New hierarchical phosphorylation pathway of the translational repressor eIF4E-binding protein 1 (4E-BP1) in ischemia-reperfusion stress.

Eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) is a translational repressor that is characterized by its capacity to bind specifically to eIF4E and inhibit its interaction with eIF4G. Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap-dependent translation, in cell stress. This study reports a physiological study of 4E-BP1 regulation by phosphorylation using control conditions and a stress-induced translational repression condition, ischemia-reperfusion (IR) stress, in brain tissue. In control conditions, 4E-BP1 was found in four phosphorylation states that were detected by two-dimensional gel electrophoresis and Western blotting, which corresponded to Thr(69)-phosphorylated alone, Thr(69)- and Thr(36)/Thr(45)-phosphorylated, all these plus Ser(64) phosphorylation, and dephosphorylation of the sites analyzed. In control or IR conditions, no Thr(36)/Thr(45) phosphorylation alone was detected without Thr(69) phosphorylation, and neither was Ser(64) phosphorylation without Thr(36)/Thr(45)/Thr(69) phosphorylation detected. Ischemic stress induced 4E-BP1 dephosphorylation at Thr(69), Thr(36)/Thr(45), and Ser(64) residues, with 4E-BP1 remaining phosphorylated at Thr(69) alone or dephosphorylated. In the subsequent reperfusion, 4E-BP1 phosphorylation was induced at Thr(36)/Thr(45) and Ser(64), in addition to Thr(69). Changes in 4E-BP1 phosphorylation after IR were according to those found for Akt and mammalian target of rapamycin (mTOR) kinases. These results demonstrate a new hierarchical phosphorylation for 4E-BP1 regulation in which Thr(69) is phosphorylated first followed by Thr(36)/Thr(45) phosphorylation, and Ser(64) is phosphorylated last. Thr(69) phosphorylation alone allows binding to eIF4E, and subsequent Thr(36)/Thr(45) phosphorylation was sufficient to dissociate 4E-BP1 from eIF4E, which led to eIF4E-4G interaction. These data help to elucidate the physiological role of 4E-BP1 phosphorylation in controlling protein synthesis.

Assessment of protein expression levels after transient global cerebral ischemia using an antibody microarray analysis.

An antibody microarray was used to analyze potential modifications in brain protein levels induced by ischemic reperfusion. Total brain extracts from rats subjected to 15 min of transient global ischemia followed by 3 days of reperfusion and sham control animals were compared within the same array. Separate arrays were run to analyze resistant (cortex) and vulnerable (CA1) regions to ischemia. Candidate components distinguishing the two cellular populations were selected under stringent criteria. IR significantly decreased the expression of Bcl-x, caspase 11, GADD153, Cdk4, E2F1, Retinoblastoma-P, SMAD4, AP-1/c-jun, ATF2, PCAF, MAP1b and cofilin within both regions. NGF and NMDA 2A receptors and IkappaB were specifically down-regulated in CA1, while Pyk2-P, b-NOS, and tyrosine hydroxylase were slightly up-regulated in the same region. Some of the array results were validated by western blot. Both the array and western blot results suggested a relevant IR induced activation of calpain specifically at CA1.

Analysis of the protein expression changes during taxol-induced apoptosis under translation inhibition conditions.

Taxol is currently used in chemotherapeutic treatments of different types of cancers. In this article, we demonstrate that taxol induces apoptosis and translation down-regulation in human embryonic kidney (HEK293T) cells. Antibody arrays are a promising new tool for the analysis of protein levels changes in cells responding to different stimuli. Using this approach, we have identified changes in the expression of 38 proteins (20 down-regulated and 18 up-regulated), implicated in several cellular processes mainly in apoptosis, cell cycle and signal transduction pathways, and also cytoskeleton proteins. Among them, we have confirmed a considerable decrease in the expression of p14(ARF) and a significant increase in the levels of dystrophin and c-Myc. It is known that c-Myc mRNA has an internal ribosome entry segment (IRES) element in its 5'UTR that could regulate its expression under global protein synthesis inhibition conditions. We demonstrate that after taxol treatment, the c-Myc IRES activity is maintained meanwhile cap-dependent activity is inhibited. In addition, an increase in c-Myc mRNA was also observed after taxol treatment. We conclude that taxol-induced c-Myc expression is regulated at both transcriptional and translational levels, the last of them by a mechanism mediated by IRES.

