NMDA-responsible, APV-insensitive receptor in cultured human astrocytes

The present study was conducted to assess N-methyl-D-aspartate (NMDA)-responsible receptors in cultured human astrocytes by monitoring whole-cell membrane currents. NMDA generated currents, that are potentiated by glycine and blocked by Mg2+, with the current/voltage relation showing a reversal potential of +/- 0 mV. The currents were not inhibited by either the selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV), or the non-selective ionotropic glutamate receptor antagonist, kynurenic acid. The currents were inhibited only by 19% in Ca2+-free extracellular solution. Furthermore, GDPbetaS, a broad G-protein inhibitor, inhibited NMDA-induced currents to 82% of original levels. The results of the present study thus suggest that an NMDA-responsible, APV-insensitive receptor with low Ca2+ permeability, distinct from the neuronal NMDA receptors, is expressed in human astrocytes and that the receptor is regulated in part by an unknown G-protein-linked receptor.     

Amyloid-beta(1-42) rapidly forms protofibrils and oligomers by distinct pathways in low concentrations of sodium dodecylsulfate

Alzheimer's disease (AD) is characterized by large numbers of senile plaques in the brain that consist of fibrillar aggregates of 40- and 42-residue amyloid-beta (Abeta) peptides. However, the degree of dementia in AD correlates better with the concentration of soluble Abeta species assayed biochemically than with histologically determined plaque counts, and several investigators now propose that soluble aggregates of Abeta are the neurotoxic agents that cause memory deficits and neuronal loss. These endogenous aggregates are minor components in brain extracts from AD patients and transgenic mice that express human Abeta, but several species have been detected by gel electrophoresis in sodium dodecylsulfate (SDS) and isolated by size exclusion chromatography (SEC). Endogenous Abeta aggregation is stimulated at cellular interfaces rich in lipid rafts, and anionic micelles that promote Abeta aggregation in vitro may be good models of these interfaces. We previously found that micelles formed in dilute SDS (2 mM) promote Abeta(1-40) fiber formation by supporting peptide interaction on the surface of a single micelle complex. In contrast, here we report that monomeric Abeta(1-42) undergoes an immediate conversion to a predominant beta-structured conformation in 2 mM SDS which does not proceed to amyloid fibrils. The conformational change is instead rapidly followed by the near quantitative conversion of the 4 kDa monomer SDS gel band to 8-14 kDa bands consistent with dimers through tetramers. Removal of SDS by dialysis gave a shift in the predominant SDS gel bands to 30-60 kDa. While these oligomers resemble the endogenous aggregates, they are less stable. In particular, they do not elute as discrete species on SEC, and they are completed disaggregated by boiling in 1% SDS. It appears that endogenous oligomeric Abeta aggregates are stabilized by undefined processes that have not yet been incorporated into in vitro Abeta aggregation procedures.     

Signaling induced by hop/STI-1 depends on endocytosis

The co-chaperone hop/STI-1 is a ligand of the cell surface prion protein (PrP(C)), and their interaction leads to signaling and biological effects. Among these, hop/STI-1 induces proliferation of A172 glioblastoma cells, dependent on both PrP(C) and activation of the Erk pathway. We tested whether clathrin-mediated endocytosis affects signaling induced by hop/STI-1. Both hyperosmolarity induced by sucrose and monodansyl-cadaverine blocked Erk activity induced by hop/STI-1, without affecting the high basal Akt activity typical of A172. The endocytosis inhibitors also affected the sub-cellular distribution of phosphorylated Erk, consistent with blockade of the latter's activity. The data indicate that signaling induced by hop/STI-1 depends on endocytosis. These findings are consistent with a role of sub-cellular trafficking in signal transduction following engagement by PrP(C) by ligands such as hop/STI-1, and may help help unravel both the functions of the prion protein, as well as possible loss-of-function components of prion diseases.     

Expression of fasciculation and elongation protein zeta-1 (FEZ1) in cultured rat neonatal astrocytes

Astrocytes play a more important role than simply providing physical support for neurons, however, the function(s) of type 1 and type 2 astrocytes (T1As, T2As), remains unclear. A DNA microarray was used to identify gene expression in cultured T1As and T2As isolated from postnatal day 1 rat cortex. Ninety-nine of the 138 differentially expressed genes were involved in a diverse number of processes. The fasciculation and elongation protein zeta-1 (FEZ1) gene was studied further because it has been suggested that it is not expressed by astrocytes. RT-PCR and Western blots confirmed the microarray data and showed that FEZ1 was present in T1 and T2As and is more highly expressed in T2As. Immunocytochemistry revealed that FEZ1 was located in the astrocytic cytoplasm and cell processes but not the nucleus. The results contribute to a clearer understanding of the two types of astrocytes.     

