NEUROCHEMICAL ASPECTS OF NEURONAL ONTOGENESIS IN THE DEVELOPING RAT CEREBELLUM: CHANGES IN NEUROTRANSMITTER AND POLYAMINE SYNTHESIZING ENZYMES

Abstract— Changes in the activities of several specific enzymes were measured in the cerebellum during development. Early transient increases were found in both ornithine decarboxylase and S -adenosylmethionine decarboxylase, enzymes involved in the initial steps of polyamine synthesis. Different patterns of changes were found in neurotransmitter synthesizing enzymes. Tyrosine hydroxylase activity achieved adult levels very early, by 3 days after birth, and remained at this level. Glutamic acid decarboxylase activity, while very low at early stages, increased rapidly before birth and then after a lag period of 10 days started to increase rapidly, directly related to the general growth of cerebellar weight and protein content. Choline acetyltransferase activity started to increase rapidly, reaching a peak of about 100% of adult levels at 3-7 days after birth; the activity then gradually declined and at 20 days, after reaching a low of about 55% of adult values, gradually started to increase, reaching adult levels later than 40 days after birth. The development of protein carboxymethylase activity was similar to that of glutamic acid decarboxylase, directly related to the general growth of the cerebellum. Several interpretations of the results are discussed.     

Ornithine Decarboxylase in Human Brain: Influence of Aging, Regional Distribution, and Alzheimer's Disease

Abstract: Although experimental animal data have implicated ornithine decarboxylase, a key regulatory enzyme of polyamine biosynthesis, in brain development and function, little information is available on this enzyme in normal or abnormal human brain. We examined the influence, in autopsied human brain, of postnatal development and aging, regional distribution, and Alzheimer's disease on the activity of ornithine decarboxylase. Consistent with animal data, human brain ornithine decarboxylase activity was highest in the perinatal period, declining sharply (by ∼60%) during the first year of life to values that remained generally unchanged up to senescence. In adult brain, a moderately heterogeneous regional distribution of enzyme activity was observed, with high levels in the thalamus and occipital cortex and low levels in cerebellar cortex and putamen. In the Alzheimer's disease group, mean ornithine decarboxylase activity was significantly increased in the temporal cortex (+76%), reduced in occipital cortex (−70%), and unchanged in hippocampus and putamen. In contrast, brain enzyme activity was normal in patients with the neurodegenerative disorder spinocerebellar ataxia type I. Our demonstration of ornithine decarboxylase activity in neonatal and adult human brain suggests roles for ornithine decarboxylase in both developing and mature brain function, and we provide further evidence for the involvement of abnormal polyamine system activity in Alzheimer's disease.     

Ornithine decarboxylase activity in developing rat brain

—Total ornithine decarboxylase (ODC) (EC 4.1.1.17) activity per rat brain was elevated markedly from 14 days after conception to 12 days postnatum. ODC activity in the brainstem was very low and changed little during postnatal development. Activity in the cerebral hemispheres declined from a high level at birth to the low adult level by 8 days postnatum. Conversely activity in the cerebellum increased markedly from 3 days until 11 days postnatum, then suddenly decreased. Hence, the periods of greatest ODC activity paralleled those of maximal cell proliferation in each brain region. During perinatal brain development ODC activity changed considerably; it declined at about one day prior to term, and then increased rapidly to its highest level of activity at 4 h postnatum. Premature birth by caesarian section or lack of maternal care and nutrition did not affect this early postnatal response. The postnatal burst in ODC activity appears to be unique for brain tissue, since this response did not occur in heart, skeletal muscle or liver. Data from studies in which portions of fractions characterized by high or low enzymatic activity, respectively, were mixed or in which the supernatant enzyme fraction was dialysed are not consistent with the presence of direct inhibitors or activators of the enzyme. In addition, administration of cycloheximide to newborn rats abolished the 4-h postnatal burst in ODC activity. Our results suggest that the increase in ODC activity reflects enzyme synthesis de novo.     