Eukaryotic initiation factors (eIF) 2alpha and 4E expression, localization, and phosphorylation in brain tumors.

Increased protein synthesis is regulated, in part, by two eukaryotic translation initiation factors (eIFs): eIF4E and eIF2alpha. One or both of these factors are often overexpressed in several types of cancer cells; however, no data are available at present regarding eIF4E and eIF2alpha levels in brain tumors. In this study, we analyzed the expression, subcellular localization and phosphorylation states of eIF4E and eIF2alpha in 64 brain tumors (26 meningiomas, 16 oligodendroglial tumors, and 22 astrocytomas) and investigated the correlation with the expression of MIB-1, p53, and cyclin D1 proteins as well. There are significant differences in the phosphorylated eIF4E levels between the tumors studied, being the highest in meningiomas and the lowest in the oligodendroglial tumors. Relative to subcellular localization, eIF4E is frequently found in the nucleus of the oligodendroglial tumors and rarely in the same compartment of the meningiomas, whereas eIF2alpha showed an inverse pattern. Finally, cyclin D1 levels directly correlate with the phosphorylation status of both factors. The different expression, phosphorylation, or/and subcellular distribution of eIF2alpha and eIF4E within the brain types of tumors studied could indicate that different pathways are activated for promoting cell cycle proliferation, for instance, leading to increased cyclin D1 expression.

Nitric oxide mediates NMDA-induced persistent inhibition of protein synthesis through dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 and eukaryotic initiation factor 4G proteolysis.

Cerebral ischaemia causes long-lasting protein synthesis inhibition that is believed to contribute to brain damage. Energy depletion promotes translation inhibition during ischaemia, and the phosphorylation of eIF (eukaryotic initiation factor) 2alpha is involved in the translation inhibition induced by early ischaemia/reperfusion. However, the molecular mechanisms underlying prolonged translation down-regulation remain elusive. NMDA (N-methyl-D-aspartate) excitotoxicity is also involved in ischaemic damage, as exposure to NMDA impairs translation and promotes the synthesis of NO (nitric oxide), which can also inhibit translation. In the present study, we investigated whether NO was involved in NMDA-induced protein synthesis inhibition in neurons and studied the underlying molecular mechanisms. NMDA and the NO donor DEA/NO (diethylamine-nitric oxide sodium complex) both inhibited protein synthesis and this effect persisted after a 30 min exposure. Treatments with NMDA or NO promoted calpain-dependent eIF4G cleavage and 4E-BP1 (eIF4E-binding protein 1) dephosphorylation and also abolished the formation of eIF4E-eIF4G complexes; however, they did not induce eIF2alpha phosphorylation. Although NOS (NO synthase) inhibitors did not prevent protein synthesis inhibition during 30 min of NMDA exposure, they did abrogate the persistent inhibition of translation observed after NMDA removal. NOS inhibitors also prevented NMDA-induced eIF4G degradation, 4E-BP1 dephosphorylation, decreased eIF4E-eIF4G-binding and cell death. Although the calpain inhibitor calpeptin blocked NMDA-induced eIF4G degradation, it did not prevent 4E-BP1 dephosphorylation, which precludes eIF4E availability, and thus translation inhibition was maintained. The present study suggests that eIF4G integrity and hyperphosphorylated 4E-BP1 are needed to ensure appropriate translation in neurons. In conclusion, our data show that NO mediates NMDA-induced persistent translation inhibition and suggest that deficient eIF4F activity contributes to this process.

Characterization of the activity of human MAP kinase-interacting kinase Mnk1b.

