Autophagic processes increase during senescence in cultured sheep neurons and astrocytes.

A possible response to aging is autophagy, a self-digestion process in which portions of cytoplasm are encapsulated by double-membrane-bound structures and delivered to lysosome for degradation. A previous work of our group showed that astrocytes under starving conditions are characterized by a higher upregulation of the marker of autophagy LC3 II than neurons. Aim of the present work was to evaluate LC3 II expression in an aging model consisting in fetal sheep neurons and astrocytes at 10, 20 and 30 days of culture. Such model has been validated by a remarkable activity of ß-galactosidase, commonly used to reveal cell aging. LC3 II immunoreactivity in neurons and astrocytes progressively increased with time but differences were observed on the basis of cell density. Indeed, LC3 II immunoreactivity is higher in clusters of neurons and astrocytes and this may be due to the fact that cell-cell contact would represent a second stress in addition to aging itself. Both cell types displayed a reduction in LC3 II signal in nuclei, and a corresponding strengthening in the cytoplasm with time. This may be due to the need of aged cells to remove damaged cytoplasmic components through autophagic processes. Such variation in LC3 II localization could be caused by migration from the nucleus to cytoplasm as well as possible de novo intracytoplasmic production. The present work based on sheep neural cells in vitro may represent a helpful tool in the studies on aging processes in which autophagy plays a remarkable role.

Sheep primary astrocytes under starvation conditions express higher amount of LC3 II autophagy marker than neurons.

Autophagy is a general term for the degradation of cytoplasmic components within lysosomes. Recent studies have clearly demonstrated that autophagy has a greater variety of physiological and pathophysiological roles than expected, such as starvation adaptation, intracellular protein and organelle clearance, development, anti-aging, elimination of microorganisms, cell death, tumor suppression and antigen presentation. MAP-LC3 is one of the most common markers  to evaluate autophagic processes. In our study, the autophagic activity in neurons and astrocytes from sheep brain under starving conditions was evaluated. In order to detect LC3 immunoreactivity, confocal analysis by double immunofluorescence was performed together with the cell type markers: GFAP to identify astrocytes, ß-III tubulin to identify neurons. The results show that astrocytes are characterized by LC3-positive areas, which increase in a time-dependent manner. In contrast, LC3 immunoreactivity was very weak in neurons. Therefore, it can be assumed that astrocytes show a higher capability than neurons to cope with stress and exhibit a stronger autophagic response.

Brain aging and testosterone-induced neuroprotection: studies on cultured sheep cortical neurons.

OBJECTIVE: This research reports the expression of topoisomerase ßII in fetal sheep neuronal cells. The ß isoform of DNA topoisomerase II plays a role in DNA repair process in non proliferating cells as neurons and its expression tends to be downregulated with senescence. METHODS: Cortical neurons from 60-day-old sheep embryos underwent two protocols: the former based on rising time of culture (10, 20 and 30 days); the latter based on the 72hrs exposure to 3-nitro-L-tyrosine (oxidative/nitrosative stressor) and/or testosterone. RESULTS: Our results showed an increase in ß-galactosidase activity and, in contrast, a reduction in topoisomerase ßII expression with time (first protocol). The exposure of sheep primary neurons to 3-nitro-L-tyrosine led to an upregulation of ßII topoisomerase expression to be likely seen as a reaction to nitrosative stress. Testosterone addition to 3-nitro-L-tyrosine-exposed cells results in topoisomerase ßII decrease possibly due to the neuroprotective properties of testosterone (second protocol). No significant variations in the marker of aging ß-galactosidase were observed in the cells exposed to 3-nitro-L-tyrosine and testosterone. CONCLUSION: The protocol based on time could be of some interest as a model of neuronal senescence in vitro. Topoisomerase ßII decrease with aging likely indicates a reduced ability to repair DNA during neuronal senescence. In contrast, the second protocol may not be seen as a reliable model of aging since 3-nitro-L-tyrosine does not lead to a topoisomerase ßII decrease. Testosterone was able to cope with oxidative/nitrosative damage, allowing cells to reduce their needs in DNA repair which in turn leads to a downregulation of topoisomerase IIß expression.