CAG*CTG repeat instability in cultured human astrocytes

Cells of the central nervous system (CNS) are prone to the devastating consequences of trinucleotide repeat (TNR) expansion. Some CNS cells, including astrocytes, show substantial TNR instability in affected individuals. Since astrocyte enrichment occurs in brain regions sensitive to neurodegeneration and somatic TNR instability, immortalized SVG-A astrocytes were used as an ex vivo model to mimic TNR mutagenesis. Cultured astrocytes produced frequent (up to 2%) CAG·CTG contractions in a sequence-specific fashion, and an apparent threshold for instability was observed between 25 and 33 repeats. These results suggest that cultured astrocytes recapitulate key features of TNR mutagenesis. Furthermore, contractions were influenced by DNA replication through the repeat, suggesting that instability can arise by replication-based mechanisms in these cells. This is a crucial mechanistic point, since astrocytes in the CNS retain proliferative capacity throughout life and could be vulnerable to replication-mediated TNR instability. The presence of interruptions led to smaller but more frequent contractions, compared to a pure repeat, and the interruptions were sometimes deleted to form a perfect tract. In summary, we suggest that CAG·CTG repeat instability in cultured astrocytes is dynamic and replication-driven, suggesting that TNR mutagenesis may be influenced by the proliferative capacity of key CNS cells.     

Selective cytotoxicity of intracellular amyloid beta peptide1-42 through p53 and Bax in cultured primary human neurons

Extracellular amyloid beta peptides (Abetas) have long been thought to be a primary cause of Alzheimer's disease (AD). Now, detection of intracellular neuronal Abeta1--42 accumulation before extracellular Abeta deposits questions the relevance of intracellular peptides in AD. In the present study, we directly address whether intracellular Abeta is toxic to human neurons. Microinjections of Abeta1--42 peptide or a cDNA-expressing cytosolic Abeta1--42 rapidly induces cell death of primary human neurons. In contrast, Abeta1--40, Abeta40--1, or Abeta42--1 peptides, and cDNAs expressing cytosolic Abeta1--40 or secreted Abeta1--42 and Abeta1--40, are not toxic. As little as a 1-pM concentration or 1500 molecules/cell of Abeta1--42 peptides is neurotoxic. The nonfibrillized and fibrillized Abeta1--42 peptides are equally toxic. In contrast, Abeta1--42 peptides are not toxic to human primary astrocytes, neuronal, and nonneuronal cell lines. Inhibition of de novo protein synthesis protects against Abeta1--42 toxicity, indicating that programmed cell death is involved. Bcl-2, Bax-neutralizing antibodies, cDNA expression of a p53R273H dominant negative mutant, and caspase inhibitors prevent Abeta1--42-mediated human neuronal cell death. Taken together, our data directly demonstrate that intracellular Abeta1--42 is selectively cytotoxic to human neurons through the p53--Bax cell death pathway.     

Regulation of protein accumulation in cultured cells.

1. A technique is described whereby protein synthesis, protein breakdown and net protein accumulation are measured separately in monolayer cultures of mammalian cells. All rates are expressed as microgram of protein per 18 h incubation. 2. Under most incubation conditions with either L6 rat myoblasts or T47D human breast carcinoma cells the rates of protein accumulation, determined directly, agreed with the rates obtained by subtracting protein breakdown from protein synthesis. 3. Foetal calf serum, human and bovine colostrum, human milk and insulin increased protein accumulation in both cell lines, mainly as a consequence of effects on protein synthesis. 4. NH4Cl, in addition to inhibiting protein breakdown in both cell lines in the presence and in the absence of serum, stimulated protein synthesis in L6 myoblasts. 5. Leupeptin slightly inhibited protein breakdown without affecting protein-synthesis rates. 6. Cycloheximide almost completely inhibited protein synthesis, but restricted the net loss of cell proteins under most conditions because protein-breakdown rates were also decreased. 7. The assumptions, limitations and potential application of this technique for evaluating changes in protein turnover are described.     

