Neuroprotection by NMDA preconditioning against glutamate cytotoxicity is mediated through activation of ERK 1/2, inactivation of JNK, and by prevention of glutamate-induced CREB inactivation.

N-methyl-D-aspartate (NMDA) preconditioning is a major endogenous brain protective mechanism, activated by sub-lethal stimulation of the NMDA glutamate receptors. Selective drug activation of this mechanism is considered to be a promising neuroprotective treatment against stroke and other traumatic brain insults. We have established an experimental in vitro model of NMDA preconditioning in primary rat neuronal cultures composed of three consecutive periods: preconditioning (NMDA 50 9M for 18 h), insult (glutamic acid 200 9M for 1 h), and reperfusion (regular medium for 24 h). The insulted neuronal cultures exhibited a 2.8-fold increase in LDH release into the media during the post-insult reperfusion period, which was completely abolished in the preconditioned cultures. The alterations in the activity level of the pro-survival kinase extracellular signal-regulated kinase (ERK) 1/2, the death machine activator c-Jun N-terminal kinase (JNK), and the pro-survival transcription factor cAMP responsive element binding (CREB) were monitored in preconditioned neuronal cultures in comparison to non-preconditioned cells during the three periods of the experimental model. The preconditioned neurons exhibited increased activity levels of ERK 1/2 and decreased activity levels of JNK during all periods of the model. In addition, the non-preconditioned neurons exhibited a marked reduction in the activity level of CREB during the insult period, which was totally prevented in the preconditioned cultures. These results suggest that the neuroprotection conferred by NMDA preconditioning against glutamate cytotoxicity is mediated (at least in part) through activation of ERK 1/2, inactivation of JNK and by prevention of glutamate-induced CREB inactivation.

Molecular alterations associated with the NMDA preconditioning-induced neuroprotective mechanism against glutamate cytotoxicity.

Exposure of the brain to sub-lethal concentrations of glutamate activates, through stimulation of the glutamate N-methyl-D: -aspartate (NMDA) receptors, an endogenous brain protective mechanism (NMDA preconditioning) against glutamate cytotoxicity and various other injurious stimuli. Selective drug activation of this mechanism is considered to be a promising neuroprotective treatment against the devastating consequences of stroke and other traumatic brain insults. Although some properties of this mechanism have been characterized, many aspects concerning it are yet to be elucidated. In order to improve our understanding of the NMDA preconditioning mechanism, we have established an experimental in vitro model of primary rat neuronal cultures, in which NMDA preconditioning completely abolishes the glutamic acid insult-induced neuronal damage. Employing this model, we have monitored in the present study the level of activation or expression of several signal transducing proteins, assumed to be involved in the NMDA-activated protective mechanism, at various time points during the three successive periods of the model, preconditioning, insult, and reperfusion. The results demonstrated that the NMDA preconditioning-induced neuroprotective mechanism is associated with inactivation of p66ShcA, prevention of the insult-induced inactivation of Src, activation of AKT, inactivation followed by reactivation of FKHR-L1, and with increased expression of p52ShcA, EGFR, and MnSOD. The essential role of Src activity in the protective mechanism was further indicated by the demonstration that decreasing Src activation level by the Src inhibitor PP2 attenuated the NMDA preconditioning-induced protection. The alterations detailed above in the activation status or level of expression of the studied proteins are suggested to be part of the NMDA preconditioning-induced neuroprotective mechanism.

Involvement of Src tyrosine kinases (SFKs) and of focal adhesion kinase (FAK) in the injurious mechanism in rat primary neuronal cultures exposed to chemical ischemia.

Src family of kinases (SFKs) and focal adhesion kinase (FAK) are two important cellular signaling components known to act cooperatively in the transduction of death and survival signals. We investigated the involvement of these proteins in the mechanism of the injurious response in rat primary neuronal cultures exposed to an insult composed of chemical ischemia (poisoning with iodoacetic acid; 100 muM, for 150 min) followed by 1 h of incubation in the regular medium, an insult shown before to be associated with generation of reactive oxygen species and with the depletion of adenosine triphisphate. The exposure of the neuronal cultures to the insult resulted in cell injury, assessed by the increased release of cytoplasmic lactate dehydrogenase (LDH) into the culture media, which could be attenuated markedly by the presence of the antioxidant LY 231617. The insult resulted in the decreased level of phosphorylation of the SFKs members Src, Fyn, and Yes at the Src Y416-equivalent activation sites and of the FAK Y397 activation site, degradation of FAK to a p85 fragment, and disassembling of the FAK-SFKs complexes. The inhibition of SFKs was found to be responsible for part of the insult-induced cell damage manifested in increased LDH release. Pervanadate, an inhibitor of the phosphotyrosine phosphatases (PTPs), abrogated the inactivation of SFKs and attenuated cell injury, indicating that insult-induced activation of PTPs is involved in SFKs inhibition and the ensued damage. The inhibition of SFKs and FAK is probably the cause of the disassembling of SFKs-FAK complexes, a process known to be associated with apoptosis.

