Ontogenetic changes in protein level of amyloid precursor protein (APP) in growth cones and synaptosomes from rat brain and prenatal expression pattern of APP mRNA isoforms in developing rat embryo.

To elucidate the functional role of the amyloid precursor protein (APP) during brain ontogeny, developmental changes of APP levels in growth cones and synaptosomes were studied from embryonic day 14 up to postnatal day (PD) 400 using Western analysis. APP level in growth cones was low during prenatal stages of development, but demonstrating a continuous increase from PD 3 up to PD 10. Highest concentration of APP in synaptosomes was found between PD 7 and 10, followed by a considerable decrease up to PD 30 and persisting at this level up to PD 400. In situ hybridization to differentiate between APP695 mRNA, APP751 mRNA and APP770 mRNA revealed distinct age-related expression pattern of various APP isoforms. During prenatal brain development APP695 mRNA is maximally expressed in brain structures, containing differentiating nerve cells. APP751 and APP770 mRNA isoforms are diffusely distributed in the embryo throughout the prenatal period examined and their expression is higher in peripheral organs such as skin, lung, liver and bones as compared to the brain. The increase of APP level during synaptogenesis suggests a functional role of APP in the processes of neurite outgrowth and cell targeting as well as in the maintenance of the functional integrity of synapses in the mature brain. The APP695 isoform seems to be the major form involved in embryonic brain maturation.

Phosphorylation in vivo of chick brain microtubule-associated phospholipids.

Microtubules were prepared by temperature-dependent cycles of assembly/disassembly from chick brain labeled in vivo with 32Pi and the distribution of labeled phospholipids extracted from cold-insoluble and soluble microtubular protein fractions was analyzed by thin-layer and paper chromatography. While 32P-labeling was associated with all of the phospholipids identified after 2-D TLC, it was found that all of the relatively high radioactivity associated with phosphatidylserine (PS) was in fact associated with a minor co-migrating component which was subsequently identified as phosphatidylinositol(PI) by three independent separation procedures. It was estimated that the relative specific radioactivity in PI was several-fold higher than that associated with other microtubule-associated phospholipids. Additional experiments, in which the protein components of once-cycled microtubules were fractionated by gel permeation chromatography, provided evidence that the 36S component containing ring-like tubulin oligomers (36S) appears to be selectively associated with phospholipid components that were specifically enriched in 32P-PI. The possible significance of these findings is discussed in relation to the effects of phospholipids on microtubule dynamics and to the function of microtubules in their interactions with membranes.

Changes in the neuronal plasma membrane during synaptogenesis.

During synaptogenesis the plasma membrane of neurons undergoes considerable changes and large portions of it develop to synaptic membranes. This transformation is brought about by biochemical and morphological changes. The aim of the present investigation was to study by morphological methods the in vivo changes of some basic components of the neuronal membranes during the early postnatal period, when active synaptogenesis occurs. The cerebral cortex of Wistar rats was used for preparation of either growth cones (up to postnatal day 10) or synaptosomes (postnatal days 14-30). Our studies were focused on the changes occurring in integral membrane proteins and cholesterol domains, and in certain carbohydrate residues and anionic sites. In the first days after birth relatively few intramembranous particles are found in the plasma membranes of growth cones, few and small cholesterol domains, scarce lectin-binding and anionic sites. During the following days there is a clear tendency for increase of the number of all of the studied structures until the appearance of typical synaptic membranes. Throughout the studied developmental period the protein and the cholesterol molecules were found to occupy distinct membrane domains. The structure of the developing neuronal membrane, poor on proteins and cholesterol, and its maturation to the fully functional synaptic membrane is discussed.

Comparison of the Lowry and the Bradford protein assays as applied for protein estimation of membrane-containing fractions.

