Inflammatory signalling pathways involved in astroglial activation by unconjugated bilirubin

During neonatal hyperbilirubinaemia, astrocytes activated by unconjugated bilirubin (UCB) may contribute to brain toxicity through the production of cytokines. As a first step in addressing the signal transduction cascades involved in the UCB-induced astroglial immunological response, we tested whether tumour necrosis factor (TNF)-alpha receptor 1 (TNFR1), mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB) would be activated in astrocytes exposed to UCB, and examined the profile of cytokine production. Astrocyte cultures stimulated with UCB showed a rapid rise in TNFR1 protein levels, followed by activation of the MAPKs p38, Jun N-terminal kinase1/2 and extracellular signal-regulated kinase1/2, and NF-kappaB. Interestingly, the induction of these signal effectors preceded the early up-regulation of TNF-alpha and interleukin (IL)-1beta mRNAs, and later secretion of TNF-alpha, IL-1beta and IL-6. Treatment of astrocytes with UCB also induced cell death, with levels comparable to those obtained after exposure of astrocytes to recombinant TNF-alpha and IL-1beta. Moreover, loss of cell viability and cytokine secretion were reduced when the NF-kappaB signal transduction pathway was inhibited, suggesting a key role for NF-kappaB in the astroglial response to UCB. These results demonstrate the complexity of the molecular mechanisms involved in cell injury by UCB during hyperbilirubinaemia and provide a basis for the development of novel therapeutic strategies.     

Role of astrocytes in trimethyltin neurotoxicity

Although the neurotoxicity of trimethyltin (TMT) is well known, mechanisms are still not clear. Glia have been proposed to mediate the toxic action of TMT on nerve cells. Accordingly, the effects of TMT were tested in primary neuronal cultures from rat cerebellum and compared to effects in astrocytes and mixed cultures. Neuronal damage observed following TMT exposure was less in the presence of astrocytes and astrocytes alone were resistant to TMT. Thus, astrocytes have a protective effect against TMT-induced neurotoxicity. TMT caused an oxidative stress in granule cell cultures involving a variety of oxidative species (O2)*-, H2O2, NO), but astrocytes were less sensitive to TMT-induced oxidative species generation. Antioxidants, glutathione and 7-nitroindazole attenuated neuronal cell death induced by TMT. It appears that oxidative stress mediates a large part of the destructive action of TMT in neuronal cultures. The presence of astrocytes appears to modulate TMT-induced oxidative stress so that TMT causes only a small increase in lipid peroxidation in mouse brain after systemic administration. Thus, TMT induces a pronounced oxidative stress in cultured neurons, but when astrocytes are present, oxidative species play a lesser role in the neurotoxic action of TMT.     

The protective effect of docosahexaenoic acid on the bilirubin neurotoxicity

Usually, all newborns demonstrate high serum unconjugated bilirubin (UCB) level. UCB may induce adverse effects in the central nervous system. We aimed to evaluate the cytotoxic effects of UCB and the protective effects of docosahexaenoic acid (DHA) on astrocyte cell cultures. The viability of astrocyte cells decreased after UCB treatment in a dose-dependent manner. Pre-incubation of DHA prevents the cells from UCB-mediated neurotoxicity. Our results shown that UCB leads to inhibition of antioxidant enzymes superoxide dismutase (SOD), catalase and GPx activity and induction of apoptosis. But only 4-h pretreatment of DHA can suppress of UCB-mediated inhibition of antioxidant enzymes SOD, catalase and GPx activity and induction of apoptosis in astrocytes. Our results strongly indicated that DHA has a protective effect on UCB-mediated neurotoxicity through inhibition apoptosis and antioxidant enzymes activity of SOD, CAT and GPx in rat primer astrocyte cell line     

The presence of astrocytes enhances beta amyloid-induced neurotoxicity in hippocampal cell cultures.

A characteristic feature of neuritic plaques in Alzheimer's disease is represented by the presence of activated astrocytes, surrounding dystrophic neurons and beta-amyloid deposition. To explore the role of astrocytes in in vitro beta-amyloid neurotoxicity, we studied the effect of beta-amyloid treatment in hippocampal neurons in two different cell models: pure cultures, where neurons were grown in absence of astrocytes and mixed cultures, where neurons were seeded on a confluent layer of astrocytes. We evaluated two characteristic aspects of in vitro beta-amyloid neurotoxicity: reduction of cell viability and degeneration of the neuritic tree. We demonstrated that neurons growing on astrocytes were more prone to the detrimental effect of the amyloid peptide, with respect to neurons grown in absence of the glial component. Our results support the hypothesis that beta-amyloid-astrocyte interaction can adversely condition neurons and contribute to neuronal damage in Alzheimer's disease.     

