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Increased levels of neuron-specific enolase (NSE), a key glycolytic enzyme, in either the cerebrospinal fluid or the serum is correlated with both the duration and the outcome of status epilepticus. To further understand the molecular basis of seizure-induced elevations in NSE protein, we investigated NSE mRNA expression in the adult rat brain following systemic administration of kainic acid. The findings demonstrated either no change or a decrease in NSE gene expression during, and following, status epilepticus, suggesting that posttranscriptional mechanisms are responsible for seizure-induced increases in NSE protein., (Copyright 1999 Academic Press.)

The p53 tumor suppressor gene encodes a cell cycle regulatory protein that is induced by DNA damage and has been implicated in apoptosis. To investigate whether excitotoxic cell death due to kainic acid (KA) and cell death due to N-methyl-D-aspartate (NMDA) share similar molecular mechanisms, we studied p53 expression and DNA fragmentation in organotypic hippocampal slice cultures following excitotoxin treatment. Cellular analyses showed that both p53 induction and DNA fragmentation occurred only in injured neurons following exposure to either excitotoxin. The temporal profiles of these changes demonstrated that p53 induction preceded DNA fragmentation. The extent of regional alterations in p53 expression and DNA fragmentation correlated with drug-related toxicity (i.e., NMDA > KA). These results support the hypothesis that p53 is a marker of neuronal death in the CNS and suggest the possibility that excitotoxin-mediated neuronal death may occur through a p53-dependent pathway.

We explored the constitutive expression, maturational regulation, and relation to kainic-acid-induced apoptosis of cyclooxygenase (COX)-2 mRNA in rat brain. In adult rats, COX-2 mRNA was expressed primarily in limbic structures. Constitutive COX-2 mRNA expression increased markedly between Postnatal Day 7 (P7) and P14, reaching adult levels by P21. Despite intense KA-induced seizures, no COX-2 mRNA induction was found before P14 in any brain region examined. During response to KA-induced seizures in adult brain, COX-2 mRNA induction paralleled temporally and overlapped anatomically the appearance of cellular morphological features of apoptosis in subsets of cells of the pyramidal neuron layer of the hippocampal formation, amygdaloid complex, and pyriform cortex. While COX-2 mRNA showed KA-induced elevation in the granule cell layer of the dentate gyrus, no detectable morphological features of apoptosis were found in this region. Finally, monotypic culture of rat corticohippocampal neurons confirmed the neuronal expression of COX-2 in vitro and revealed that COX-2 is induced during response to glutamate treatment, leading to neuron death. These studies may provide novel insights into the role of COX-2 and mechanisms of action of nonsteroidal anti-inflammatory drugs in Alzheimer's disease.

The excitotoxic effects of N-methyl-D-aspartic acid (NMDA) and kainic acid (KA) were studied in organotypic hippocampal slices maintained in vitro for various periods of time. Cultures aged to equivalent Postnatal Day (EPD) 10-12, 15-17, and 23-26 were exposed to 50 microM KA or 50 microM NMDA and were analyzed at 0, 3, 6, 9, 12, 24, 48 h, or 5 days after the initiation of the excitotoxin exposure. Neuronal injury was determined by: (1) propidium iodide (PI) uptake; (2) lactate dehydrogenase (LDH) release; (3) morphological damage in hematoxylin and eosin (H/E) stained sections; (4) loss of Nissl stain. Changes in PI uptake and LDH release after KA or NMDA treatment indicated that there was a developmental shift towards increasing sensitivity to KA toxicity during in vitro development, whereas cultures of all ages were equally sensitive to NMDA toxicity. The profile of damage in H/E-stained sections after treatment with KA or NMDA indicated a transient phase of damaged morphology at 12 and 24 h that was not evident after 5 days. To determine whether the disappearance of morphological manifestations of neuronal damage 5 days after treatment was due to recovery of morphology or to neuronal death, neuronal loss in Nissl-stained sections was also quantified. KA treatment did not cause significant neuronal loss in any hippocampal region in EPD 10-12 cultures, indicating that the neurons were able to successfully recover from the damage demonstrated in H/E sections at 12 and 24 h in these cultures. KA treatment in mature cultures (EPD 23-26) and NMDA treatment in all cultures produced a marked loss of identifiable Nissl-stained neurons at 5 days, indicating neuronal death and disintegration. The results provide further support for the similarities between the organotypic hippocampal culture model and in vivo excitotoxic models and also confirm that excitotoxic neuronal injury can be reversible under some conditions.