Aromatase expression in cultured fetal sheep astrocytes after nitrosative/oxidative damage.

Aromatase, the enzyme converting androgens into estrogens, is involved in many brain processes such as neural differentiation and plasticity or the prevention of cell death. We have previously observed an increase in aromatase immunoreactivity in sheep neurons exposed in vitro to the oxidant 3-nitro-L: -tyrosine. However, little is known regarding the way that sheep astrocytes cope with nitrosative stress, a condition occurring in sheep in the pathogenesis of neurodegenerative disorders such as scrapie and Maedi-Visna. Our aim has been to evaluate the effects of 3-nitro-L-tyrosine on astrocyte primary cultures from 90-day-old fetal sheep brain. Living cells were observed and characterized by immunofluorescence with a GFAP antibody, which indicated that the majority of the cells were astrocytes. A viability assay was performed on both untreated and treated cells. Reverse transcription with the polymerase chain reaction was undertaken to monitor time- and dose-dependent variations in aromatase gene expression. Stressed astrocytes showed signs of deterioration, were reduced in number, and appeared round with few short processes; the cell death rate was ∼30%. Aromatase expression was detected starting from a 24-h exposure to 1 mM 3-nitro-L-tyrosine and reached the highest levels at 72 h. Thus, oxidative damage probably results in the local production of neuroprotective estradiol by reactive astrocytes via the aromatization of testosterone.

Expression and distribution of P450-aromatase in the ovine hypothalamus at different stages of fetal development.

OBJECTIVES: An important step of sexual differentiation is the conversion of testosterone to estrogen by aromatase leading to masculinization and defeminization of the fetal brain areas crucial for normal sexual behavior and reproduction. Brain sexual differentiation occurs throughout a critical period starting from different prenatal stages depending on the species. Such period goes on from gestation day (GD) 30 to 100GD in the sheep. The fetal sheep brain is reported to aromatize androgens to estrogens at 64GD. The main goal of this work was to evaluate aromatase expression in sheep hypothalami during the whole period of sexual differentiation (35GD, 55GD, 80GD, 115GD) and whether differences may be observed depending on gestational stage and sex. METHODS: Sections at the hypothalamic level underwent immunoperoxidase technique employing anti-aromatase and anti-androgen receptor antibodies. Samples from 35GD and 55GD were also processed with in situ hybridization using aromatase cDNA probe. Blot analyses were performed to quantify possible aromatase immunoexpression differences between sexes. For sexing, samples at 35GD and 55GD underwent DNA extraction and SRY amplification. RESULTS: Our results revealed aromatase and androgen receptor immunoreactivity along the whole period of sexual differentiation. Both molecules were detected in many brain regions and markedly in the periventricular area. The highest aromatase and androgen receptor amounts were observed at 35GD and 55GD, when aromatase was more abundant in females than in males. CONCLUSIONS: In conclusion, the sheep can be included among the species where aromatase is highly expressed in the hypothalamus during the whole period of sexual differentiation.

Different nitrosative-induced microtubular modifications and testosterone neuroprotective effects on high-D-glucose-exposed neuroblastoma and glioma cells.

OBJECTIVES: Diabetic complications can often affect the central nervous system since the chronic exposure to hyperglycemia can result in the production of high concentration of reactive oxygen species with subsequent damage of several cell structures such as the cytoskeleton. In order to antagonize the oxidative status many substances have been tested as antioxidants. In the present work attention has been focused on the possible nitrosative effect of hyperglycemia on microtubular network of neuroblastoma and glioma mortalized cell lines, testing the possible neuroprotective effect of testosterone. METHODS: Neuroblastoma (C1300) and glioma (C6) cell lines were cultured in the presence of 300 mM (C1300) or 310 mM (C6) D-glucose, with or without 50 nM testosterone. After 72 hrs, morphology, growth rate, cell viability and catalase activity were evaluated. In addition, with the aim to detect any changes in the amount of tubulin isoforms, Western blot analysis was performed. RESULTS: In D-glucose-exposed cells, it was found a down-regulation of tubulin isoforms and an increase in 3-nitro-L-tyrosine and subsequent modifications in cell morphology, growth rate, viability and catalase activity. All these changes were more severe in neuroblastoma than in glioma cell line. When testosterone was added to the medium, all the parameters were very similar to controls. This neuroprotective action was well-detectable in C1300 cells, whereas testosterone was not able to recover significantly in C6 cells. CONCLUSION: Our results displayed: i) a selective action of high glucose on microtubules; ii) a different sensitivity to oxidative stress in neuronal and glial cells; iii) a different neuroprotective action of testosterone on neuronal and glial cells.

