Effect of Oligodendroglial Microdomain Components on NGF Signaling

Abstract

Recent results indicate that the integrity of the myelin sheath is compromised with age [1], and that myelin destruction could be a precipitating event in age-related disorders [2]. Furthermore, oligodendrocytes (OL), which furnish the CNS myelin, do not support remyelination in older animals to the same extent as in younger animals. An increased process formation reflects an OL response to NGF, however, differentially depending on age of the donor [3]. We were interested to know as to whether NGF signaling is modulated by microdomain components such as caveolin and cholesterol, which might undergo age-related changes. Immunocytochemistry of cultured pig OL revealed the co-expression of caveolin-1 and the 140 kDa NGF receptor TrkA. Caveolin-containing microdomains were isolated via previously published buoyant density centrifugation methods (±Triton X-100) and by using MACS technology. Western blotting showed a co-labeling of the caveolar protein, flotillin-1, in addition to TrkA, which is enriched in the Triton X-100 insoluble fraction, p75 NTR, and p21 Ras. Preliminary results indicate that oligodendroglial caveolin and cholesterol are up-regulated from 8 DIV to 16 DIV, a period of extensive oligodendroglial process regeneration. Cells exposed to PEG-cholesterol increased their process formation; PEG-cholesterol plus NGF accelerated the NGF response; under both conditions, an in-gel-kinase assay demonstrated an increased MAPK activity, a step of the downstream TrkA signaling. On the other hand, exposure to cyclodextrin (1.2 mM), which disrupts caveolar microdomains by removing cholesterol from the plasma membrane, resulted in a less effective NGF response. The results at present indicate that NGF signaling is modulated by cholesterol; the effect of caveolin-1 siRNA is under investigation.