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Biomedical subjects

R Guarneri

Publications and source records attributed to R Guarneri.

At least 19 recordsLinked to original sources
PubMed2003

Neurosteroids in the retina: neurodegenerative and neuroprotective agents in retinal degeneration.

Steroids may have a powerful role in neuronal degeneration. Recent research has revealed that steroids may influence the onset and progression of some retinal disorders as well as neurodegenerative diseases and, as in brain, they accumulate in the retina via a local synthesis (neurosteroids) and metabolism of blood-circulating steroid hormones. Their crucial role as neurodegenerative and neuroprotective agents has been also upheld in a retinal excitotoxic paradigm. These findings are reviewed especially from the emerging perspective that after an insult local changes in steroidogenic responses and consequent neurosteroid availability might turn out to be offensive or defensive cellular adaptations for the potentiation or prevention of neuronal death.

Animals↗
PubMed2002

Mitochondrial oxidative metabolism in motor neuron degeneration (mnd) mouse central nervous system.

The mnd mouse spontaneously develops slowly evolving motoneuron pathology leading to progressive motor impairment. There is strong evidence that a complex interplay between oxidative stress, mitochondria abnormalities and alteration of glutamate neurotransmission plays an important role in the pathogenesis of motor neuron diseases. Therefore, we investigated the presence of mitochondrial dysfunction in frontal, central (comprising the motor area) and occipital regions of the cerebral cortex and in the spinal cord of 35-week-old mnd mice. Lipid peroxide derivatives reacting with thiobarbituric acid (TBARS) were measured in the cervical, thoracic and lumbar spinal cord. In addition biochemical and behavioural analyses were carried out in mnd mice chronically treated with l-carnitine from the 11th to the 34th week of life (mndT mice). Slight but significant alterations of mitochondrial enzyme activities were seen in the mnd cortical regions. The central area was the most affected and both complex I, IV and citrate synthase were decreased with respect to controls. The rate of oxygen consumption (QO2) was markedly decreased in both the upper (cervical + upper portion of the thoracic region) and lower (lumbar + lower portion of the thoracic region) mnd spinal cord. The level of TBARS showed a rostro-caudal trend to increase, being 30% higher in the lumbar tract of mnd mice in comparison with controls. L-carnitine treatment increased the mitochondrial enzyme activities in cortical regions towards control value and was effective in enhancing QO2 and decreasing TBARS levels in the spinal cord of mndT. Behavioural testing showed that L-carnitine significantly delayed the onset of motor behaviour impairment. This beneficial effect was declining at 35 week of age, when the biochemical measurements were performed.

Age Factors↗
PubMed2002

A caspase-3-dependent pathway is predominantly activated by the excitotoxin pregnenolone sulfate and requires early and late cytochrome c release and cell-specific caspase-2 activation in the retinal cell death.

This study investigates the implication of mitochondria- and caspase-dependent pathways in the death of retinal neurones exposed to the neurosteroid pregnenolone sulfate (PS) shown to evoke apoptosis and contribute to amplification and propagation of excitotoxicity. After a brief PS challenge of intact retinas, caspase-3 and caspase-2 activation and cytochrome c release occur early and independent of changes in the oxidative state measured by superoxide dismutase activity. The temporal and spatial relationship of these events suggests that a caspase-3-dependent pathway is activated in response to cytochrome c release and requires caspase-2 activation and a late cytochrome c release in specific cellular subsets of retinal layers. The protection by caspase inhibitors indicates a predominant role of the pathway in PS-induced retinal apoptosis, although a limited use of caspase inhibitors is upheld on a conceivable shift from apoptosis toward necrosis. Conversely, 3alpha-hydroxy-5beta-pregnan-20-one sulfate and 17beta-oestradiol provide complete prevention of PS-induced retinal death.

Animals↗
PubMed2001

Identification of an antigen related to the sea urchin RNA-binding protein LP54 in mammalian central nervous system.

LP54 is an RNA-binding protein involved in localization of maternal messengers in sea urchin egg and embryos. Using a polyclonal antibody directed against Paracentrotus lividus LP54 we detected a 66-kDa cross-reacting antigen in undifferentiated and differentiated SH-SY5Y human neuroblastoma cells. After treatment of undifferentiated cells with detergent, the 66-kDa antigen was found to be enriched in the cytoskeletal fraction. By Western blot the expression of this antigen was also analyzed in regions of the CNS and in tissues of the adult rat and its exclusive presence in the hippocampus and thalamus was revealed. The immunoreactivity with P. lividus antibody against LP54 in hippocampal lysate was also confirmed throughout anti-LP54 immunoaffinity column and competition experiments. The results indicates that a related protein to the sea urchin LP54 is evolutionary conserved in mammalian CNS.

Animals↗
PubMed2000

Human neuroblastoma SH-SY5Y cell line: neurosteroid-producing cell line relying on cytoskeletal organization.