Two variants of ornithine decarboxylase activity in the mouse liver. The nature of enzyme induction

Ornithine decarboxylase activity in mouse liver is predominantly located in the cell nuclei. After injection of some inducing agents (thioacetamide, diethylnitrosamine, hydrocortisone) the enzyme leaves the nucleus for cytosol. A circadian rhythm of ornithine decarboxylase activity has been observed in nucleus and cytosol, the decrease of enzyme activity in the nucleus being accompanied by its increase in cytosol. The enzyme obtained from intact mice with a minimal level of ornithine decarboxylase activity in the cytosol differs in ion-exchange properties, pH-optimum and Km for ornithine from the thioacetamide stimulated (nucleus enzyme).     

Modulation of ornithine decarboxylase activity and ornithine decarboxylase-antizyme complex in rat heart by hormone and putrescine treatment

Ornithine decarboxylase was present in a cryptic, complexed form in an amount approximately equivalent to that of free ornithine decarboxylase activity in adult rat heart. Addition of isoproterenol (10 mg/kg) caused a notable rise in ornithine decarboxylase activity and a simultaneous decrease in the amount of the complexed enzyme. During the period of ornithine decarboxylase decay, when cardiac putrescine content had reached high values, the level of the complex increased above that of the control. Administration of putrescine (1.5 mmol/kg, twice) or dexamethasone (4 mg/kg) produced a decrease of heart ornithine decarboxylase activity, while it did not remarkably affect the level of complexed ornithine decarboxylase, therefore raising significantly the ratio of bound to total ornithine decarboxylase. Putrescine also elicited the appearance of free antizyme, concomitantly with the disappearance of free ornithine decarboxylase activity after 3-4 h of treatment. These results indicate that a significant amount of ornithine decarboxylase occurs in an inactive form in the heart under physiological conditions and that its absolute and relative levels may vary following stimuli which affect heart ornithine decarboxylase activity.     

Translational regulation of mammalian ornithine decarboxylase by polyamines

Ornithine decarboxylase, which catalyses the formation of putrescine, is the first and rate-limiting enzyme in the biosynthesis of polyamines in mammalian cells. The enzyme is highly regulated, as indicated by rapid changes in its mRNA and protein during cell growth. Here we report that ornithine decarboxylase is regulated at the translational level by polyamines in difluoromethylornithine-resistant mouse myeloma cells that overproduce the enzyme due to amplification of an ornithine decarboxylase gene. When such cells are exposed to putrescine or other polyamines, there is a rapid and specific decrease in the rate of synthesis of ornithine decarboxylase, assayed by pulse-labeling. Neither the cellular content of ornithine decarboxylase mRNA nor the half-life of ornithine decarboxylase protein is affected. Our results indicate that polyamines negatively regulate the translation of ornithine decarboxylase mRNA, thereby controlling their own synthesis.     

Transgenic mice aberrantly expressing human ornithine decarboxylase gene.

We have generated transgenic mice carrying human ornithine decarboxylase gene. Two different transgene constructs were used: (i) a 5'-truncated human ornithine decarboxylase gene and (ii) an intact human ornithine decarboxylase gene. Transgenic mice carrying the 5'-truncated gene did not express human ornithine decarboxylase-specific mRNA. Transgenic mice carrying the intact human ornithine decarboxylase gene expressed human-specific ornithine decarboxylase mRNA in all tissues studied. However, as indicated by actual enzyme assays, the expression pattern was highly unusual. In comparison with their wild-type littermates, the transgenic mice exhibited greatly elevated enzyme activity in almost every tissue studied. Ornithine decarboxylase activity was moderately elevated in parenchymal organs such as liver, kidney, and spleen. Tissues like heart, muscle, lung, thymus, testis, and brain displayed an enzyme activity that was 20 to 80 times higher than that in the respective tissues of nontransgenic animals. The offspring of the first transgenic male founder animal did not show any overt abnormalities, yet their reproductive performance was reduced. The second transgenic founder animal, showing similar aberrant expression of ornithine decarboxylase in all tissues studied, including an extremely high activity in testis, was found to be infertile. Histological examination of the tissues of the latter animal revealed marked changes in testicular morphology. The germinal epithelium was hypoplastic, and the spermatogenesis was virtually totally shut off. Similar examination of male members of the first transgenic mouse line revealed comparable, yet less severe, histological changes in testis.     

