Andreia Barateiro

Temporal oligodendrocyte lineage progression: In vitro models of proliferation, differentiation and myelination

Oligodendrocytes are neuroglial cells responsible, within the central nervous system, for myelin sheath formation that provides an electric insulation of axons and accelerate the transmission of electrical signals. In order to be able to produce myelin, oligodendrocytes progress through a series of differentiation steps from oligodendrocyte precursor cells to mature oligodendrocytes (migration, increase in morphologic complexity and expression pattern of specific markers), which are modulated by cross talk with other nerve cells. If during the developmental stage any of these mechanisms is affected by toxic or external stimuli it may result into impaired myelination leading to neurological deficits. Such being the case, several approaches have been developed to evaluate how oligodendrocyte development and myelination may be impaired. The present review aims to summarize changes that oligodendrocytes suffer from precursor cells to mature ones, and to describe and discuss the different in vitro models used to evaluate not only oligodendrocyte development (proliferation, migration, differentiation and ability to myelinate), but also their interaction with neurons and other glial cells. First we discuss the temporal oligodendrocyte lineage progression, highlighting the differences between human and rodent, usually used as tissue supply for in vitro cultures. Second we describe how to perform and characterize the different in vitro cultures, as well as the methodologies to evaluate oligodendrocyte functionality in each culture system, discussing their advantages and disadvantages. Finally, we briefly discuss the current status of in vivo models for oligodendrocyte development and myelination.

Features of bilirubin-induced reactive microglia: From phagocytosis to inflammation

Microglia constitute the brains immunocompetent cells and are intricately implicated in numerous inflammatory processes included in neonatal brain injury. In addition, clearance of tissue debris by microglia is essential for tissue homeostasis and may have a neuroprotective outcome. Since unconjugated bilirubin (UCB) has been proven to induce astroglial immunological activation and neuronal cell death, we addressed the question of whether microglia acquires a reactive phenotype when challenged by UCB and intended to characterize this response. In the present study we report that microglia primary cultures stimulated by UCB react by the acquisition of a phagocytic phenotype that shifted into an inflammatory response characterized by the secretion of the pro-inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, upregulation of cyclooxygenase (COX)-2 and increased matrix metalloproteinase (MMP)-2 and -9 activities. Further investigation upon upstream signalling pathways revealed that UCB led to the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB at an early time point, suggesting that these pathways might underlie both the phagocytic and the inflammatory phenotypes engaged by microglia. Curiously, the phagocytic and inflammatory phenotypes in UCB-activated microglia seem to alternate along time, indicating that microglia reacts towards UCB insult firstly with a phagocytic response, in an attempt to constrain the lesion extent and comprising a neuroprotective measure. Upon prolonged UCB exposure periods, either a shift on global microglia reaction occurred or there could be two distinct sub-populations of microglial cells, one directed at eliminating the damaged cells by phagocytosis, and another that engaged a more delayed inflammatory response. In conclusion, microglial cells are relevant partners to consider during bilirubin encephalopathy and the modulation of its activation might be a promising therapeutic target.

Anti-inflammatory effect of rosmarinic acid and an extract of Rosmarinus officinalis in rat models of local and systemic inflammation.

Rosmarinic acid is a polyphenolic compound and main constituent of Rosmarinus officinalis and has been shown to possess antioxidant and anti‐inflammatory properties. We aimed to evaluate the anti‐inflammatory properties of rosmarinic acid and of an extract of R. officinalis in local inflammation (carrageenin‐induced paw oedema model in the rat), and further evaluate the protective effect of rosmarinic acid in rat models of systemic inflammation: liver ischaemia–reperfusion (I/R) and thermal injury models. In the local inflammation model, rosmarinic acid was administered at 10, 25 and 50 mg/kg (p.o.), and the extract was administered at 10 and 25 mg/kg (equivalent doses to rosmarinic acid groups) to male Wistar rats. Administration of rosmarinic acid and extract at the dose of 25 mg/kg reduced paw oedema at 6 hr by over 60%, exhibiting a dose–response effect, suggesting that rosmarinic was the main contributor to the anti‐inflammatory effect. In the liver I/R model, rosmarinic acid was administered at 25 mg/kg (i.v.) 30 min. prior to the induction of ischaemia and led to the significant reduction in the serum concentration of transaminases (AST and ALT) and LDH. In the thermal injury model, rosmarinic acid was administered at 25 mg/kg (i.v.) 5 min. prior to the induction of injury and significantly reduced multi‐organ dysfunction markers (liver, kidney, lung) by modulating NF‐κB and metalloproteinase‐9. For the first time, the anti‐inflammatory potential of rosmarinic acid has been identified, as it causes a substantial reduction in inflammation, and we speculate that it might be useful in the pharmacological modulation of injuries associated to inflammation.