Androgen receptor immunoreactivity in rat occipital cortex after callosotomy.

Gonadal steroidogenesis can be influenced by direct neural links between the central nervous system and the gonads. It is known that androgen receptor (AR) is expressed in many areas of the rat brain involved in neuroendocrine control of reproduction,such as the cerebral cortex.It has been recently shown that the occipital cortex exerts an inhibitory effect on testicular stereoidogenesis by a pituitary-independent neural mechanism. Moreover, the complete transection of the corpus callosum leads to an increase in testosterone (T) secretion of hemigonadectomized rats. The present study was undertaken to analyze the possible corticocortical influences regulating male reproductive activities. Adult male Wistar rats were divided into 4 groups: 1) intact animals as control; 2) rats undergoing sham callosotomy; 3) posterior callosotomy; 4) gonadectomy and posterior callosotomy. Western blot analysis showed no remarkable variations in cortical AR expression in any of the groups except in group I where a significant decrease in AR levels was found. Similarly, both immunocytochemical study and cell count estimation showed a lower AR immunoreactivity in occipital cortex of callosotomized rats than in other groups. In addition, there was no difference in serum T and LH concentration between sham-callosotomized and callosotomized rats. In conclusion, our results showthat posterior callosotomy led to a reduction in AR in the right occipital cortex suggesting a putative inhibiting effect of the contralateral cortical area.

Effects of testosterone on differentiation and oxidative stress resistance in C1300 neuroblastoma cells.

OBJECTIVES: Using undifferentiated mouse neuroblastoma cells (C1300), we have previously observed that testosterone (T) exerts a neuroprotective action against oxidative stress. Nitrogen intermediates induce the production of 3-nitro-L-tyrosine (3NT), an amino acid analogue involved in many neurodegenerative disorders. The aim of our work is to investigate T capability on C1300 cell differentiation. It is also evaluated whether differentiation could mitigate the nitrosative effects of 3NT. METHODS: The effects of both T and 3NT were studied on an undifferentiated cell line of neural origin (C1300). For this purpose, cell cultures underwent morphometric investigation, blot analyses and catalase activity assay. All data obtained were expressed as mean+/-SD and tested by one-way ANOVA or Student's t test. RESULTS: The results were compared with those gathered by means of N6,2'-O-dibutyryl-adenosine-3',5'-cyclic-mono-phosphate (db-cAMP), a well-known differentiating agent. T-exposed cells showed an irregular shape and exhibited long branching cytoplasmic extensions, which were longer than in db-cAMP cells. Moreover, T-exposure induced an increase in the expression of tyrosinated and acetylated alpha-tubulin while 3NT-incorporation into tubulin was markedly reduced. The action of antioxidant defence systems, namely catalase activity, was enhanced in cells exposed to T. CONCLUSION: This work highlighted the effects of db-cAMP on differentiation and neuroprotection, but even indicated that T exposure induced differentiation in C1300 cells and this process matches a significant neuroprotective effect. This action seemed to be more effective than in db-cAMP-treated cells. T is suggested, like other substances having antioxidant properties, to be of potential interest in the experimental therapy of neuropathological conditions.

Right occipital cortex suppresses male rat testosterone secretion by a pituitary-independent mechanism.

