Erk1/2 MAPK and mTOR signaling sequentially regulates progression through distinct stages of oligodendrocyte differentiation.

Myelination is the culmination of a complex process in which oligodendrocyte (OL) progenitors transition through defined stages in a well-coordinated differentiation program. The signaling mechanisms that regulate this progression are poorly understood. Here we investigate the role of extracellular signal-regulated-kinase-1,-2 (Erk1/2) and the mammalian target of rapamycin (mTOR), downstream effectors of the Ras/Raf/Mek/Erk and PI3K/Akt/mTOR pathways, at specific stages of OL development in vitro. Using a panel of developmental stage-specific antigenic markers and pharmacological inhibitors, we provide evidence that Erk1/2 signaling regulates transition of early progenitors to the late progenitor stage and, as a consequence, to the immature OL stage, but not the transition of immature OL to the mature OL stage. In contrast, mTOR signaling is not required for early progenitor transition to late progenitor stage. Surprisingly, it is also not required for the transition of late progenitors to terminally differentiated immature OLs, as has been reported previously, but is required for the next sequential transition of immature OLs to the mature OL stage. Furthermore, mTOR signaling regulates OL cytoskeletal organization and major myelin protein expression. These in vitro findings correlate with our in vivo data showing that inhibition of mTOR by rapamycin injection attenuated the onset of myelination in the early postnatal brain. Thus, these studies demonstrate that Erk1/2 and mTOR signaling sequentially regulates distinct stages of OL progenitor differentiation and suggest that cells in the OL-lineage require distinct signaling mechanisms to transition through specific stages of their development.

MT FdR: a ferredoxin reductase from M. tuberculosis that couples to MT CYP51.

We report the molecular cloning, expression and partial characterization of MT FdR, an FAD-associated flavoprotein, from Mycobacterium tuberculosis similar to the oxygenase-coupled NADH-dependent ferredoxin reductases (ONFR). We establish, through kinetic and spectral analysis, that MT FdR preferentially uses NADH as cofactor. Furthermore, MT FdR forms a complex with mycobacterial ferredoxin (MT Fdx) and MT CYP51, a cytochrome P450 (CYP) from M. tuberculosis that is similar to lanosterol 14alpha-demethylase isozymes. This reconstituted system transfers electrons from the cofactor to the heme iron of MT CYP51 and effects the demethylation of lanosterol.

Characterization of the mouse peroxisome proliferator-activated receptor delta gene.

Peroxisome proliferator-activated receptors (alpha, delta and gamma) are ligand-activated transcription factors that are involved in multiple cellular responses. The PPARdelta subtype is the least understood of all PPAR subtypes. PPARdelta is activated by unsaturated fatty acids, PGI2, and by synthetic ligands. PPARdelta-regulated genes have not been identified and the factors that control PPARdelta expression are not known. The gene that encodes the mouse PPARdelta gene is contained in >30 kb DNA sequence and organized in eight exons, six of which encode the PPARdelta receptor. A PPARdelta-luciferase reporter containing 694 bp 5' upstream regulatory and 127 bp untranslated was introduced to primary brain cultures to begin a characterization of the DNA sequences that mediate transcriptional regulation of PPARdelta. PPARdelta-luciferase expression was 10 times higher in oligodendrocyte-containing mature cultures than in immature cultures, indicating that PPARdelta may play a role during oligodendrocyte migration, proliferation, and/or maturation.