Ammonia-treated Astrocytes Research Articles

The Role of Nrf2 Transcription Factor and Sp1-Nrf2 Protein Complex in Glutamine Transporter SN1 Regulation in Mouse Cortical Astrocytes Exposed to Ammonia.

Ammonia toxicity in the brain primarily affects astrocytes via a mechanism in which oxidative stress (OS), is coupled to the imbalance between glutamatergic and GABAergic transmission. Ammonia also downregulates the astrocytic N system transporter SN1 that controls glutamine supply from astrocytes to neurons for the replenishment of both neurotransmitters. Here, we tested the hypothesis that activation of Nrf2 is the process that links ammonia-induced OS formation in astrocytes to downregulation and inactivation of SN1 and that it may involve the formation of a complex between Nrf2 and Sp1. Treatment of cultured cortical mouse astrocytes with ammonia (5 mM NH4Cl for 24 h) evoked Nrf2 nuclear translocation, increased its activity in a p38 MAPK pathway-dependent manner, and enhanced Nrf2 binding to Slc38a3 promoter. Nrf2 silencing increased SN1 mRNA and protein level without influencing astrocytic [3H]glutamine transport. Ammonia decreased SN1 expression in Nrf2 siRNA treated astrocytes and reduced [3H]glutamine uptake. In addition, while Nrf2 formed a complex with Sp1 in ammonia-treated astrocytes less efficiently than in control cells, treatment of astrocytes with hybrid-mode inactivated Sp1-Nrf2 complex (Nrf2 silencing + pharmacological inhibition of Sp1) did not affect SN1 protein level in ammonia-treated astrocytes. In summary, the results document that SN1 transporter dysregulation by ammonia in astrocytes involves activation of Nrf2 but does not require the formation of the Sp1-Nrf2 complex.

Activation of Protein Kinase Cδ Contributes to the Induction of Src/EGF Receptor/ERK Signaling in Ammonia-treated Astrocytes.

Previously, we showed that Src-mediated EGF receptor transactivation/ERK activation mediates ammonia-induced astrocyte swelling, which represents a major component of brain edema in hyperammonemic disorders. Here, we tested the role of PKC in the induction of this signaling pathway and its involvement in ammonia-mediated cell swelling. We found that incubating astrocytes with bisindolylmaleimide (BIM, an inhibitor of classical and novel PKC isoforms) or rottlerin, a PKCδ-specific inhibitor, attenuated the ammonia-induced phosphorylation of EGFR, while GF109203X had no effect on this pathway. We further found that BIM or rottlerin pretreatment inhibited the ammonia-induced phosphorylation of Src and that ammonia significantly increased the level of PKCδ pulled down by a Src antibody. AG1478, a specific EGFR kinase activity inhibitor, effectively inhibited phosphorylation at Tyr1068 but had no discernable effect on phosphorylation at Tyr845. Moreover, BIM or rottlerin abrogated ammonia-induced ERK phosphorylation. BIM-, rottlerin-, or GF109203X-treated astrocytes showed a significant reduction in cell swelling compared to that observed after treatment with ammonia alone. Finally, it was found that AG1478 attenuated ammonia-induced PKCα translocation to the particulate fraction. Taken together, our results indicate that PKCδ mediates ammonia-induced astrocyte swelling by activating Src and downstream EGF receptor/ERK signaling, which may contribute to the pathogenesis of neuropsychiatric disorders associated with hyperammonemia.

P: 78 Mechanism of Alzheimer Type II Astrocyte Development: Implication for the Defective Neuronal Integrity and Neurobehavioral Deficits Associated With Chronic Hepatic Encephalopathy