Rho protein inactivation induced apoptosis of cultured human endothelial cells

Small GTP-binding Rho GTPases regulate important signaling pathways in endothelial cells, but little is known about their role in endothelial cell apoptosis. Clostridial cytotoxins specifically inactivate GTPases by glucosylation [Clostridium difficile toxin B-10463 (TcdB-10463), C. difficile toxin B-1470 (TcdB-1470)] or ADP ribosylation (C. botulinum C3 toxin). Exposure of human umbilical cord vein endothelial cells (HUVEC) to TcdB-10463, which inhibits RhoA/Rac1/Cdc42, or to C3 toxin, which inhibits RhoA, -B, -C, resulted in apoptosis, whereas inactivation of Rac1/Cdc42 with TcdB-1470 was without effect, suggesting that Rho inhibition was responsible for endothelial apoptosis. Disruption of endothelial microfilaments as well as inhibition of p160ROCK did not induce endothelial apoptosis. Exposure to TcdB-10463 resulted in activation of caspase-9 and -3 but not caspase-8 in HUVEC. Moreover, Rho inhibition reduced expression of antiapoptotic Bcl-2 and Mcl-1 and increased proapoptotic Bid but had no effect on Bax or FLIP protein levels. Caspase-3 activity and apoptosis induced by TcdB-10463 were abolished by cAMP elevation. In summary, inhibition of Rho in endothelial cells activates caspase-9- and -3-dependent apoptosis, which can be antagonized by cAMP elevation.     

Insulin increases glutamate transporter GLT1 in cultured astrocytes

The astroglial cell-specific glutamate transporter subtype 2 (excitatory amino acid transporter 2, GLT1) plays an important role in excitotoxicity that develops after damage to the central nervous system (CNS) is incurred. Both the protein kinase C signaling pathway and the epidermal growth factor (EGF) pathway have been suggested to participate in the modulation of GLT1, but the modulatory mechanisms of GLT1 expression are not fully understood. In the present study, we aimed to evaluate the effects of insulin on GLT1 expression. We found that short-term stimulation of insulin led to the upregulation of both total and surface expressions of GLT1. Akt phosphorylation increased after insulin treatment, and triciribine, the inhibitor of Akt phosphorylation, significantly inhibited the effects of insulin. We also found that the upregulation of GLT1 expression correlated with increased kappa B motif-binding phosphoprotein (KBBP) and GLT1 mRNA levels. Our results suggest that insulin may modulate the expression of astrocytic GLT1, which might play a role in reactive astrocytes after CNS injuries.     

Widespread immunoreactivity for neuronal nuclei in cultured human and rodent astrocytes

The monoclonal antibody (mAb) neuronal nuclei (NeuN) labels the nuclei of mature neurons in vivo in vertebrates. NeuN has also been used to define post-mitotic neurons or differentiating neuronal precursors in vitro . In this study, we demonstrate that the NeuN mAb labels the nuclei of astrocytes cultured from fetal and adult human, newborn rat, and embryonic mouse brain tissue. A non-neuronal fibroblast cell line (3T3) also displayed NeuN immunoreactivity. We confirmed that NeuN labels neurons but not astrocytes in sections of P10 rat brain. Western blot analysis of NeuN immunoreactive species revealed a distribution of bands in nucleus-enriched fractions derived from the different cell lines that was similar, but not identical to adult rat brain homogenates. We then examined the hypothesis that the glial fibrillary acidic protein/NeuN-double positive population of cells might correspond to neuronal precursors. Although the NeuN-positive astrocytes were proliferating, no evidence of neurogenesis was detected. Furthermore, expression of additional neuronal precursor markers was not detected. Our results indicate that primary astrocytes derived from mouse, rat, and human brain express NeuN. Our findings are consistent with NeuN being a selective marker of neurons in vivo , but indicate that studies utilizing NeuN-immunoreactivity as a definitive marker of post-mitotic neurons in vitro should be interpreted with caution.     

Polyamine uptake in cultured astrocytes: characterization and modulation by protein kinases

The properties and regulation of the polyamine transport system in brain are still poorly understood. The present study shows, for the first time, the existence of a polyamine transport system in cerebellar astrocytes and suggests that polyamine uptake is mediated by a single and saturable high-affinity transport system for putrescine, spermine, and spermidine (K:(m) = 3.2, 1.2, and 1.8 microM:, respectively). Although substitution of NaCl by choline chloride produced a decrease in the putrescine, spermine, and spermidine uptake, it seems that polyamine transport in cerebellar astrocytes is not mediated by an Na(+) cotransport as in the presence of Na(+) and cholinium, polyamine uptake was much lower than when measured in a sucrose-based medium. On the other hand, ouabain, gramicidin (a Na(+) ionophore), and ionomycin (a Ca(2+) ionophore) produced a strong inhibition of polyamine uptake, suggesting that membrane potential could have an important role in the functioning of the astroglial polyamine uptake system. Moreover, protein kinase C inhibition produced an enhancement of polyamine uptake, whereas stimulation of protein kinase C with phorbol esters inhibited polyamine uptake. Alternatively, the tyrosine kinase inhibitor genistein caused a marked reduction in the uptake. No effects on polyamine uptake were observed with inhibitors and activators of cyclic AMP-dependent protein kinase or when Ca(2+)/calmodulin-dependent protein kinase II was inhibited with KN-62. These results suggest that the polyamine uptake system in cerebellar astrocytes could be modulated by protein kinase C and tyrosine kinase activities.     

