Araceli Espinosa de los Monteros

Alternative splicing prevents transferrin secretion during differentiation of a human oligodendrocyte cell line.

Transferrin, the iron‐transport protein of vertebrate serum, is synthesized mainly in the liver, from which it is secreted into the blood. Transferrin is also synthesized in oligodendrocytes and is an early marker of their differentiation. We have analyzed the regulation of transferrin expression in HOG cells, a human oligodendrocyte cell line. Transferrin expression was correlated with the appearance of oligodendrocyte differentiation markers when cells were exposed to differentiation medium. In contrast to the protein expressed in hepatocytes or in Sertoli cells, transferrin was secreted by neither HOG cells nor immature rat primary oligodendrocytes in vitro. Moreover, transferrin appears to be localized in the cytosol and not in the secretory compartment, as is expected for secreted proteins. This transferrin localization was correlated with the synthesis of a specific transcript, resulting from an alternative splicing, which leads to the elimination of the signal peptide sequence. These results suggest the existence of a functional difference between transferrin synthesized in the brain and in other organs such as liver and testis. They are in accordance with the hypothesis that transferrin plays a specific role, other than iron transport, in oligodendrocyte maturation and in the myelination process. J. Neurosci. Res. 61:388–395, 2000.

Myelination and motor coordination are increased in transferrin transgenic mice

Myelin deficiency in the central nervous system (CNS) can cause severe disabling conditions. Most of the transgenic mice models overexpressing myelin components have limitations for investigators of myelin deficiency and myelin therapy as they severely alter CNS architecture. It has been postulated that transferrin (Tf) is involved in oligodendrocyte (OL) maturation and myelinogenesis. Because Tf is not an intrinsic myelin constituent, we decided to investigate if its overexpression could have an impact on the myelination process without affecting myelin integrity. We generated transgenic mice containing the complete human Tf gene specifically overexpressed in OLs. This overexpression leads to more than a 30% increase in myelin components, such as galactolipids, phospholipids, and proteins. Electron microscopy showed that myelin is structurally normal in terms of thickness and compaction. Behavior analysis showed that mice do not display significant modifications in their locomotion and cognitive and emotional abilities. Furthermore, in one of the genetic background, animals presented a significant increase in motor coordination. We did not find any modification in OL number during early postnatal development, suggesting that Tf does not act on OL proliferation. In addition, the levels of iron and ferritin remained unchanged in the brain of transgenic mice compared to control mice. Our findings indicate that, besides its known iron transport function, Tf is able to influence myelination process and induce behavioral improvements in mice.

Transplantation of CG4 oligodendrocyte progenitor cells in the myelin‐deficient rat brain results in myelination of axons and enhanced oligodendroglial markers

Transplantation of oligodendrocyte (Ol) progenitor cells into the central nervous system is a promising approach for the treatment of myelin disorders. This approach requires providing adequate numbers of healthy cells with myelinating potential. We recently showed the successful transplantation of Ol progenitors into the myelin‐deficient (md) rat brain. In the present work, CG4 cells, a cell line with properties of Ol progenitors, were labeled with fast blue and grafted into P3–P5 pups born to carrier mothers. Examination of host brains 2 weeks posttransplant indicated that CG4 cells display a much more extensive migration capacity than their wild‐type counterparts. These cells synthesized myelin components. In addition, ultrastructural analysis showed myelin formation along axons of md hosts in various brain regions, including corpus callosum, cerebellum, and brainstem. Furthermore, in situ hybridization studies performed on sagittal sections revealed extensive expression of transferrin‐mRNA within the md host parenchyma. The high survival and functional features displayed by CG4 cells after transplantation, together with their striking wide distribution within the host parenchyma, as assessed by the presence of myelinated fibers in mutant hosts, emphasizes the importance of using highly motile and proliferative Ol progenitor cells. Strategies to improve the condition and life span of md rat pups are currently under investigation. J. Neurosci. Res. 50:872–887, 1997.

Transferrin: An early marker of oligodendrocytes in culture

In this study, the developmental pattern of transferrin expression, the iron transporting glycoprotein, was investigated morphologically and immunocytochemically in mixed glial cultures as well as pure cultures of mature oligodendrocytes, both derived from newborn rat brain.

Transplantation of Cultured Premyelinating Oligodendrocytes into Normal and Myelin-Deficient Rat Brain

Cultures of oligodendroglial cells at various stages of maturation, from progenitors to maturing oligodendrocytes, were prepared from neonatal rat brain primary cultures and then were prelabeled in the culture dish with the fluorescent dye, fast blue (FB). Single cell suspensions were grafted into normal or myelin-deficient rat brains. The normal as well as the myelin-deficient in vivo environment allowed cell survival, migration, and differentiation. The FB+ cells expressed the oligodendroglial markers, glycerol phosphate dehydrogenase, galactocerebroside, and myelin basic protein. In the normal rat transplanted cells were identifiable at all times studied up to 24 weeks. Extensive migration of FB+ cells was observed in whole-brain sagittal sections. Our results show that the plasticity of oligodendroglia differentiation, extensively studied in vitro, can now be investigated in the normal and myelin-deficient in vivo environment.