Tumor suppressor p53 encodes a protein that is an important regulator of the cell cycle. However, under certain conditions increased p53 expression results in programmed cell death or apoptosis. We used in situ hybridization histochemistry to investigate the role of p53 in the adrenalectomy-induced degeneration of hippocampal granule cells. Three days after adrenalectomy, a subpopulation of granule cells exhibiting morphological features of apoptosis expressed increased amounts of p53 mRNA. Both adrenalectomy-induced p53 expression and granule cell degeneration were prevented by daily administration of corticosterone. In situ end-labeling of nuclei containing fragmented DNA revealed a distribution similar to that of cells with increased p53 expression. These results demonstrate an association between p53 induction and apoptosis in the central nervous system and support the idea that cell cycle-related genes play a role in neuronal death pathways.

Heterotopic cardiac isografts are vasculary perfused organs that maintain structural and functional integrity. We have used this model to study the time course of change in total heart and left ventricular size which results from mechanical unloading of the myocardium. When compared to the in situ (working) heart there is a 19% decrease in the size of the heterotopic (non-working) heart (P less than 0.01) as early as 3 days post-transplantation. By 14 days there is 50% decrease in the size of the transplanted heart which is maintained at this atrophic size when measured after 4 weeks. Left ventricular protein synthesis was assayed by the simultaneous in vitro perfusion of the host and transplanted hearts under identical hemodynamic conditions. The hourly incorporation of 14C-lysine into total left ventricular protein was 21 nmol in the transplant compared to 50 nmol in the in situ heart (P less than 0.01). This incorporation remained significantly lower throughout the period of study. In contrast, both the total (mumol lysine/g protein nitrogen/h) and fractional rates of protein synthesis which were lower in the transplanted left ventricle at days 3 and 7 returned to control values by day 28. The present studies demonstrate that heterotropic cardiac transplantation leads to a prompt and reproducible decline in cardiac mass and in total protein synthesis. These studies further support the role of cardiac work as an important determinant in the regulation of cardiac protein synthesis.

Human coronaviruses (HCV) are ubiquitous pathogens which cause respiratory, gastrointestinal, and possibly neurological disorders. To better understand the molecular biology of the prototype HCV-229E strain, the complete nucleotide sequence of the membrane protein (M) gene was determined from cloned cDNA. The open reading frame is preceded by a consensus transcriptional initiation sequence UCUAAACU, identical to the one found upstream of the N gene. The M gene encodes a 225-amino acid polypeptide with a molecular weight (MW) of 25,822, slightly higher than the apparent MW of 19,000-22,000 observed for the unprocessed M protein obtained after in vitro translation and immunoprecipitation. The M amino acid sequence presents a significant degree of homology (38%) with its counterpart of transmissible gastroenteritis coronavirus (TGEV). The M protein of HCV-229E is highly hydrophobic and its hydropathicity profile shows a transmembranous region composed of three major hydrophobic domains characteristic of a typical coronavirus M protein. About 10% (20 amino acids) of the HCV-229E M protein constitutes a hydrophilic and probably external portion. One N-glycosylation and three potential O-glycosylation sites are found in this exposed domain.

Human coronaviruses are important human pathogens and have also been implicated in multiple sclerosis. To further understand the molecular biology of human coronavirus 229E (HCV-229E), molecular cloning and sequence analysis of the viral RNA have been initiated. Following established protocols, the 3'-terminal 1732 nucleotides of the genome were sequenced. A large open reading frame encodes a 389 amino acid protein of 43,366 Da, which is presumably the nucleocapsid protein. The predicted protein is similar in size, chemical properties, and amino acid sequence to the nucleocapsid proteins of other coronaviruses. This is especially evident when the sequence is compared with that of the antigenically related porcine transmissible gastroenteritis virus (TGEV), with which a region of 46% amino acid sequence homology was found. Hydropathy profiles revealed the existence of several conserved domains which could have functional significance. An intergenic consensus sequence precedes the 5'-end of the proposed nucleocapsid protein gene. The consensus sequence is present in other coronaviruses and has been proposed as the site of binding of the leader sequence for mRNA transcriptional start. This region was also examined by primer extension analysis of mRNAs, which identified a 60-nucleotide leader sequence. The 3'-noncoding region of the genome contains an 11-nucleotide sequence, which is relatively conserved throughout the Coronavirus family and lends support to the theory that this region is important for the replication of negative-strand RNA.