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Somatostatin (SRIF) exerts a modulatory function on neuronal transmission in the CNS. It has been proposed that a reduction of calcium currents is the major determinant of the inhibitory activity of this peptide on synaptic transmission. Because the neurotoxicity induced by activation of the NMDA subtype of glutamate receptor is mediated through excessive Ca2+ influx, we investigated whether SRIF counteracted NMDA-induced neuronal cell death. Neurons from embryonic rat cerebral cortex were cultured for 7-10 days and then exposed to 0.5 and 1 mM NMDA for 24 h. The neuronal viability, as assessed by the colorimetric method, decreased by 40 and 60%, respectively, compared with the control condition. Morphological and biochemical evidence indicated that cell death occurred by necrosis and not through an apoptotic mechanism. SRIF (0.5-10 microM), simultaneously applied with excitatory amino acid, significantly reduced in a dose-dependent manner the neurotoxic effect of NMDA but not that of KA (0.25-0.5 mM). GABA (10 microM) partially protected neurons to a similar extent from NMDA- or KA-induced toxicity. SRIF type 2 receptor agonists, octreotide (SMS 201-995; 10 microM) and vapreotide (RC 160; 10 microM), did not influence the NMDA-dependent neurotoxicity. The intracellular mechanism involved in SRIF neuroprotection was investigated. Pertussin toxin (300 ng/ml), a G protein blocker, antagonized the protective effect of SRIF on NMDA neurotoxicity. Furthermore, the neuroprotective effect of SRIF was mimicked by dibutyryl-cyclic GMP (10 microM), a cyclic GMP analogue, whereas 8-(4-chlorphenylthio)-cyclic AMP (10 microM), a cyclic AMP analogue, was ineffective. The cyclic GMP content was increased in a dose-dependent manner by SRIF (2.5-10 microM). Finally, both specific (Rp-8-bromoguanosine 3',5'-monophosphate, 10 microM) and nonspecific [1-(5 isoquinolinylsulfonyl)-2-methylpiperazine (H7), 10 microM] cyclic GMP-dependent protein kinase (cGMP-PK) inhibitors did not interfere with NMDA toxicity but substantially reduced SRIF neuroprotection. Our data suggest a selective neuroprotective role of SRIF versus NMDA-induced nonapoptotic neuronal death in cortical cells. This effect is likely mediated by cGMP-PK presumably by regulation of the intracellular Ca2+ level.

Heme oxygenase (HO), which catalyzes the degradation of heme, has two isozymes (HO-1 and HO-2). In brain the noninducible HO-2 isoform is predominant, whereas the inducible HO-1 is a marker of oxidative stress. Because brain oxidative stress might be present in prion-related encephalopathies (PREs), as in other neurodegenerative diseases, we investigated whether HO-1 mRNA was induced in neuronal and astroglial cell cultures by a peptide corresponding to residue 106-126 of human prion protein (PrP). This peptide is amyloidogenic, and when added in vitro to cultured cells it reproduces the neuronal death and astroglial proliferation and hypertrophy occurring in PREs. HO-1 mRNA did not accumulate in rat cultured neurons from hippocampus or cortex exposed to PrP 106-126 (50 microM for 5 days). PrP 106-126 induced HO-1 mRNA accumulation in rat astroglial cultures depending on the exposure time and concentration, being maximal (33-fold) after 7 days of exposure at 50 microM. The nonamyloidogenic amidated or amidated-acetylated PrP 106-126 was ineffective, as was a scrambled peptide used as control. N-Acetylcysteine reduced (50%) the accumulation of HO-1 mRNA in astroglial cells after PrP 106-126 (25 microM) given for 5 days. Thus, oxidative stress is apparently a feature of the toxicity of PrP 106-126, and it might also occur in PREs; induction of HO-1 could contribute to the greater resistance of astrocytes compared with neurons to PrP 106-126 toxicity.

