Terry C. Johnson

THE EFFECTS OF MATURATION ON IN VITRO PROTEIN SYNTHESIS BY MOUSE BRAIN CELLS

PROTEIN metabolism by brain tissue has recently been studied by several in cirro techniques (Acs, NEIDLE and WAELSCH, 1961; MOKRASCH and MANNER, 1963; ROBERTS and BAXTER, 1963; RUBIN and STENZEL, 1965). Although many investigators have employed cerebral cortex slices and cell-free homogenates, there have been very few attempts to use brain cell suspensions. Cell suspensions offer an intermediate between the complex intercellular relationships present in the cortical slices and the cell free systems which eliminate factors such as cell membrane permeability. The present studies were undertaken to investigate the feasibility of employing brain cell suspensions to determine the effect of brain development on cellular permeability and subsequent protein synthesis.

REGULATION OF PROTEIN SYNTHESIS DURING POSTNATAL MATURATION OF THE BRAIN

THE HISTOLOGIC and cellular features that characterize the postnatal development of the brain of many mammals are extremely complex, and represent a series of events that are associated with both cellular growth and differentiation. For instance, postnatal neurogenesis has been characterized by developmental features that include both cell division and the cellular movement, or migration, or neuronal and glial elements (ALTMAN, 1966; SHWADA & LANGMAN, 1970; HERNDON, 1971). At birth, the cerebal cortex is primarily composed of closely packed cell bodies and during the first week of development the cortex grows to reach its major thickness (FOLCH-PI. 1971). Growth during this critical period of postnatal life is characterized by the extension of axons from the cell bodies and the growth and arborization of dendritic processes. During this time the blood circulatory system is also found to be actively extending and branching, and the formation of functional synapses result in the postnatal neuropil. Associated with this complex development of the neuropil is an alteration in the volume of the extracellular spaces which may provide suitable pathways for axon and dendrite extension and arborization (BONDAREFF & PYSH, 1968). However. changes are not limited to neurons and their supporting elements since this period of neuronal maturation is also characterized by an active proliferation and differentiation of glial cells (VAUGHN, 1969; VAUGHN & PETERS, 1971). In addition to these dramatic histologic changes, postnatal development of the mammalian brain is distinguished by numerous biochemical events that include the formation of synaptic vesicles and their respective transmitter substances, neurosecretory granules, increases in the specific activity of numerous enzymes, and the active deposition of myelin (BALAZS, 1971; FOLCH-PI, 1971; GROVE et al., 1973).

The effects of phenylalanine on amino acid metabolism and protein synthesis in brain cells in vitro.

Incubation of brain cell suspensions with 14 mM‐phenylalanine resulted in rapid alterations of amino acid metabolism and protein synthesis. Both thc rate of uptake and the final intracellular concentration of several radioactively‐labelled amino acids were decreased by high concentrations oi phenylalanine. By prelabelling cells with radioactive amino acids, phenylalanine was also shown to effect a rapid loss of the labelled amino acids from brain cells. Amino acid analysis after the incubation of the cells with phenylalanine indicated that several amino acids were decreased in their intracellular concentrations with effects similar to those measured with radioisotopic experiments (large neutral > small and large basic > small neutral > acidic amino acids). Although amino acid uptake and efflux were altered by the presence of 14 mwphenylalanine, little or no alteration was detected in the resulting specific activity of the intracellular amino acids. High levels of phenylalanine did not significantly altcr cellular catabolism of either alanine, lysine, leucine or isoleucine. As determined by the isolation of labcllcd aminoacyl‐tRNA from cells incubated with and without phenylalanine, there was little or no alteration in the level of this precursor for radioactive alanine and lysine. There was, however, a detectable decrease in thc labelling of aminoacyl‐tRNA for leucine and isoleucine. Only aftcr correcting for the changes of the specific activity of the precursors and thcir availability to translational events, could the effects of phenylalanine on protein synthesis be established. An inhibition of the incorporation into protein for each amino acid was approximately 20%.

LEVEL AND AMINO ACID ACCEPTOR ACTIVITY OF MOUSE BRAIN tRNA DURING NEURAL DEVELOPMENT

Abstract— The level of tRNA in mouse brain tissue was measured at various stages of postnatal development. The amount of tRNA per unit of brain wet weight was little, if at all, altered during the first 22 days after birth and decreased by 26 and 32 per cent by 56 days and maturity, respectively. On a DNA or cellular basis, there was no maturation‐dependent decrease in tRNA content. The total amino acid acceptor activity of tRNA for seven different amino acids was measured during neural development. There were considerable differences in the tRNA acceptor activities of individual amino acids within an age group; however on a DNA basis, there was little difference between tRNA preparations obtained from newborn and adult mouse brain tissue. The in vivo levels of aminoacylated‐tRNA for the seven amino acids of interest, were measured in brain tissue of 1–, 9–, 34, 70–day‐old and adult (over 9 months old) mice. Alterations in tRNA level, total tRNA acceptor activity, for each amino acid, and the levels of in uivo aminoacylation of tRNA were shown to be independent of developmental alterations in brain amino acid pool sizes. The results are discussed with regard to the availability of cellular amino acids for translational events during early mammalian brain development.