Molecular mechanisms of the synergistic induction of ornithine decarboxylase by asparagine and glucagon in primary cultured hepatocytes

In primary cultures of adult rat hepatocytes maintained in a salts/glucose medium, a more than 100-fold increase in ornithine decarboxylase (EC 4.1.1.17) activity was caused by asparagine and glucagon in a synergistic manner. The synthesis rate of ornithine decarboxylase was determined by [35S]methionine incorporation into the enzyme protein, and the amount of ornithine decarboxylase-mRNA was measured by hybridization with a cloned rat liver ornithine decarboxylase-cDNA. The synthesis rate of ornithine decarboxylase was stimulated more than 20-fold by asparagine and glucagon together, but the amount of ornithine decarboxylase-mRNA was increased only 3-4-fold, indicating that translational stimulation was involved in the induction process. Asparagine alone stimulated the synthesis of ornithine decarboxylase without substantial effect on the amount of ornithine decarboxylase-mRNA, whereas glucagon alone increased the amount of ornithine decarboxylase-mRNA about 3-fold without a detectable change in either enzyme activity or enzyme synthesis. Asparagine, at least in part, also suppressed degradation of ornithine decarboxylase.     

Developmental changes in mouse brain polyamine metabolism

Mouse brain ornithine decarboxylase (ODC) activity is high at the time of birth, whereas S-adenosyl-l-methionine decarboxylase (SAM-DC) activity is low. ODC activity, and putrescine, spermidine and spermine concentrations decline rapidly during postnatal development to the low level characteristic of mature brains, while SAM-DC activity behaves in the opposite manner. The fluctuations in mouse brain polyamine metabolism are in accord with those found in the rat. The apparentK _m values of ODC and SAM-DC for their substrates decline parallel with the decrease of substrate and product concentrations during ontogeny suggesting substrate and/or product dependent regulation of polyamine synthesis in the developing brain.     

Changes in ornithine transcarbamylase activity and protein level during perinatal period in rat liver. Effects of actinomycin D

Ornithine transcarbamylase activity and immunoreactive enzyme level are compared during perinatal period and in adult rat. Ornithine transcarbamylase activity regularly rises during late fetal period and presents a marked increase 24 hours after birth. Immunoreactive enzyme level does not correlate with this developmental pattern. Ornithine transcarbamylase level increases from 0.06 mg on day 19.5 of pregnancy to 0.417 mg/g liver on day 21.5 and remains constant after birth (0.418 mg/g liver). These results suggest that inactive mitochondrial ornithine transcarbamylase accumulates before birth and that the postnatal increase in enzyme activity is mainly associated with an activation. Furthermore, the paradoxical effect of actinomycin D on ornithine transcarbamylase activity is associated with an increase in enzyme level (about 25%).     

Changes in ornithine decarboxylase and antizyme activities in developing mouse brain.

A macromolecular inhibitor to ornithine decarboxylase (ODC) present in mouse brain was identified as ODC antizyme [Fong, Heller & Canellakis (1976) Biochim. Biophys. Acta 428, 456-465; Heller, Fong & Canellakis (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1858-1862] on the basis of kinetic properties, Mr and reversal of its inhibition by antizyme inhibitor. The brain antizyme, however, did not cross-react immunochemically with any of seven monoclonal antibodies to rat liver antizyme. ODC activity in mouse brain rapidly decreased after birth, in parallel with putrescine content, and almost disappeared by 3 weeks of age. Free antizyme activity appeared shortly after birth and increased gradually, whereas ODC-antizyme complex already existed at birth and then gradually decreased. Thus total amount of antizyme remained about the same throughout the developmental period in mouse brain. In addition to ODC-antizyme complex, inactive ODC protein was detected by radioimmunoassay in about the same level as the complex at 3 weeks of age. Upon cycloheximide treatment, both free ODC activity and ODC-antizyme complex rapidly disappeared, although free antizyme and the inactive ODC protein were both quite stable.     