Beta-amyloid accumulates in cerebral deposits in Alzheimer's disease, so to test the correlation between the neurotoxic and fibrillogenic capacity of beta-amyloid, we synthesized a peptide homologous to fragment 25-35 of beta-amyloid (beta25-35) and amidated at the C-terminus (beta25-35-NH2). As the amidation strongly reduced the amyloidogenic capacity of beta25-35, we compared its neurotoxic activity in the amidated (beta25-35-NH2) and nonamidated forms. The viability of primary cultures from fetal rat hippocampus was reduced in a dose-related manner (10-100 microM) similarly by beta25-35 and beta25-35-NH2, whereas a scrambled peptide, amidated or nonamidated, did not alter the neuronal viability. The neurotoxic activity of beta25-35-NH2 is mediated by apoptosis as demonstrated by morphological and biochemical investigations. Electron microscopy examination of culture media with beta25-35 or beta25-35-NH2 incubated with neuronal cells for 7 days confirmed the high level of fibrillogenic activity of beta25-35 and the almost total absence of fibrils in the solution with beta25-35-NH2. Furthermore, staining with thioflavine S was used to identify amyloid fibrils, and only the cultures exposed to beta25-35 exhibited intense staining associated with neuronal membranes. These data indicate that the neurotoxic activity of the beta-amyloid fragment is independent of the aggregated state of the peptide.

Cell viability and gene expression were studied in primary astroglial cells cultured in a nominally calcium-free medium. Ca2+ deprivation reduced progressively the astrocytes' viability, starting from 12 h; the restoration of a normal Ca2+ concentration (1.8 mM) in the medium after 12-h deprivation reversed the degenerative effect within 24 h. Biochemical and morphological examinations indicated that cell death induced by Ca2+ deprivation was mediated by apoptosis. This was associated with the expression of c-fos, c-jun, and c-myc, which, with different time courses, were induced in astrocytes after Ca2+ deprivation. Furthermore, shifting to a Ca2+-free medium modified the expression of Ich-1S transcript and rapidly increased intracellular cyclic AMP, which has been implicated in the transcriptional activation of immediate-early genes. The absence of Ca2+ in the medium reduced the expression of constitutive proteins such as alpha-actin, clusterin, glial fibrillary acidic protein, amyloid precursor protein, and glucose-6-phosphate dehydrogenase. The expression of these mRNAs was reduced >50% after 8 h of Ca2+ deprivation, when the effect on cell viability was negligible. When Ca2+ deprivation was prolonged for 24 h the expression of mRNA dropped completely, and restoration of the Ca2+ ions in the medium for 48 h did not reverse this effect. In contrast with general assumption, the apoptotic machinery in astrocytes is activated similarly not only by increased Ca2+ influx but also with the extracellular Ca2+ deprivation.

The effect of a peptide homologous to the biologically active fragment of beta amyloid 25-35 (beta 25-35) was studied on interleukin 6 (IL-6) and tumour necrosis factor (TNF-alpha) secretion induced by lipopolysaccharide (LPS) in primary rat astrocytes and microglia. Twenty-four hour exposure to LPS (50 ng/ml) induced IL-6 and TNF-alpha both in astrocytes and in microglial cells, while the effect of beta 25-35 (50 microM) per se was negligible in both cell types. In microglial cells, the application of beta peptide did not alter the production of either cytokine induced by LPS. However, beta 25-35 strongly amplified the production of both IL-6 and TNF-alpha in astrocytes. These findings confirm the complex interaction between cytokines and amyloidogenesis in Alzheimer's disease and indicate that astrocytes rather than microglia respond to the beta amyloid fragment, suggesting that these cells may be actively involved in cytokine-mediated events in AD.

Crinoid echinoderms can completely and rapidly regenerate arms lost following self-induced or traumatic amputation. Arm regeneration in these animals therefore provides a valuable experimental model for studying all aspects of regenerative processes, particularly with respect to the nervous system and its specific contribution to regenerative phenomena. Taking into account the primary role of the nervous system in regeneration in other invertebrates, we have investigated the specific involvement of neural factors, viz. the monoamine neurotransmitters dopamine and serotonin, in arm regeneration of Antedon mediterranea. In the present work, the presence of classical monoamines has been revealed by employing specific immunocytochemical and histofluorescence tests in association with biochemical detection by means of high pressure liquid chromatography. The distribution pattern of these neurohumoral molecules at standard regenerative stages has been compared with that of normal non-regenerating arms. Results indicate that both dopamine and serotonin dramatically change in both their distribution and concentration during the repair and regenerative processes. Their remarkably enhanced pattern during regeneration and widespread presence at the level of both nervous and non-nervous tissues indicates that they are important neural growth-promoting factors in crinoid arm regeneration.

