Caspase-3 activation in oligodendrocytes from the myelin-deficient rat.

The myelin-deficient (MD) rat has a point mutation in its proteolipid protein (PLP) gene that causes severe dysmyelination and oligodendrocyte cell death. Using an in vitro model, we have shown that MD oligodendrocytes initially differentiate similarly to wild-type cells, expressing galactocerebroside, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin basic protein. However, at the time when PLP expression would normally begin, the MD oligodendrocytes die via an apoptotic pathway involving caspase activation. The active form of caspase-3 was detected, along with the cleavage products of poly-(ADP-ribose) polymerase (PARP) and spectrin, major targets of caspase-mediated proteolysis. A specific inhibitor of casapse-3, Ac-DEVD-CMK, reduced apoptosis in MD oligodendrocytes, but the rescued cells did not mature fully or express myelin-oligodendrocyte glycoprotein. These results suggest that mutant PLP affects not only cell death but also oligodendrocyte differentiation.

Stage-specific effects of bone morphogenetic proteins on the oligodendrocyte lineage.

Oligodendrocyte maturation is regulated by multiple secreted factors present in the brain during critical stages of development. Whereas most of these factors promote oligodendrocyte proliferation and survival, members of the bone morphogenetic protein family (BMPs) recently have been shown to inhibit oligodendrocyte differentiation in vitro. Oligodendrocyte precursors treated with BMPs differentiate to the astrocyte lineage. Given that cells at various stages of the oligodendrocyte lineage have distinct responses to growth factors, we hypothesized that the response to BMP would be stage-specific. Using highly purified, stage-specific cultures, we found that BMP has distinct effects on cultured oligodendrocyte preprogenitors, precursors, and mature oligodendrocytes. Oligodendrocyte preprogenitors (PSA-NCAM+, A2B5-) treated with BMP2 or BMP4 developed a novel astrocyte phenotype characterized by a morphological change and expression of glial fibrillary acidic protein (GFAP) but little glutamine synthetase expression and no labeling with A2B5 antibody. In contrast, treating oligodendrocyte precursors with BMPs resulted in the accumulation of cells with the traditional type 2 astrocyte phenotype (GFAP+, A2B5+). However, many of the cells with an astrocytic morphology did not express GFAP or glutamine synthetase unless thyroid hormone was present in the medium. The addition of fibroblast growth factor along with BMP to either oligodendrocyte preprogenitor or the oligodendrocyte precursor cells inhibited the switch to the astrocyte lineage, whereas platelet-derived growth factor addition had no effect. Treatment of mature oligodendrocytes with BMP elicited no change in morphology or expression of GFAP. These data suggest that as cells progress through the oligodendrocyte lineage, they show developmentally restricted responses to the BMPs.

Complexity analysis of oligodendroglial processes expressing myelin-associated glycoprotein.

Oligodendroglia synthesize myelin in the mammalian central nervous system. Mature oligodendroglia have been identified in culture by two criteria; the expression of molecules characteristic of myelin, such as galactocerebroside (galC) and myelin-associated glycoprotein (MAG), and the elaboration of complex processes. Myelin gene expression can be documented by the binding of specific antibodies and antisera to the myelin-specific molecules; process complexity can be described by the fractal dimension, D. In this study, anti-MAG antisera was used to document MAG expression in the processes of oligodendroglia. Eighty percent of the galC+ oligodendroglia bound anti-MAG antiserum. With time in culture, MAG immunoreactivity seemed to extend from the cell soma into the oligodendroglial processes. To quantify this observation, fractal dimensions were calculated using either galC or MAG immunoreactivity to visualize oligodendroglial processes. A fractal dimension of 1.5 was calculated for O1+ processes by day 4 of culture; this value for D remained constant over the course of 1 month in culture. The fractal dimension calculated for MAG+ processes increased from 1.2 to 1.5 over the course of 28 days in culture. This change in fractal dimension confirms our visual impression that galC-containing processes acquire MAG slowly over the course of several weeks in culture.