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.     

Unconjugated Bilirubin Exposure Impairs Hippocampal Long-Term Synaptic Plasticity

Background

Jaundice is one of the most common problems encountered in newborn infants, due to immaturity of hepatic conjugation and transport processes for bilirubin. Although the majority of neonatal jaundice is benign, some neonates with severe hyperbilirubinemia develop bilirubin encephalopathy or kernicterus. Accumulation of unconjugated bilirubin (UCB) in selected brain regions may result in temporary or permanent impairments of auditory, motor, or cognitive function; however, the molecular mechanisms by which UCB elicits such neurotoxicity are still poorly understood. The present study is undertaken to investigate whether prolonged exposure of rat organotypic hippocampal slice cultures to UCB alters the induction of long-term synaptic plasticity.

Methodology/Principal Findings

Using electrophysiological recording techniques, we find that exposure of hippocampal slice cultures to clinically relevant concentrations of UCB for 24 or 48 h results in an impairment of CA1 long-term potentiation (LTP) and long-term depression (LTD) induction in a time- and concentration-dependent manner. Hippocampal slice cultures stimulated with UCB show no changes in the secretion profiles of the pro-inflammatory cytokines, interleukin-1β and tumor necrosis factor-α, or the propidium ioide uptake. UCB treatment produced a significant decrease in the levels of NR1, NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) receptors through a calpain-mediated proteolytic cleavage mechanism. Pretreatment of the hippocampal slice cultures with NMDA receptor antagonist or calpain inhibitors effectively prevented the UCB-induced impairment of LTP and LTD.

Conclusion/Significance

Our results indicate that the proteolytic cleavage of NMDA receptor subunits by calpain may play a critical role in mediating the UCB-induced impairment of long-term synaptic plasticity in the hippocampus. These observations provide new insights into the molecular mechanisms underlying UCB-induced impairment of hippocampal synaptic plasticity which, in turn, might provide opportunities for the development of novel therapeutic strategies that targets these pathways for treatment.     

Bilirubin selectively inhibits cytochrome c oxidase activity and induces apoptosis in immature cortical neurons: assessment of the protective effects of glycoursodeoxycholic acid

High levels of unconjugated bilirubin (UCB) may initiate encephalopathy in neonatal life, mainly in pre-mature infants. The molecular mechanisms of this bilirubin-induced neurologic dysfunction (BIND) are not yet clarified and no neuroprotective strategy is currently worldwide accepted. Here, we show that UCB, at conditions mimicking those of hyperbilirubinemic newborns (50 μM UCB in the presence of 100 μM human serum albumin), rapidly (within 1 h) inhibited cytochrome c oxidase activity and ascorbate-driven oxygen consumption in 3 days in vitro rat cortical neurons. This was accompanied by a bioenergetic and oxidative crisis, and apoptotic cell death, as judged by the collapse of the inner-mitochondrial membrane potential, increased glycolytic activity, superoxide anion radical production, and ATP release, as well as disruption of glutathione redox status. Furthermore, the antioxidant compound glycoursodeoxycholic acid (GUDCA) fully abrogated UCB-induced cytochrome c oxidase inhibition and significantly prevented oxidative stress, metabolic alterations, and cell demise. These results suggest that the neurotoxicity associated with neonatal bilirubin-induced encephalopathy occur through a dysregulation of energy metabolism, and supports the notion that GUDCA may be useful in the treatment of BIND.     