Prion-related encephalopathies are characterized by the accumulation of an abnormal prion protein isoform (PrPSc) associated with neuronal degeneration and astrogliosis. The synthetic peptide homologous to PrP fragment 106-126 (PrP 106-126) induced in vitro neuronal apoptosis and glial proliferation. We used Northern blot analysis and the RNA polymerase chain reaction to assess the expression of several genes associated with programmed cell death and proliferation. Blots of total RNA extracted from neuronal and astroglial cells exposed to PrP 106-126 for between 1 h and 7 days were hybridized with probes recognizing c-fos, c-jun, c-myc, p53, hsp-70 and bcl-2 mRNA. Except for a slight decrease in bcl-2 mRNA in neuronal cells, no change in other transcripts was evident. Since clusterin (apolipoprotein J) mRNA levels are increased in prion-related encephalopathies and clusterin immunoreactivity has been located in association with PrPSc in Gerstmann-Sträussler-Scheinker brain, the expression of clusterin was determined in neuronal and astroglial cells chronically exposed to PrP 106-126. Although the induction of clusterin has been involved in the apoptotic mechanism in other experimental conditions, its expression was unchanged in PrP 106-126-treated neurons, while a three-fold induction of clusterin mRNA was observed in astrocytes exposed to PrP 106-126. To investigate whether the clusterin up-regulation was simply associated with the astroglial proliferative stimulus of PrP 106-126 or was specifically induced by the peptide, we measured clusterin expression in astrocytes cultured in fetal calf serum-free medium and exposed to PrP 106-126 or fetal calf serum restoration. In this condition the PrP peptide, like fetal calf serum, increased the glial proliferation rate, but only PrP 106-126 doubled clusterin mRNA. The selectivity of this effect indicates that PrPSc is directly involved in the clusterin up-regulation seen in prion-related encephalopathies and is associated with astroglial cells.

Arm regeneration in the crinoid echinoderm Antedon mediterranea can provide a very useful model for studying regenerative development. This process can be described as an epimorphic process in which new structures develop from migratory actively proliferating cells. Both the repair and the regenerative phases are due to this fundamental mechanism, which is the only way of recruiting cells to form the cicatricial layer and the blastema. In order to identify the source, the proliferation sites and the recruitment times of these cells, cell proliferation was monitored by employing an immunocytochemical system to detect the thymidine analogue bromodeoxyuridine (BrdU) incorporated by S-phase cells of the regenerating tissues. On the basis of BrdU incorporation the actively proliferating elements can be identified both close and far from the amputation 1) in the populations of migratory cells which are produced at the level of the brachial nerve and the coelomic wall and 2) in the coelomic epithelium itself. These structures are preferential sources of new cells and give a differential contribution during the repair and the regenerative phases in terms of potential of cell proliferation. © 1995 Wiley-Liss, Inc.

Crinoids have remarkable regenerative capacities. After traumatic or self-induced amputation they can replace one or several arms through a complex sequence of morphogenetic events which lead quickly to the complete reconstruction of the lost part. These phenomena are well known in their general aspects and a comprehensive description of arm regeneration was previously provided (Candia Carnevali et al., Recent Trends in Regeneration Research. Plenum Press, New York, '89). However, there is a considerable gap in knowledge of the early regenerative phases which are particularly significant for the following development. The aim of the present work is to describe and interpret the different aspects of these early stages of arm regeneration in Antedon mediterranea. According to our results the regenerative development of Antedon arm seems to consist of an epimorphic process which essentially implies the involvement of proliferating undifferentiated cells. The early stages of this process can be distinguished in a first repair phase, which includes the overall 24 hr post-amputation period and is characterized mainly by the processes of wound healing and tissue repair, and in a second regenerative phase, which includes the 24–72 hr post-amputation period and is characterized by the first regenerative events. The different arm components involved in the amputation/regeneration phenomena seem to play different and specific roles in these initial phases. © 1993 Wiley-Liss, Inc.