Identification and characterization of the first mutation (Arg776Cys) in the C-terminal domain of the Human Molybdenum Cofactor Sulfurase (HMCS) associated with type II classical xanthinuria.

Classical xanthinuria type II is an autosomal recessive disorder characterized by deficiency of xanthine dehydrogenase and aldehyde oxidase activities due to lack of a common sulfido-olybdenum cofactor (MoCo). Two mutations, both in the N-terminal domain of the Human Molybdenum Cofactor Sulfurase (HMCS), were reported in patients with type II xanthinuria. Whereas the N-terminal domain of HMCS was demonstrated to have cysteine desulfurase activity, the C-terminal domain hypothetically transfers the sulfur to the MoCo. We describe the first mutation in the C-terminal domain of HMCS identified in a Bedouin-Arab child presenting with urolithiasis and in an asymptomatic Jewish female. Patients were diagnosed with type II xanthinuria by homozygosity mapping and/or allopurinol loading test. The Bedouin-Arab child was homozygous for a c.2326C>T (p.Arg776Cys) mutation, while the female patient was compound heterozygous for this and a novel c.1034insA (p.Gln347fsStop379) mutation in the N-terminal domain of HMCS. Cosegregation of the homozygous mutant genotype with hypouricemia and hypouricosuria was demonstrated in the Bedouin family. Haplotype analysis indicated that p.Arg776Cys is a recurrent mutation. Arg776 together with six surrounding amino acid residues were found fully conserved and predicted to be buried in homologous eukaryotic MoCo sulfurases. Moreover, Arg776 is conserved in a diversity of eukaryotic and prokaryotic proteins that posses a domain homologous to the C-terminal domain of HMCS. Our findings suggest that Arg776 is essential for a core structure of the C-terminal domain of the HMCS and identification of a mutation at this site may contribute clarifying the mechanism of MoCo sulfuration.

Molecular, biochemical, and genetic characterization of a female patient with Lesch-Nyhan disease.

Lesch-Nyhan disease (LND) is a rare X-linked recessive disorder caused by virtually complete deficiency of activity of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8). Human HPRT is encoded by a single structural gene located on the long arm of the X-chromosome (Xq26). The classical LND phenotype occurs almost exclusively in males, manifested in excessive purine production and characteristic neurological manifestations, including compulsive self-mutilation, choreoathetosis, spasticity, and occasionally developmental delay. Heterozygous females are usually phenotypically normal, due to the random inactivation of the X chromosome (Lyonization mechanism). However, six females were reported to be affected with the full biochemical and clinical manifestations of LND. All these cases were heterozygous for an HPRT mutation. Absence of transcription of the normal HPRT allele was attributed in all of them to non-random inactivation of the X chromosome carrying the normal allele. Here we describe an additional LND female, who presented with acute renal failure at the age of two months, in whom absence of transcription of the two HPRT alleles occurred due to as yet undescribed mechanism in LND females: the transcription of one HPRT allele was blocked due to a de novo X chromosome-autosome translocation 46,XX,t(X:2)(q26:p25), with a breaking point encompassing the HPRT gene locus, whereas the transcription of the normal allele was inhibited due to non-random inactivation of the second X-chromosome. Cultured fibroblasts from this patient exhibited the biochemical alterations in purine nucleotide metabolism characteristic of male LND fibroblasts.

Oxidative stress activates transcription factor NF-kB-mediated protective signaling in primary rat neuronal cultures.