Investigation of structure and rate of synthesis of ornithine decarboxylase protein in mouse kidney

An immunoblotting technique was used to study the forms of ornithine decarboxylase present in androgen-induced mouse kidney. Two forms were detected which differed slightly in isoelectric point but not in subunit molecular weight (approximately 55 000). Both forms were enzymatically active and could be labeled by reaction with radioactive alpha-(difluoromethyl)-ornithine, an enzyme-activated irreversible inhibitor. On storage of crude kidney homogenates or partially purified preparations of ornithine decarboxylase, the enzyme protein was degraded to a smaller size (Mr approximately 53 000) without substantial loss of enzyme activity. The synthesis and degradation of ornithine decarboxylase protein were studied by labeling the protein by intraperitoneal injection of [35S]methionine and immunoprecipitation using both monoclonal and polyclonal antibodies. The fraction of total protein synthesis represented by renal ornithine decarboxylase was increased at least 25-fold by testosterone treatment of female mice and was found to be about 1.1% in the fully induced androgen-treated female. Both forms of the enzyme were rapidly labeled in vivo, and the immunoprecipitable ornithine decarboxylase protein was almost completely lost after 4-h exposure to cycloheximide, confirming directly the very rapid turnover of this enzyme. Treatment with 1,3-diaminopropane which is known to cause a great reduction in ornithine decarboxylase activity did not greatly selectively inhibit the synthesis of the enzyme. However, 1,3-diaminopropane did produce an increase in the rate of degradation of ornithine decarboxylase and a general reduction in protein synthesis. These two factors, therefore, appear to be responsible for the loss of ornithine decarboxylase activity and protein in response to 1,3-diaminopropane.     

Polyamines and their biosynthetic decarboxylases in various tissues of the young rat during recovery from undernutrition.

1. Weanling male and female rats were undernourished for 4 weeks and then rehabilitated by allowing ad libitum feeding. 2. During rehabilitation polyamine-biosynthetic enzymes were examined in the liver, spleen and quadriceps and gastrocnemius muscles. 3. During the first few hours of rehabilitiation there was a marked increase in liver weight, accompanied by a very marked increase in ornithine decarboxylase activity. Increases in the activity of this enzyme in other tissues did not occur until between 2 and 7 days of rehabilitation, at which time there were further increases in enzyme activity in the liver. 4. S-Adenosylmethionine decarboxylase activity also showed marked fluctuations in activity in all the tissues examined. 5. Hepatic putrescine and spermidine concentrations also varied during rehabilitation, but permine concentration remained relatively constant. Both spermine and spermidine were at normal concentrations in the liver from the 10th days of rehabilitation onwards. 6. In all of the tissues examined there were marked sex differences in the parameters studied, particularly in splenic and muscular ornithine decarboxylase activity. 7. In the tissues of the male rats, changes in polyamine synthesis paralled changes in nucleic acid and protein synthesis.     

POLYAMINE METABOLISM IN THE BRAIN AND LIVER OF THE DEVELOPING MONKEY

Abstract— The concentrations of putrescine and the polyamines, spermidine and spermine, along with the specific activities of the enzymes involved in their biosynthesis, ornithine decarboxylase, S -adenosylmethionine decarboxylase and spermidine synthase have been measured in brain and liver of the developing Rhesus monkey from mid-gestation, through birth and neonatal life to maturity. The results suggest that it is an increased concentration of putrescine and an increased specific activity of ornithine decarboxylase which are associated with the rapid growth of fetal brain during the middle of gestation. By the end of two-thirds of gestation, both of these parameters have attained values similar to those found in mature brain. The concentration of spermidine in brain and the specific activities of S -adenosylmethionine decarboxylase and spermidine synthase are lower in fetal brain than adult brain and increase slowly after birth to reach values similar to those of the adult only after several months. These results provide additional evidence that in the mature brain spermidine serves some function other than one associated with rapid growth.
Fetal liver at mid-gestation was characterized by increased concentrations of both putrescine and spermidine and increased specific activities of the enzymes which synthesize them. By two-thirds of gestation, values similar to those found in adult liver had been attained. Liver has thus reached maturity with regard to polyamine metabolism by this time.     

