Testosterone metabolism in brain cells and membranes

The central nervous system (CNS) is considered a target structure for the action of all the classes of hormonal steroids produced by the organism. Well-characterized genomic and less well-understood membrane mechanisms of action are probably involved in the steroid modulation of brain activities. Moreover, some classes of steroids need to be converted into “active” metabolites before interacting with their effector systems. In particular, testosterone (T) exerts many of its effects after conversion to 5-dihydrotestosterone (DHT) and estrogens. The CNS possesses both the 5-reductase, the enzyme which produces DHT and the aromatase which transforms T into estrogens; however, the relative role and distribution of these enzymes in the various structural components of the CNS has not been clarified so far. The 5-reductase has been found to be present in high concentrations in brain white matter structures because these are particularly rich in myelin membranes, to which the enzymatic activity appears to be associated. This membrane localization might suggest a possible involvement of steroidal 5-reduced metabolites in membrane-mediated events in the CNS. Moreover, the distribution of 5-reductase was studied in neurons, astrocytes and oligodendrocytes isolated from the brain of male rats by density gradient ultracentrifugation, as well as in neurons and glial cells grown in culture. The aromatase activity was also evaluated in neurons and glial cells grown in culture and in isolated oligodendrocytes. Among the three cell types isolated, neurons appear to be more active than oligodendrocytes and astrocytes, respectively, in converting T into DHT. Also, in cell culture experiments, neurons are more active in forming DHT than glial cells. Only neurons possess aromatase activity, while glial cells are apparently unable to aromatize T.     

Aromatase expression and cell proliferation following injury of the adult zebra finch hippocampus

Estrogens can be neuroprotective following traumatic brain injury. Immediately after trauma to the zebra finch hippocampus, the estrogen-synthetic enzyme aromatase is rapidly upregulated in astrocytes and radial glia around the lesion site. Brain injury also induces high levels of cell proliferation. Estrogens promote neuronal differentiation, migration, and survival naturally in the avian brain. We suspect that glia are a source of estrogens promoting cell proliferation after neural injury. To explore this hypothesis, we examined the spatial and temporal relationship between glial aromatase expression and cell proliferation after neural injury in adult female zebra finches. Birds were ovariectomized and given a blank implant or one filled with estradiol; some birds were also administered an aromatase inhibitor or vehicle. All birds received penetrating injuries to the right hippocampus. Twenty-four hours after lesioning, birds were injected once with BrdU to label mitotically active cells and euthanized 2 h, 24 h, or 7 days later. The brains were processed for double-label BrdU and aromatase immunocytochemistry. Injury-induced glial aromatase expression was unaffected by survival time and aromatase inhibition. BrdU labeling was significantly reduced at 24 h by ovariectomy and by aromatase inhibition; effects were partially reversed by E2 replacement. Irrespective of ovariectomy, the densities of aromatase immunoreactive astrocytes and BrdU-labeled cells at known distances from the lesion site were highly correlated. These data suggest that injury-induced glial aromatization may influence the reorganization of injured tissue by providing a rich estrogenic environment available to influence cellular incorporation.     

A cyp19a1b‐gfp (aromatase B) transgenic zebrafish line that expresses GFP in radial glial cells

Aromatase is an enzyme that catalyzes the synthesis of estrogen in gonads and brain. Teleost fish express aromatase (AroB) strongly in the brain facilitating its detailed examination. To understand the function of AroB in the brain, we generated transgenic zebrafish that expresses green fluorescent protein (GFP) driven by the brain aromatase cyp19a1b promoter. GFP was found in the radial glial cells of transgenic larvae and adult fish that overlap with AroB immunoreactivity in the correct temporal and spatial pattern. GFP was also coexpressed with radial cell marker BLBP, but was not in neurons. In addition, GFP expression in the radial glial cells was stimulated by estrogen, same as endogenous AroB expression. Thus, this transgenic line faithfully mimics the regulation of AroB expression in radial glial cells. It provides a powerful tool to further characterize progenitor radial cells in adult and developing fish and to evaluate estrogenic activities of xenoestrogens and phytoestrogens. genesis 47:67–73, 2009. © 2008 Wiley‐Liss, Inc.     

