Rat brain fatty acid-binding protein during development.

Cytosolic fatty acid-binding proteins (FABPs) have been described in rat and bovine whole brain. In the present study we investigated the distribution of FABP among white matter and gray matter as well as its changes during development. Fatty acid binding activity was similar in white and gray matter up to 40 days of age. In white matter it showed an age dependent increase thereafter, while in gray matter it remained constant throughout. Gel filtration (Sephadex G-75) of white matter cytosol of adult female rats resolved the fatty acid-binding activity in two peaks: A (Vo) and B (12-14 KDa; FABP). The specific binding activity in the FABP fraction was 10.4 pmol/micrograms of protein. The activity in peak A showed an age-dependent increase which paralleled myelin deposition. In contrast, the activity in the FABP fraction (peak B) remained undetectable up to 40 days of age, increasing thereafter. The differential distribution of cellular brain proteins with the capacity to bind fatty acids in gray matter and white matter suggests that this activity could be related to glial cells or to cell related structures such as myelin.     

Glycosaminoglycans of disaggregated foetal mouse brain tissue cultures

Synopsis Disaggregated foetal mouse brain tissue cultures were examined for glycosaminoglycans using Alcian Blue and periodic acid-Schiff staining techniques. It was found that spongioblasts (neuron and glial cell precursors) were rich in sulphated glycosaminoglycans, while astrocytes contained little or no sulphated polymers. The chief acid glycosaminoglycans of the brain reportedin vivo, hyaluronic acid, chondroitin sulphate and sialic acid-bearing polymers, were not demonstrated in the mouse brain cultures. There was a decline in glycosaminoglycan content over two weeks in culture, but during the corresponding periodin vivo an increase has been reported. These deficiencies are possibly correlated with the failure of the cultures to myelinate.     

Glycosaminoglycans and glycoproteins associated with rat brain nuclei

The concentration, composition and sulfate labeling of glycosaminoglycans and glycoproteins have been studied in purified nuclei isolated in bulk from rat brain. The concentration of total glycosaminoglycans is 0.142 μmol hexosamine/100 mg protein, comprising 57% chrondroitin 4-sulfate, 7% chondroitin 6-sulfate, 29% hyaluronic acid and 7% heparan sulfate. Control experiments demonstrated that less than 5% of the sulfated glycosaminoglycans associated with nuclei could be accounted for by the nonspecific adsorption of soluble acidic proteoglycans to basic nuclear proteins. Glycoprotein carbohydrate is present at a level of 206 μg/100 mg protein, and has an average composition of 30% N-acetylglucosamine, 29% mannose, 19% N-acetylneuraminic acid, 15% galactose, 4% N-acetylgalactosamine, and 3% fucose. Labeling studies also indicated the presence of ester sulfate residues on the glycoprotein oligosaccharides.     

Studies on myelin-basic-protein methylation during mouse brain development.

The synthesis and methylation in vivo of myelin basic protein (MBP) during the mouse brain development has been investigated. When mice ranging in age from 13 to 60 days were injected intracerebrally with L-[methyl-3H]methionine, the incorporation of radioactivity into MBP isolated from youngest brain was found to be the highest and declined progressively in mature brains. This pattern of radioactivity incorporation was inversely correlated with the total amount of MBP in the brains, suggesting a higher ratio of MBP methylation to synthesis in younger brain. To differentiate the relative rate of protein synthesis and methylation, animals were given intracerebral injections of a L-[methyl-3H]methionine and L-[35S]methionine mixture and the ratio of 3H/35S (methylation index) was determined. The ratios in the isolated MBP fractions were higher than those of 'acid extracts' and 'breakthrough' fractions, with a maximal ratio in the youngest brain. This high ratio was well correlated with the higher protein methylase I (PMI) activity in younger brains. The MBP fractions were further separated on SDS/polyacrylamide-gel electrophoresis into several species with apparent Mr ranging from 32,400 to 14,500. The results indicated that each protein species accumulated at a characteristic rate as a function of age. The high-Mr (32,400) species was predominant in younger brain, whereas the smaller MBP was the major species in older brain tissue. The importance of this developmental pattern of MBP synthesis and methylation is discussed in relation to PMI activity.     