Bilirubin as a determinant for altered neurogenesis,neuritogenesis, and synaptogenesis

Elevated levels of serum unconjugated bilirubin (UCB) in the first weeks of life may lead to long‐term neurologic impairment. We previously reported that an early exposure of developing neurons to UCB, in conditions mimicking moderate to severe neonatal jaundice, leads to neuritic atrophy and cell death. Here, we have further analyzed the effect of UCB on nerve cell differentiation and neuronal development, addressing how UCB may affect the viability of undifferentiated neural precursor cells and their fate decisions, as well as the development of hippocampal neurons in terms of dendritic and axonal elongation and branching, the axonal growth cone morphology, and the establishment of dendritic spines and synapses. Our results indicate that UCB reduces the viability of proliferating neural precursors, decreases neurogenesis without affecting astrogliogenesis, and increases cellular dysfunction in differentiating cells. In addition, an early exposure of neurons to UCB decreases the number of dendritic and axonal branches at 3 and 9 days in vitro (DIV), and a higher number of neurons showed a smaller growth cone area. UCB‐treated neurons also reveal a decreased density of dendritic spines and synapses at 21 DIV. Such deleterious role of UCB in neuronal differentiation, development, and plasticity may compromise the performance of the brain in later life. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Altered glial function causes neuronal death and increases neuronal susceptibility to 1-methyl-4-phenylpyridinium- and 6-hydroxydopamine-induced toxicity in astrocytic/ventral mesencephalic co-cultures

Altered glial function in the substantia nigra in Parkinson's disease may lead to the release of toxic substances that cause dopaminergic cell death or increase neuronal vulnerability to neurotoxins. To investigate this concept, we examined the effects of subjecting astrocytes to lipopolysaccharide (LPS)-induced activation alone or combined with L-buthionine-[S,R]-sulfoximine-induced glutathione depletion or inhibition of complex I activity by 1-methyl-4-phenylpyridinium (MPP+) on the viability of primary ventral mesencephalic neurones or susceptibility to MPP+ and 6-hydroxydopamine (6-OHDA) in co-cultures. LPS-activated astrocytes caused neuronal death in a time-dependent manner, but glutathione-depleted or complex I-inhibited astrocytes had no effect on neuronal viability. The neurotoxicity of LPS-activated astrocytes was inhibited by the inducible nitric oxide synthase inhibitor aminoguanidine, by the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and by reduced glutathione (GSH). MPP+-induced neuronal death was greater in ventral mesencephalic cultures previously cultured with LPS-activated, glutathione-depleted, or complex I-inhibited astrocytes compared with co-cultures containing normal astrocytes. The increased neuronal susceptibility to MPP+ caused by LPS-activated or complex I-inhibited astrocytes and glutathione-depleted astrocytes was inhibited by the NMDA/glutamate antagonist MK-801 and by GSH, respectively. Neuronal death caused by 6-OHDA was increased in ventral mesencephalic cultures previously cultured with LPS-activated and glutathione-depleted, but not complex I-inhibited astrocytes, compared with co-cultures containing normal astrocytes. Treatment of co-cultures with GSH prevented the increased neuronal susceptibility to 6-OHDA. These findings suggest that glial dysfunction may cause neuronal death or render neurones susceptible to toxic insults via a mechanism involving the release of free radicals and glutamate. Such a mechanism may play a role in the development or progression of nigrostriatal degeneration in Parkinson's disease.     

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.     

Dynamics of neuron-glia interplay upon exposure to unconjugated bilirubin

Microglia are the main players of the brain immune response. They act as active sensors that rapidly respond to injurious insults by shifting into different activated states. Elevated levels of unconjugated bilirubin (UCB) induce cell death, immunostimulation and oxidative stress in both neurons and astrocytes. We recently reported that microglial phagocytic phenotype precedes the release of pro-inflammatory cytokines upon UCB exposure. We investigated whether and how microglia microenvironment influences the response to UCB. Our findings revealed that conditioned media derived from UCB-treated astrocytes reduce microglial inflammatory reaction and cell death, suggesting an attempt to curtail microglial over activation. Conditioned medium from UCB-challenged neurons, although down-regulating tumor necrosis factor-α and interleukin-1β promoted the release of interleukin-6 and nitric oxide, the activation of matrix metalloproteinase-9, and cell death, as compared with UCB-direct effects on microglia. Moreover, soluble factors released by UCB-treated neurons intensified the phagocytic properties manifested by microglia under direct exposure to UCB. Results from neuron-microglia mixed cultures incubated with UCB evidenced that sensitized microglia were able to prevent neurite outgrowth impairment and cell death. In conclusion, our data indicate that stressed neurons signal microglial clearance functions, but also overstimulate its inflammatory potential ultimately leading to microglia demise.     