The Aristotle's lantern, or masticatory apparatus, of regular sea-urchins is a complex musculo-skeletal system which is thought to have contributed significantly to the evolutionary success of these animals. This paper gives an account of the antomical relationships and functional morphology of both skeletal and soft tissue components in the lantern and related structures of the sea-urchin Stylocidaris affinis (Cidaridae), and compares these features with their equivalent in the previously described lantern of the sea-urchin Paracentrotus lividus (Echinidae, Camarodonta). There are major differences in the skeletons of these lanterns which involve mostly the arrangement and morphology of elements participating in movement, i.e. joints and articular surfaces, and which highlight the generally heavier and less mobile nature of the lantern in the Cidaridae. There are remarkably few differences, however, in the microstructure of the skeletal stereom. Significant dissimilarities were found in the anatomical arrangement of muscles and ligamentous structures and in their macro- and microstructure. The implications of these morphological features for the functioning of the lantern of the Cidaridae are discussed in the context of an integrated model of lantern biomechanics.

A new method is presented for the quantification of cell viability based on densitometry with computerized image analysis. Neuronal cells were stained with crystal violet and densitometric analysis was performed with an IBAS 2.0 image analyzer (Kontron/ Zeiss), using specially implemented dedicated software which integrates the optical density of the culture in each well with the area covered by the stained cells. To test the reliability of the densitometric method cortical cells were plated at different concentrations (5 x 10(4)-10(6)/ml); the standard curve obtained by analysis of crystal violet staining showed a linear proportion between cell number and optical density signal. The validation and accuracy of the method were assessed and compared with other methods using rat cortical cells cultured in vitro for 10 days and exposed to kainic acid (250 microM) for 24 h. Neuronal viability was reduced by 40-50% and comparison with direct cell counting, MTT assay, and spectrophotometric analysis confirmed that the method is simple, quick, and reliable.

The mechanisms of cell death of rat cortical neurons chronically exposed to the beta-amyloid (betaA) biologically active fragment beta-(25-35) involve oxidative stress. We examined the influence of culture conditions on the neuroprotective activity of antioxidants against beta-(25-35) toxicity. Common radical scavengers such as N-acetylcysteine (250 microM) and N-t-butyl-phenylnitrone (500 microM) only protected cortical cells cultured in the presence of fetal calf serum (FCS) from betaA insult. The neuroprotective effect of lazaroids (U74389G and U83836E), 21-aminosteroids with antioxidant activity, was tested in cells grown with or without FCS. U74389G did not interfere with beta-(25-35) toxicity in either condition, while U83836E at a very low concentration (15 nM) protected cortical cells exposed to the beta peptide only when the neurons were cultured in the presence of FCS. These data show that a lazaroid can prevent beta-(25-35) toxicity and that the antioxidants exerted their protective effect in certain conditions.

The aim of this work was to investigate whether free radical reactions play a role in beta-amyloid neurotoxicity. Rat cortical neurons were exposed acutely (24 h) or chronically (3, 7 days) to beta-amyloid biologically active fragment beta 25-35 (50 microM). In these conditions, where only the longest exposure induced neuronal death, superoxide dismutase activity was increased after acute exposure but no change was detected after chronic treatments, whereas a different pattern was observed for glutathione peroxidase. In the basal condition, there was an eight-fold increase in dichlorofluoroscein, used as peroxide production marker, in neuronal cells after 7 days treatment with beta 25-35. Moreover, the intracellular peroxide production induced by Fe2+/ascorbate stimulation was amplified by beta 25-35, increasingly up to 7 days of exposure, by which time the dichlorofluoroscein-stimulated levels were 33 times higher than in controls. In conclusion, our results show that oxidative stress and free radical production are linked to beta 25-35 exposure and may contribute to neurodegenerative events associated with beta-amyloid deposits in Alzheimer's disease.

To investigate the role of IL-6 in the pathogenesis of Alzheimer's disease (AD) its effect on amyloid precursor protein (APP) mRNA expression was evaluated. The levels of APP mRNA were determined by Northern blot analysis in primary cultured rat cortical neurons and glial cells exposed to IL-6 (50-200 ng/ml). The cytokine increased neuronal APP mRNA expression about 100% at the highest dose after 6 h of exposure. APP mRNA expression was unaffected in astroglial cells exposed to IL-6. Since IL-1 beta also increased neuronal APP mRNA, the combination of IL-1 beta and IL-6 was tested. The effects were partially additive. The ability of beta-amyloid fragment 25-35 to induce IL-1 or IL-6 mRNA was also investigated in astroglial cells. IL-1 beta mRNA was strongly induced by beta 25-35 (25-100 microM) while the expression of IL-6 mRNA remaining unchanged. The results suggest roles for both IL-1 and IL-6 in the neuronal mechanisms related to beta-amyloid protein deposition in AD.