S. Reale

Stress Proteins and SH-Groups in Oxidant-Induced Cellular Injury After Chronic Ethanol Administration in Rat

It is generally agreed that lipid peroxides play an important role in the pathogenesis of ethanol-induced cellular injury and that free sulfhydryl groups are vital in cellular defense against endogenous or exogenous oxidants. It has been observed that oxidative stress induces the synthesis of the 70-kDa family of heat-shock proteins (HSPs). Induction of HSPs represents an essential and highly conserved cellular response to a variety of stressful stimuli. In the present study we measured in various brain areas and in liver the intracellular levels of HSP70 proteins, sulfhydryl groups and the antioxidant enzyme status after chronic administration of mild intoxicating doses of ethanol to rats. Expression of HSP70 in response to alcohol administration was particularly high in the hippocampus and striatum. In these brain areas, the increase in HSP70 protein levels occurred in absence of significant changes of antioxidant enzyme activities and was correlated with a marked depletion of intracellular bound thiols and with a decreased susceptibility to lipid peroxidation. Lower levels of HSP70 induction were found in cortex and cerebellum and were associated to decreases in SOD and CAT enzyme activities, with a lower depletion of protein bound thiols and with an increased susceptibility to lipid peroxidation. This study agrees with our previous results performed on acute alcohol intoxication and supports the hypothesis that HSP70 induction protects the different brain areas against oxidative stress.

Changes of mitochondrial cytochrome c oxidase and FoF1 ATP synthase subunits in rat cerebral cortex during aging.

The contents of subunits I, II/III, and IV of cytochrome c oxidase and of subunits α, β and γ of FoF1 ATP synthase in inner mitochondrial membrane proteins purified from cerebral cortex of rat at 2, 6, 12, 18, 24, and 26 months of age were analyzed by western blot. Age-related changes in the content of subunits, either of mitochondrial or nuclear origin, were observed. All the cytochrome c oxidase (COX) subunits examined showed an age-related increase from 2-month-old rats up to 24 months with a decrease at the oldest age (26 months). The same pattern of age-dependent changes was observed for γ ATP synthase, while the α and β subunits increased progressively up to 26 months.

Effect of growth factors on nuclear and mitochondrial ADP-ribosylation processes during astroglial cell development and aging in culture

Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I) and insulin (INS) are powerful mitogens and may regulate gene expression in cultured astrocytes by ADP-ribosylation process. Nuclear poly-ADP ribose polymerase (PARP) and mitochondrial monoADP-ribosyltransferase (ADPRT) are the key enzymes involved in poly-ADP-ribosylation and mono ADP-ribosylation, respectively. In this investigation the effect of EGF, bFGF, IGF-I or INS on nuclear PARP and mitochondrial ADPRT activities were assessed in nuclei and mitochondria purified from developing (30 DIV) or aging (90 and 190 DIV) primary rat astrocyte cultures. A marked increase of PARP activity in bFGF or IGF-I treated astroglial cell cultures at 30 DIV was found. Nuclear PARP and mitochondrial ADPRT activities were greatly stimulated by treatment with EGF or INS alone or together in astrocyte cultures at 30 DIV. Nuclear PARP and mitochondrial ADPRT activities showed a more remarkable increase in control untreated astrocyte cultures at 190 DIV than at 90 DIV. These findings suggest that ADP-ribosylation process is involved in DNA damage and repair during cell differentiation and aging in culture. Twelve hours treatment with EGF, INS or bFGF significantly stimulated nuclear PARP and mitochondrial ADPRT activities in 190 DIV aging astrocyte cultures. The above results indicate that EGF, INS and bFGF may play a crucial role in the post-translational modification of chromosomal proteins including ADP-ribosylation process in in vitro models. This suggests that growth factors regulate genomic stability in glial cells during development and maturation, stimulating nuclear and mitochondrial ADP-ribosylation processes in developing or aging astrocyte cultures.

Immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons induce a functional switch in the growth factor responsiveness of astroglia: involvement of basic fibroblast growth factor.

Recent evidence indicates that astroglial‐derived growth factors (GFs) participate in the development of luteinizing hormone‐releasing hormone (LHRH) neurons, but it is still unknown whether LHRH neurons may exert a reciprocal modulation of glial cell function. Using immortalized hypothalamic LHRH (GT1‐1) neurons in co‐culture with glial cells, we have recently shown that basic fibroblast growth factor (bFGF) plays a prominent role in the glial‐induced acquisition of the mature LHRH phenotype by GT1‐1 cells. We have resorted to this model and combined biochemical and morphological approaches to study whether the response of glial cells to a number of GFs (including bFGF, insulin‐like growth factor I, IGF‐I, epidermal growth factor, EGF and insulin) expressed during LHRH neuron differentiation, is modulated by co‐culture with pure LHRH neurons. Pre‐treatment of hypothalamic astrocytes with an inactive (‘priming’) dose of bFGF for 12 h powerfully increased astroglia proliferative response to IGF‐I (10 ng/ml), EGF (10 g/ml) and insulin (10 μg/ml), inducing a 65–100% increase in the [3H]thymidine incorporation compared to untreated cultures. When astroglial cells and developing GT1‐1 neurons were co‐cultured for 5 days in vitro (DIV), the [3H]thymidine incorporation was significantly higher than in astroglial cells cultured without neurons. Application of the different GFs to the co‐culture for either 12 or 24 h further stimulated DNA synthesis to various extent according to the GF applied and the time of application. Localization of the proliferating cells by dual immunohistochemical staining, followed by cell counting and bromodeoxiuridine (BrdU) labeling index calculation, revealed that the incorporation of BrdU was restricted to the nuclei of LHRH‐immunopositive neurons. Such changes were accompanied by extensive morphological alterations of astroglial and LHRH fiber networks, whereas neutralization of bFGF activity in GT1‐1 neuron–glial co‐cultures by a bFGF‐antibody, dramatically counteracted the observed effects. The functional switch of astroglia proliferative response to GFs coupled to the potent morphological and functional modifications of developing glia and pure LHRH neurons observed in vitro, support a bidirectional interaction between immortalized LHRH neurons and astroglial cells and identify bFGF as a key player in this crosstalk.