Spermine induced phosphorylation of a 42 kD/pI 5.9 nuclear protein in different human tumor cell lines

In vitro phosphorylation of 5 M urea extracts from nuclei obtained from different human tumor cell lines leads to incorporation of phosphate from 32P-gamma-ATP in more than 20 polypeptides with an acidic pI. Whereas heparin at a concentration of 1 microgram had no effect on the phosphorylation pattern, spermine stimulated the total phosphorylation up to twofold. Furthermore, in the presence of this polyamine, the two-dimensional polyacrylamide gel revealed an additional phosphoprotein with an apparent pI of 5.9 and a relative molecular mass of 42 000. Phosphoamino acid analysis of the most prominent phosphoproteins showed serine and threonine as phosphoacceptors.     

Amyloid-beta1-42 treatment does not have a specific effect on cholinergic neurons in in vitro basal forebrain neuronal cultures of rat

The neurotoxic effect of amyloid-beta peptide (1-42) was investigated in cultures of neuronal tissue derived from the basal forebrain of embryonic rat. The axonal varicosities of the cholinergic cells were revealed by vesicular acetylcholine transporter staining, and the axonal varicosities in general by synaptophysin immunohistochemistry. The results demonstrate that the treatment of in vitro neuronal cultures with 20 microM amyloid-beta peptide (1-42) for 2 days on day 5, 12 or 15 exerted a neurotoxic effect on both the cholinergic and the non-cholinergic neurons. In the same cultures, the absolute number of synaptophysin-positive axon varicosities was reduced to greater extent (control: 203 +/- 37/field vs treated: 101 +/- 16/field) than the number of vesicular acetylcholine transporter-immunoreactive (control: 48 +/- 4/field vs treated: 0/field) structures. It is concluded that amyloid-beta peptide (1-42) does not have a specific effect only on the cholinergic neurons, but affects non-cholinergic neurons as well.     

The production of macrophage inflammatory protein-2 induced by soluble intercellular adhesion molecule-1 in mouse astrocytes is mediated by src tyrosine kinases and p42/44 mitogen-activated protein kinase

Severe traumatic brain injury stimulates the release of soluble intercellular adhesion molecule-1 (sICAM-1) into CSF. Studies in cultured mouse astrocytes suggest that sICAM-1 induces the production of macrophage inflammatory protein-2 (MIP-2). In the present study, we investigated the underlying mechanisms for MIP-2 induction. sICAM-1 induced MIP-2 in astrocytes lacking membrane-bound ICAM-1, indicating that its action is due to heterophilic binding to an undescribed receptor rather than homophilic binding to surface ICAM-1. Signal transduction may be mediated by src tyrosine kinases, as the src tyrosine kinase inhibitors herbimycin A and PP2 abolished MIP-2 induction by sICAM-1. Phosphorylation of p42/44 mitogen-activated protein kinase (MAPK), but not of p38 MAPK, occurred further downstream, as evidenced by western blot analysis combined with the use of herbimycin A and specific MAPK inhibitors. By contrast, induction of MIP-2 by tumour necrosis factor-alpha (TNF-alpha) involved both p42/44 MAPK and p38 MAPK. Following stimulation with either sICAM-1 or TNF-alpha, astrocyte supernatants promoted chemotaxis of human neutrophils and incubation of these supernatants with anti-MIP-2 antibodies more efficiently suppressed the migration induced by sICAM-1 than by TNF-alpha. These results show that sICAM-1 induces the production of biologically active MIP-2 in astrocytes by heterophilic binding to an undefined receptor and activation of src tyrosine kinases and p42/44 MAPK.     

