Neuritic growth impairment and cell death by unconjugated bilirubin is mediated by NO and glutamate, modulated by microglia, and prevented by glycoursodeoxycholic acid and interleukin-10
Highlights
► Increased glutamate and nitric oxide by UCB trigger neuritic plasticity impairment. ► GUDCA counteracts UCB down-regulation of pre-synaptic proteins. ► IL-10 and GUDCA prevent glutamate efflux and neurodegeneration by UCB. ► Microglia exposed to UCB increasingly uptake glutamate and release NO. ► GUDCA avoids glutamate and NO increase in UCB-treated hippocampal slices.
Introduction
High levels of unconjugated bilirubin (UCB) may lead to neurological damage, mainly in neonates born prematurely. Despite decades of research on the molecular mechanisms of bilirubin-induced neurological dysfunction (BIND), the determinants of vulnerability and reversibility are still only partially understood. Most important, there are no studies on whether modulation of microglial activation might be a promising therapeutic target to prevent BIND.
Elevated levels of UCB trigger astrocytic, neuronal and microglial cell death by both necrosis and apoptosis (Falcão et al., 2006, Gordo et al., 2006, Silva et al., 2002). UCB has also been shown to inhibit glutamate uptake (Silva et al., 1999, Silva et al., 2002) and to enhance its secretion from neurons (Falcão et al., 2006), astrocytes (Falcão et al., 2005, Fernandes et al., 2004) and microglia (Gordo et al., 2006), enabling N-methyl-d-aspartic acid (NMDA)-mediated excitotoxicity (Brito et al., 2010, Grojean et al., 2000, McDonald et al., 1998). In addition, we have demonstrated that UCB increases nitric oxide (NO) production in both immature and differentiated neurons, as well as in microglia (Brito et al., 2010, Silva et al., 2011, Vaz et al., 2011a). Recently, Haustein et al. (2010) by using the organotypic culture model have shown that the degeneration of excitatory synaptic terminals in the auditory brainstem by UCB was mediated by neuronal NO synthase (NOS).
Inhibition of NOS by l-NAME and blockade of NMDA receptors by MK-801 abrogated NO formation and neurotoxicity, evidencing glutamate and NO as key determinants in UCB neurotoxicity (Brito et al., 2010). However, both l-NAME and MK-801 have important secondary effects (Tsuchiya et al., 2010, Kovacic and Somanathan, 2010). In contrast, no noticeable secondary effects are indicated for the bile acid glycoursodeoxycholic acid (GUDCA). GUDCA counteracts UCB-induced alterations in the redox status (Brito et al., 2008) as well as mitochondria dysfunction and energy impairment in neurons (Vaz et al., 2010), and reduces the immunostimulant effects of UCB on astrocytes (Fernandes and Brites, 2009). Other interesting anti-inflammatory molecule is IL-10, which besides inhibiting NO release (Ferlazzo et al., 2003, Huang et al., 2002, Ledeboer et al., 2000), and reducing glutamate-induced excitotoxicity (Bachis et al., 2001), also revealed to suppress cytokine secretion by UCB-treated astrocytes, but not astroglial demise (Fernandes et al., 2007).
Among the harmful effects produced by UCB are the reduced dendrite extension and ramification, as well as diminished dendritic spine formation and synapse establishment (Falcão et al., 2007, Fernandes et al., 2009). While the involvement of glutamate in neuritic outgrowth regulation is widely established (Mattson et al., 1988, Hoffman et al., 1996, Monnerie et al., 2003, Lee et al., 2005) that of NO was albeit indicated to play a role in neuroblastoma differentiation (Evangelopoulos et al., 2010). Whether UCB-induced changes in NO and/or glutamate homeostasis are implicated on aberrant neuronal connectivity is not known.
Microglia, when activated, release excessive NO, reactive oxygen (ROS) and nitrogen species (RNS), thus aggravating acute brain insults (Raivich et al., 1999). Interestingly, microglia were shown to be activated by UCB (Gordo et al., 2006), changing from a phagocytic phenotype to a proinflammatory state that may culminate in cell death (Silva et al., 2010, Silva et al., 2011).
Therefore, we investigated the influence of glutamate and NO in UCB-induced neurite morphogenesis impairment and neuronal demise. Also, because the crosstalk between glial cells and neurons modulate microglial activation, the production of glutamate and NO by UCB was evaluated in an ex-vivo model of hippocampal slice cultures. To determine if the effects produced could be ascribed to microglial cells we compared the data achieved with that in microglia-depleted slices. To evaluate protective effects of IL-10 and GUDCA on UCB-induced impairment of neurite sprouting, neurotransmitter release, pre-synaptic failure and neurodegeneration, we used both isolated immature neurons and hippocampal slice cultures.
Our results indicate that UCB causes a decrease in the expression of the pre-synaptic proteins, that NO and glutamate mediate neurite network dystrophia and cell death, that microglia are intervening players, and that GUDCA and IL-10 are neuroprotective.
Section snippets
Chemicals and antibodies
Neurobasal medium, B-27 Supplement (50×), Hanks’ balanced salt solution (HBSS), HBSS without Ca2+ and Mg2+, gentamicin (50 mg/mL), trypsin (0.5 g/L) and Alexa Fluor 594 chicken anti-goat IgG were acquired from Invitrogen (Carlsbad, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and l-glutamine, were purchased from Biochrom AG (Berlin, Germany). Antibiotic–antimycotic solution (20×), human serum albumin (HSA; fraction V, fatty acid free), bovine serum albumin
Neuronal morphogenesis impairment and cell demise by UCB are mediated by glutamate and NO
By using MK-801 (a NMDA glutamate-subtype receptor antagonist) and l-NAME (a non-specific NO synthase inhibitor), we have previously demonstrated that both glutamate and NO are determinants of UCB neurotoxicity (Brito et al., 2010). We wonder whether MK-801 and l-NAME were still able to prevent UCB-induced death after 24 h incubation with immature cortical neurons, since our prior studies were performed with mature neurons and only 4 h incubation. Therefore, neurons cultured for 3 DIV were
Discussion
Here we demonstrate that glutamate and NO are directly implicated in neuronal demise, in the impairment of neuritic outgrowth and in the reduced expression of pre-synaptic proteins by UCB. By using microglia-depleted and non-depleted organotypic slice cultures we also show that microglia participate in the homeostasis of glutamate and in the production of NO. The present work also demonstrates the efficacy of IL-10 and GUDCA in preventing the deleterious effects of UCB on neuritic plasticity
Acknowledgements
The authors would like to thank Prof. Helmut Kettenmann’s research group for sharing their expertise on microglia, Liliana Bernardino for her skill with organotypic-cultured hippocampal slices and Pedro Pereira and Ana Isabel Pereira for technical assistance. This work was supported by the strategic project PEst-OE/SAU/UI4013/2011 and PTDC/SAU-NEU/64385/2006 grant (to D.B.), from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal. S.L.S. was recipient of a PhD fellowship
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