Human mitogen-activated protein (MAP) kinase interacting kinase 1b (Mnk1b) is a splice variant of human Mnk1a, which has been identified in our laboratory [A. O'Loghlen, V.M. Gonzalez, D. Pineiro, M.I. Perez-Morgado, M. Salinas, M.E. Martin, Identification and molecular characterization of Mnk1b, a splice variant of human MAP kinase-interacting kinase Mnk1, Exp. Cell Res. 299 (2004) 343-355]. Mnk1b has much higher basal eIF4E kinase activity than Mnk1a. Because Mnk1b presents different features in its C-terminus with respect to Mnk1a, we have studied in this paper the potential role of these structural differences in determining the higher basal eIF4E kinase activity as well as the subcellular localization of Mnk1b. In this paper, we demonstrate that phosphorylation of the Thr209 and Thr214 in the activation loop of Mnk1b is necessary for its activation. However, the different kinase activity between Mnk1a and Mnk1b is independent of the phosphorylation status of the activation loop residues. By deletion of the C-terminal tail in Mnk1a, we confirmed that the absence of this sequence is not responsible for the higher eIF4E kinase activity present in Mnk1b. Moreover, our findings support a crucial role of the 12 amino acids, particularly the Ala344, in the C-terminal specific region of Mnk1b (Mnk1bSR), on the kinase activity of the protein.

Regulatory proteins of eukaryotic initiation factor 2-alpha subunit (eIF2 alpha) phosphatase, under ischemic reperfusion and tolerance.

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha), which is one of the substrates of protein phosphatase 1 (PP1), occurs rapidly during the first minutes of post-ischemic reperfusion after an episode of cerebral ischemia. In the present work, two experimental models of transient global ischemia and ischemic tolerance (IT) were used to study PP1 interacting/regulatory proteins following ischemic reperfusion. For that purpose we utilized PP1 purified by microcystin chromatography, as well as 2D DIGE of PP1alpha and PP1gamma immunoprecipitates. The highest levels of phosphorylated eIF2alpha found after 30 min reperfusion in rats without IT, correlated with increased levels in PP1 immunoprecipitates of the inhibitor DARPP32 as well as GRP78 and HSC70 proteins. After 4 h reperfusion, the levels of these proteins in PP1c complexes had returned to control values, in parallel to a significant decrease in eIF2alpha phosphorylated levels. IT that promoted a decrease in eIF2alpha phosphorylated levels after 30 min reperfusion induced the association of GADD34 with PP1c, while prevented that of DARPP32, GRP78, and HSC70. Different levels of HSC70 and DARPP32 associated with PP1alpha and PP1gamma isoforms, whereas GRP78 was only detected in PP1gamma immunoprecipitates. Here we suggest that PP1, through different signaling complexes with their interacting proteins, may modulate the eIF2alpha phosphorylation/dephosphorylation during reperfusion after a transient global ischemia in the rat brain. Of particular interest is the potential role of GADD34/PP1c complexes after tolerance acquisition.

Antibodies reactive to heat shock protein 90 induce oligodendrocyte precursor cell death in culture. Implications for demyelination in multiple sclerosis.

Oligodendrocyte precursor cells (OPCs) are extremely efficient at remyelination. These cells persist in the adult human central nervous system and can proliferate. However, the failure to remyelinate is a pathological characteristic of the human demyelinating disease multiple sclerosis (MS), which suggests that these cells are ineffective in this disorder. This paper reports that IgG antibodies in the cerebrospinal fluid (CSF) of MS patients specifically recognize an antigen on OPCs in culture. Control patients were found not to possess these antibodies. The antigen was immunoprecipitated in cell extracts from cultures with purified IgG from MS CSF. Peptide mass fingerprinting identified it as the beta type of heat shock protein 90 (Hsp90). Two-dimensional electrophoresis and immunoblot showed that this antigen in fact corresponds to two specific isoforms of Hsp90beta. Several control assays using monoclonal and polyclonal anti-Hsp90 antibodies confirmed the specific expression of Hsp90 on OPCs. Labeling OPCs in vivo with MS CSF and anti-Hsp90 antibodies and subsequent immunofluorescence confocal microscopy located the antigen on the cell surface. The binding of CSF antibodies from MS patients to the OPC surface led to complement activation and significant extinction of the OPC population. These results suggest that OPCs may be a target of anti-Hsp90 antibodies in MS patients and that this could prevent remyelination.

Suppression of human Mnk1 by small interfering RNA increases the eukaryotic initiation factor 4F activity in HEK293T cells.