OBJECTIVES: In addition to being regulated by the hypothalamo-hypophyseal system, testosterone (T) secretion is influenced by a number of less understood mechanisms. The aim of the present study was to examine whether defined areas of the right cerebral cortex could modulate T production. METHODS: In adult male Wistar rats right frontal or occipital decortication, anterior or posterior callosotomy and corresponding sham-operations were performed. After 7-day survival time, T secretion in vitro, serum T and LH concentrations were measured by RIA. RESULTS: Right occipital decortication and posterior callosotomy resulted in an increase in T secretion in vitro when compared to the corresponding sham-operated controls. In contrast, right frontal decortication or anterior callosotomy did not interfere with steroidogenesis. Serum LH concentration was not altered by any interventions. CONCLUSION: The right occipital but not the right frontal cortex is involved in the control of T secretion. The caudal part of the corpus callosum accommodating the fibers originating from the occipital cortex might have a similar function. The fact that LH remained unchanged in all experimental groups suggests that the right occipital cortex and the caudal part of the corpus callosum influence testicular steroidogenesis by a pituitary-independent mechanism.

Growth-associated protein expression in the frontal and occipital cortices of callosotomized rats.

Neurons confined to the central nervous system usually fail to regenerate their axons after injury, although evidence of axonal re-growth has been reported. In this study, rat transcallosal fibres were severed in the midline in order to investigate the reactive plasticity responses in the frontal and occipital cortices of both sides. The expression of growth-associated proteins, tyrosinated alpha-tubulin and GAP-43, was monitored at different time-points post-injury. Protein levels dropped during the first days post-axotomy, but subsequently returned to control levels. This initial decrease could be due to degeneration, and the subsequent increase connected to the reactive neosynaptogenesis and fibre sprouting from surrounding ipsilateral neurons, which is responsible for the reinnervation of the denervated area. Although transcallosal neurons are usually considered poorly regenerative, their axotomy may therefore induce reactive events.

Phenotypic features of the domestic pigs bred in the Roman settlements of Pompeii and Caralis.

A reconstruction of the morphological features of domestic pigs from two Roman settlements is here suggested by means of the study of skeletal and dental remains, with the aim at evaluating their degree of selection in comparison with wild boars. Material was formed by 111 bone and tooth fragments and was uncovered during the excavations of Polybius' House in Pompeii and of Roman buildings in the neighbourhood of Caralis harbour (Sardinia). The remains underwent morphological examination. The eruption of permanent teeth and ossification of epiphyseal cartilages let us establish that most animals were over 18-20 months. When possible, the determination of sex was made by detecting tusk features. The presence of anthropic signs on the bone surface provides some information about slaughtering and cooking procedure in the Roman period and supports the hypothesis that the animal remnants were food remains. Osteometric analysis was carried out on long and short bones and teeth through suitable multiplicative parameters, leading to the assessment of the withers height and other main phenotypic features. Logarithmic deviation pointed out the significant osteometric differences between the domestic pigs from the two Roman settlements. These data were also compared with those from wild boars and modern crossbred wild boars X non-selected pigs. In conclusion, our data show that pigs from Caralis bear much resemblance to wild boars, whereas those from Pompeii appear to be improved, so sharing some phenotypic features of modem improved breeds.

Morphological and functional changes induced by the amino acid analogue 3-nitrotyrosine in mouse neuroblastoma and rat glioma cell lines.

The amino acid analogue 3-nitrotyrosine (3-NT) is formed in neural cells as a result of the intense stimulation of NMDA glutamate receptors. 3-NT is involved in the pathology of diverse neurodegenerative disorders. The aim of our work is to investigate the sensitivity of cultured neural and glial cells to 3-NT. We report the morphological changes detected on mouse neuroblastoma (C1300) and rat glioma (C6) cell lines cultured in a medium supplemented with different 3-NT concentrations. Western blot displayed a selective incorporation of 3-NT into a single protein that co-migrated with tubulin. Both cell lines showed morphological changes, nuclear suffering, decreased viability and growth inhibition (starting from 90 and 360 microM for C1300 and C6, respectively). Such effects were dose-dependent, though glioma cells showed severe alterations at higher 3-NT concentrations. Our results point out a higher 3-NT sensitivity in the neural cells studied in comparison with those of glial origin. The dramatic toxicity of 3-NT in neural cells suggests further investigations focused on the biochemical mechanisms at the roots of neurodegenerative diseases.