Experimentally induced and natural recovery from the effects of phenylalanine on brain protein synthesis

The decrease in the neural polyribosomes produced during hyperphenylalaninemia could not be restored to normal levels by the injection of other single neutral amino acids. All of the neutral amino acids that are transported with phenylalanine were found to produce an alteration of neural polyribosomes similar to that measured with phenylalanine. However, the injection of a balanced mixture of 6 or 7 neutral amino acids could restore the brain polyribosomes to normal states. Although this experimentally induced recovery did not lower brain phenylalanine concentrations, it did restore the acylation levels of methionyl-tRNA, and in particular, the methionyl-tRNA initiator species. This also led to a concomitant stimulation of the elongation rate of brain polypeptide synthesis. A natural recovery of brain polyribosomal levels (occurring 2 h after 1 mg/g phenylalanine is injected) did not appear to represent a real recovery of neural protein metabolism. Phenylalanine concentrations were increased in the brain, the acylation levels of methionyl-tRNA, alanyl-tRNA and the initiator methionyl-tRNA remained altered, and the rate of ribosome translocation was decreased 28%.

Maturation-dependent events related to DNA-dependent RNA synthesis in intact mouse brain nuclei.

Summary Intact nuclei, isolated from mouse brain tissue at various stages of postnatal development, were employed to investigate the maturation-dependent events involved in DNA-dependent RNA synthesis. The incorporation of GTP into acid-insoluble RNA was shown to be enzymatic, dependent on DNA template activity and independent of homopolymer formation. Maturation-dependent alterations in nuclear activity were manifested by the incubation of nuclei in reaction mixtures and the deletion of nucleotides and spermidine. The measured rate of RNA synthesis was found to increase during the first 12 days following birth and then to rapidly decrease to a basal level by 22 days of age. The rate of GTP uptake was shown to be markedly increased during neural development although the rate of uptake was not directly related to the rate of RNA synthesis. RNA synthetized by nuclei isolated from neonatal brain tissue was relatively labile and rapidly degraded to acid-soluble products.

Developmental changes in aminoacylation of mouse brain tRNA

Abstract The concentration of tRNA for seven amino acids and the percentage of each tRNA that is aminoacylated in , vivo were determined at various stages of postnatal brain development. When normalized on a brain weight basis, the amount of amino acid acceptance of tRNAPhe, tRNAVal and tRNAGlu in 1-day old brain tissue was 50% to 100% higher than that measured in the adult brain. However, the percentage of tRNA molecules which is aminoacylated in , vivo was shown to gradually decrease during neural maturation. Age-dependent changes in the proportions of aminoacylated-tRNA were shown to be independent of the concentrations of free amino acids. This phenomenon is discussed with regard to free amino acid pools and the availibility of these precursors to protein synthesis during various stages of neural development.

FETAL DEVELOPMENT: THE EFFECTS OF MATURATION ON IN VITRO PROTEIN SYNTHESIS BY MOUSE BRAIN TISSUE

—The elucidation of the translational regulatory events which function during the critical fetal and neonatal period is an important prerequisite to our understanding of normal, as well as abnormal, brain growth and differentiation. Brain cell suspensions and cell‐free homogenates were employed to study the protein synthetic activity during the maturation of fetal‐ neural tissue. The results clearly demonstrated that while neural tissue from 1‐day postnatal mice was 10 times more active in protein synthesis than brain tissue from adult mice, the former was many fold less active in translational events than fetal neural tissue from 13‐day post‐zygotic mice. Fetal polypeptide synthetic activity was found to decrease from the 13th day to the 19th day post‐zygotic. This decrement in the translational activity was not due to amino acid availability or pools, or to differences, quantitatively or qualitatively, in polysome concentrations. The enhanced rate of protein synthetic activity measured with neural tissue from 13‐day post‐zygotic mice was shown to be due to an increase in rate of protein synthesis and not to an enhanced rate of protein degradation.

REGULATION OF PROTEIN SYNTHESIS IN DEVELOPING MOUSE BRAIN TISSUE: IN VITRO BINDING OF TEMPLATE RNA TO BRAIN RIBOSOMES

Abstract— Ribosomes, isolated from brain tissue of mice of various ages, were tested for their ability to participate in cell‐free protein synthesis and to bind polyuridylic acid. Although protein synthesis was markedly reduced by ribosomal preparations obtained from increasingly older animals, no significant differences could be measured with respect to template RNA binding. Similar binding properties were also measured with ribosomal subunits purified from young and mature brain cell ribonucleoprotein particles. In addition, no differences could be detected in the relative firmness of template RNA binding that could explain the maturation‐dependent loss in ribosomal activity.

RNA metabolism in isolated mouse brain nuclei during early postnatal development.

—RNA metabolism in isolated brain nuclei has been shown to be dramatically altered during early postnatal brain development. The present study involved an examination of the RNA products synthesized by nuclei at various stages of postnatal neural maturation. In all cases, the majority of the RNA appeared to be heterodisperse, non‐ribosomal and non‐tRNA in nature. In comparison to the RNA isolated from nuclei of neonatal tissue, the RNA from nuclei of 12‐day and 30‐day‐old mouse brain was found to be of smaller molecular weight. Despite the heterodisperse nature of these RNA molecules, the addition of α‐amanitin did not completely inhibit nuclear synthesis.