A. M. Giuffrida

RNA POLYMERASE ACTIVITY IN VARIOUS CLASSES OF NUCLEI FROM DIFFERENT REGIONS OF RAT BRAIN DURING POSTNATAL DEVELOPMENT

—The changes in the wet weight and the numbers of cell nuclei recovered from the cerebral hemispheres, the cerebellum and the brain stem of rats from the period of 5–30 days after birth have been determined. In parallel a study has been made of the RNA polymerase activity, both in the unfractionated nuclei from these regions and in the nuclei separated by zonal centrifugation. In general there is a considerable decline in activity during this period, which occurs in all class of nuclei although not to the same extent. The most dense nuclei from the cerebellum retain relatively high activity at 20 days after birth, possibly due to the contribution of the microneuronal nuclei.

Changes in Rapid Transport of Phospholipids in the Rat Sciatic Nerve During Axonal Regeneration

Abstract: Axonal transport of phospholipids in normal and regenerating sciatic nerve of the rat was studied. At various intervals after axotomy of the right sciatic nerve in the midthigh region and subsequent perineurial sutures of the transected fascicles, a mixture of 60 μCi [Me‐HC]choline and 15 μCi [2‐3H]glycerol in the region of the spinal motor neurons of the L5 and L6 segments was injected bilaterally. The amount of radioactive lipid (and in certain cases its distribution in various lipid classes) along the nerve was determined as a function of time. Three days after fascicular suture and 6 h after spinal cord injection of precursors, there was an accumulation of labeled phospholipids and sphingolipids in the transected sciatic nerve in the region immediately proximal to the site of suture. Nine days after, there was a marked increase in the accumulation of radioactivity in the distal segments of the injured nerve, which increased up to 14 days after cutting and disappeared as regeneration proceeded (21–45 days). In all segments of both normal and regenerating nerve fibers, as well as in L5 and L6 spinal cord segments, only phosphatidylcholine and sphingomyelin were labeled with [14C]choline. These results suggest that the regeneration process in a distal segment of a peripheral neuron, following cutting and fascicular repairing by surgical sutures, is sustained in the first 3 weeks by changes in the amount of phospholipids rapidly transported along the axon towards the site of nerve fiber outgrowth.

Effect of hypothyroidism on the biogenesis of free mitochondria in the cerebral hemispheres and in cerebellum of rat during postnatal development

The effect of propylthiouracil-induced neonatal hypothyroidism on some aspects of the biogenesis of free (non-synaptosomal) mitochondria in the cerebral hemispheres and in the cerebellum of developing rat has been studied. The results obtained show that in hypothyroid rats mitochondrial DNA synthesis is delayed, mitochondrial RNA synthesis is not affected and cytochrome aa3 content of mitochondria is lower than in controls. Furthermore ultrathin sections of 14-and 21-day old hypothyroid rat cerebella show mitochondria with an altered ultrastructural organization and large intracristal spaces.

Nuclear and mitochondrial DNA synthesis and energy metabolism in primary rat glial cell cultures

DNA synthesis in nuclei and mitochondria purified from serum-supplemented rat glial cell cultures at different days after plating was studied. Furthermore in mitochondria, some enzymatic activities related to energy transduction (citrate synthase, malate dehydrogenase, total NADH-cytochromec reductase, cytochrome oxidase and glutamate dehydrogenase) were measured. For DNA labeling [methyl-3H]thymidine was added to the culture medium at different days after plating. During the culture times studied the specific activity of total, nuclear, and mitochondrial DNA decreased from 8 days in vitro (DIV) to 21 DIV and increased at 30 DIV. The specific activity of nuclear DNA was always higher than that of mitochondrial DNA. The specific activity of the above mentioned mitochondrial enzymes increased from 8 DIV up to 21 DIV and decreased at 30 DIV, suggesting a relationship between the energy metabolism and the differentiation of glial cells in culture.