Disialoganglioside GDla of rat brain subcellular particles during development.

The increase observed in the amount of the disialoganglioside GDlof the rat cerebrum during development between 21 and 81 days of age accounted for nearly 40% of the overall increase in total ganglioside in the tissue during the same period. Subcellular fractionation showed the microsomal fraction to contribute by far the most towards this increase in Cerebral ganglioside GDla. It is suggested that microsomal ganglioside GDla may serve as a marker for dendritic arborization in the rat cerebrum.     

Regulation of three beta-tubulin mRNAs during rat brain development.

The nucleotide sequence of a complete rat brain beta-tubulin T beta 15 has been determined from three overlapping cDNA clones. The overall length of the T beta 15 sequence is 1589 bp and shows between 84.5% and 88.6% homology within the coding region as compared with chick and human beta-tubulin sequences. On the other hand, the 3'-non-coding region is highly divergent. Comparison of the derived amino acid sequences from different species demonstrates that the amino acid changes are not randomly distributed, but rather there are several conserved and two highly variable regions common to beta-tubulin polypeptides from various sources. The T beta 15 sequence encodes a dominant neuronal 1.8-kb beta-tubulin mRNA species. Two other minor beta-tubulin mRNA species of 2.6 and 2.9 kb are present in rat brain. By using two synthetic oligonucleotide probes complementary to the carboxyl-terminal divergent region and to the amino-terminal conserved region, we have shown that the three mRNAs are distinct species, which are developmentally regulated. The level of the 1.8-kb mRNA species increases till the age of 12 days thereafter its level decreases. The 2.9-kb mRNA is an early neuronal mRNA species, while the 2.6-kb mRNA is a late neuronal species which is detected at 30 days of rat brain development. The data illustrate that there is a differential expression of the beta-tubulin multigene family during rat brain development which may suggest different functions for the various beta-tubulin isotopes.     

Corticosterone administration and lipid metabolism in brain regions during development.

Effect of corticosterone on lipid contents of different brain regions and the effect of age on the sensitivity of these regions to corticosterone have been studied. Corticosterone administration (40 mg/kg body wt, sc) to 17-day-old rat for 3 days led to significant decrease in phospholipid content of cerebellum and increase in cholesterol contents of hippocampus and striatum. However, there was no effect on cerebral cortex and brain stem lipids. This alteration in lipids was associated with decrease in [U-14C] glucose incorporation into cholesterol and phospholipids, decrease in plasma beta-hydroxy butyrate levels and increase in beta-hydroxy butyrate dehydrogenase activity in hippocampus and striatum, thereby suggesting that suppression of glucose utilization by corticosterone was compensated by higher utilization of ketone bodies for lipid synthesis in these regions. The sensitivity to corticosterone appears to be age-specific as, at 20-day, cerebellum, hippocampus and striatum were susceptible, at 10-day only hippocampus and at 40- and 90-day none of these regions responded to the treatment.     

Tubulin and actin synthesis during brain development

Chick brain proteins from 5- through 13-day embryos were labeled with l-[³⁵S]methionine for 30 min in vitro and analyzed by two-dimensional gel electrophoresis. Autoradiographs of the gels were scanned with a computer-coupled densitometer to measure the relative rates of protein synthesis. The actins and the tubulins were the most abundant proteins and had the highest rates of synthesis. β and γ actin were synthesized at constant rates throughout this period of development, but the rate of tubulin synthesis increased fourfold. Six α tubulins and two β tubulins were distinguished, and they were all synthesized at all times. The relative rates of synthesis of these forms changed with development in a complex pattern, but the stoichiometry of α:β remained 1:1.     

Differential expression of GABAA receptor alpha-subunits in rat brain during development.

Unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor have been expressed in E. coli and used to generate polyclonal antisera specific for these subunits. The antibodies identify proteins by SDS-polyacrylamide gel electrophoresis and western blotting of molecular size 51 kDa, 53 kDa, 59 kDa and 55 kDa, respectively, which show differential patterns of expression during development. Whereas the alpha 2 and alpha 3 subunits are present at early stages, the expression of alpha 1 and alpha 3 subunits is low at birth and increases with age. This differential expression could be correlated with previous studies examining the developmental expression of BZ1 and BZ2 benzodiazepine binding sites.     