Effect of CDP-choline treatment on mitochondrial and synaptosomal protein composition in different brain regions during aging

Several age‐dependent modifications of inner mitochondrial membrane and synaptosomal plasma membrane proteins from different brain regions of 4‐, 12‐, 18‐ and 24‐month‐old male Wistar rats, were observed. Some proteins, identified by immunoblotting assay as various subunits of mitochondrial respiratory chain complexes and calmodulin, were particularly impaired. Chronic treatment with CDP‐choline at a dose of 20 mg/kg body weight per day for 28 days caused significant changes in the amounts of several of the above mentioned proteins. Most of the proteins, which decreased during aging, showed a significant increase after CDP‐choline treatment compared with the corresponding control values at the same age. The effect of CDP‐choline might be due to: — the increased availability of cytidylic nucleotides, which in the brain are present in limited amounts compared to the other nucleotides; — the increased content of total adenine nucleotides; —the improvement of brain energy metabolism.

Age-Dependent Changes of Nucleic Acid Labeling in Different Rat Brain Regions

The effects of aging on in vivo DNA and RNA labeling and on RNA content in various brain regions of 4-, 12-, and 24-month-old rats were investigated. No difference in [methyl-14C]thymidine incorporation into DNA of cerebral cortex and cerebelllum during aging was observed.The ratio of RNA/DNA content significantly decreased from 4 to 24 months of age in cerebral cortex, cerebellum and striatum. RNA labeling decreased by 15% in cerebral cortex of 24-month-old animals while in the other brain areas examined (cerebellum, hippocampus, hypothalamus, brainstem, striatum) did not change during aging.In the cerebral cortex, the ratio of the specific radioactivity of microsomal RNA to that of nuclear RNA, determined by in vivo experiments, was not affected by the aging process. A significant decrease of total, poly(A)+ RNA and poly(A)- RNA content was observed in the same brain area of 24-month-old rats compared to 4-month-old ones. Moreover, densitometric and radioactivity patterns obtained by gel electrophoresis of labeled RNA after in vitro experiments (tissue slices of cerebral cortex) showed a different ribosomal RNA processing during aging. In vivo chronic treatment with CDP-choline was able to increase RNA labeling in corpus striatum of 24-month-old animals.

Insulin-like Growth Factor-I Effects on ADP-Ribosylation Processes and Interactions with Glucocorticoids During Maturation and Differentiation of Astroglial Cells in Primary Culture

Nervous system function depends upon the extensive and intimate coupling between neuronal cells and glial cells [1, 2]. We have recently shown [3, 4] that during differentiation in vitro, astroglial cells in primary culture release polypeptide growth factors that exert dramatic effects on the differentiation of an immortalized hypothalamic LH-RH neuronal (Gt1-1 subclone) cell line [3–6]. The growth factors (GFs) have emerged as crucial intercellular signaling agents that coordinate the developmental and adult physiological processes of both astrocytes and neurons [6–8]. Insulin-like growth factors I and II (IGF-I and IGF-II) are peptide growth factors structurally related to insulin. IGF-I, IGF-II and fibroblast growth factors (FGFs) are synthesized by developing astroglial cells and exert autocrine and paracrine mitogenic actions [6–9]. Primary astroglial cells possess IGF receptors and synthesize IGFs and IGF binding proteins [9,10]. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), IGF-I and insulin are potent mitogens capable of inducing cell division in various cell types and in particular in cultured cells from the central nervous system (CNS) [6–8, 11–13]. The effects of bFGF on the morphology of cultured astrocytes prepared from various areas of newborn rat brain, and on their expression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) have also been described [14]. Furthermore, EGF acts as a neurotrophic agent preferential for dopaminergic neurons in rat embryonic mesencephalic cultures [12], and enhances the proliferation of cultured astrocytes from rat brain [7,15].

ADP-ribosylation of proteins in brain regions of rats during postnatal development

Post‐translational modifications of chromatin‐bound proteins play an important role in the regulation of eukaryotic gene expression. Processes such as acetylation, methylation, phosphorylation and ADP‐ribosylation may alter the interaction of these proteins with DNA and consequently affect chromatin conformation and the binding of enzymes and other molecules involved in the regulation of gene expression.

Age-related chances of mitochondrial cytochrome C oxidase and F0F1-ATP synthase subunit contents in rat cerebral cortex

The levels of subunits I, II/III, and IV of cytochrome c oxidase and of subunits alpha, beta and gamma of F(0)F(1)-ATP synthase in inner mitochondrial membrane proteins purified from cerebral cortex of rat at 2, 6, 12, 18, 24, 26 months of age were analyzed by Western blot. Age-related changes in the content of subunits, encoded either in mitochondrial or nuclear DNA, were observed.

Effect of EGF on the Labeling of Various RNA Species and of Proteins from Subcellular Fractions in Primary Rat Astroglial Cell Cultures

The ability of epidermal growth factor (EGF) to stimulate cell growth, proliferation and differentiation has been shown for a variety of cell types. Rat cerebral astrocytes bind high amounts of EGF and are responsive to the mitogenic stimulation of EGF when cultured in serum-free medium (1).