Jean-Marie Matthieu

Triiodothyronine Has Diverse and Multiple Stimulating Effects on Expression of the Major Myelin Protein Genes

Abstract: If the importance of triiodothyronine (T3) on brain development including myelinogenesis has long been recognized, its mechanism of action at the gene level is still not fully elucidated. We studied the effect of T3 on the expression of myelin protein genes in aggregating brain cell cultures. T3 increases the concentrations of mRNA transcribed from the following four myelin protein genes: myelin basic protein (Mbp), myelin‐associated glycoprotein (Mag), proteolipid protein (Plp), and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (Cnp). T3 is not only a triggering signal for oligodendrocyte differentiation, but it has continuous stimulatory effects on myelin gene expression. Transcription in isolated nuclei experiments shows that T3 increases Mag and Cnp transcription rates. After inhibiting transcription with actinomycin D, we measured the half‐lives of specific mRNAs. Our results show that T3 increases the stability of mRNA for myelin basic protein, and probably proteolipid protein. In vitro translation followed by myelin basic protein‐specific immunoprecipitation showed a direct stimulatory effect of T3 on myelin basic protein mRNA translation. Moreover, this stimulation was higher when the mRNA was already stabilized in culture, indicating that stabilization is achieved through mRNA structural modifications. These results demonstrate the diverse and multiple mechanisms of T3 stimulation of myelin protein genes.

Myelination in rat brain aggregating cell cultures

Abstract Aggregates of fetal rat brain were maintained in rotating culture for 30–40 days and were analyzed morphologically and biochemically. At 4 days in culture all cells were undifferentiated. At 26 days in vitro over 90% of all cells within the aggregates could be identified as neurons, astrocytes or oligodendrocytes. Myelinated axons and morphologically mature synapses were present at 26 days. Myelination started between 18 and 19 days in culture as determined biochemically. Myelin basic protein sulphatide synthesis and 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity increased with in vitro age. The amount of myelin observed within the aggregates was much lower than observed at the corresponding age in vivo . Neurons and neuronal processes were undergoing severe degeneration in the 40-day aggregates and synaptic contacts were not maintained. There were no normal myelinated axons at 40 days although multilammellar membranes were found intra- and extracellularly. The ganglioside pattern of the aggregates were qualitatively similar to rat whole brain. Quantitatively the G M3 ganglioside was elevated in comparison to whole rat brain. Our results indicate that aggregating rat brain cultures provide a useful in vitro system for the biochemical and morphological analysis of myelin formation.

BIOCHEMICAL CHARACTERIZATION OF A MYELIN FRACTION ISOLATED FROM RAT BRAIN AGGREGATING CELL CULTURES

Abstract— Subcellular fractions isolated from rat brain aggregating cell cultures were studied by electron microscopy and showed the presence of typical myelin membranes. The chemical composition of purified culture myelin was similar to the fraction isolated from rat brain in terms of CNP specific activity, protein and lipid composition. The ratio of small to large components of myelin basic protein was comparable in culture and in vivo. These two proteins incorporated radioactive phosphorus. The major myelin glycoprotein was present and during development in culture its apparent molecular weight decreased although it never reached the position observed in myelin isolated from adult rats. In culture, the yield of myelin did not increase substantially between 33 and 50 days and was comparable to that of 15‐day‐old rat brain. The ratio basic protein to proteolipid protein resembled immature myelin and the cerebroside content was very low. A ‘floating fraction’ was isolated from the cultures and contained some myelin but mostly single membranes. Although these results indicate that myelin maturation is delayed in vitro this culture system provides substantial amounts of purified myelin to allow a complete biochemical analysis and metabolic studies during development.

Myelin gene expression during demyelination and remyelination in aggregating brain cell cultures.