Activation of transcription factor nuclear factor-kappaB (NF-kappaB) can result in enhanced de novo synthesis of both proteins that confer protection and those that cause death. The present study was undertaken to clarify in primary neuronal cultures the consequences of the oxidative stress-induced activation of NF-kappaB and mediation of death or survival signals. The neuronal cultures were exposed to chemical ischemia (iodoacetic acid), followed by reperfusion (I/R insult). This insult injured the neurons, as manifested in a 7- to 10-fold increase in LDH release, and decreased the cellular content of IkappaBalpha by 55-65 %, indicating NF-kappaB activation. The antioxidants LY231617, melatonin, and sodium salicylate and the antioxidant and inhibitor of NF-kappaB activation pyrrolidine dithiocarbamate, protected the neurons against the insult and prevented the decrease in cellular IkappaBalpha content. In contrast, inhibition of NF-kappaB translocation by SN50 in both uninsulted and insulted neuronal cultures resulted in a 2.9- and 2.4-fold increase in LDH release, respectively. The results indicate that the insult-induced oxidative stress activates transcription factor NF-kappaB associated with induction of protection and suggest that constitutive activation of NF-kappaB under physiological conditions acts to protect the neurons against physiological injury.

Phospholipase C is involved in the adenosine-activated signal transduction pathway conferring protection against iodoacetic acid-induced injury in primary rat neuronal cultures.

We have demonstrated before that exposure of neuronal cultures to poisoning by iodoacetic acid, followed by "reperfusion" (iodoacetate-"reperfusion" insult; IAA-R insult), results in severe cytotoxicity. This insult was found to be associated with ATP depletion and generation of reactive oxygen species. The cultured neurons could be protected against the insult by activation of the adenosine A1 receptors and by presence of antioxidants. Previous studies in our laboratory demonstrated that the adenosine-activated signal transduction pathway (Ado-STP) conferring protection against the IAA-R insult, involves activation of protein kinase C-epsilon (PKCepsilon) and opening of ATP sensitive potassium (K(ATP)) channels. In this respect, the adenosine-activated protective mechanism against the IAA-R insult is similar to the Ado-STP in the neurons and in cardiomyocytes against ischemia-reperfusion injury. Phospholipase C (PLC) is an additional component demonstrated recently to participate in the myocardial Ado-STP protecting against ischemia-reperfusion. Here we provide proof for the involvement of PLC also in the neuronal Ado-STP protecting against the IAA-R insult. Primary rat neuronal cultures were exposed to the IAA-R insult. The neurons could be protected against this insult by activation of the adenosine A1 receptors by N6-(R)-phenylisopropyladenosine (R-PIA), a specific A1 adenosine receptor agonist. Exposure of the cultures to the PLC inhibitor U73122, abrogated the protection. The exposure of the cultures to R-PIA was found to enhance PLC activity, an effect that could be abrogated by presence of U73122. The R-PIA-induced increase in PLC activity was short-lived, in the range of minutes. These results demonstrate that activation of PLC is a vital step in the neuronal protective Ado-STP, but that it does not contribute directly to the relatively long time window of the protection signal shown previously to characterize the neuronal mechanism. The results also support the suggestion that the Ado-STP protecting against the IAA-R insult and that protecting against ischemia-reperfusion may represent the same mechanism.

What picture is worth a thousand words? A comparative evaluation of a burn prevention programme by type of medium in Israel.

Burns are associated with longer hospital stay, permanent disfigurement and emotional stress disorders, and represent a health problem, especially among economically and socially deprived populations, such as the Bedouin population in Israel, hence the importance of intervention programmes. The objective of this research was to examine the extent to which the effect of a visual one-session burn prevention programme was determined by the type of medium used. We also examined the possibility that fear motivates action only when someone feels confident in his/her ability to control the threat. Data were based on the pre-/post-programme self-report questionnaires administered to a randomly selected three-group sample of 12- to 13-year-old Bedouin children (n=179). All three sessions were identical, but differed in the type of medium used: slides (S), video (V), or slides and video consecutively (S + V). We measured health beliefs (perceived threat, internal/external control, self-efficacy) and sense of coherence (SOC), both before and 2 months after completion of the intervention. We also measured post-programme fear reaction and the improvement in burn-related knowledge, understanding and safety behaviour as the outcome measure. No significant post-programme differences between intervention groups were found, either in terms of outcome measure or in terms of health beliefs and SOC. However, within- person analysis indicated that the S group participants had the highest level of post-exposure fear and a decrease in luck control over injuries. The S + V group demonstrated the lowest within change. The hierarchical regression analysis revealed that self-efficacy, fear, higher socio-economic status and female gender predicted improvement. As hypothesized, the interaction between fear and self-efficacy added significantly to prediction. It seems that health beliefs and demographic characteristics were more powerful in predicting the effect of the intervention than the choice of medium per se. A multifaceted approach and more comprehensive interventions are needed in order to promote health among disadvantaged populations.