Glucocorticoids Exacerbate Hypoxic and Hypoglycemic Hippocampal Injury In Vitro: Biochemical Correlates and a Role for Astrocytes

The acute secretion of glucocorticoids is critical for responding to physiological stress. Under normal circumstances these hormones do not cause acute neuronal injury, but they have been shown to enhance ischemic and seizure-induced neuronal injury in the rat brain. Using fetal rat hippocampal cultures, we asked whether hypoxic and hypoglycemic cell damage in vitro could be exacerbated by direct exposure to corticosterone (CORT). Each of these insults alone damaged neuronal cells, whereas 4-6 h of hypoxic treatment could damage age-matched astrocytes if glucose was reduced or omitted. Ischemic-like injury to both cell types could be attenuated by pretreatment with high (30 mM) glucose. Exposure to 100 nM CORT did not affect cell viability under control conditions but enhanced both hypoxic and hypoglycemic neuronal injury. In both cases, pretreatment with high glucose abolished this CORT-mediated synergy. In astrocyte cultures, CORT exacerbated both hypoxic and hypoglycemic injury and this effect was also attenuated by high-glucose pretreatment. Identical 24-h CORT treatment caused a 13% reduction in glucose uptake in astrocytes and a 38% reduction in glycogen content, without affecting the level of intracellular glucose. Thus, CORT could endanger both neurons and astrocytes in mixed hippocampal cultures and this effect emerged only under conditions of substrate depletion. The metabolic disruption in astrocytes by CORT further suggests that the ability of CORT to exacerbate neuronal injury may be due in part to impaired glial cell function.     

NT2 derived neuronal and astrocytic network signalling

A major focus of stem cell research is the generation of neurons that may then be implanted to treat neurodegenerative diseases. However, a picture is emerging where astrocytes are partners to neurons in sustaining and modulating brain function. We therefore investigated the functional properties of NT2 derived astrocytes and neurons using electrophysiological and calcium imaging approaches. NT2 neurons (NT2Ns) expressed sodium dependent action potentials, as well as responses to depolarisation and the neurotransmitter glutamate. NT2Ns exhibited spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling. Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, NT2 astrocytes (NT2As) exhibited morphology and functional properties consistent with this glial cell type. NT2As responded to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. NT2As also generated propagating calcium waves that were gap junction and purinergic signalling dependent. Our results show that NT2 derived astrocytes exhibit appropriate functionality and that NT2N networks interact with NT2A networks in co-culture. These findings underline the utility of such cultures to investigate human brain cell type signalling under controlled conditions. Furthermore, since stem cell derived neuron function and survival is of great importance therapeutically, our findings suggest that the presence of complementary astrocytes may be valuable in supporting stem cell derived neuronal networks. Indeed, this also supports the intriguing possibility of selective therapeutic replacement of astrocytes in diseases where these cells are either lost or lose functionality.     

Astrocytes Protect Neurons from Ammonia Toxicity

Ammonia is a neurotoxin that is implicated in the CNS dysfunction associated with hepatic encephalopathy, urea cycle disorders, Reye’s syndrome and other neurological conditions. While in vivo studies suggest that astrocytes are the principal target of ammonia toxicity, recent in vitro investigations suggest that neurons may also be directly affected by ammonia. To further examine the issue of neural cell sensitivity to ammonia, pure rat cortical neuronal cultures, as well as co-cultures of neurons and astrocytes, were exposed to 5 mM NH₄Cl for 48 h. Cultures were examined for morphological changes by light microscopy, measures of cell death, free radical production and changes in the mitochondrial inner membrane potential. Ammonia caused extensive degenerative changes in pure cultured neurons, while such neuronal changes were minor in the co-cultures. Similarly, processes of pure cultured neurons displayed a significant loss of the mitochondrial inner membrane potential, as compared to neurons in co-cultures. Cell death (LDH release) in ammonia-treated neuronal cultures was twice as great as untreated controls, while in co-cultures ammonia did not significantly increase cell death. Free radical production at 3 min was increased (69%, P<0.05) in pure neuronal cultures but not in co-cultures. The neuroprotective effects observed in co-cultures may have been mediated by the astrocyte’s ability to scavenge free radicals, by their detoxification of ammonia and/or by their neurotrophic actions. The neuroprotective action of astrocytes may explain the failure to detect significant pathological changes in neurons in ammonia toxicity in vivo. Special issue dedicated to Dr. Bernd Hamprecht.     