Remyelination can be studied in aggregating rat brain cell cultures after limited demyelination. Demyelination was induced using a monoclonal antibody against myelin/oligodendrocyte glycoprotein (MOG mAb), in the presence of complement. De- and remyelination were assessed by measuring myelin basic protein (MBP). Two days after removing the MOG mAb, MBP levels reached 50% of controls and after 7 days 93%. During this period, cell proliferation determined by [14C]thymidine incorporation was similar in remyelinating and control cultures. Hormones and growth factors were tested for possible stimulatory effect on remyelinating cultures. Bovine growth hormone (bGH), triiodothyronine (T3), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) did not improve remyelination. Only epidermal growth factor (EGF) increased the level of remyelination. PDGF increased the rate of cell proliferation in both control and remyelinating cultures. A significant proportion of oligodendrocytes entered the cell division cycle and were not available for remyelination. The results obtained with PDGF and FGF (inhibition) support the idea that a pool of progenitor cells was still present and able to proliferate and differentiate into myelinating oligodendrocytes. The levels of myelin protein mRNAs were investigated during de- and remyelination. During demyelination, myelin protein mRNA levels decreased to approximately 50% of control cultures and returned to normal during remyelination. These preliminary results indicate that normal levels of gene transcription are sufficient to meet the increased need for newly synthesized myelin proteins during remyelination.

GLIAL VERSUS NEURONAL ORIGIN OF MYELIN PROTEINS AND GLYCOPROTEINS STUDIED BY COMBINED INTRAOCULAR AND INTRACRANIAL LABELLING

Abstract— —The contribution of axonal transport to the production of myelin proteins and glycoproteins was investigated using the double labelling technique of combined intraocular and intracerebral injections in the same animal. Myelin and an axolemma‐enriched fraction were isolated from pooled optic nerves, chiasma and optic tracts. Separation by gel electrophoresis showed that typical myelin proteins and glycoproteins were only significantly labelled by intracerebral injection. Intraocular injection labelled high molecular weight proteins other than the major Wolfgram protein and the major myelin glycoprotein. Fifteen days after intraocular injection the label was concentrated in a high molecular weight protein which migrated slightly more slowly than the major Wolfgram protein. The pattern of proteins and glycoproteins in myelin labelled by intraocular injection was very similar to that obtained in the axolemma‐enriched fraction by the same route. These results indicate that neuronal metabolism and axonal transport do not contribute significantly to the synthesis of specific myelin proteins and glycoproteins, but suggest that the components of myelin fractions which are labelled by intraocular injection are contaminants of axolemmal origin. One of these glycoproteins may prove a useful marker of axolemma membranes.

Immunocytochemical investigation of myelin deficient (mld) mutant mice

In the P.N.S., GFAP fi laments have been detected Immmunohlstochemlcal ly In the enteric 91101 cel Is and somme Schwann ce l l s . ItBre we study the molecular ident i ly of th is P.N.S. GFAP Immunoresctlvlty, and compare I t lo GFAP in the C.N.S., using Immuno¢hemlcal and ImunGhlslDchemical methods. Immunoblafftlng, with antiserum lo brain GFAP, shows that the peripheral GFAP I lmunore~ct lv l ly res i des in a polypeptlde with • molecular weight of 49 kdw identical lo that of r a t brain GFAP. Furthermore, th is GFAP reac t i v i t y can he detected Ilmunahlsl~chemlcel ly in Schwann and satel l i f e ce l l s in several peripheral nerves and ganglia, in addit ion to enteric g i l a . The d l s l r l bu t i on of GFAP among Schwann eel Is suggests that , In the nerves surveyed, they may be expressed by most or al I non-myelin forming Schwann cel Is, but not by myelln-for~lng Schwann eel Is. We also show, using a monoclonal antibody to GFAP (antI-GFAP-3, made by Albrachtsen, GJrster4)erg end Beck) In both Immunohlslochemlcal end Imunoblott lng sl~dlws, that the GFAP found in most peripheral g i la Is not identical to that of eslTocytes. Act exception lo th is f inding Is seen in the myenteric plexuses where Immunohlstochemlcal ly detectable GFAP Is found In aclep but not al I , of the enteric g i l a , using the monoclonal antibody. 111us, the rmu i t s suggest that GFA polyl)eptldes may be a heterogenous group, that share some common deter~inants and a cmmon molecular weight, and show a widespread end complex dls lTIbut lon In the g i la of both the C.N.S. and P.N.S.