Dual functions of Dab1 during brain development

Reelin coordinates the movements of neurons during brain development by signaling through the Dab1 adaptor and Src family tyrosine kinases. Experiments with cultured neurons have shown that when Dab1 is phosphorylated on tyrosine, it activates Akt and provides a scaffold for assembling signaling complexes, including the paralogous Crk and CrkL adaptors. The roles of Akt and Dab1 complexes during development have been unclear. We have generated two Dab1 alleles, each lacking two out of the four putative tyrosine phosphorylation sites. Neither allele supports normal brain development, but each allele complements the other. Two tyrosines are required for Reelin to stimulate Dab1 phosphorylation at the other sites, to activate Akt, and to downregulate Dab1 levels. The other two tyrosines are required to stimulate a Crk/CrkL-C3G pathway. The absence of Crk/CrkL binding sites and C3G activation causes an unusual layering phenotype. These results show that Reelin-induced Akt stimulation and Dab1 turnover are not sufficient for normal development and suggest that Dab1 acts both as a kinase switch and as a scaffold for assembling signaling complexes in vivo.     

Alterations of CaMKII after hypoxia-ischemia during brain development

Transient brain hypoxia-ischemia (HI) in neonates leads to delayed neuronal death and long-term neurological deficits. However, the underlying mechanisms are incompletely understood. Calcium-calmodulin-dependent protein kinase II (CaMKII) is one of the most abundant protein kinases in neurons and plays crucial roles in synaptic development and plasticity. This study used a neonatal brain HI model to investigate whether and how CaMKII was altered after HI and how the changes were affected by brain development. Expression of CaMKII was markedly up-regulated during brain development. After HI, CaMKII was totally and permanently depleted from the cytosol and concomitantly deposited into a Triton-insoluble fraction in neurons that were undergoing delayed neuronal death. Autophosphorylation of CaMKII-Thr286 transiently increased at 30 min of reperfusion and declined thereafter. All these changes were mild in P7 pups but more dramatic in P26 rats, consistent with the development-dependent CaMKII expression in neurons. The results suggest that long-term CaMKII depletion from the cytosolic fraction and deposition into the Triton-insoluble fraction may disable synaptic development, damage synaptic plasticity, and contribute to delayed neuronal death and long-term synaptic deficits after transient HI.     

Elongation of mitochondrial fatty acids during brain development

Abstract— Elongation of mitochondrial fatty acids was studied in whole brain samples from rats before, during and after the period of myelination. The mitochondria were isolated by centrifugation in a discontinuous sucrose gradient and incubated under N₂ in a medium containing NADH, NADPH, ATP and acetyl-[1-¹⁴C]coenzyme A. Fatty acids were extracted, methylated and analysed by gas-liquid chromatography. A distinct pattern emerged in which brain mitochondria from rats undergoing myelination synthesized longer chain fatty acids preferentially, particularly C_22:4. Mitochondria from brains of mature rats synthesized shorter chain fatty acids preferentially, mainly C_18:0 and C_20:4. We suggest that eicosamonoenoic acid (C_22:1) is a precursor in vivo of nervonic acid (C_24:1).     

Differential substrate oxidation by dissociated brain cells and homogenates during development.

The rates of oxidation of 3-hydroxy[3-14C]butyrate, [3-14C]acetoacetate and [6-14C]glucose were compared by using two different preparations of brain from the same animals (i.e. whole homogenates and dissociated brain cells) at various ages during development. In homogenates the rates of oxidation of 3-hydroxy[3-14C]butyrate and [3-14C]acetoacetate were high in young rats and low in adults, and were significantly higher at most ages during development than those obtained for intact cells. In contrast, rates of [6-14C]glucose oxidation by homogenates and intact cells were essentially the same at early ages; however, the rate by homogenates did not change throughout development, whereas that by intact cells increased severalfold by adulthood. In adult animals the initial glucose concentration affected the rate of glucose oxidation in homogenates, but not in intact cells. These data suggest a role for the intact cell membrane in the regulation of alternative substrate utilization by brain cells and that this process changes during development. However, the data may reflect selective differences in the cellular and subcellular components in these two preparations.