Short-interfering RNAs (siRNAs) have proved to be a useful tool in studying gene function in plants, invertebrates and mammalian systems. Herein, we report the use of siRNAs for targeting the human MAP kinase-interacting kinase Mnk1 gene. This study demonstrates the efficacy of the designed siRNA in silencing Mnk1 in the human cell line HEK293T and shows that Mnk1 suppression decreases eukaryotic initiation factor 4E phosphorylation without causing any change in global protein synthesis rate and cell proliferation. Interestingly, suppression of Mnk1 results in a significant increase in eukaryotic initiation factor 4F complex formation after 72 h of transfection.

Low concentrations of glutamate induce apoptosis in cultured neurons: implications for amyotrophic lateral sclerosis.

Evidence is accumulating that excessive glutamate concentration in the extracellular space is neurotoxic and plays a role in amyotrophic lateral sclerosis (ALS). However, the published results on glutamate levels in cerebrospinal fluid (CSF) and on glutamate-mediated toxicity of CSF in ALS disease remain controversial. In this report, we studied CSF from patients with sporadic ALS and controls to determine glutamate concentrations, and then analyzed the neurotoxic effect of glutamate at the concentrations present in CSF from ALS patients on cultured cortical neuronal cells. Our study shows that glutamate, at the concentrations found in CSF from ALS patients (5.8 microM), diminished cell viability and increased apoptosis determined by the fluorescent DNA-binding dye Hoechst 33342 as well as by Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick End-Labeling (TUNEL) reaction in cultured neuronal cells. However, glutamate concentrations as those found in CSF from controls (2.8 microM or below) did not induce any effect. Both significant glutamate-induced effects were inhibited in the presence of NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione), an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate-sensitive glutamate receptor antagonist. These results demonstrate that AMPA/kainate receptors are involved in the glutamate-mediated neurotoxic effects on cultured neurons, according to reports that implicate these receptors in ALS disease. We conclude that the glutamate-mediated neuronal apoptosis through AMPA/kainate receptors could occur in ALS patients who have elevated CSF glutamate concentration.

Neuronal apoptosis induced by cerebrospinal fluid from multiple sclerosis patients correlates with hypointense lesions on T1 magnetic resonance imaging.

Neuronal damage seems to be a major source of disability in multiple sclerosis (MS) patients and at present magnetic resonance imaging (MRI) is a sensitive method to evaluate lesion and disease activity. We studied the potential correlation between changes in MS patients' disability after relapse, the degree of T1 lesion hypointensity on MRI in vivo and neuronal apoptosis induced by cerebrospinal fluid (CSF) on neuron cultures. In this study, we included 24 MS patients with relapsing disease. Clinical recovery from relapse was measured by the Expanded Disability Status Scale (EDSS). T1-weighted MRI studies were done according to established standards and neuronal apoptosis was induced by treatment of neuronal cultures with CSF from patients while relapsing. Recovery after relapse is inversely correlated with neuronal apoptosis (r=-0.725, p<0.0001). A correlation was found between T1 lesion hypointensity and a poor recovery from relapse (r=0.656, p=0.0005) and such hypointensity correlated strongly with neuronal apoptosis (r=-0.779, p<0.0001). CSF from all patients with hypointense T1 lesions caused significantly increased neuronal apoptosis, whereas all CSF that did not induced such effects corresponded to patients without T1 lesions. The recovery from an acute MS relapse is significantly worse in patients with hypointense T1 lesions in MRI and in those whose CSF damaged neurons on cultures in vitro, phenomena that closely correlated each other.

The proteome of human brain after ischemic stroke.

Although stroke is among the most common causes of death and chronic disability worldwide, the proteome of the ischemic human brain remains unknown. Only a few studies have investigated the ischemic brain proteome in rodent stroke models. We performed a proteomic study of the human brain after ischemic stroke using a 2-dimensional differential gel electrophoresis-based proteomic approach. In brain samples from 6 deceased stroke patients and 3 control subjects, there was an average of 1,442 ± 231 protein spots in the gels. Changes of at least 1.5-fold in the relative expression of 132 protein spots between different cerebral areas (infarct core, peri-infarct, and contralateral tissue) were identified (p < 0.05); 39 of these were successfully identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Among the identified protein spots, we validated the results of 10 proteins by Western blot and determined the cellular localization in brain parenchyma for 3 of the identified proteins: dihydropyrimidinase-related protein 2, vesicle-fusing ATPase, and Rho dissociation inhibitor 1. These results contribute to understanding the processes that follow cerebral ischemia; moreover, some of the identified proteins may be therapeutic targets or biologic markers for determining the diagnosis and prognosis of stroke.