Rapid axonal transport of glycerophospholipids in regenerating hypoglossal nerve of the rabbit.

Abstract: The intraaxonal transport of phospholipids in regenerating hypo‐glossal nerve of the rabbit was investigated by administration of labeled lipid precursors into the medulla oblongata. At various time intervals after crushing the left hypoglossal nerve at the level of the digastric muscle, a mixture of 60 MCi of [2‐3H]glycerol and 15 /μCi of [1‐14C]palmitate, dissolved in 15% bovine serum albumin, was injected into the calamus scriptorius of the fourth ventricle. The amount and the pattern of labeling of glycerophospholipids synthesized in the motor neurons were determined. Three days after nerve crush there was an accumulation of labeled glycerophospholipids immediately proximal to the injury site. Seven days after crushing, the regenerating nerve incorporated rapidly transported labeled lipids in greater amounts than the contralateral normal nerve; the incorporation was elevated along the entire length of the nerve containing both regenerating axons and the post‐crush sprouting terminals. The difference between the two sides increased up to 14 days, but disappeared as regeneration proceeded (21‐45 days). The “pool” of radioactive lipids remaining in the cell bodies of hypoglossal nuclei, in the segments of nerve, both proximal and distal to the crush site, and in all the segments of uncrushed nerve was similar 6–12 h after labeling. Among the phospholipids, the highest 3H and 14C radioactivity was observed in phos‐phatidylcholine and phosphatidylethanolamine. These results support the hypothesis than an increase in the amount of glycerophospholipids, conveyed by rapid axonal transport, takes place in the first 2 weeks during nerve regeneration. The increased transport of lipids presumably reflects an augmented demand for membrane precursors during the sprouting process.

Acetylation and phosphorylation of histones and nonhistone chromosomal proteins in neuronal and glial nuclei purified from cerebral hemispheres of developing rat brain.

The processes of acetylation and phosphorylation of histones and nonhistone proteins (NHPs) in neuronal and glial nuclei purified from cerebral hemispheres of rats at 1, 10, and 30 days of age were investigated. Purified neuronal and glial nuclei were incubated in the presence of [3H]acetyl‐CoA and of [γ‐32P]ATP. Histones and NHPs were extracted and fractionated by gel electrophoresis. Densitometric and radioactive patterns were obtained. The results showed an increase of acetylation and phosphorylation from 1 to 10 and 30 days of age in both neuronal and glial nuclei in almost all histone and NHP fractions. Among the histones, the H3 fraction was always more labeled than the other fractions and showed the most remarkable differences during postnatal development. In the NHP fractions, the increase in acetylation from 1 to 10 and 30 days of age was more evident in the low‐molecular‐weight region of neuronal nuclei than in the corresponding fraction of glial nuclei. The appearance of highly phosphorylated proteins (70,000–90,000 daltons)—absent at 1 day, appearing at 10 days, and more evident at 30 days of age—was observed in both neuronal and glial nuclei.

