Exploring the death pathway of oligodendrocyte precursor cell induced by unconjugated bilirubin
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1
University of Lisbon, Centro de Patogénese Molecular/iMed.UL, Faculty of Pharmacy, Portugal
Myelination initiates in the last half of gestation and proceed in postnatal life. Hence, oligodendrocyte damage along this time may disturb the myelination process resulting in severe malfunction of the human CNS. Interestingly, neonatal jaundice has been associated to the onset of neurological defects and to white matter abnormalities [1], suggesting that unconjugated bilirubin (UCB) may compromise myelination. To date, in only one study UCB was shown to decrease the viability of mature oligodendrocytes [2]. Hence, here we aimed to investigate whether and how UCB determines the demise of cultured oligodendrocyte precursor cells (OPCs). Mixed glial cultures were obtained from cerebral cortex of neonatal rat pups. At 10 days in vitro (DIVs) OPCs were isolated by shaking after dislodging of microglia [3]. Enriched OPCs cultures were achieved by the use of specific proliferating factors. After 6 DIVs cells were treated with 50 µM UCB plus 100 µM of human serum albumin for 4 and 8 h. Evaluation of the purity of OPCs cultures were performed by immunocytochemistry using antibodies directed to OPCs (A2B5 and O4) and astrocytes (GFAP). Assessment of cell viability was performed by LDH leakage, mitochondrial abnormalities by MitoTracker Red (500 nM) and endoplasmic reticulum (ER) stress by the activity of calcium-dependent calpain using a specific substrate BOC-LM-CMAC (10 µM).
Approximately 80% of our enriched OPCs cultures were A2B5/O4 positive with less than 3% GFAP positive, and the remaining representing oligodendrocytes in varying stages of differentiation. Exposure of OPCs to UCB increased the release of LDH at 4 (17% or ~1.7-fold, p<0.01) and 8 h (36% or ~2.2-fold, p<0.01). Interestingly, while a decrease in mitochondrial viability (~0.8-fold, p<0.01) was observed at 4 h, enhancement of calpain activity was only detected at 8 h (~1.5-fold, p<0.01) pointing that mitochondrial dysfunction by UCB occurs prior to ER stress.
Our results suggest that UCB exerts cytotoxicity in OPCs through both mitochondrial dysfunction and ER stress cell death pathways, although the susceptibility of both cellular compartments showed to be time-dependent. Targeting OPCs dysfunction by UCB may lead to new therapeutic strategies aimed at preventing abnormal myelination following neonatal jaundice.
Funded by FCT-PTDC/SAU-NEU/64385/2006 (to DB)
References
1. Gkoltsiou et al. Early Hum Dev 2008;84:829-38.
2. Genc et al. Brain Res 2003;985:35-41.
3. Chen et al. Nat Protoc 2007;2:1044-51.
Conference:
11th Meeting of the Portuguese Society for Neuroscience, Braga, Portugal, 4 Jun - 6 Jun, 2009.
Presentation Type:
Poster Presentation
Topic:
Neurodegenerative Disorders
Citation:
Barateiro
A,
Vaz
AR,
Silva
SL,
Fernandes
A and
Brites
D
(2009). Exploring the death pathway of oligodendrocyte precursor cell induced by unconjugated bilirubin.
Front. Neurosci.
Conference Abstract:
11th Meeting of the Portuguese Society for Neuroscience.
doi: 10.3389/conf.neuro.01.2009.11.043
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Received:
06 Aug 2009;
Published Online:
06 Aug 2009.
*
Correspondence:
Dora Brites, University of Lisbon, Centro de Patogénese Molecular/iMed.UL, Faculty of Pharmacy, Lisbon, Portugal, dbrites@ff.ul.pt