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

Abstract

Neuronal oxidative damage and cell death by unconjugated bilirubin (UCB) showed to be mediated by overstimulation of glutamate receptors and nitric oxide (NO) production, which was abrogated by the bile acid glycoursodeoxycholic acid (GUDCA). Microglia, a crucial mediator of CNS inflammation, evidenced to react to UCB by releasing glutamate and NO before becoming senescent. Our studies demonstrated that neurite outgrowth deficits are produced in neurons exposed to UCB and that conditioned media from these UCB-treated neurons further stimulate NO production by microglia. Nevertheless, microglia protective and/or harmful effects in neonatal jaundice are poorly understood, or unrecognized. Here, we investigated the role of microglia, glutamate and NO in the impairment of neurite sprouting by UCB. Therapeutic potential of the anti-inflammatory cytokine interleukin (IL)-10 and GUDCA was also evaluated. By using MK-801 (a NMDA glutamate-subtype receptor antagonist) and l-NAME (a non-specific NO synthase inhibitor) we found that glutamate and NO are determinants in the early and enduring deficits in neurite extension and ramification induced by UCB. Both GUDCA and IL-10 prevented these effects and decreased the production of glutamate and NO. Only GUDCA was able to counteract neuronal death and synaptic changes. Data from organotypic-cultured hippocampal slices, depleted or non-depleted in microglia, supported that microglia participate in glutamate homeostasis and contribute to NO production and cell demise, which were again abrogated by GUDCA. Collectively our data suggest that microglia is a key player in UCB-induced neurotoxicity and that GUDCA might be a valuable preventive therapy in neonates at risk of UCB encephalopathy.

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.