Pregnenolone, the precursor of all steroids, is synthesized by CNS structures. The synthesis requires an obligatory step involving cholesterol transport to mitochondrial cytochrome P450-cholesterol side chain cleavage (cytP450scc), although the underlying mechanism(s) are still mostly unknown. We used the human neuroblastoma SH-SY5Y cell line to investigate cytP450scc expression and activity and to establish a role of cytoskeleton in pregnenolone synthesis. Immunocytochemical and biochemical approaches revealed that undifferentiated as well as differentiated cells either by retinoic acid (RA) or phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), possess cytP450scc and rapidly synthesize pregnenolone in the presence of a NADPH-generating system. The newly neurosteroid formation by SH-SY5Y cells was increased by 22R-hydroxycholesterol and blocked by the cytP450scc inhibitor, aminoglutethimide. When trilostane was used to inhibit 3beta-hydroxysteroid dehydrogenase catalyzing pregnenolone conversion into progesterone, a higher pregnenolone accumulation occurred in TPA-differentiated cells than in RA-differentiated ones. Although SU 10603, a blocker of 17alpha-hydroxylase/c17,20-lyase enzyme involved in DHEA formation from pregnenolone, gave rise to an elevated neurosteroid content only in RA-differentiated cells. No difference in pregnenolone levels was found in undifferentiated cells treated with each inhibitor. Thus, differentiation seems to promote pregnenolone-metabolizing enzyme activities that may vary upon phenotypic changes induced by RA or TPA. Treatments of differentiated cells with the microtubule-depolymerizing drug colchicine and the actin microfilament-altering agent cytochalasin D decreased pregnenolone synthesis without affecting cell viability or cytP450scc amount. Addition of the cell-permeant cholesterol analogue 22R-hydroxycholesterol known to elude cholesterol transport systems induced pregnenolone synthesis, however, indicating that perturbations in cytoskeleton likely affect endogenous cholesterol transport. The relevance of this finding may rest on the observed involvement of cytoskeletal organization in such events as neuronal plasticity, cognitive function and also neurodegenerative disorders in which neurosteroids have been shown to have a part.

Central Nervous System↗
PubMed2000

Pregnenolone sulfate, a naturally occurring excitotoxin involved in delayed retinal cell death.

The present study was designed to investigate the neurosteroid pregnenolone sulfate (PS), known for its ability to modulate NMDA receptors and interfere with acute excitotoxicity, in delayed retinal cell death. Three hours after exposure of the isolated and intact retina to a 30-min PS pulse, DNA fragmentation as assessed by genomic DNA gel electrophoresis and a modified in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method appeared concurrently with an increase in superoxide dismutase (SOD) activity and thiobarbituric acid-reactive substances (TBARS) levels. At 7 h, the increased amount of DNA laddering was accompanied by a higher number of TUNEL-positive cells in the inner nuclear and ganglion cell layers. Necrotic signs were characterized by DNA smear migration, lactate dehydrogenase (LDH) release, and damage mainly in the inner nuclear layer. PS-induced delayed cell death was markedly reduced by the NMDA receptor antagonists 4-(3-phosphonopropyl)-2-piperazinecarboxylic acid and 3alpha-hydroxy-5beta-pregnan-20-one sulfate but completely blocked after concomitant addition of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Steroids with antioxidant properties (progesterone, dehydroepiandrosterone and its sulfate ester, and 17beta-estradiol) differently prevented PS-induced delayed cell death. Cycloheximide treatment protected against DNA fragmentation and LDH release but failed to prevent the rise in SOD activity and TBARS level. We conclude that a brief PS pulse causes delayed cell death in a slowly evolving apoptotic fashion characterized by a cycloheximide-sensitive death program downstream of reactive oxygen species generation and lipid peroxidation, turning into secondary necrosis in a retinal cell subset.

Adjuvants, Immunologic↗
PubMed1998

Pregnenolone sulfate modulates NMDA receptors, inducing and potentiating acute excitotoxicity in isolated retina.

Pregnenolone sulfate (PS) acts as a positive allosteric modulator of N-methyl-D-aspartate (NMDA) receptor-mediated responses. In the retina, we previously observed that the synthesis of pregnenolone and PS increases after stimulation of NMDA receptors and blockade of the synthesis reduces retinal cell death. This study was carried out to explore in the isolated and intact retina the possible role of PS in NMDA-induced excitotoxicity. Lactate dehydrogenase (LDH) measurements and morphological analysis revealed that a 90-min exogenous application of PS at 0.1-500 microM concentrations potentiated NMDA-induced cell death and at 50-500 microM concentrations caused cytotoxicity. After 45 min, either NMDA or PS caused no significant LDH release; but their co-application resulted in a high degree of toxicity. In addition, we found that a mild NMDA insult developed into serious damage when even low PS concentrations (0.1-10 microM) were used. Toxicity-inducing and -potentiating effects were specific to PS modulatory action on NMDA receptors, in that they were blocked by 4-(3-phosphonopropyl)2-piperazinecarboxylic acid (CPP) and MK-801 but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and neither dehydroepiandrosterone sulfate nor pregnenolone caused LDH release. Prevention of degenerative signs was seen in retinae pretreated with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a Cl- channel blocker, thus indicating a Na+/Cl--dependent acute mode of excitotoxic cell death responsible for PS toxicity. The positive interaction between the neurosteroid and NMDA receptors was further proved by a PS dose-dependent increase in NMDA-induced stimulation of [3H] MK-801 binding to retinal membranes. The results suggest a crucial role of PS in retinal vulnerability and propose the toxicity-potentiating effects as an important key in linking NMDA-induced endogenous synthesis to acute excitotoxicity.