Changes of Synaptosomal Energy Metabolism Induced by Hypoxia During Aging

Synaptosomes were isolated from the motor area of the cerebral cortex of normoxic or hypoxic (PaO2=17–19 mmHg, for 15 min) beagle dogs of different ages. Synaptosomes were incubated in Krebs-Henseleit-Hepes buffer (for 10 min at 24°C) and the energetic state was defined by: the balance of the labile phosphates (ATP, ADP, AMP, and creatine phosphate); the respiratory rate; the redox state of the intramitochondrial NAD-couple. By the present experimental model, it is possible to evaluate the potential damage (induced by the “in vivo” hypoxic insult) that synaptosomes cannot reverse under optimal incubation. Aging affected the phosphorylation state of the post-hypoxic incubated synaptosomes. The oxygen consumption rate was quite similar in the synaptosomal fractions from the motor area of hypoxic beagle dogs of different ages, but the cytochromec anda contents were lower in the preparations from hypoxic older brains. In dogs of different ages, hypoxia always lowered the respiration of the synaptosomes, but aging affected the oxygen consumption rates only in post-hypoxic synaptosomes incubated with succinate. The synaptosomal energetic state was defined also by the redox state of the intramitochondrial NAD-couple (ΔGox-red) and the phosphorylation state of adenine nucleotide system (ΔGATP). The free-energy change (ΔΔG) for the coupled reactions was calculated, too. In synaptosomes isolated from the cerebral cortex of dogs submitted to hypoxia, the equilibrium (calculated for the mitochondrial electron transfer chain and the phosphorylation of adenine nucleotides) was markedly altered as function of aging. The extensive age-related ΔΔG changes were largely supported by alteration of the phosphorylation state of adenine nucleotides, rather than by modification of the redox state of the electron transfer chain.All present data suggest that the bioenergetic derangement caused by hypoxia and aging may be interpreted also in terms of modification of the biophysical and biochemical mechanisms involving the mitochondrial membranes and particularly the inner mitochondrial membrane.

Remodeling and Sorting Process of Ethanolamine and Choline Glycerophospholipids During Their Axonal Transport in the Rabbit Optic Pathway

Abstract: The existence of a mechanism by which the ester‐ and ether‐linked aliphatic chains of the major phospholipids are retailored during their axonal transport and sorted to specific membrane systems along the optic nerve and tract was investigated. A mixture of [I‐14C]hexadecanol and [3H]arachidonic acid was injected into the vitreous body of albino rabbits. At 24 h and 8 days later, the distribution (as measured by the 3H/14C ratio) and the positioning (as monitored by hydrolytic procedures) of radioactivity in the various phospholipid classes of retina, purified axons, and myelin of the optic nerve and tract were determined. At the two intervals after labeling, the 3H/14C ratios of each diradyl type of phosphatidylethanolamine and phosphatidylcholine were (a) substantially unchanged all along the axons within the optic nerve and tract and (b) markedly modified in comparison with those found in the retina and axons for molecular species selectively restricted to myelin sheath. Evidence is thus available that intraxonally moving ethanolamine and choline glycerophospholipids, among others, are added to axonal membranes most likely without extensive modifications. In contrast, they are transferred into myelin after retailoring. Through these two processes, the sorting and targeting of newly synthesized phospholipids to their correct membrane domains. such as axoplasmic organelles, axolemma, or periaxonal myelin, could be controlled.

RNA synthesis in neuronal and glial cell nuclei from rat cerebral hemispheres during early postnatal development

RNA synthesis in rat cerebral hemispheres at 1, 5, and 10 days of age and the relative contribution brought by neuronal and glial nuclei to RNA synthesis was investigated. The experiments were carried out both in vivo (by i.p. injection of [3H]uridine) and in vitro (either by incubation of tissue slices with [3H]uridine or by determination of RNA polymerase activities). The labeling of RNA decreases from 1 to 10 days of age both in vivo and in vitro; the decrease is of the same extent in neuronal and glial nuclei. RNA polymerase activity Mg2+-dependent does not change significantly from 1 to 10 days of age either in total, in neuronal, or in glial nuclei, whereas the Mn2+-dependent activity increases significantly over the same developmental period studied. The significance of RNA polymerase assay as an index of in vivo RNA synthesis is discussed.

LABELING OF RNA IN YOUNG AND ADULT RAT BRAIN: Evidence for Different RNA Processing

The labeling of RNA in young and adult rat brain has been studied by measuring in vitro (tissue slices incubation) the incorporation of labeled uridine into RNA of total tissue and of the various subcellular fractions purified from cerebral hemispheres of 1- and 10-month-old rats. Gel electrophoretic analysis of the newly synthesized nuclear and microsomal RNA was also accomplished. An active metabolism of RNA in adult animals was found; moreover, distinct differences in ribosomal RNA processing in cerebral hemispheres of 1- and 10-month-old rats, with a more rapid processing in the brain of adult animals, were obtained.