ER stress, mitochondrial dysfunction and calpain/JNK activation are involved in oligodendrocyte precursor cell death by unconjugated bilirubin.

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.

Pro-inflammatory cytokines intensify the activation of NO/NOS, JNK1/2 and caspase cascades in immature neurons exposed to elevated levels of unconjugated bilirubin.

Hyperbilirubinemia may lead to encephalopathy in neonatal life, particularly in premature infants. Although the mechanisms were never established, clinicians commonly consider sepsis as a risk factor for bilirubin-induced neurological dysfunction (BIND). Our previous studies showed that elevated levels of unconjugated bilirubin (UCB) have immunostimulant effects, which are potentiated by lipopolysaccharide (LPS), and that immature neural cells are more vulnerable to UCB. The present study was undertaken to explore the role of nitric oxide (NO)/NO synthase (NOS), c-Jun N-terminal kinases (JNK) 1/2 and caspase activation in BIND, as well as the additional effects of inflammation, in immature neurons, incubated from 1 h to 24 h, at 37°C. UCB, at conditions mimicking those of jaundiced newborns (UCB/serum albumin=0.5), induced NO production, neuronal NOS (nNOS) expression and JNK1/2 activation in 3 days in vitro neuron cultures. As a consequence of these events, mitochondrial and extrinsic pathways of apoptosis were initiated, ultimately leading to neuronal dysfunction. Co-incubation with TNF-α+IL-1β intensified the activation of NO/NOS, JNK1/2, caspase-8, caspase-9 and caspase-3 by UCB. Cleavage of Bid into truncated Bid (tBid), as well as increased cytotoxic potential, were also observed. Interestingly, both L-NAME (NOS inhibitor) and SP600125 (JNK1/2 inhibitor) reversed the effects produced by UCB either alone, or in association with pro-inflammatory cytokines. Taken together, our data reveal not only that activation of NO/NOS, JNK1/2 and caspase cascades are important determinants of BIND, but also that the association of TNF-α+IL-1β have cumulative effects. These events provide a reason for the risk of sepsis in BIND and point to potential targets for therapeutic intervention.

Selective vulnerability of rat brain regions to unconjugated bilirubin

Hippocampus is one of the brain regions most vulnerable to unconjugated bilirubin (UCB) encephalopathy, although cerebellum also shows selective yellow staining in kernicterus. We previously demonstrated that UCB induces oxidative stress in cortical neurons, disruption of neuronal network dynamics, either in developing cortical or hippocampal neurons, and that immature cortical neurons are more prone to UCB-induced injury. Here, we studied if immature rat neurons isolated from cortex, cerebellum and hippocampus present distinct features of oxidative stress and cell dysfunction upon UCB exposure. We also explored whether oxidative damage and its regulation contribute to neuronal dysfunction induced by hyperbilirubinemia, considering neurite extension and ramification, as well as cell death. Our results show that UCB induces nitric oxide synthase expression, as well as production of nitrites and cyclic guanosine monophosphate in immature neurons, mainly in those from hippocampus. After exposure to UCB, hippocampal neurons presented the highest content of reactive oxygen species, disruption of glutathione redox status and cell death, when compared to neurons from cortex or cerebellum. In particular, the results indicate that cells exposed to UCB undertake an adaptive response that involves DJ-1, a multifunctional neuroprotective protein implicated in the maintenance of cellular oxidation status. However, longer neuronal exposure to UCB caused down-regulation of DJ-1 expression, especially in hippocampal neurons. In addition, a greater impairment in neurite outgrowth and branching following UCB treatment was also noticed in immature neurons from hippocampus. Interestingly, pre-incubation with N-acetylcysteine, a precursor of glutathione synthesis, protected neurons from UCB-induced oxidative stress and necrotic cell death, preventing DJ-1 down-regulation and neuritic impairment. Taken together, these data point to oxidative injury and disruption of neuritic network as hallmarks in hippocampal susceptibility to UCB. Most importantly, they also suggest that local differences in glutathione content may account to the different susceptibility between brain regions exposed to UCB.