BACKGROUND: Chronic hepatic encephalopathy (CHE) is a major neurological condition that occurs following chronic liver failure (CLF) following drug-induced hepatotoxicity, viral hepatitis, or exposure to various hepatotoxins. CHE is characterized by mental confusion, behavioral changes, and motor disturbances. The molecular basis for CHE remains elusive. The presence of Alzheimer type II astrocytes (AT2A) is the only histopathological findings observed in CHE. However, little is currently known regarding its development, and its involvement in the pathogenesis of CHE. METHODS: Astrocytic glia maturation factor and other inflammatory factors were measured by western blots, immunohistochemistry, and by ELISA. AT2A was analyzed by histopathology. RESULTS: We identified increased levels of astrocytc glia maturation factor (GMF), a factor strongly implicated in neuroinflammation and in the overexpression of various inflammatory factors (IL-1β, TNF-α, IL-6, chemokine (C-X-C motif) ligand 1 (CXCL1), matrix metalloproteinase-3 (MMP-3), prostaglandin E2 (PGE2) and cyclooxygenase 2 (COX2), as well as reduced levels of α-tubulin and glial fibrillary acidic protein, along with increased levels of aggregated nuclear protein lamin a/c in the thioacetamide-induced rat model of CHE. Further, we identified reduced levels of neuronal proteins, PSD95, synaptophysin, and NMDA-nr1. Moreover, synaptic density and dendritic complexity are reduced post-CLF. Since elevated blood/brain ammonia levels have been strongly implicated in the pathogenesis of CHE, while exposure of cultured astrocytes to ammonia was shown to develop AT2A, we utilized this in vitro system to delineate mechanisms by which ammonia contributes to the development of AT2A. We found increased levels of GMF, aggregated nuclear protein lamin a/c, and inflammatory factors (IL-1β, TNF-α, IL-6, CXCL1, MMP-3, PGE2 and COX2), as well as reduced levels and oxidized forms of α-tubulin in astrocytes post-ammonia treatment, which was similar to that found in vivo. Further, exposure of cultured neurons to conditioned media (CM) from ammonia-treated astrocytes (AT2A), but not ammonia per se, resulted in reduced levels of neuronal PSD95, synaptophysin and NMDA-nr1, as well as reduced synaptic density and dendritic complexity. Noteworthy, pharmacological inhibition of GMF, or silencing GMF by CRISPR reversed the defective neuronal integrity post-exposure of neurons to CM from ammonia-treated astrocytes in vitro. CONCLUSIONS: These findings strongly suggest that increased levels of GMF post-CLF may negatively impact neuronal integrity that may ultimately contribute to the neurobehavioral/cognitive and motor deficits observed in CHE. We anticipate that our studies aimed at a better understanding of the molecular mechanisms involved in the development of AT2A, and its impact on neuronal integrity in CHE, will greatly facilitate the identification of agents capable of ameliorating this debilitating condition.

Silencing of Transcription Factor Sp1 Promotes SN1 Transporter Regulation by Ammonia in Mouse Cortical Astrocytes.

The involvement of the astrocytic SN1 (SNAT3) transporter in ammonia-induced l-glutamine retention was recently documented in mouse-cultured astrocytes. Here we investigated the involvement of specificity protein 1 (Sp1) transcription factor in SN1 regulation in ammonium chloride (“ammonia”)-treated astrocytes. Sp1 expression and its cellular localization were determined using real-time qPCR, Western blot, and confocal microscopy. Sp1 binding to Snat3 promoter was analyzed by chromatin immunoprecipitation. The role of Sp1 in SN1 expression and SN1-mediated [3H]glutamine uptake in ammonia-treated astrocytes was verified using siRNA and mithramycin A. The involvement of protein kinase C (PKC) isoforms in Sp1 level/phosphorylation status was verified using siRNA technology. Sp1 translocation to the nuclei and its enhanced binding to the Snat3 promoter, along with Sp1 dependence of system N-mediated [3H]glutamine uptake, were observed in astrocytes upon ammonia exposure. Ammonia decreased the level of phosphorylated Sp1, and the effect was reinforced by long-term incubation with PKC modulator, phorbol 12-myristate 13-acetate, which is a treatment likely to dephosphorylate Sp1. Furthermore, silencing of the PKCδ isoform appears to enhance the ammonia effect on the Sp1 level. Collectively, the results demonstrate the regulatory role of Sp1 in regulation of SN1 expression and activity in ammonia-treated astrocytes and implicate altered Sp1 phosphorylation status in this capacity.

Decreased STAT3 Phosphorylation Mediates Cell Swelling in Ammonia-Treated Astrocyte Cultures.

