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ER Stress, Mitochondrial Dysfunction and Calpain/JNK Activation are Involved in Oligodendrocyte Precursor Cell Death by Unconjugated Bilirubin

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Abstract

Research on the mechanisms of bilirubin-induced neurological dysfunction focuses mainly on neuronal death, astrocyte-mediated events and microglia activation. Although myelin damage by unconjugated bilirubin (UCB) has been documented in neonatal kernicterus cases, the events leading to myelination impairment were never explored. This condition may occur by reduced oligodendrocyte precursor cells (OPC) number, or failure of OPC to differentiate in myelinating oligodendrocytes. We have shown that UCB elicits an inflammatory response, glutamate release and reactive oxygen species (ROS) generation in neurons and glial cells, biomolecules with toxic properties on OPC. Hence, we propose to examine whether UCB determines OPC demise and, if so, which signaling pathways are involved. Our results show that OPC display increased apoptosis and necrosis-like cell death upon UCB exposure, mediated by early signals of endoplasmic reticulum (ER) stress [e.g. upregulation of glucose-regulated protein (GRP)78, inositol-requiring enzyme (IRE)-1α and activation transcription factor (ATF)-6, as well as activation of caspase-2 and c-Jun N-terminal kinase (JNK)], followed by mitochondrial dysfunction (e.g. loss of mitochondria membrane potential and caspase-9 activation). The later calpain activation points to intracellular Ca2+ overload and intervention of both ER and mitochondria. Downstream production of ROS may derive from mitochondria damage and secondary injuries, possibly determining the second cycle of GRP78, IRE-1α, caspase-2 and JNK activation. Moreover, inhibition of caspases, calpains and oxidative stress, by using specific inhibitors, prevented UCB-induced OPC death. UCB did not induce the release of cytokines or glutamate by OPC. These results indicate that UCB by reducing OPC survival, through a cascade of programmed intracellular events triggered by ER stress and mitochondria dysfunction, can compromise myelinogenesis.

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Acknowledgments

This work was supported by the strategic project PEst-OE/SAU/UI4013/2011 and PTDC/SAU-NEU/64385/2006 grants from Fundação para a Ciência e a Tecnologia (FCT), Lisbon, Portugal (to D. B.). A. B. was recipient of a PhD fellowship (SFRH/BD/43885/2008) from FCT. The funding organization had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Authors and Affiliations

  1. Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Avenida Professor Gama Pinto, 1649-003, Lisbon, Portugal

    Andreia Barateiro, Ana Rita Vaz, Sandra Leitão Silva, Adelaide Fernandes & Dora Brites

  2. Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Avenida Professor Gama Pinto, 1649-003, Lisbon, Portugal

    Adelaide Fernandes & Dora Brites

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  1. Andreia Barateiro
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  2. Ana Rita Vaz
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  3. Sandra Leitão Silva
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  4. Adelaide Fernandes
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  5. Dora Brites
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Correspondence to Dora Brites.

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Inhibition of caspases, calpains and oxidative stress prevented unconjugated bilirubin (UCB)-induced oligodendrocyte precursor cell (OPC) death. Rat cortical OPC were exposed to 50 μM UCB in the presence of 100 μM human serum albumin and to specific inhibitors for caspases (Z-VAD-FMK, 1 μM), calpains (calpeptin, 25 μM) or oxidative stress (N-acetylcysteine – NAC, 100 μM). a Caspase-9, caspase-2 and caspase-3 activities with or without Z-VAD-FMK incubation were determined by colorimetric substrate cleavage assay as indicated in Materials and Methods at 4, 8 and 24 h, respectively. Graph bars represent the fold change from vehicle (mean ± SEM), from at least three independent experiments. b Reactive oxygen species (ROS) were determined at 6 h by fluorescence assay with dihydrorhodamine 123 as described in Materials and Methods after incubation in the presence or absence of NAC. Representative images of one experiment and graph bars with fold change from vehicle (mean ± SEM) for ROS production from at least three independent experiments are shown. c Calpain activity was determined at 4 h of incubation using a specific fluorogenic substrate as indicated in Materials and Methods after treatment with or without calpeptin. Representative images of one experiment and graph bars with fold change from vehicle (mean ± SEM) for calpain activation from at least three independent experiments are shown. **P < 0.01 vs. respective vehicle; ## P < 0.01 vs. respective UCB. (TIFF 9060 kb)

12017_2012_8187_MOESM2_ESM.tiff

Unconjugated bilirubin (UCB) induces an increase in ATF-6 and IRE-1α, chaperones usually involved in endoplasmic reticulum stress, in oligodendrocyte precursor cells (OPC). Rat cortical OPC were exposed to 50 μM UCB in the presence of 100 μM human serum albumin for the indicated time periods. a Representative results of ATF-6 and IRE-1α protein expression by Western blot using specific antibodies are shown. b Graph bars represent the fold increase from vehicle (mean ± SEM) obtained for protein band intensity by scanning densitometry standardized with respect to β-actin protein for ATF-6 and IRE-1α, determined as indicated in Materials and Methods, from at least four independent experiments. **P < 0.01 and *P < 0.05 vs. respective vehicle. (TIFF 2855 kb)

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Barateiro, A., Vaz, A.R., Silva, S.L. et al. ER Stress, Mitochondrial Dysfunction and Calpain/JNK Activation are Involved in Oligodendrocyte Precursor Cell Death by Unconjugated Bilirubin. Neuromol Med 14, 285–302 (2012). https://doi.org/10.1007/s12017-012-8187-9

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  • DOI: https://doi.org/10.1007/s12017-012-8187-9

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