G. Shanker

Investigations on Myelination In Vitro: Regulation of 2′,3′-Cyclic Nucleotide 3′-Phosphohydrolase by Thyroid Hormone in Cultures of Dissociated Brain Cells from Embryonic Mice

Abstract: The direct influence of l‐3,3′,5‐triiodothyronine (T3) on the development of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (EC 3.1.4.37, CNPase) is demonstrated by using an in vitro culture system of dissociated embryonic mouse brain cells. Serum from a thyroidectomized calf, which contained low levels of T3 (31 ng/100 ml), and thyroxine, T4 (<1 μg/ml), was used in the culture medium in place of normal calf serum (T3, 103 ng/100 ml; T4, 5.7 μg/ml) to render the culture responsive to exogenously added T3. The lower levels of enzyme activity observed in the presence of such a deficient medium could be restored to normal values by T3 supplementation. Half‐maximal effect was obtained with 2.5 ± 10−9m‐T3. Three days of hormone treatment resulted in the maximal stimulation of CNPase. T4 was less effective in inducing CNPase activity and the inactive analog of the hormone, reverse T3 (3,3′,5′‐T3) was ineffective. The morphological appearance of the cells was characterized by deformed (smaller size and less in number) reaggregates in the cultures, lacking hormone.

Investigations on myelinogenesis in vitro: A study of the critical period at which thyroid hormone exerts its maximum regulatory effect on the developmental experession of two myelin associated markers in cultured brain cells from embryonic mice

Cultures of cells dissociated from embryonic mouse brain were used to assess the period in which thyroid hormone exerts its maximum influence on the regulation of the expression of two myelin associated metabolites, sulfolipids and 2′3′-cyclic nucleotide 3′-phosphohydrolase (CNP-ase). Cultures were grown for a specified number of days on a medium containing normal calf serum and then a portion were switched to a medium containing hypothyroid calf serum for 2 days. One half of these cultures were then supplemented with 50 nM triiodothyronine and growth was continued in all cultures for 3 more days. The cells were then assayed for CNP-ase activity and for their ability to incorporate35SO4 into sulfolipids. Studies with both myelin markers showed that in the earlier culture ages of 5, 8, and 11 days, thyroid hormone was able to fully restore the activities when abbed to cultures grown on hypothyroid calf-serum. In contrast, in the intermediate age range (15, 19, and 22 days) the restoration was partial, while in the higher ages, there was practically negligible restoration with T3. Since the culture system eliminates the possibility of a blood brain barrier and drastically decreases the complicity of other hormones, the lack of a myelinogeni response to thyroid hormone after a certain age must be attributed to the loss of sensitivity of the oligodendroglia to T3 possibly through genetic programming.

Regulation of myelin basic protein (arginine) methyltransferase by thyroid hormone in myelinogenic cultures of cells dissociated from embryonic mouse brain.

Abstract: The ontogenetic expression of myelin basic protein (arginine) methyltransferase in myelinogenic cultures of cells dissociated from embryonic mouse brain is highly dependent on the presence of thyroid hormone. Restoration of myelin basic protein methyltransferase to normal activities occurred 16 h after the addition of 100 nMl‐3,5,3′‐triiodothyronine to hypothyroid medium. These data demonstrate that thyroid hormone can regulate a posttranslational event. On the other hand, histone (arginine) methyltransferase has a different temporal activity pattern, which is not coordinated with myelination, and is not influenced by the lack of thyroid hormone. These data, which suggest the existence of two methyltransferases, were substantiated by demonstrating that the total amount of methylation of added myelin basic protein and histone is the same whether they are incubated together or separately. The requirement of thyroid hormone for the expression of the myelin basic protein methyltransferase and not for histone methyltransferase suggests that thyroid hormone preferentially regulates myelin‐associated events in these cultures.

Degrees of Cooperativity between Triiodothyronine and Hydrocortisone in Their Regulation of the Expression of Myelin Basic Protein and Proteolipid Protein during Brain Development

Cultures of cells dissociated from embryonic mouse cerebra were used to demonstrate: (1) that the developmental expression of the mRNA of proteolipid protein is dependent on thyroid hormone; (2) that the expression of the mRNA of proteolipid protein is stimulated not only by triiodothyronine but also by hydrocortisone, which achieve their respective stimulations by an additive and uncompetitive mechanism; (3) the stimulation of the net accumulation of the mRNA of myelin basic protein by hydrocortisone and triiodothyronine is also cooperative, additive, and uncompetitive, and (4) the stimulation of the net accumulation of myelin basic protein, during development by hydrocortisone, is completely dependent on the presence of thyroid hormone. These results suggest that the regulation of the synthesis of myelin basic protein by hydrocortisone requires the presence of triiodothyronine at a posttranscriptional event, but not for transcription itself.