Brain edema, due largely to astrocyte swelling, and the subsequent increase in intracranial pressure and brain herniation, are major complications of acute liver failure (ALF). Elevated level of brain ammonia has been strongly implicated in the development of astrocyte swelling associated with ALF. The means by which ammonia brings about astrocyte swelling, however, is incompletely understood. Recently, oxidative/nitrosative stress and associated signaling events, including activation of mitogen-activated protein kinases (MAPKs), as well as activation of the transcription factor, nuclear factor-kappaB (NF-κB), have been implicated in the mechanism of ammonia-induced astrocyte swelling. Since these signaling events are known to be regulated by the transcription factor, signal transducer and activator of transcription 3 (STAT3), we examined the state of STAT3 activation in ammonia-treated cultured astrocytes, and determined whether altered STAT3 activation and/or protein expression contribute to the ammonia-induced astrocyte swelling. STAT3 was found to be dephosphorylated (inactivated) at Tyrosine705 in ammonia-treated cultured astrocytes. Total STAT3 protein level was also reduced in ammonia-treated astrocytes. We also found a significant increase in protein tyrosine phosphatase receptor type-1 (PTPRT-1) protein expression in ammonia-treated cultured astrocytes, and that inhibition of PTPRT-1 enhanced the phosphorylation of STAT3 after ammonia treatment. Additionally, exposure of cultured astrocytes to inhibitors of protein tyrosine phosphatases diminished the ammonia-induced cell swelling, while cultured astrocytes over-expressing STAT3 showed a reduction in the astrocyte swelling induced by ammonia. Collectively, these studies strongly suggest that inactivation of STAT3 represents a critical event in the mechanism of the astrocyte swelling associated with acute liver failure.

Decreased astrocytic thrombospondin-1 secretion after chronic ammonia treatment reduces the level of synaptic proteins: in vitro and in vivo studies.

Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin-1 (TSP-1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH₄Cl, 0.5-2.5 mM) for 1-10 days, and TSP-1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra- and extracellular TSP-1 levels. Exposure of cultured neurons to conditioned media from ammonia-treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP-1 over-expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP-1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP-1 synthesis in other cell types, also reversed the ammonia-induced TSP-1 reduction. Likewise, we found a significant decline in TSP-1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP-1 may represent an important therapeutic target for CHE. Defective release of astrocytic factors may impair synaptic integrity in chronic hepatic encephalopathy. We found a reduction in the release of the astrocytic matricellular proteins thrombospondin-1 (TSP-1) in ammonia-treated astrocytes; such reduction was associated with a decrease in synaptic proteins caused by conditioned media from ammonia-treated astrocytes. Exposure of neurons to CM from ammonia-treated astrocytes, in which TSP-1 is over-expressed, reversed (by approx 75%) the reduction in synaptic proteins. NF-kB = nuclear factor kappa B; PSD95 = post-synaptic density protein 95; ONS = oxidative/nitrative stress.

Ammonia induces aquaporin-4 rearrangement in the plasma membrane of cultured astrocytes

Aquaporin-4 (AQP4) is a water channel protein mainly located in the astroglial plasma membrane, the precise function of which in the brain edema that accompanies hepatic encephalopathy (HE) is unclear. Since ammonia is the main pathogenic agent in HE, its effect on AQP4 expression and distribution in confluent primary astroglial cultures was examined via their exposure to ammonium chloride (1, 3 and 5mM) for 5 and 10days. Ammonia induced the general inhibition of AQP4 mRNA synthesis except in the 1mM/5day treatment. However, the AQP4 protein content measured was dependent on the method of analysis; an apparent increase was recorded in treated cells in in-cell Western assays, while an apparent reduction was seen with the classic Western blot method, perhaps due to differences in AQP4 aggregation. Ammonia might therefore induce the formation of insoluble AQP4 aggregates in the astroglial plasma membrane. The finding of AQP4 in the pellet of classic Western blot samples, plus data obtained via confocal microscopy, atomic force microscopy (using immunolabeled cells with gold nanoparticles) and scanning electron microscopy, all corroborate this hypothesis. The effect of ammonia on AQP4 seems not to be due to any osmotic effect; identical osmotic stress induced by glutamine and salt had no significant effect on the AQP4 content. AQP4 functional analysis (subjecting astrocytes to a hypo-osmotic medium and using flow cytometry to measure cell size) demonstrated a smaller water influx in ammonia-treated astrocytes suggesting that AQP4 aggregates are representative of an inactive status; however, more confirmatory studies are required to fully understand the functional status of AQP4 aggregates. The present results suggest that ammonia affects AQP4 expression and distribution, and that astrocytes change their expression of AQP4 mRNA as well as the aggregation status of the ensuing protein depending on the ammonia concentration and duration of exposure.