Modifications in energy metabolism during the development of chick glial cells and neurons in culture

Developmental changes in lactate dehydrogenase (LDH), enolase, hexokinase (HK), malate dehydrogenase (MDH), and glutamate dehydrogenase (GDH) activities were measured in cultures of pure neurons and glial cells prepared from brains of chick embryos (8 day-old for neurons, 14 day-old for glial cells) as a function of cellular development with time in culture. The modifications observed in culture were compared to those measured in brain extracts during the development of the nervous tissue in the chick embryo and during the post-hatching period. A significant increase of MDH, GDH, LDH, and enolase activities are observed in neurons between 3 and 6 days of culture, whereas simultaneously a decrease of HK values occurs. In the embryonic brain between 11 and 14 days of incubation, which would correspond for the neuronal cultures to day 3 through 6, modifications of MDH, GDH, HK, and enolase levels are similar to those observed in neurons in culture. Only the increase of LDH activity is less pronounced in vivo than in cultivated cells. The evolution of the tested enzymatic activities in the brain of the chick during the period between 7 days before and 10 days after hatching is quite similar to that observed in cultivated glial cells (prepared from 14 day-old embryos) between 6 and 18 days of culture. All tested activities increased in comparable proportions. The modifications of the enzymatic profile indicate that some maturation phenomena affecting energy metabolism of neuronal and glial elements in culture, are quite similar to those occuring in the total nervous tissue. A relationship between the development of the energy metabolism of the brain and differentiation processes affecting neuroblasts and the glial-forming cells is discussed.     

Aspects of biochemical differentiation in the central nervous system.

A demonstration of cell-specific patterns of development in the immature CNS is provided by examples of characteristic, cell-specific time-courses of enzyme development in different classes of brain cells isolated in highly purified form by bulk-separation from the cerebral and cerebellar cortex of the growing rat. The enzymatic analysis was carried out at the level of the nerve and glial cell lysosomes and mitochondria, two subcellular organelles crucial to the economy of all cells. The findings reveal rather similar developmental patterns for the lysosomal hydrolase N-acetyl-beta-D-glucosaminidase in neurons and glial cells of the cerebral cortex as well as in two different cerebellar nerve cell types, the Purkinje and the granule cell. However, significant differences in the post-natal chronology of development of the mitochondrial enzyme alpha-glycerophosphate dehydrogenase were noted between cortical nerve and glial cells, the glial enzyme exhibiting 6-fold higher levels of activity than the neuronal one throughout the first month of postnatal life. The findings emphasize the feasibility as well as the necessity of studies aimed at the elucidation of the cell-specific aspects of the biochemistry of developing nerve and glial cells.     

Glycosaminoglycan Storage in Cultured Neonatal Murine Mucopolysaccharidosis Type VII Neuroglial Cells and Correction by β-Glucuronidase Gene Transfer

Abstract: The inherited deficiency of β-glucuronidase activity causes the lysosomal storage disorder mucopolysaccharidosis (MPS) type VII (Sly disease). The sequential catabolism of glycosaminoglycans in lysosomes is blocked, and undegraded substrates accumulate in cells of many tissues, including neurons and glia in the brain. To evaluate the deficient metabolic pathway, primary cultures of mixed brain cells were established from newborn MPS VII mice. β-Glucuronidase levels and glycosaminoglycan accumulation were studied in normal, carrier, and MPS VII cells. Retroviral vector-mediated transfer of a normal β-glucuronidase cDNA corrected the enzymatic deficiency in MPS VII cells and restored glycosaminoglycan catabolism to normal. High levels of β-glucuronidase expression were sustained in vector-corrected nondividing glial cell cultures for >2 months. These studies provide an in vitro model for evaluating somatic gene transfer in neural cells affected in mucopolysaccharidoses.     