Calpain inhibition stimulates caspase-dependent apoptosis induced by taxol in NIH3T3 cells.

Taxol is an anticancer drug that triggers apoptosis in a wide spectrum of cancers such as ovarian, breast, lung, head and neck, and bladder carcinoma by both caspase-dependent and -independent apoptosis mechanisms. However, the exact signaling pathways involved in taxol-induced apoptosis strongly depend on the cellular background and they are not completely established yet. In this study we demonstrate that taxol induces caspase-3-independent apoptosis in NIH3T3 cells by a calpain-mediated mechanism. Taxol treatment produced changes in the mitochondrial membrane potential (Delta Psi m) which could be responsible of Ca(2+) release from the mitochondria and the consequent calpain activation. Interestingly, we show that calpain produced proteolysis of caspase-3 and demonstrate that, accordingly, calpain inhibition increased taxol-induced apoptosis. In addition, we reveal that poly (ADP-ribose) polymerase (PARP) was processed by calpain in taxol-treated cells and by caspase-3 after calpain inhibition. In conclusion, these results demonstrate for the first time that calpain could play an important role modulating taxol-induced apoptosis. Further studies are needed to address the potentiality of inducing apoptosis by a combined use of taxol and calpain inhibitors in cells with increased calpain activity.

A DNA aptamer population specifically detects Leishmania infantum H2A antigen.

Aptamers are short single-stranded DNA or RNA oligonucleotides that are selected in vitro by their affinity and specificity for the target. Binding is a consequence of the particular tertiary structure that they are able to acquire, depending on their sequence. Parasites of the genus Leishmania belongs to the lower eukaryote order Kinetoplastida that causes leishmaniosis in man and animals. Histone genes in Leishmania are of considerable interest because these flagellates do not condense their chromatin during mitosis. Thus, the study of the structural features of histones has been considered of particular interest and, as a result, in recent years a great number of histone genes have been characterized in trypanosomatids. Histones are extremely conserved proteins, reflecting their apparent universality of function. Sequence similarity of kinetoplastid core histones those of higher eukaryotes is found predominantly in the globular region with high sequence divergences in the N- and in the C-terminal domains. These divergences indicate that they may be potential diagnostic and/or therapeutics targets. We have successfully isolated a pool of DNA sequences, named SELH2A, which specifically binds to Leishmania infantum H2A. When tested in an enzyme-linked oligonucleotide assay, slot blot and Western blot analysis, the aptamer pool exhibited specificity in its ability to bind only to H2A antigen but not to other proteins from L. infantum including other histones. Thus, it appears that this novel anti-H2A aptamer population may be of potential application as a diagnostic system for leishmaniosis.

Proteomic characterization of protein phosphatase 1 complexes in ischemia-reperfusion and ischemic tolerance.

Serine/threonine protein phosphatase 1 (PP1) regulates multiple cellular processes. Protein phosphorylation-dephosphorylation is largely altered during ischemia and subsequent reperfusion. The brain is particularly vulnerable to stress resulting from ischemia-reperfusion (IR), however, the acquisition of ischemic tolerance (IT) protects against IR stress. We studied PP1 complexes in response to IR stress and IT in brain using proteomic characterization of PP1 complexes in animal models of IR and IT. PP1alpha and PP1gamma were immunoprecipitated and resolved by 2-D. DIGE analysis detected 14 different PP1-interacting proteins that exhibited significant changes in their association with PP1alpha or PP1gamma. These proteins were identified by MALDI-TOF MS. Seven had the PP1-binding RVxF motif. IR altered the interaction of heat shock cognate 71 kDa-protein, creatine kinase B, and dopamine- and cAMP-regulated phosphoprotein 32 kDa (DARPP32) with both PP1alpha and PP1gamma, and the interaction of phosphodiesterase-6B, transitional ER ATPase, lamin-A, glucose-regulated 78 kDa-protein, dihydropyrimidinase-related protein-2, gamma-enolase, neurofilament-L, and ubiquitin ligase SIAH2 with PP1gamma. IT prevented most of the IR-induced effects. This study identifies novel PP1alpha- and PP1gamma-interacting proteins and reveals an in vivo modularity of PP1 holoenzymes in response to physiological ischemic stress. It supports a potential role of PP1 in IR stress and as a target of the endogenous protective mechanisms induced by IT.