Although the Bradford protein estimation assay has found wide distribution, it has a number of drawbacks, which in some cases have been shown to produce erroneous results upon comparison to other, more precise, methods for protein estimation. It was found that the underestimation of the protein content of membrane-containing fractions cannot be overcome by pretreatment with NaOH or the detergents employed (Triton X-100, sodium dodecyl sulfate, 3[(3-cholamidopropyl)-dimethylammonio]propanesulfonic acid) and the protein estimates obtained do not agree with estimates obtained by the Lowry assay. Upon storage of fractions at -20 degrees C there is a considerable loss of dye binding activity, varying in accordance with the membrane content of the fractions, reaching up to 58% in the case of membrane-enriched fractions stored at -20 degrees C for 15 days. Pretreatment with the employed agents brought about an equal increase of dye binding capacity, specific for the individual fractions; however, none of these agents could recover the dye binding activity lost during several days of storage at -20 degrees C. It is suggested that the straightforward Bradford procedure has a rather limited scope of application, particularly concerning membrane-containing samples, and requires preliminary studies to determine its applicability according to the nature of the biological material examined.

Developmental changes of proteins and concanavalin A reactive glycoproteins in growth cones from rat forebrain.

Growth cones were isolated from the forebrains of 1, 5 and 9 days-old rats. The ultrastructural characterization of the obtained subcellular fractions reveals that two of them (GC1 and GC2) contain predominantly growth cones. It was found that the protein content of the membranes contained in these fractions increases 7.5 times, while in whole forebrain the increase is only 3 times, showing that during the studied developmental period there is a predominant protein enrichment of the specialized brain structures (e.g. growth cones). Electrophoretic studies show that there are characteristic changes of the Coomassie Brilliant Blue R250 staining and concanavalin A reactive protein profiles. Comparison of the protein patterns of growth cones to those of synaptosomes from mature forebrain reveal a number of bands, which appear to be characteristic for one of these structures. The possible roles of the developmentally controlled proteins in the processes of synaptogenesis is discussed.

Changes of soluble and membrane proteins of rat brain during pre- and postnatal development.

Membrane and soluble protein fractions were obtained from forebrain, midbrain and hindbrain of embryos or neonatal rats. The amount of the protein of the corresponding brain parts was followed up as a function of DNA content. Age-related changes of the concentration of over 50 protein bands were observed in all three brain parts. There are also bands which tend to disappear (or appear) at distinct stages of development. In each of the brain parts there are bands showing age-dependent changes characteristic for this part. A common feature of the changes of the membrane protein patterns of forebrain and midbrain observed during development is a decrease of the concentration of proteins with lower molecular mass (below 40 kD), while proteins of higher molecular mass become better pronounced. Compared to forebrain and midbrain the hindbrain has a relatively conservative protein composition throughout development.

Effects of vanadate on the assembly and disassembly of purified tubulin.

Sodium-orthovanadate (100-700 microM) added to purified pig brain microtubule protein (molar ratios 13-90 moles vanadate/mole tubulin) inhibits to a considerable extent the assembly (up to 65%) and the disassembly rates (up to 60%) of microtubules, as determined by turbidimetry. Vanadate added to preformed microtubules did not appreciably alter the turbidity level of the samples, however, the disassembly rates were decreased in the same manner as when vanadate was added prior to polymerization. Microtubule protein kept on ice for 3-6 hours became more susceptible to vanadate than freshly prepared protein. The effect of vanadate was independent of the GTP concentration at which the polymerization assays were performed (0.025 to 1 mM GTP). In the presence of taxol, which increases the rate and extent of microtubule formation, vanadate had no effect on assembly rates. Disassembly was inhibited, however, much less than in the presence of vanadate alone. Electron microscopy and polyacrylamide gel electrophoresis did not reveal differences between microtubules prepared in the presence or in the absence of vanadate. This is consistent with the notion that vanadate does not interfere with the interaction between tubulin and the high-molecular weight microtubule-associated proteins. Apparently vanadate brings about an allosteric change of the microtubule protein(s) resulting in the abnormal polymerization kinetics of tubulin found in our study. The above results may be relevant for studies where the effects of vanadate on intracellular motility are interpreted as being solely due to a specific inhibition of ATPases.