RNA oxidation and zinc in hepatic encephalopathy and hyperammonemia

Hepatic encephalopathy is a neuropsychiatric manifestation of acute and chronic liver failure. Ammonia plays a key role in the pathogenesis of hepatic encephalopathy by inducing astrocyte swelling and/or sensitizing astrocytes to swelling by a heterogeneous panel of precipitating factors and conditions. Whereas astrocyte swelling in acute liver failure contributes to a clinically overt brain edema, a low grade glial edema without clinically overt brain edema is observed in hepatic encephalopathy in liver cirrhosis. Astrocyte swelling produces reactive oxygen and nitrogen oxide species (ROS/RNOS), which again increase astrocyte swelling, thereby creating a self-amplifying signaling loop. Astroglial swelling and ROS/RNOS increase protein tyrosine nitration and may account for neurotoxic effects of ammonia and other precipitants of hepatic encephalopathy. Recently, RNA oxidation and an increase of free intracellular zinc ([Zn(2+)](i)) were identified as further consequences of astrocyte swelling and ROS/RNOS production. An elevation of [Zn(2+)](i) mediates mRNA expression of metallothionein and the peripheral benzodiazepine receptor (PBR) induced by hypoosmotic astrocyte swelling. Further, Zn(2+) mediates RNA oxidation in ammonia-treated astrocytes. In the brain of hyperammonemic rats oxidized RNA localizes in part to perivascular astrocyte processes and to postsynaptic dendritic spines. RNA oxidation may impair postsynaptic protein synthesis, which is critically involved in learning and memory consolidation. RNA oxidation offers a novel explanation for multiple disturbances of neurotransmitter systems and gene expression and the cognitive deficits observed in hepatic encephalopathy.

Ammonia affects the activity and expression of soluble and particulate GC in cultured rat astrocytes

Neurotoxic effects of ammonia are mediated by increased accumulation of nitric oxide (NO), which combines with free radicals to form a highly toxic compound, peroxynitrite. Previous experiments in vivo and in vitro have suggested that this phenomenon engages neuron-derived NO and is coupled to changes in the accumulation of cGMP. The present study accounted for the facts that: (i) astrocytes, not neurons are the morphological target of ammonia, and (ii) both NO-dependent, soluble (sGC) and NO-independent, particulate guanylate cyclase (pGC) mediate cGMP production in the cells. Neocortical rat astrocytes were treated for 1 or 24 h with 5 mM ammonium chloride (“ammonia”) and then subjected to: (i) cGMP measurement, and (ii) mRNA and/or protein expression analysis of α1 and β1 subunits of sGC and two pGC forms: pGC-A and pGC-B. Treatment with ammonia for 1 h increased accumulation of cGMP and sGCβ1 mRNA expression, without producing significant changes in the protein expression. This was followed by a decrease of cGMP level at 24 h treatment, associated with a decreased expression of sGCβ1 and sGCα1 mRNA and sGCβ1 protein. Expression of pGC-A and pGC-B mRNA was elevated in ammonia-treated astrocytes after 24 h. Accordingly, increased cGMP accumulation was noted in the presence of a specific sGC inhibitor (ODQ). The results show that ammonia affects cGMP production in astrocytes, and that this may involve not only sGC but also pGC.

