Gliotoxic actions of excitatory amino acids.

Cultures of neonatal Type I astrocytes of the rat were exposed to a series of excitatory amino acid analogs to identify those compounds that were gliotoxic. In addition to L-alpha-aminoadipate, a previously identified gliotoxin, L-homocysteate, L-serine-O-sulfate, L-alpha-amino-4-phosphonobutyrate and L-alpha-amino-3-phosphono-propionate were also found to induce a sequence of degenerative events that led to the lysis of the astrocytes. Cellular injury was assessed by quantifying the activity of lactate dehydrogenase present in the surviving astrocytes. Prior to lysis, the cells went through a succession of distinctive morphological changes, the most prominent of which involved nuclear alterations. The nuclei appeared swollen, contained "pale" or "watery" nucleoplasm and exhibited a very prominent nuclear membrane and obvious nucleoli. These astrocytes appeared quite similar in appearance to the Alzheimer's Type II astrocytes, principally associated with the pathology of hepatic encephalopathy. The nuclear anomalies, which are thought to be indicative of cellular damage and compromised function, were also produced by the endogenous transmitters L-glutamate and L-aspartate, although with time, the affected astrocytes appeared to recover and return to normal morphology, without lyzing. These findings suggest that excessive levels of excitatory amino acids may induce cellular damage to astrocytes, as well as neurons. Once damaged, the resulting reductions in astrocyte function may further contribute to CNS losses and the overall pathology attributed to the excitatory amino acids.

Gliotoxic properties of the Lathyrus excitotoxin beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (beta-L-ODAP).

beta-N-Oxalyl-L-alpha,beta-diaminopropionic acid (beta-L-ODAP) is an excitatory amino acid agonist found in the seeds of Lathyrus sativus that is believed to be the major causative agent in the pathology of human lathyrism. We have found that in addition to its previously recognized neurotoxic properties, beta-L-ODAP is also gliotoxic. When added to cultures of neonatal rat astrocytes, beta-L-ODAP induced a series of morphological changes (e.g., extensive vacuole formation, pale and swollen nuclei with obvious nucleoli, and cellular swelling) that led to the eventual lysis of the glial cells. If the beta-L-ODAP was removed prior to the lysis of the astrocytes, many of the early morphological changes appeared to be reversible. When quantitated by a loss of the lactate dehydrogenase activity, beta-L-ODAP lysed the astrocytes with an LD50 of 2.1 +/- 0.2 mM following 48 h of exposure. Lower concentrations of beta-L-ODAP were found to be more toxic if the duration of the exposure was increased. The results suggest that the overall impact of the toxin on the CNS may represent the cumulative action of beta-L-ODAP at a number of distinct points on both neurons and astrocytes. The potential that these multiple sites of action may affect the normal regulation of extracellular glutamate and, consequently, disturb the balance between its normal and pathological roles is discussed.

Identification of the novel K15 gene at the rightmost end of the Kaposi's sarcoma-associated herpesvirus genome.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a distinct open reading frame called K15 at a position equivalent to the gene encoding LMP2A of Epstein-Barr virus (EBV). K15 isolates from body cavity-based lymphoma (BCBL) cells exhibited a dramatic sequence variation and a complex splicing pattern. However, all K15 alleles are organized similarly with the potential SH2 and SH3 binding motifs in their cytoplasmic regions. Northern blot analysis showed that K15 was weakly expressed in latently infected BCBL-1 cells, and the level of its expression was significantly induced by tetradecanoyl phorbol acetate stimulation. K15 encoded 40- to 55-kDa proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was localized at the cytoplasm and plasma membrane. To demonstrate the signal-transducing activity of the K15 protein, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8alpha polypeptide was replaced with that of KSHV K15. While the CD8-K15 chimera was not capable of eliciting cellular signal transduction upon stimulation with an anti-CD8 antibody, it significantly inhibited B-cell receptor signaling, as evidenced by a suppression of tyrosine phosphorylation and intracellular calcium mobilization. This inhibition required the putative SH2 or SH3 binding motif in the cytoplasmic region of K15. Biochemical study of CD8-K15 chimeras showed that the cytoplasmic region of K15 was constitutively tyrosine phosphorylated and that the tyrosine residue within the putative SH2 binding motif of K15 was a primary site of phosphorylation. These results demonstrate that KSHV K15 resembles LMP2A in genomic location, splicing pattern, and protein structure and by the presence of functional signal-transducing motifs in the cytoplasmic region. Thus, KSHV K15 is likely a distant evolutionary relative of EBV LMP2A.