Unconjugated Bilirubin Restricts Oligodendrocyte Differentiation and Axonal Myelination

High levels of serum unconjugated bilirubin (UCB) in newborns are associated with axonal damage and glial reactivity that may contribute to subsequent neurologic injury and encephalopathy (kernicterus). Impairments in myelination and white matter damage were observed at autopsy in kernicteric infants. We have recently reported that UCB reduces oligodendrocyte progenitor cell (OPC) survival in a pure OPC in vitro proliferative culture. Here, we hypothesized that neonatal hyperbilirubinemia may also impair oligodendrocyte (OL) maturation and myelination. We used an experimental model of hyperbilirubinemia that has been shown to mimic the pathophysiological conditions leading to brain dysfunction by unbound (free) UCB. Using primary cultures of OL, we demonstrated that UCB delays cell differentiation by increasing the OPC number and reducing the number of mature OL. This finding was combined with a downregulation of Olig1 mRNA levels and upregulation of Olig2 mRNA levels. Addition of UCB, prior to or during differentiation, impaired OL morphological maturation, extension of processes and cell diameter. Both conditions reduced active guanosine triphosphate (GTP)-bound Rac1 fraction. In myelinating co-cultures of dorsal root ganglia neurons and OL, UCB treatment prior to the onset of myelination decreased oligodendroglial differentiation and the number of myelinating OL, also observed when UCB was added after the onset of myelination. In both circumstances, UCB decreased the number of myelin internodes per OL, as well as the myelin internode length. Our studies demonstrate that increased concentrations of UCB compromise myelinogenesis, thereby elucidating a potential deleterious consequence of elevated UCB.

Rat Cerebellar Slice Cultures Exposed to Bilirubin Evidence Reactive Gliosis, Excitotoxicity and Impaired Myelinogenesis that Is Prevented by AMPA and TNF-α Inhibitors

The cerebellum is one of the most affected brain regions in the course of bilirubin-induced neurological dysfunction. We recently demonstrated that unconjugated bilirubin (UCB) reduces oligodendrocyte progenitor cell (OPC) survival and impairs oligodendrocyte (OL) differentiation and myelination in co-cultures of dorsal root ganglia neurons and OL. Here, we used organotypic cerebellar slice cultures, which replicate many aspects of the in vivo system, to dissect myelination defects by UCB in the presence of neuroimmune-related glial cells. Our results demonstrate that treatment of cerebellar slices with UCB reduces the number of myelinated fibres and myelin basic protein mRNA expression. Interestingly, UCB addition to slices increased the percentage of OPC and decreased mature OL content, whereas it decreased Olig1 and increased Olig2 mRNA expression. These UCB effects were associated with enhanced gliosis, revealed by an increased burden of both microglia and astrocytes. Additionally, UCB treatment led to a marked increase of tumor necrosis factor (TNF)-α and glutamate release, in parallel with a decrease of interleukin (IL)-6. No changes were observed relatively to IL-1β and S100B secretion. Curiously, both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist and TNF-α antibody partially prevented the myelination defects that followed UCB exposure. These data point to a detrimental role of UCB in OL maturation and myelination together with astrocytosis, microgliosis, and both inflammatory and excitotoxic responses, which collectively may account for myelin deficits following moderate to severe neonatal jaundice.

Dynamics of neuron-glia interplay upon exposure to unconjugated bilirubin

J. Neurochem. (2011) 117, 412–424.

Oligodendrocyte Development and Myelination in Neurodevelopment: Molecular Mechanisms in Health and Disease.

Oligodendrocytes are the myelinating cells of the central nervous system that constitute about 5 to 10% of the total glial population. These cells are responsible for myelin sheath production, which is essential not only for the rapid and efficient conduction of the electrical impulses along the axons, but also for preserving axonal integrity. Oligodendrocytes arise from oligodendrocyte progenitor cells that proliferate and differentiate just before and after birth, under a highly-regulated program. Both oligodendrocytes and their precursors are very susceptible to injury by several mechanisms, including excitotoxic damage, oxidative stress and inflammatory events. In this review, we will cover not only several important aspects of oligodendrocyte development and regulatory mechanisms involved in this process, but also some of the most important pathways of injury associated to oligodendrogenesis. Moreover, we will also address some neurological disorders along life journey that present impairment in oligodendrocyte function and in myelination during neurodevelopment, such as periventricular leukomalacia, hypoxia/ischemia and hyperbilirubinemia that in turn can potentiate the emergence of neurological and neurodegenerative diseases like schizophrenia, multiple sclerosis and Alzheimers disease.