The group C of Sry-related high-mobility group (HMG) box (Sox) transcription factors has three members in most vertebrates: Sox4, Sox11 and Sox12. Sox4 and Sox11 have key roles in cardiac, neuronal and other major developmental processes, but their molecular roles in many lineages and the roles of Sox12 remain largely unknown. We show here that the three genes are co-expressed at high levels in neuronal and mesenchymal tissues in the developing mouse, and at variable relative levels in many other tissues. The three proteins have conserved remarkable identity through evolution in the HMG box DNA-binding domain and in the C-terminal 33 residues, and we demonstrate that the latter residues constitute their transactivation domain (TAD). Sox11 activates transcription several times more efficiently than Sox4 and up to one order of magnitude more efficiently than Sox12, owing to a more stable α-helical structure of its TAD. This domain and acidic domains interfere with DNA binding, Sox11 being most affected and Sox4 least affected. The proteins are nevertheless capable of competing with one another in reporter gene transactivation. We conclude that the three SoxC proteins have conserved overlapping expression patterns and molecular properties, and might therefore act in concert to fulfill essential roles in vivo.

The pro-form of nerve growth factor (pro-NGF) has been shown to be a high affinity ligand for p75NTR and to induce apoptosis through this receptor. It has been reported that pro-NGF, rather than mature NGF, is the predominant form of this neurotrophin in human brain. In the present work we studied the potential involvement of pro-NGF purified from human brains affected by Alzheimer's disease (AD), where it is especially abundant, in the neuronal apoptosis observed in this disease. Western blot analysis of human brain tissue showed the existence of several pro-NGF forms. Some of these pro-NGF forms were significantly increased in AD brain cortex in a disease stage-dependent manner. Pro-NGF, purified by chromatography from human AD brains, induced apoptotic cell death in sympathetic neurons and in a p75NTR stably transfected cell line. Blocking p75NTR in cell culture abolished neuronal apoptosis caused by pro-NGF. p75NTR-transfected cells underwent apoptosis in the presence of pro-NGF while control wild-type cells did not. Taken together, these results indicate that pro-NGF purified from AD human brains can induce apoptosis in neuronal cell cultures through its interaction with the p75NTR receptor.

The pro form of neurotrophic growth factor (pro-NGF), purified by chromatography from human Alzheimer's disease (AD)-affected brains (ADhbi-pro-NGF), has been shown to induce apoptotic cell death in neuronal cell cultures through its interaction with the p75 neurotrophin receptor (p75NTR). In the present work, we report that ADhbi-pro-NGF stimulates processing of p75NTR with ␣-and ␥-secretases, yielding a 20-kd intracellular domain (p75 ICD ) that translocates to the nucleus. This process was accompanied by delayed apoptosis. In AD, p75 ICD was significantly increased in human entorhinal cortex. Although human frontal cortex has been described as showing a higher pro-NGF increase in AD, the increase in the entorhinal cortex paralleled p75NTR processing in its intracellular domain. In addition, pro-NGF isolated from AD-affected brains differed functionally from pro-NGF isolated from comparably aged control brains, with pro-NGF isolated from control brains being unstable and undergoing degradation to NGF when added to cell culture. As p75 ICD and pro-NGF are both mediators of apoptosis and are both found in increased levels in the cerebral cortex in AD, the present data have implications for understanding neuronal degeneration in AD.

Much current knowledge of oligodendrocyte biology, the myelin-forming cells in the central nervous system (CNS), comes from cell culture studies mainly from postnatal rat tissue but mouse cells have been much more difficult to produce in large quantities. We have developed a high yield protocol for production of oligodendrocyte precursor cells from mouse embryonic neural progenitors grown as neurospheres. Neurospheres can be maintained and expanded for long periods in culture in the presence of EGF. When floating neurospheres were plated on substrate-coated dishes in media supplemented with PDGF and bFGF, the spheres attached and generated migrating cells that were predominantly oligodendrocyte-lineage cells. Furthermore, cells in spheres could be shifted to the oligodendrocyte phenotype prior to plating on substrate, by incubation in suspension with PDGF/bFGF. Single cell suspensions plated after dissociation of either EGF-treated neurospheres or PDGF/bFGF-treated oligospheres had the bipolar, elongated morphology characteristic of oligodendrocyte precursor cells. mRNA and protein expression analysis of the cells generated by this method confirmed their oligodendrocyte lineage. Oligodendrocyte precursors generated by this method matured in response to ciliary neurotrophic factor treatment, producing cells with multiple processes and myelin-like membranes. The most important aspect of this protocol is the ability to generate very high numbers of relatively pure mouse oligodendrocyte progenitor cells, which can be easily transfected. These studies open up many kinds of investigations on transgenic and mutant mouse oligodendrocytes, thereby providing a valuable tool to study oligodendrocyte biology and development.

No abstract