Astrocytic but not neuronal increased expression and redistribution of parkin during unfolded protein stress

Parkin is a ubiquitin ligase that facilitates proteasomal protein degradation and is involved in a common autosomal recessive form of Parkinson's disease. Its expression is part of the unfolded protein response in cell lines where its overexpression protects against unfolded protein stress. How parkin expression is regulated in brain primary cells under stress situations is however, less well established. Here, the cellular and subcellular localization of parkin under basal conditions and during unfolded protein stress was investigated in primary cultures of rat astrocytes and hippocampal neurons. Immunofluorescense microscopy and biochemical analysis demonstrated that parkin is mainly associated with the endoplasmic reticulum (ER) in hippocampal neurons while it is associated with Golgi membranes, the nuclei and light vesicles in astrocytes. The constitutive parkin expression was high in neurons as compared with astrocytes. However, unfolded protein stress elicited a selective increase in astrocytic parkin expression and a change in distribution, whereas neuronal parkin remained largely unmodified. The cell specific differences argue in favour of different cellular binding sites and substrates for the protein and a pathogenic role for astrocytes in Parkinson's disease caused by parkin dysfunction.     

Neuroprotective immunity: T cell-derived glutamate endows astrocytes with a neuroprotective phenotype

A well-controlled T cell response to CNS injury may result in increased neuronal survival. However, the precise mechanism of T cell-induced neuroprotection is unknown. In this study, we report the unexpected finding that during culture of T cells, high levels of glutamate accumulate, which are efficiently cleared if T cells are cocultured with astrocytes. The T cell-derived glutamate elicits in turn, the release of neuroprotective thiols (cysteine, glutathione, and cysteinyl-glycine) and lactate from astrocytes. Media obtained from astrocytes conditioned in the presence of T cells reduce neuronal apoptosis induced by oxidative stress in primary neuronal cultures from 48 +/- 14 to 9 +/- 4% (p < 0.001). Inhibition of glutamate-dependent signaling during astrocyte-T cell cocultivation by a glutamate uptake inhibitor, l-aspartic acid beta-hydroxamate, abolishes this neuroprotective effect. The ability of astrocytes to clear extracellular glutamate is impaired under conditions of oxidative stress. We demonstrate that T cells, via secreted cytokines, restore glutamate clearance capacity of astrocytes under oxidative conditions. Furthermore, under normoxic conditions, glutamate-buffering capacity of astrocytes is increased upon cocultivation with T cells. It is known that, following CNS injury, astrocytes can respond with beneficial or destructive effects on neurons. However, the context and signaling mechanisms for this dual astrocytic response are unknown. Our results implicate T cells as potential determinants of the context that elicits a protective role for astrocytes in the damaged CNS.     

Poly(ADPR)polymerase expression and activity during proliferation and differentiation of rat astrocyte and neuronal cultures.

Poly(ADPR)polymerase (poly(ADPR)P) mRNA and enzymatic activity levels were investigated in primary cultures of rat astrocytes and neurons in the absence or presence of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), respectively. In cultured rat astrocytes, a biphasic increase in poly(ADPR)P mRNA, associated with enhanced nuclear poly(ADPR)P enzymatic activity, were observed. The first rise in poly(ADPR)P mRNA and enzymatic activity is at the beginning of cell proliferation and the second with the occurrence of cell differentiation. In the presence of bFGF (5 ng/ml) the mRNA peaks and the differentiation-associated poly(ADPR)P enzymatic activity undergoes a 2-fold increase. In neuronal cultures an initial high level of poly(ADPR)P mRNA is followed by a decrease while differentiation is progressively achieved. A limited increase of poly(ADPR)P activity is observed during this phase. In the presence of NGF (50 ng/ml), similar poly(ADPR)P mRNA expression and enzymatic activity patterns are observed. The results suggest that poly(ADPR)P is involved at the onset of nerve-cell proliferation and differentiation.     

Neonatal astrocyte damage is sufficient to trigger progressive striatal degeneration in a rat model of glutaric acidemia-I

Background

We have investigated whether an acute metabolic damage to astrocytes during the neonatal period may critically disrupt subsequent brain development, leading to neurodevelopmental disorders. Astrocytes are vulnerable to glutaric acid (GA), a dicarboxylic acid that accumulates in millimolar concentrations in Glutaric Acidemia I (GA-I), an inherited neurometabolic childhood disease characterized by degeneration of striatal neurons. While GA induces astrocyte mitochondrial dysfunction, oxidative stress and subsequent increased proliferation, it is presently unknown whether such astrocytic dysfunction is sufficient to trigger striatal neuronal loss.