Translation regulation after taxol treatment in NIH3T3 cells involves the elongation factor (eEF)2.

Changes to the translational machinery that occur during apoptosis have been described in the last few years. The two principal ways in which translational factors are modified during apoptosis are: (i) changes in protein phosphorylation and (ii) specific proteolytic cleavages. Taxol, a member of a new class of anti-tubulin drugs, is currently used in chemotherapeutic treatments of different types of cancers. We have previously demonstrated that taxol induces calpain-mediated apoptosis in NIH3T3 cells [Piñeiro et al., Exp. Cell Res., 2007, 313:369-379]. In this study we found that translation was significantly inhibited during taxol-induced apoptosis in these cells. We have studied the phosphorylation status and expression levels of eIF2a, eIF4E, eIF4G and the regulatory protein 4E-BP1, all of which are implicated in translation regulation. We found that taxol treatment did not induce changes in eIF2alpha phosphorylation, but strongly decreased eIF4G, eIF4E and 4E-BP1 expression levels. MDL28170, a specific inhibitor of calpain, prevented reduction of eIF4G, but not of eIF4E or 4E-BP1 levels. Moreover, the calpain inhibitor did not block taxol-induced translation inhibition. All together these findings demonstrated that none of these factors are responsible for the taxol-induced protein synthesis inhibition. On the contrary, taxol treatment increased elongation factor eEF2 phosphorylation in a calpain-independent manner, supporting a role for eEF2 in taxol-induced translation inhibition.

Delayed postconditionig initiates additive mechanism necessary for survival of selectively vulnerable neurons after transient ischemia in rat brain.

1. The aim of this study was to validate the role of postconditioning, used 2 days after lethal ischemia, for protection of selectively vulnerable brain neurons against delayed neuronal death. 2. Eight, 10, or 15 min of transient forebrain ischemia in rat (four-vessel occlusion model) was used as initial lethal ischemia. Fluoro Jade B, the marker of neurodegeneration, and NeuN, a specific neuronal marker were used for visualization of changes 7 or 28 days after ischemia without and with delayed postconditioning. 3. Our results confirm that postconditioning if used at right time and with optimal intensity can prevent process of delayed neuronal death. At least three techniques, known as preconditioners, can be used as postconditioning: short ischemia, 3-nitropropionic acid and norepinephrine. A cardinal role for the prevention of death in selectively vulnerable neurons comprises synthesis of proteins during the first 5 h after postconditioning. Ten minutes of ischemia alone is lethal for 70% of pyramidal CA1 neurons in hippocampus. Injection of inhibitor of protein synthesis (Cycloheximide), if administered simultaneously with postconditioning, suppressed beneficial effect of postconditioning and resulted in 50% of CA1 neurons succumbing to neurodegeneration. Although, when Cycloheximide was injected 5 h after postconditioning, this treatment resulted in survival of 90% of CA1 neurons. 4. Though postconditioning significantly protects hippocampal CA1 neurons up to 10 min of ischemia, its efficacy at 15 min ischemia is exhausted. However, protective impact of postconditioning in less-sensitive neuronal populations (cortex and striatum) is very good after such a damaging insult like 15 min ischemia. This statement also means that up to 15 min of ischemia, postconditioning does not induce cumulation of injuries produced by the first and the second stress.

Anti-heat shock protein 90beta antibodies decrease pre-oligodendrocyte population in perinatal and adult cell cultures. Implications for remyelination in multiple sclerosis.