Insulin: its binding to specific receptors and its stimulation of DNA synthesis and 2',3'-cyclic nucleotide phosphohydrolase activity in cerebral cells cultured from embryonic mouse brain.

The presence and specificity of insulin receptors was investigated in cultured cells obtained from 15–16 days old embryonic mouse cerebra. Developmental studies suggested that the maximum insulin binding occurred at about 11 days in vitro (DIV). Scatchard analysis of binding data revealed two types of binding sites. One type of receptor was the high affinity type (Kd=7.77×10−9 M; number of receptor sites,Bmax=350 fmol/mg protein) while the other type was of low affinity type (Kd=5.75×10−8 M;Bmax=1150 fmol/mg protein). The specificity of receptors for insulin was also confirmed by showing that [125I]insulin was displaced by non-radioactive insulin but not by glucagon or growth hormone. Insulin displayed a clear dose-dependent stimulation of thymidine incorporation. It also stimulated the activity of the enzyme 2′,3′-cyclic nucleotide phosphohydrolase (CNPase), which is specifically associated with myelin produced from oligodendroglia. Thus insulin has a positive influence on the proliferation and differentiation of brain cells.

Development study on the regulation of neurotransmitter-sensitive adenylate cyclase systems in primary cerebral cell cultures from embryonic mice

An ontogenetic study of the effect of various neuhormones and other activators on adenylate cyclase systems was carried out using cultures of ceils from 15-d-old embryonic mouse brain. Dopamine stimulated the enzyme activity at earlier culture ages (i.e. 4 and l0 d) but had little stimulatory effect at later ages (i.e. 20 and 33 d). Further, this stimulation at the earlier ages was blocked by the dopaminergic blocker, fluphenazine, but not by α and β-adrenergic antagonists. In contrast to dopamine, isoproterenol (a β-adrenergic agonist) had little stimulatory effect at earlier ages, but its ability to stimulate cyclase activity increased with age. This increase in all age groups was blocked by propranolol ( afg - adrenergic antagonists) . Epinephrine-sensitive enzyme activity showed a steady increase with age, which could be blocked with propranolol except in β-d-old cultures, where it was blocked instead by fluphenazine. Because the cultures are relatively enriched in neurons at earlier ages and in glia in later ages, the results suggest a predominantly neuronal localization for the dopamine sensitive adenylate cyclases and a glial localization of the isoproterenol and epinephrine sensitive adenylate cyclases. Histamine, serotonin, calcium/calmodulin and chloroadenosine were either only slightly or not at all stimulatory.

ReducedS-adenosylmethionine: Protein-lysineN-methyltransferase activity (protein methylase III) in shiverer mutant mouse brain

Mice with the dysmyelinating mutation shiverer were studied by measuring the activity of two protein methylases and myelin marker enzymes in the brain. It was observed thatS-adenosylmethionine: protein-lysineN-methyltransferase (protein methylase III, EC. 2.1.1.43) activity is significantly reduced in phenotypically affected homozygous shiverer (shi/shi) mutant mouse brain compared to the unaffected heterozygous littermate brain. This reduction in enzyme activity is manifested mainly by reduced formation of trimethyllysine during the in vitro methylation of histone. In contrast, myelin marker enzymes such as 2′,3′-cyclic nucleotide 3′-phosphohydrolase and 5′-nucleotidase as well asS-adenosyl-methionine: protein-carboxylO-methyltransferase (protein methylase II, EC. 2.1.1.24) activities were not significantly affected in these strains of mice.

Investigations on myelinogenesis in vitro: I. The occurrence of endogenous protein kinase and its role in the phosphorylation of myelin basic proteins in “Myelin-like membranes” isolated from cerebral cell cultures

The presence of a protein kinase capable of phosphorylating endogenous as well as exogenously added myelin basic proteins has been demonstrated in a myelin-like membrane fraction isolated from reaggregating and surface adhering, primary cultures of cells dissociated from embryonic mouse brain. Only the large and small components of myelin basic proteins were found to be phosphorylated when myelin-like membrane fraction was incubated with [γ-32P]ATP. The protein kinase endogenous to the myelin-like membrane fraction was mainly of the cyclic AMP independent type. There was very little cyclic AMP dependent or cyclic GMP dependent protein kinase activities in this myelin-like fraction. Although the myelin basic proteins were the only endogenous proteins phosphorylated, protein kinase of the myelin-like membrane was capable of catalyzing the phosphorylation of exogenous substrates, such as histones.