cultures, spontaneous Ca2+ transient duration was not altered by UCB. The protein content of GRP78, AT6, CHOP, Calpain and Caspase-12 treated with UCB was twofold higher compared to controls. Ca2+-dependent Calpain and Caspase-12 induction by UCB were significantly reduced when cells were pretreated with the ER-stress inhibitor 4-PBA. We showed the direct interference of UCB with neuronal intracellular Ca2+ dynamics, suggesting ER Ca2+ stores as a primary target of UCB toxicity with the activation of the apoptotic ER-stress-dependent pathway.