Lesions in the CNS of patients with multiple sclerosis (MS) often fail to remyelinate, resulting in neurological dysfunction. A key factor seems to be the inefficiency of oligodendrocyte precursor cells (OPCs). We recently reported antibodies against heat shock protein 90beta (Hsp90beta) in MS patients that recognized the antigen on the OPC surface. This study investigates the mechanism and result of anti-Hsp90beta antibody attack. These antibodies induced OPC death in culture in a complement-dependent fashion. Anti-Hsp90beta antibody-induced, complement-mediated OPC death only operated in these cells and caused a significant reduction in the number of O4-positive pro-oligodendrocytes (pre-oligodendrocytes). Adult cultured OPCs also expressed Hsp90beta on their cell surface and were attacked by anti-Hsp90beta antibodies leading to a significant decrease in the pre-oligodendrocyte population. In the presence of low levels of anti-Hsp90beta antibody--i.e. in the range seen in the CSF of MS patients--the complement concentration was critical to reduce the pre-oligodendrocyte population (via attack to OPCs). Higher concentrations of anti-Hsp90beta antibodies and complement became extinct the pre-oligodendrocytes. Complement 1-esterase inhibitor prevented these effects in the pre-oligodendrocyte population. These findings demonstrate, for the first time in vitro, a feasible mechanism to decrease the production of new oligodendrocytes, thus limiting the possibility of remyelination.

Evidence for a role of second pathophysiological stress in prevention of delayed neuronal death in the hippocampal CA1 region.

In ischemic tolerance experiment, when we applied 5-min ischemia 2 days before 30-min ischemia, we achieved a remarkable (95.8%) survival of CA1 neurons. However, when we applied 5-min ischemia itself, without following lethal ischemia, we found out 45.8% degeneration of neurons in the CA1. This means that salvage of 40% CA1 neurons from postischemic degeneration was initiated by the second pathophysiological stress. These findings encouraged us to hypothesize that the second pathophysiological stress used 48 h after lethal ischemia can be efficient in prevention of delayed neuronal death. Our results demonstrate that whereas 8 min of lethal ischemia destroys 49.9% of CAI neurons, 10 min of ischemia destroys 71.6% of CA1 neurons, three different techniques of the second pathophysiological stress are able to protect against both: CA1 damage as well as spatial learning/memory dysfunction. Bolus of norepinephrine (3.1 micromol/kg i.p.) used two days after 8 min ischemia saved 94.2%, 6 min ischemia applied 2 days after 10 min ischemia rescued 89.9%, and an injection of 3-nitropropionic acid (20 mg/kg i.p.) applied two days after 10 min ischemia protected 77.5% of CA1 neurons. Thus, the second pathophysiological stress, if applied at a suitable time after lethal ischemia, represents a significant therapeutic window to opportunity for salvaging neurons in the hippocampal CA1 region against delayed neuronal death.

Protective effect of L-trans-pyrrolidine-2,4-dicarboxilic acid preload against cell death induced by oxygen/glucose deprivation in differentiated PC12 cells.

It has been postulated that cellular glutamate is released into the extracellular fluid when the energy supply of the brain is compromised (i.e., anoxia or oxygen/glucose deprivation), and there the amino acid triggers the so-called excitotoxic cascade, causing neuronal death. Several mechanisms for this release have been postulated, and, by using glutamate transporter inhibitors, several authors have established that reversed uptake is the major mechanism through which glutamate is released in acute oxygen/glucose deprivation. We have studied the effect of the slowly transported glutamate analogue L-trans-pyrrolidine-2,4-dicarboxilic acid (PDC) preload on glutamate release and cell death in an in vitro model of oxygen plus glucose deprivation with differentiated PC12 cells. As expected, we found that PDC preload inhibits glutamate release induced by oxygen/glucose deprivation, supporting the conclusion that it occurs via reverse transport. In addition, we show that PDC preload but not the nontransportable glutamate uptake inhibitor DL-threo-beta-benzyloxyaspartate (TBOA) protects cells against the death induced by oxygen/glucose deprivation, indicating that PDC entry into the cell is necessary for this protective effect. This protection does not correlate with the extracellular glutamate concentration or changes in proteins synthesis rate and eukaryotic initiation 2 phosphorylation. Oxygen/glucose deprivation induces a significant increase in glutathione levels in both unloaded and PDC-preloaded cells, but this increase is not due to up-regulation of glutamate cysteine ligase levels. Intracellular glutathione disulfide (GSSG) significantly increased after oxygen/glucose deprivation. It was also interesting that intracellular GSSG levels in PDC-preloaded cells under oxygen/glucose deprivation strongly correlate with the protection exerted by this compound against cell death.