Transferrin is an early marker of hepatic differentiation, and its expression correlates with the postnatal development of oligodendrocytes in mice.

Transferrin (Tf), the iron transport protein, is essential for the growth and differentiation of cells. Therefore, it provides an excellent model to analyze the regulatory mechanisms controlling the expression of a eukaryotic gene in different cell types and during fetal and adult life. In this study, the tissue‐specific and developmental regulation of the Tf gene in vivo were analyzed. Human Tf mRNA was detected mainly in fetal and adult liver. A weaker expression was observed in adult and fetal brain and in fetal spleen. By in situ hybridization the presence of mouse Tf mRNA was detected in the hepatic primordia. This is the first observation pointing out Tf as an early marker of hepatic differentiation, prior to the formation of the liver. Thus, TF may be an important tool to follow the hepatic specification of the gut endoderm. Mouse Tf mRNA was also detected in the liver bud and subsequently in the liver throughout fetal life, and in newborn and adult animals. No expression of the Tf gene was observed in the mouse fetal central nervous system (CNS). In contrast, Tf mRNA was detected from the 5th day after birth in the derivatives of the caudal part of the neural tube and subsequently in the derivatives of the rhomboencephalon and that of the prosencephalon. These results indicate that Tf gene expression correlates with the postnatal development of oligodendrocytes in the mouse CNS. To test whether the control elements of the human gene previously found in ex vivo experiments were also active in vivo during fetal and adult life, we fused the −4000/+39 5′ flanking region of the human gene to the coding region of the lacZ gene and generated transgenic mice. The expression of the reporter gene during development was analyzed. J. Neurosci. Res. 50:421–432, 1997.

PROXIMAL PROMOTER REGION IS SUFFICIENT TO REGULATE TISSUE-SPECIFIC EXPRESSION OF UDP-GALACTOSE : CERAMIDE GALACTOSYLTRANSFERASE GENE

UDP‐galactose:ceramide galactosyltransferase (CGT) is the enzyme which catalyzes the final step of the biosynthesis of galactocerebroside (GalC), the most abundant glycolipid in myelin. We identified regulatory elements which are related to the tissue‐specific expression of the mouse CGT gene by promoter assay using chimeric CGT‐luciferase constructs. By comparing promoter activity in oligodendroglial CG4 cells and NIH3T3 fibroblasts, only a few hundred base pairs spanning from −309 to −98 were shown to be necessary for the tissue‐specific activity of CGT promoter. A negative regulatory element was found in a more distal region, from −709 to −527, and it also worked in tissue‐specific manner. Sequence analysis suggests that several known elements found commonly in myelin‐related genes may explain these tissue‐specific regulations of the transcriptional activity. J. Neurosci. Res. 52:757–765, 1998.

Ontogeny of glycerol phosphate dehydrogenasepositive oligodendrocytes in rat brain. Impaired differentiation of oligodendrocytes in the myelin deficient mutant rat

The ontogeny of oligodendrocytes in the myelin deficient (md) rat mutant and in control rats was explored immunohistochemically using an antiserum against the oligodendrocyte specific enzyme, glycerol phosphate dehydrogenase (GPDH), and the avidin‐biotin complex technique. In control rats, GPDH was demonstrated to be expressed relatively early in oligodendrocyte differentiation, prior to either myelin basic protein or proteolipid protein expression. With development, oligodendrocytes containing GPDH increased in number, apparent staining intensity, cell soma area and process elaboration. Fewer GPDH+oligodendrocytes were observed in the brain of mutant rats than in unaffected littermates at all developmental ages, and major developmental increases in oligodendrocyte density were delayed. The density of GPDH+oligodendrocytes was reduced by about 40% in both the corpus callosum and in the cingulate cortex of P22–25 md mutants compared with control rats. The oligodendrocyte cell soma area was not influenced by the md condition, and increased 2‐fold with development in rats of both genotypes. The area of coronal sections occupied by the corpus callosum increased about 2.5‐fold with development, and was 30% smaller in mutant rats late in their lifespan than in unaffected littermates. The reductions in oligodendrocyte density reported here are of insufficient magnitude to fully account for biochemically measured reductions in oligodendrocyte gene expression accompanying the md trait, indicating that gene expression per oligodendrocyte is also impaired. Cell counts in control rats also revealed that oligodendrocytes are overproduced during development. Cell density and the total number of corpus callosum GPDH+oligodendrocytes per section were maximal at P22–25 and then decreased to adult values. These results suggest that glial cells, like neurons, may be generated in excessive numbers, and some subsequently die, as a normal concomitant of development.