Enhanced expression of Harvey ras induced by serum deprivation in cultured astrocytes

Trophic deprivation contributes to astrocyte damage that occurs in acute and chronic neurodegenerative disorders. Unraveling the underlying mechanisms may pave way to novel cytoprotective strategies. Cultured mouse astrocytes responded to trophic deprivation with a large and transient increase in the expression of p21^ras, which was secondary to an enhanced formation of reactive oxygen species (ROS) detected by cytofluorimetric analysis after preloading with 2',7'-dichlorofluorescein diacetate. The increase in p21^ras levels was largely attenuated by the reducing agent, N -acetylcysteine, which was proven to reduce ROS formation in astrocytes subjected to serum deprivation. We extended the analysis to the Ha-Ras isoform, which has been implicated in mechanisms of cytotoxicity. We found that serum deprivation enhanced the expression and activity of Ha-Ras without changing Ha-Ras mRNA levels. The increase in Ha-Ras levels was sensitive to the protein synthesis inhibitor, cycloheximide, suggesting that serum deprivation increases translation of preformed Ha-Ras mRNA. The late decline in Ha-Ras levels observed after 60 min was prevented by the proteasome inhibitor, MG132, as well as by the selective mitogen-activated protein kinase (MAPK) inhibitor, PD98059. Serum deprivation led to the activation of the MAPK pathway in cultured astrocytes, as shown by an increase in phosphorylated extracellular signal-regulated kinase 1/2 levels after 5 and 30 min. Finally, using the siRNA technology, we found that an acute knock-down of Ha-Ras was protective against astrocyte damage induced by serum deprivation. We conclude that cultured astrocytes respond to trophic deprivation with an increased expression in Ha-Ras, which is limited by the concomitant activation of the MAPK pathway, but is nevertheless involved in the pathophysiology of cell damage.     

Involvement of protein kinase C in prostaglandin D2 synthesis by cultured astrocytes

The role of protein kinase C (PKC) and calcium in the stimulation of prostaglandin D₂ (PGD₂) synthesis was investigated in primary rat astroglial cultures using the phorbol esters phorbol 12-myristate, 13-acetate (PMA), phorbol 12,13-dibutyrate (PDB) and the calcium ionophore A₂₃₁₈₇. Both phorbol esters and the ionophore were able to stimulate PGD₂ synthesis in a concentration dependent manner. The inactive stereoisomers of PMA and PDB had no significant effect. Combinations of subthreshold concentrations of phorbol esters (10 nM PMA or 10 nM PBD) potentiated PG formation induced by 100 nM A₂₃₁₈₇. An even more pronounced effect was observed when phorbol ester concentrations were increased to 100nM. The contribution of extra- and intracellular calcium in phorbol ester or A₂₃₁₈₇ stimulated PGD₂ synthesis was evaluated by carrying out experiments with calcium-free media plus EGTA or with the intracellular calcium-chelating agent TMB-8. Ionophore stimulated PGD₂ release was shut down to basal values upon removal of extracellular calcium, whereas phorbol ester stimulated PGD₂ formation persisted at a reduced level. It was unabated also upon further addition of EGTA. In the presence of TMB-8, however, phorbol ester stimulated PGD₂ synthesis was completely suppressed. These data strongly suggest that PKC has an additional effect on the activation of phospholipase A₂ and subsequent prostanoid synthesis, which is independent from extracellular calcium and, thus, support the concept of more than one metabolic pathway in astrocytes that synergistically regulate phospholipase A₂ activity.     

Beta-amyloid (1-42) affects MTT reduction in astrocytes: implications for vesicular trafficking and cell functionality

Beta-amyloid (Abeta) peptide deposition in the brains of Alzheimer's disease patients results in reactive astrogliosis which may enhance neuronal cell death. Abeta has also been reported to impair important supportive astrocyte functions, such as glutamate uptake in vitro. We studied the effect of amyloid beta-peptide (Abeta) on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, cellular ATP content, lactate release, and proliferation using neonatal rat astrocyte cultures. Abeta(1-42) decreased MTT reduction potently in the absence of cell death, but did not affect cellular ATP levels or lactate release. Moreover, the cells displayed increased proliferation after incubation with Abeta(1-42), confirming that the decreased MTT reduction was not deleterious to cell viability. Abeta(1-42) enhanced transfer of MTT dye to the cell surface leading to cessation of MTT reduction and cell death. Bafilomycin A1, but not brefeldin A, prevented the enhanced trafficking of MTT, suggesting that lysosomes, but not Golgi apparatus, are involved. Our results show that the viability of astrocytes themselves is not diminished by beta-amyloid peptide. However, Abeta alters vesicular trafficking in astrocytes, which may disturb the supportive function of astrocytes in the brains of patients with Alzheimer's disease.