Glial cells phagocytose neuronal debris during the metamorphosis of the central nervous system in Drosophila melanogaster

 Using electron microscopy we demonstrate that degenerating neurons and cellular debris resulting from neuronal reorganization are phagocytosed by glial cells in the brain and nerve cord of the fruitfly Drosophila melanogaster during the first few hours following pupariation. At this stage several classes of glial cells appear to be engaged in intense phagocytosis. In the cell body rind, neuronal cell bodies are engulfed and phagocytosed by the same glial cells that enwrap healthy neurons in this region. In the neuropil, cellular debris in tracts and synaptic centres resulting from metamorphic re-differentiation of larval neurons is phagocytosed by neuropil-associated glial cells. Phagocytic glial cells are hypertrophied, produce large amounts of lysosome-like bodies and contain a large number of mitochondria, condensed chromatin bodies, membranes and other remains from neuronal degeneration in phagosomes. Received: 23 January 1996 / Accepted in revised form: 21 May 1996     

Glial cells mediate target layer selection of retinal axons in the developing visual system of Drosophila

In the fly visual system, each class of photoreceptor neurons (R cells) projects to a different synaptic layer in the brain. R1-R6 axons terminate in the lamina, while R7 and R8 axons pass through the lamina and stop in the medulla. As R cell axons enter the lamina, they encounter both glial cells and neurons. The cellular requirement for R1-R6 targeting was determined using loss-of-function mutations affecting different cell types in the lamina. nonstop (encoding a ubiquitin-specific protease) is required for glial cell development and hedgehog for neuronal development. Removal of glial cells but not neurons disrupts R1-R6 targeting. We propose that glial cells provide the initial stop signal promoting growth cone termination in the lamina. These findings uncover a novel function for neuron-glial interactions in regulating target specificity.     

Fine structure of the neural sheath,glia and neurons in the brain of the housefly,Musca domestica

Summary The fine structure of the neural sheath, glial cells and nerve cells in the brain of adult male houseflies is described. The neural sheath is composed of neural lamella and perineurium. The neural lamella consists of an external lamina and collagen-like fibrils which are embedded in an amorphous matrix. The perineurial cells form a continuous layer around the brain. On their inner surface, perineurial cells form junctional complexes with glial cell processes. A cortical cellular layer composed of neurons and glial cells surrounds the centrally located neuropil. Three types of glial cells are identified. Glial cells differ in size and in relative development and distribution of organelles. Thin processes of glioplasm completely surround the cell bodies of the neurons. Five types of neurons are described. Most of the neurons are monopolar, a few are bipolar.Supported by a grant from the National Science Foundation     

Phospholipid metabolism in neuronal and glial cells during aging

The incorporation of cytidine-containing precursors (CDP-Cho and CDP-Etn) into the main phospholipid classes of cellular fractions enriched in neurons and glial cells from whole rat brains of different ages was examined. The rate of synthesis of choline phosphoglycerides in neuronal homogenates significantly decreased with age up to 18 months; after this time no additional decrease was found. The decrease of CDP-Etn incorporation in neurons was found to be less significantly affected by age up to 18 months, but the enzymic activity decreased after 18 months of age. No changes were found in the corresponding glial activity at any age. Biochemical phenomena that occur in 18-month-old rat brain (aged animals) were compared with phenomena occurring in 2-month-old rat brain (adult animals). No significant variations of lipid composition were found in neurons from either 18-month-old or 2-month-old rat brain. These results, together with some kinetic parameters, suggest that ethanolamine and choline phosphotransferases are affected differently by aging.     

Quantitation of DNA repair in brain cell cultures: implications for autoradiographic analysis of mixed cell populations.

Quantitation of DNA repair in the mixed cell population of mouse embryo brain cultures has been assessed by autoradiographic analysis of unscheduled DNA synthesis following UV-irradiation. The proportion of labelled neurons and the grain density over neuronal nuclei are both less than the corresponding values for glial cells. The nuclear geometries of these two classes of cell are very different. Partial correction for the different geometries by relating grain density to nuclear area brings estimates of neuronal and glial DNA repair synthesis more closely in line. These findings have general implications for autoradiographic measurement of DNA repair in mixed cell populations and in differentiated versus dividing cells.     

