Age‐dependent accumulation of ubiquitinated 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase in myelin lipid rafts

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Abstract
Changes in brain white matter are prominent features of the aging brain and include glial cell activation, disruption of myelin membranes with resultant reorganization of the molecular components of the node of Ranvier, and loss of myelinated fibers associated with inflammation and oxidative stress. In previous studies, overexpression of CNP, a key myelin protein, was implicated in age-related changes in myelin and axons. Here we examine the extent of CNP accumulation in brain white matter and isolated myelin of aged rhesus monkeys and its relationship to CNP degradation and partitioning in myelin. With age, excess CNP is found in myelin and throughout brain white matter accompanied by proteolytic fragments of CNP. These increases occur in the absence of changes in CNP mRNA levels. Using a combination of 2D electrophoresis, immunoprecipitation, and mass spectrometry analysis, ubiquitinated CNP was demonstrable in the Triton X-100 insoluble lipid raft associated fractions of myelin isolated from rhesus monkeys. Further, using ubiquitin-mediated fluorescence complementation (UbFC), ubiquitinated CNP was visualized by microscopy in both COS-7 and MO3.13 cells and by immunoblot in MO3.13 cells and appears to at least partially localize within lipid rafts. The findings suggest that incomplete degradation of CNP due to failure of the proteasomal system and aberrant degradation by calpain-1 leads to age-related CNP accumulation and proteolysis. In sum, we suspect these phenomena result in age-related dysfunction of CNP in the lipid raft, which may lead to myelin and axonal pathology.