Methodology/Principal Findings

A single intracerebroventricular dose of GA was administered to rat pups at postnatal day 0 (P0) to induce an acute, transient rise of GA levels in the central nervous system (CNS). GA administration potently elicited proliferation of astrocytes expressing S100β followed by GFAP astrocytosis and nitrotyrosine staining lasting until P45. Remarkably, GA did not induce acute neuronal loss assessed by FluoroJade C and NeuN cell count. Instead, neuronal death appeared several days after GA treatment and progressively increased until P45, suggesting a delayed onset of striatal degeneration. The axonal bundles perforating the striatum were disorganized following GA administration. In cell cultures, GA did not affect survival of either striatal astrocytes or neurons, even at high concentrations. However, astrocytes activated by a short exposure to GA caused neuronal death through the production of soluble factors. Iron porphyrin antioxidants prevented GA-induced astrocyte proliferation and striatal degeneration in vivo, as well as astrocyte-mediated neuronal loss in vitro.

Conclusions/Significance

Taken together, these results indicate that a transient metabolic insult with GA induces long lasting phenotypic changes in astrocytes that cause them to promote striatal neuronal death. Pharmacological protection of astrocytes with antioxidants during encephalopatic crisis may prevent astrocyte dysfunction and the ineluctable progression of disease in children with GA-I.     

Endothelial microsomal prostaglandin E synthase-1 facilitates neurotoxicity by elevating astrocytic Ca2+ levels

Recurrent seizures may cause neuronal damage in the hippocampus. As neurons form intimate interactions with astrocytes via glutamate, this neuron-glia circuit may play a pivotal role in neuronal excitotoxicity following such seizures. On the other hand, astrocytes contact vascular endothelia with their endfeet. Recently, we found kainic acid (KA) administration induced microsomal prostaglandin E synthase-1 (mPGES-1) and prostaglandin E(2) (PGE(2)) receptor EP3 in venous endothelia and on astrocytes, respectively. In addition, mice deficient in mPGES-1 exhibited an improvement in KA-induced neuronal loss, suggesting that endothelial PGE(2) might modulate neuronal damage via astrocytes. In this study, we therefore investigated whether the functional associations between endothelia and astrocytes via endothelial mPGES-1 lead to neuronal injury using primary cultures of hippocampal slices. We first confirmed the delayed induction of endothelial mPGES-1 in the wild-type (WT) slices after KA-treatment. Next, we examined the effects of endothelial mPGES-1 on Ca(2+) levels in astrocytes, subsequent glutamate release and neuronal injury using cultured slices prepared from WT and mPGES-1 knockout mice. Moreover, we investigated which EP receptor on astrocytes was activated by PGE(2). We found that endothelial mPGES-1 produced PGE(2) that enhanced astrocytic Ca(2+) levels via EP3 receptors and increased Ca(2+)-dependent glutamate release, aggravating neuronal injury. This novel endothelium-astrocyte-neuron signaling pathway may be crucial for neuronal damage after repetitive seizures, and hence could be a new target for drug development.     

Astrocyte-neurone communication following oxygen-glucose deprivation

We looked at the possible interactions between astrocytes and neurones during reperfusion using an in vitro model of ischaemia-reperfusion injury, as a controlled environment that lends itself easily to manipulation of the numerous variables involved in such an insult. We constructed a chamber in which O2 can be lowered to a concentration of 1 microm and developed a primary cortical neuronal culture that is 99% pure and can survive to at least 10 days in vitro. We also established a novel system for the co-culture of astrocytes and neurones in order to study the communication between these cells in a manner that allows the complete separation of one cell type from another. Neurone cultures showed profound cell death following an ischaemic period of only 15 min. We co-cultured neurones that had been subjected to a 15-min ischaemic insult with either non-insulted astrocytes or astrocyte-conditioned medium during the reperfusion stage. Both astrocytes and astrocyte-conditioned medium enhanced neuronal survival. Our data also suggest that astrocyte-sourced neuronal glutathione synthesis may play a role in preventing neuronal death.