Abnormal ornithine decarboxylase activity in transgenic mice increases tumor formation and infertility

A transgenic mouse line carrying ornithine decarboxylase cDNA as the transgene under the control of a mouse mammary tumor virus long terminal repeat (MMTV LTR) promoter was generated in order to study whether ornithine decarboxylase transgene expression will have any physiological or pathological effect during the entire life of a transgenic mouse. The high frequency of infertile animals and the loss of pups made the breeding of homozygous mice unsuccessful. However, a colony of heterozygous transgenic mice was followed for 2 years. In adult heterozygous transgenic mice, ornithine decarboxylase activity was significantly increased in the testis, seminal vesicle and preputial gland when compared to non-transgenic controls. In contrast, ornithine decarboxylase activity was decreased in the kidney and prostate of transgenic mice. No significant changes in ornithine decarboxylase activity were found in the ovary and mammary gland and only moderate changes in ornithine decarboxylase activity were detected in the heart, brain, pancreas and lung. The most common abnormalities found in adult animals (12 males and 20 females) of the transgenic line were inflammatory processes, including pancreatitis, hepatitis, sialoadenitis and pyelonephritis. Spontaneous tumors were observed in eight animals, including two benign tumors (one dermatofibroma, one liver hemangioma) and six malignant tumors (one lymphoma, one intestinal and three mammary adenocarcinomas and one adenocarcinoma in the lung). No significant pathological changes were found in 17 nontransgenic controls.     

Sequential changes in ornithine decarboxylase thymidine kinase, and other enzyme activities in regenerating liver in rats on controlled feeding schedules.

The changes in activity of five enzymes including ornithine decarboxylase (ODC), tyrosine aminotransferase (TAT), thymidine kinase (TK), ornithine aminotransferase (OAT) and serine dehydratase (SDH) in the early stage of the regenerating rat liver have been studied under a controlled feeding and lighting schedule. The first three enzyme activities were stimulated sequentially by partial hepatectomy. The earliest response was observed in ODC activity. A significant increase in this enzyme activity was observed at 2 hrs and the maximal level was at 4 hrs after the operation. TAT began to increase at 4 hrs and the maximal level was at 8 hrs. The TK activity was induced at about 24 hrs and the highest value was at 48 hrs after partial hepatectomy.A significant decrease in OAT activity was observed at 24 hrs after the operation and subsequently. Although a decrease in SDH activity was also observed this decrease did not seem to correlate directly with the regeneration process, since a lowered level of the enzyme activity was also found in the sham operated group.     

Effect of experimental diabetes on ornithine decarboxylase activity of rat tissues

Measurements have been made of the activity of ornithine decarboxylase of liver, heart, kidney and brain in alloxan-diabetic and control rats. In all these tissues this enzyme had decreased markedly at four weeks after induction of diabetes. These results are discussed in relation to the hormonal control and cyclic nucleotide regulation of ornithine decarboxylase.     

Expression of high activity of ornithine decarboxylase and occurrence of unusual chromophobic cells in anterior pituitary gland of a novel growth-retarded strain of mice, grm/grm

Extremely high activity of ornithine decarboxylase (ODC) was detected in the pituitary gland of growth- retarded mice, grm/grm at 2 months after birth. The elevated enzyme activity gradually decreased to the control level in 14 months after birth. In the pituitary gland of the growth-retarded mice, unusual chromophobic cells were also present from the early stages after birth. The chromophobic cells showed conspicuous proliferations and resulted in a distinct hyperplasia of the tissue after 4 months after birth. These findings suggest that OCD is correlated to the progressive transformation of pituitary cells into the chromophobic cells.