Neuronal protection of oligodendrocytes from antibody-independent complement lysis.

Cultured rat oligodendrocytes are lysed by complement via antibody-independent activation of the classical pathway. This susceptibility to complement lysis has been demonstrated to be due to lack of CD59, a complement regulatory protein which inhibits assembly of the membrane attack complex. In this study the effects of homologous and heterologous complement were examined in a co-culture system of rat oligodendrocytes and peripheral neurones, where axonal ensheathment was observed as early as 4 days after the addition of glial progenitors to the neurones. Following exposure to complement, ensheathing oligodendrocytes were markedly less sensitive to antibody-independent but not antibody-dependent complement lysis than were cells grown without neurones. Immunocytochemical data revealed that co-cultured oligodendrocytes remained CD59 negative, but in contrast to oligodendrocytes cultured alone, were negative for C3b when incubated with C7-deficient serum. Taken together these data indicate that the decreased sensitivity of co-cultured oligodendrocytes to complement lysis is not attributed to the increased expression of CD59, but rather in a failure to activate complement. Incubation of oligodendrocytes with neurone-conditioned medium afforded significant protection (68%), against antibody-independent complement attack, suggesting that soluble neuronal factors can protect oligodendrocytes from complement-mediated lysis.

CD59 expression and complement susceptibility of human neuronal cell line (NTera2).

The present study analysed the susceptibility of neuronal cells to human complement. A human neuronal precursor cell line (NTera2) which can be induced to differentiate into post-mitotic neuronal cells was employed. Using immunocytochemistry, NTera2 neurones, but not their precursors (stem cells), were shown to activate complement in the absence of antibody and to lack CD59, an inhibitor of the terminal stages of the complement cascade. Following exposure to human serum, neurones but not stem cells were lysed by complement, as demonstrated by a cell viability assay and consistent rise in intracellular calcium. This response was abrogated when cells were treated with complement-depleted serum by heat inactivation or cobra venom factor. Protection from lysis was observed following incorporation of CD59 with its glycolipid anchor in the neuronal cell membrane.

Gene structure and expression of the Corynebacterium flavum N13 ask-asd operon.

Two promoters required for expression of the ask-asd genes, encoding aspartokinase (AK) and aspartate-semialdehyde dehydrogenase (ASD), in Corynebacterium flavum N13, askP1 and askP2, have been identified by deletion analysis and S1 nuclease mapping. Transcription from askP1 initiates 35 and 38 bp upstream of the ask structural gene. A second promoter, askP2, lies within the ask coding region, upstream of the translation start site of the AK beta subunit and can direct the expression of AK beta and ASD. Western immunoblot analysis and heterologous expression in Escherichia coli demonstrate that two separate polypeptides, a 44.8-kDa alpha subunit and an 18.5-kDa beta subunit, are expressed from the C. flavum N13 ask gene from distinct, in-frame translation initiation sites. A second AK mutation, G345D, which reduces the sensitivity of AK to concerted feedback inhibition by threonine plus lysine, was identified.