Molecular and cellular physiology of aromatase in the brain and retina

Due to exceptionally high brain aromatase activity, teleost fish are advantageous for studying neural aromatase regulation, localization, and physiology. To determine the molecular mechanism of enhanced expression, we have isolated, cloned and sequenced a 3 kb full-length aromatase cDNA from a goldfish (Carassius auratus) brain library using a human placental aromatase cDNA as probe. The deduced sequence of goldfish aromatase is 510 amino acids (predicted M_W, 58 kDa) with 69% overall sequence similarity, when compared to human placental aromatase, and higher hormologies in presumptive functional domains. A major 3 kb mRNA species was abundant in brain and low or non-detectable in non-neural tissues, reflecting the order of enzyme activities. To determine the cellular basis of high enzyme activity in goldfish brain, a human placental aromatase antibody was used to immunolocalize labeled cells. This antibody immunoprecipitated a single 56 kDa in vitro translation product of goldfish brain poly(A⁺)RNA and revealed discrete clusters of intensely stained neurons, processes, and terminals concentrated in, but not limited to, reproductive brain centers. Close proximity of aromatase- and androgen receptor-positive neurons in certain regions provides anatomic evidence of a functional relationship between direct and indirect pathways of neural androgen action. Aromatase-positive neurons and fibers formed interconnected networks in novel loci (e.g. retina→optic tract→optic tectum), and catalytic activity was confirmed biochemically in these tissues, indicating that neuroestrogen may have a role in visual input and integration. Availability of goldfish-specific nucleotide and antibody probes will facilitate further studies using this model.     

Biosynthesis of Galactocerebrosides and Glucocerebrosides in Glial Cell Lines

UDP-galactose:ceramide galactosyltransferase (CGalT, EC 2.4.1.45) and UDP-glucose:ceramide glucosyltransferase (CGlcT, EC 2.4.1.80) were determined in the glial cell lines G26-20, G26-24, C6, and C6TK-. The enzymatic assay for CGalT in cultured glial cells was complicated by a rapid conversion of UDP-galactose to UDP-glucose, due to the elevated UDP-galactose-4'-epimerase activity in certain glial cell clones. It seems that mechanisms regulating UDP-galactose-4'-epimerase activity and levels of UDP sugars in the glial cell lines differ from those in brain tissue. Compared with the maximum activity of CGalT in the myelinating rat brain, the enzyme activities in the oligodendroglioma clonal cell lines G26-20 and G26-24 were 16-30 times lower. On the other hand, CGalT levels in G26-20 and G26-24 cells were comparable to the values found in young rat brain before myelination starts. No CGalT activity could be detected in C6 or C6TK- cells by the method used in this study, whereas CGlcT activity was found in all glial cell lines tested and its levels were close to the values observed in the young rat brain.     

Differential Distribution of Purine Metabolizing Enzymes Between Glia and Neurons

Abstract: Previous studies showed that in cultured chick ciliary ganglion neurons and CNS glia, adenosine can be synthesized by hydrolysis of 5'-AMP and that the accumulation of the adenosine degradative products inosine and hypoxanthine was significantly greater in glial than in neuronal cultures. Furthermore, previous immunochemical and histochemical studies in brain showed that adenosine deaminase and nucleoside phosphorylase are localized in endothelial and glial cells but are absent in neurons; however, adenosine deaminase may be found in a few neurons in discrete brain regions. These results suggested that adenosine degradative pathways may be more active in glia. Thus, we have determined if there is a differential distribution of adenosine deaminase, nucleoside phosphorylase, and xanthlne oxidase enzyme fluxes in glia, comparing primary cultures of central and ciliary ganglion neurons and glial cells from chick embryos. Hypoxanthine-guanine phosphoribosyltransferase and production of adenosine by S-adenosylhomocysteine hydrolase activity were also examined. Our results show that there is a distinct profile of purine metabolizing enzymes for glia and neurons in culture. Both cell types have an S-adenosylhomocysteine hydrolase, but it was more active in neurons than in glia. In contrast, in glia the enzymatic activities of xanthine oxidase (443 ± 61 pmol/min/10⁷ cells), nucleoside phosphorylase (187 ± B pmol/min/10⁷ cells), and adenosine deaminase (233 ± 32 pmol/min/10⁷ cells) were more active at least 100, 20, and five times, respectively, than in ciliary ganglion neurons and 100, 100, and nine times, respectively, than in central neurons.     

