Neuron, astrocyte, and oligodendrocyte cultures which were established from developing rat brain were examined for their utilization of glucose, ketone bodies, and free fatty acids by oxidative processes. 14CO2 production was measured in these cells from [1-14C] or [6-14C]glucose; [1-14C]octanoate and [1-14C], [6-14C], or [16-14C]palmitate; and [3-14C]acetoacetate and D(-)-3-hydroxy[3-14C]butyrate. Pyruvate dehydrogenase (EC 1.2.4.1.) and 3-oxoacid-CoA transferase (EC 2.8.3.5) activities were found at high levels in each of the cell populations. Astrocytes and oligodendrocytes produced much more 14CO2 from [1-14C]glucose than from [6-14C]glucose, indicating substantial hexose monophosphate shunt activity. This process was not as active in neurons. All three cell populations readily utilized the ketone bodies for oxidative metabolism at rates 7-9 times greater than they utilized glucose. Only astrocytes were able to utilize fatty acids for 14CO2 production, and the rate of utilization was greater than that of the ketone bodies. We found that the metabolic patterns of these brain cells which were derived from the developing brain complement the nature of the diet of the suckling animal which is rich in fat and low in carbohydrate. They readily utilized the ketone bodies or fatty acids and spared glucose for processes that metabolites of fat cannot fulfill.
During neonatal development glucocorticoids potentiate oligodendrocyte differentiation and myelinogenesis by regulating the expes of myelin basic protein, proteolipid protein, and glycerol phosphate dehydrogenase (snglycerol-3-phosphate: NAD' 2-oxidoreductase, EC 1.1.1.8).The actual locus at which hydrocortisone exerts its developmental influence on glal physiology is, however, not well understood. Gycerol phosphate dehydrogenase is glucocorticoid-inducible in oligodendrocytes at all stages of development both in vivo and in vitro. In newborn rat cerebral cultures, between 9 and 15 days in vitro, a 2-to 3-fold increase in myelin basic protein and proteolipid protein mRNA levels occurs in oligodendrocytes within 12 hr of hydrocortisone treatment. Immunotaining demonstrates that this increase in mRNAs is followed by a 2-to 3-fold increase in the protein levels within 24 hr. In vitro transcription assays performed with oligodendrocyte nuclei show an 11-fold increase in the transcriptional activity of glycerol phosphate dehydrogenase in response to hydrocortisone but no increase in transcription of myelin basic protein or proteolipid protein. These results indicate that during early myelinogenesis, glucocorticoids influence the expression of key oligodendroglial markers by different processes: The expression of glycerol phosphate dehydrogenase is regulated at the transcriptional level, whereas the expression of myelin basic protein and proteolipid protein is modulated via a different, yet uncharacterized, mechanism involving posttranscriptional regulation.The expression of glycerol phosphate dehydrogenase (GPDH; sn-glycerol-3-phosphate: NAD' 2-oxidoreductase, EC 1.1.1.8), myelin basic protein (MBP), and proteolipid protein (PLP) marks the differentiation of oligodendrocytes, the myelinating cells in the central nervous system (1, 2). MBP and PLP are components of the myelin sheath, constituting 30%o and 50%o of the proteins in myelin, respectively. The expression and biosynthesis of MBP and PLP in oligodendrocytes have been shown to be coordinately expressed with the process of myelination (3)(4)(5)(6).Peak expression of GPDH during neonatal development also coincides with the active period of myelination (7,8). Localized in oligodendrocytes (9), GPDH provides glycerol phosphate for the biosynthesis of phosphatides (10), a precursor for membrane components (11). The developmental regulation of GPDH gene expression and its modulation by glucocorticoids in the rat brain and in primary cultures of oligodendrocytes have been well characterized (12).Glucocorticoids and other hormones have been implicated in the stimulation of myelinogenesis (13)(14)(15)(16). During glial differentiation, the expression of 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) has been shown to be increased by dexamethasone, a potent glucocorticoid agonist, both in reaggregating (13) and dissociated (14) The ability ofglucocorticoids to influence the developmental expression of genes in the brain correlates highly with the ontogen...
Neural stem cells (NSCs) were isolated from embryonic day 16 Sprague-Dawley rats and cultured in a novel serum-free stem cell medium that selected for the growth of NSCs and against the growth of GFAP(+) cells (astrocytes). NSCs maintained in culture for extended periods of time retained immunoreactivity for both nestin and PSA-NCAM, two markers characteristic of the stem cell phenotype. Moreover, using an oligodendrocyte (OL) specification medium, NSCs differentiated into OL as evidenced by their morphology and expression of multiple oligodendrocyte/myelin-specific markers. In addition, NSCs are capable of acquiring a neuronal phenotype as evidenced by expressing neuronal markers, such as neurofilament (NF) and NeuN when cultured in a defined medium for neurons indicating that these cells are also a good source of neuroblasts, which could be used to replace neuronal populations in the brain. We also showed successful propagation and differentiation of NSCs into OL after cryostorage, allowing for the later use of stored NSCs. The long-term goal of culturing NSCs and committed oligodendrocyte progenitors (OLP) is to obtain homogeneous populations for transplantation with the goal of remyelinating the myelin-deficient CNS. Our preliminary experiments carried out on normal and myelin deficient rats demonstrate that these cells survive and migrate extensively in both types of hosts. NSCs grafted as such, as well as cells derived from NSCs exposed to selective specification before grafting, are able to differentiate within the host brain. As expected, NSCs are capable of giving rise to astrocytes in a medium favoring this phenotype.
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