Glycogen synthesis in the astrocyte: from glycogenin to proglycogen to glycogen

The astrocyte of the newborn rat brain has proven to be a versatile system in which to study glycogen biogenesis. We have taken advantage of the rapid stimulation of glycogen synthesis that occurs when glucose is fed to astrocytes, and the marked limitation on this synthesis that occurs in astrocytes previously exposed to ammonium ions. These observations have been related to our earlier reports of the initiation of glycogen synthesis on a protein primer, glycogenin, and the discovery of a low-molecular-weight form of glycogen, proglycogen. The following conclusions have been drawn: 1) In the ammonia-treated astrocytes starved of glucose, free glycogenin is present. 2) When these astrocytes are fed with glucose, proglycogen is synthesized from the glycogenin primer by a glycogen-synthase-like UDPglucose transglucosylase activity (proglycogen synthase) distinct from the well-recognized glycogen synthase, and synthesis stops at this point. 3) Proglycogen is the precursor of macromolecular glycogen, which is synthesized from proglycogen by glycogen synthase when glucose is fed to untreated astrocytes, accounting for the much greater accumulation of total glycogen. 4) The stimulus to proglycogen and macroglycogen synthesis that occurs on feeding glucose to untreated or ammonia-treated astrocytes is the result of the activation of proglycogen synthase, not of glycogen synthase. 5) Therefore, in the synthesis of macromolecular glycogen from glycogenin via proglycogen, the step between glycogenin and proglycogen is rate-limiting. 6) The discovery of additional potential control points in glycogen synthesis, now emerging, may assist the identification of so-far-unexplained aberrations of glycogen metabolism.

The role of astrocytes in hepatic encephalopathy.

The Alzheimer type II astrocyte change is the distinctive morphologic alteration in brain of humans and experimental animals succumbing to hepatic encephalopathy (HE). Whether this change is a primary event in the pathogenesis of HE or whether it is secondary to injury of some other component(s) of the CNS has not been clarified. Studies in a rat model of HE have revealed early reactive changes in astrocytes characterized by cytoplasmic hypertrophy. During the later phases, degenerative changes ensue corresponding to the Alzheimer type II change observed by light microscopy. In view of the role of astrocytes in ammonia detoxification and the importance of ammonia in the pathogenesis of HE, we have suggested that the initial astrocytic changes are the morphological correlates of ammonia detoxification. We have speculated that the later degenerative alterations could lead to failure by astrocytes to carry out key functions (e.g., neurotransmitter uptake, ion regulation, and the like) and contribute the development of the encephalopathy. Recently, the potential involvement of astrocytes in HE has been further investigated, using primary astrocyte cultures. Exposure of cultures to ammonia at clinically relevant concentrations has shown morphologic changes closely resembling those observed in experimental HE in vivo. These deleterious effects can partly be prevented by raising cyclic AMP levels in cells. Other potential toxins (octanoic acid, phenol) have shown pathologic changes as well. Although some alterations were common to all three, they each possessed distinctive pathological effects. A synergistic interaction has also been demonstrated with these toxins. Functional studies of ammonia-treated astrocytes have shown the following: With low doses or short-term exposure, the uptakes of K+, glutamate, and GABA remained unchanged or slightly increased, whereas with higher doses or longer treatment, those activities diminished. A fall in ATP values occurred with prolonged ammonia treatment. Preliminary findings have shown no significant derangements in the beta-adrenergic receptor, except for a slight decrease in receptor affinity. However, cyclic AMP production was diminished following stimulation with isoproterenol. A slight rise in the number of benzodiazepine receptors was found. These studies indicate that profound changes occur in astrocytes following exposure to ammonia and other putative toxins. It is proposed that toxins and factors involved in the precipitation of HE do so by affecting astroglial properties. Derangements in such properties may lead to glial dysfunction (primary gliopathy), resulting in an encephalopathic state.

Effect of cyclic AMP on ammonia-induced alterations in primary astrocyte cultures.

Current evidence suggests that astrocytes may be the target of ammonia toxicity. Consistent with this view are recent investigations which have shown morphologic alterations in primary astrocyte cultures following exposure to ammonia. In the present study, these alterations became severely aggravated when the cultures were not grown or maintained in dibutyryl cyclic adenosine monophosphate (AMP). Cyclic AMP analogues and agents that increase intracellular cyclic AMP levels significantly inhibited the toxic effects of ammonia. The exact mechanism responsible for this apparent protective effect of cyclic AMP on ammonia-treated astrocytes is not known. The possible means by which cyclic AMP may serve to ameliorate ammonia-induced toxicity are discussed.