Sexual dimorphism in the developmental regulation of brain aromatase

Steroid sex hormones have an organizational role in gender-specific brain development. Aromatase (cytochrome P450_AR), converting testosterone (T) to estradiol-17β (E₂) is a key enzyme in brain development and the regulation of aromatase determines the availability of E₂ effective for neural differentiation. Gender differences in brain development and behaviour are likely to be influenced by E₂ acting during sensitive periods. This differentiating action has been demonstrated in rodent and avian species, but also probably occurs in primates including humans. In rodents, E₂ is formed in various hypothalamic areas of the brain during fetal and postnatal development. The question considered here is whether hypothalamic aromatase activity is gender-specific during sensitive phases of behavioural and brain development, and when these sensitive phases occur. In vitro preoptic and limbic aromatase activity has been measured in two strains of wild mice, genetically selected for behavioural aggression based on attack latency, and in the BALB/c mouse. Short attack latency males show a different developmental pattern of aromatase activity in hypothalamus and amygdala to long attack latency males. Using primary brain cell cultures of the BALB/c mouse, sex differences in hypothalamic aromatase activity during both early embryonic and later perinatal development can be demonstrated, with higher E₂ formation in males. The sex dimorphisms are brain region specific, since no differences between male and female are detectable in cultured cortical cells. Immunoreactive staining with a polyclonal aromatase antibody identifies a neuronal rather than an astroglial localization of the enzyme. T increases fetal brain aromatase activity and numbers of aromatase-immunoreactive hypothalamic neuronal cell bodies. T appears to influence the growth of hypothalamic neurons containing aromatase. Differentiation of sexually dimorphic brain mechanisms may involve maturation of a gender-specific network of estrogen-forming neurons which are steroid-sensitive in early development.     

An In Vivo Model for Studying Function of Brain Tissue Temporarily Devoid of Glial Cell Metabolism: The Use of Fluorocitrate

The effect of intrastriatal injection of fluorocitrate on amino acid pattern, cell enzyme markers, and ultrastructural appearance was investigated. A dose of 1 nmol of fluorocitrate resulted in temporarily decreased levels of glutamine, glutamate, and aspartate, whereas the level of alanine was increased. The glutamine level was severely reduced after 4 h but was reversed after 24 h. The activity of different cellular enzyme markers did not change markedly after this dose. Ultrastructural changes in glial cells were observed, concomitant with the biochemical changes. A dose of greater than or equal to 2 nmol of fluorocitrate resulted in more marked and irreversible changes in amino acid levels. By 24-72 h after the injection of this dose, several marker enzyme activities decreased markedly. The ultrastructural changes affected the neurons as well as the glial cells and were not reversible. The use of microinjection of 1 nmol of fluorocitrate into the neostriatum of the rat to provide a model for studying transmitter amino acid metabolism in brain devoid of glial cell activity is discussed.     

Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule

By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50- fold. This fraction was injected into mice to produce monoclonal antibodies; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an Mr = 135,000 band in immunoblots of embryonic chick brain membranes. These results suggest that this molecule is present on the surfaces of neurons and that it directly or indirectly mediates adhesion between neurons and glial cells. Because the monoclonal antibody as well as the original polyspecific antibodies that were active in the assay did not bind to glial cells, we infer that neuron- glial interaction is heterophilic, i.e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells.     

胶质源性神经营养因子对不同组织细胞的影响

GDNF来自于小胶质神经元,首先作为中脑多巴胺能神经元的复活因子被发现,可促进细胞存活,并有增加多巴胺神经元细胞大小及轴突长度的作用。GDNF通过与锚定蛋白细胞表面受体糖基磷脂酰肌醇的相互作用来调节细胞活性。GDNF家族a-1受体,通过跨膜酪氨酸受体或者神经元细胞黏附分子,来促进细胞存活,神经突生长,以及突触发育。后续的研究提示,无论未成年还是成体大脑,GDNF对多种神经细胞都有复活的作用,并与一些周围神经复活、迁移、分化相关。不同的脑缺血实验模型均证实了外源性GDNF对于病灶部位及全脑的神经保护作用,包括局部应用营养因子,利用病毒载体运载GDNF基因以及移植表达GDNF的细胞。近来研究还证实,GDNF不仅对多巴胺能神经元,中枢和周围神经系统的运动、感觉神经元,以及自主神经元有营养和保护作用,对于非神经系统也有不同调节作用。本文将重点讨论这些GDNF作用的不同策略以及机制。