Bilirubin reduces axonal arborization through alterations of cytoskeletal dynamics
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1
University of Lisbon, Centro de Patogénese Molecular/iMed.UL, Faculty of Pharmacy, Portugal
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2
University of Minnesota, Department of Neuroscience, Faculty of Health,Medicine and Life Sciences, United States
Moderate levels of unconjugated bilirubin (UCB) are associated with an increase in minor neurologic dysfunction, contributing to a high risk for behavioural and learning problems [1]. The elaboration of neuronal axons and dendrites is dependent on a functional cytoskeleton, which is strictly regulated by associated proteins such as Tau1 and MAP2 [2]. Here we investigate if UCB interferes on axonal arborization and whether its effects are related with changes of cytoskeletal dynamics.
Primary cultures of mouse hippocampal neurons were incubated at 1 day-in-vitro (DIV) with 50μM purified UCB plus 100μM human serum albumin, for 24 h at 37ºC, and additionally cultured until 3 DIV. For evaluation of axon arborization neurons were electroporated with pCAG-EGFP to identify individual neurons. MAP2 and Tau1 expression and localization along the axon were determined by immunostaining with specific antibodies and their binding to microtubules (MT) assessed after extraction of soluble cytoplasmic components prior to fixation. For evaluation of MT dynamics cells were electroporated with pCAG-EB3-EGFP to identify the MT plus end-binding protein 3 (EB3) an indicator of MT polymerization.
An early exposure of neurons to UCB decreased axonal arborization (10%) mainly by reducing the number of branches (>20%, p<0.05). In normal conditions, MT binding of MAP2 is stronger in the cell body and dendrites, while that of Tau is tighter in the axon. However, UCB induced an increased axonal entry of MAP2 (~30%, p<0.05) with a higher expression in the apical portion of the axon (>20%, p<0.05). Tau1 expression increased after UCB treatment (>60%, p<0.05), namely at the middle and terminal portions of the axon. These results indicate an increased UCB-induced binding of MAP2 and Tau1 to MT (~20% and ~40% respectively, p<0.05), confirmed by immunostaining following extraction. In addition, UCB reduced the presence of EB3 at the apical portion of the axon (~30%, p<0.05), pointing to a deficient polymerization of MT at this level.
Our results show that exposure of neurons to UCB during early development modify axonal arborization with specific changes at the MT cytoskeletal dynamics, features that contribute to elucidate the mechanisms leading to neuronal abnormalities upon neonatal hyperbilirubinemia.
Funded by FCT-PTDC/SAU-NEU/64385/2006 (to DB)
References
1. Soorani-Lunsing et al. Pediatr Res 2001;50:701-5.
2. Kwei et al. Neuroreport 1998;9:1035-40.
Conference:
11th Meeting of the Portuguese Society for Neuroscience, Braga, Portugal, 4 Jun - 6 Jun, 2009.
Presentation Type:
Poster Presentation
Topic:
Neuronal Communication
Citation:
Fernandes
A,
Coutinho
E,
Lanier
LM and
Brites
D
(2009). Bilirubin reduces axonal arborization through alterations of cytoskeletal dynamics.
Front. Neurosci.
Conference Abstract:
11th Meeting of the Portuguese Society for Neuroscience.
doi: 10.3389/conf.neuro.01.2009.11.114
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Received:
11 Aug 2009;
Published Online:
11 Aug 2009.
*
Correspondence:
Dora Brites, University of Lisbon, Centro de Patogénese Molecular/iMed.UL, Faculty of Pharmacy, Lisboa, Portugal, dbrites@ff.ul.pt