Reactive oxygen species play an important role in iodoacetate-induced neurotoxicity in primary rat neuronal cultures and in differentiated PC12 cells.

The role of reactive oxygen species in the pathogenesis of the neurotoxicity associated with ischemia-reperfusion, was investigated in a model of primary rat neuronal cultures and of differentiated PC12 cells, subjected to chemical ischemia by iodoacetic acid (IAA, 2.5 h) followed by a short period of reperfusion (1 h). The injury to the cells was assessed by lactate dehydrogenase (LDH) release into the culture media. The PC12 cells exhibited relative resistance to IAA cytotoxicity. Therefore these cells were studied at a 4-fold higher IAA concentration (400 microM instead of 100 microM for the neurons). The injury to both cell types was significantly greater in the short post-insult reperfusion (PIR) period than during the insult period. The presence, during the combined insult and PIR periods, of alpha-tocopherol (100 microM), melatonin (1 mM) and LY231617 (5 microM), conferred to both cell types considerable protection against the injury occurring during the insult and during the PIR periods (assessed separately). Superoxide dismutase (SOD; 500 IU/ml) conferred protection to the neurons, but not to the PC12 cells. When exposure to the antioxidants was limited to the short (15 min) pre insult period, only LY231617 conferred protection. In the neurons the protection occurred only during the insult period, whereas in the PC12 cells during both the insult and PIR periods. When the exposure to the antioxidants was limited to the PIR period, only SOD conferred protection and only in the neuronal cultures. These findings suggest that neuronal damage caused during ischemia-reperfusion can be diminished markedly by co-presence of antioxidants during the insult period. Certain antioxidants may protect the neurons even when present only before or after the insult.

A novel point mutation (I137T) in the conserved 5-phosphoribosyl-1-pyrophosphate binding motif of hypoxanthine-guanine phosphoribosyltransferase (HPRTJerusalem) in a variant of Lesch-Nyhan syndrome.

We identified a novel point mutation (I137T) in the hypoxanthine-guanine phosphoribosyltransferase (HPRT; EC 2.4.2.8) encoding gene, in a patient with partial deficiency of the enzyme (variant of Lesch-Nyhan syndrome). The mutation, ATT to ACT, resulting in substitution of isoleucine to threonine, occurred at codon 137 (exon 6), which is within the region encoding the binding site for 5-phosphoribosyl-1-pyrophosphate (PRPP). We suggest the mechanism by which the mutation-induced structural alteration of HPRT reduced the affinity of the enzyme for PRPP.

Protein kinase C-epsilon is involved in the adenosine-activated signal transduction pathway conferring protection against ischemia-reperfusion injury in primary rat neuronal cultures.

Adenosine activates a signal transduction pathway (STP) in the heart and the brain, conferring protection against ischemia-reperfusion insult. Activation of protein kinase C (PKC), probably mainly PKC-epsilon, has been demonstrated to be part of the heart STP, but its role in the neuronal pathway is less clear. Here, we provide proof for the participation of PKC-epsilon in the neuronal adenosine-activated STP. Primary rat neuronal cultures were exposed to chemical ischemia by iodoacetate, followed by reperfusion. The cultured neurons were protected against this insult by activation of the adenosine mechanism, by N6-(R)-phenylisopropyladenosine [R(-)-PIA], a specific A1 adenosine receptor agonist. Exposure of the cultures to bisindolylmaleimide I, a highly selective PKC inhibitor, abrogated the protection. The exposure of the cultures to R(-)-PIA was found to result in phosphorylation (activation) of PKC-epsilon. Furthermore, insertion into the cells of a specific peptide inhibitor of PKC-epsilon translocation (epsilonV1-2), also abrogated the protection conferred by R(-)-PIA. These results demonstrate that activation of PKC-epsilon is a vital step in the neuronal adenosine-activated STP.