Animals↗
PubMed1998

Induction of neurosteroid synthesis by NMDA receptors in isolated rat retina: a potential early event in excitotoxicity.

Here we investigated the possible regulation of neurosteroidogenesis by N-methyl-D-aspartic acid (NMDA) receptor activation and addressed the hypothesis that neurosteroid synthesis may be involved in acute excitotoxicity. In the isolated retina, exposure to NMDA modified pregnenolone and pregnenolone sulphate formation. This effect was dose and time dependent, the synthesis being increased by relatively moderate NMDA doses (1-100 microM) within 30 min exposure and reduced to its control value by 60 min or by raising drug concentrations. NMDA-stimulated neurosteroid synthesis was blocked by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine hydrogen maleate (MK-801) and 3(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP), depended on extracellular calcium and reproduced by glutamate. Lactate dehydrogenase (LDH) release and morphological analysis revealed that retinal cell viability was not significantly affected after 30 min exposure to 50 microM NMDA, but severe cell damage occurred by 60 min. When the GABAA (gamma-aminobutyric acid) receptor agonist muscimol (1-1000 microM), known to activate retinal neurosteroidogenesis, was added together with NMDA, no additional increase in neurosteroid synthesis was observed, and NMDA-induced LDH release remained unchanged. However, exposure to a high concentration of muscimol alone (500 microM) provoked a similar degree of toxicity to NMDA. By contrast, bicuculline abolished the increase in neurosteroidogenesis and LDH release. Similarly, pretreatment with R (+)-p-aminoglutethimide (AMG), an inhibitor of cholesterol side-chain cleavage cytochrome P450, attenuated acute retinal cell damage. The inhibitory nature of AMG on NMDA-stimulated neurosteroidogenesis was confirmed in the observation that drug treatment reduced pregnenolone content and did not affect the bindings of [3H] MK-801 and [3H] muscimol. The results demonstrate that NMDA receptors regulate neurosteroidogenesis through a transneuronal mechanism, which implies GABAA receptor activation. The early NMDA-mediated stimulation of neurosteroid synthesis seems to play a critical role in acute excitotoxicity; consequently, its inhibition is likely to delay neuronal cell death.

Animals↗
PubMed1995

gamma-Aminobutyric acid type A/benzodiazepine receptors regulate rat retina neurosteroidogenesis.

It has been previously shown that retinal ganglion cells have the ability to synthesize steroids including neuroactive steroids such as pregnenolone sulfate. Since ganglion cells possess GABAA/benzodiazepine (BZ) receptors and neurosteroids modulate retinal GABAA receptor function, we investigated the role of these receptors in isolated rat retina neurosteroidogenesis. Ligands for central-type BZ receptors stimulated retinal pregnenolone synthesis. Clonazepam was the most potent ligand examined acting at nanomolar concentrations. Moreover, the effective steroidogenesis stimulatory dose (ED50) for these ligands and the Ki to inhibit [3H]flunitrazepam binding showed a coefficient of correlation of r = 0.87, suggesting the involvement of the central-type BZ receptors in this event. Ro 5-4864, which preferentially binds to peripheral-type BZ receptors, was less efficacious and potent whereas PK 11195 did not affect the basal pregnenolone formation and did not antagonize the Ro 5-4864 stimulated steroid synthesis. The GABAergic agonist muscimol, stimulated neurosteroid synthesis and this effect was reversed by the GABAergic antagonists bicuculline and picrotoxinin. In addition, these antagonists decreased basal pregnenolone formation, suggesting a tonic GABAergic control of the steroidogenic pathway, and reduced clonazepam-stimulated steroidogenesis. These results, together with the reported ability of neurosteroids to modulate GABAA receptor function, suggest a novel regulatory mechanism to control the inhibitory transmission.

Animals↗
PubMed1994

Neurosteroidogenesis in rat retinas.

Neurosteroids (steroids synthesized in the CNS) function by modulating neurotransmission. To establish an experimental model for investigation of neurosteroid synthesis and regulation, independent of blood-borne steroids, we examined the steroidogenic activity of isolated rat retinas. We identified progesterone, pregnenolone, dehydroepiandrosterone, desoxycorticosterone, 3 alpha,5 alpha-tetrahydrodesoxycorticosterone, 3 alpha-hydroxy-5 alpha-dihydroprogesterone, 17-hydroxyprogesterone, and 17-hydroxypregnenolone together with their esterified forms. As pregnenolone is the precursor of all steroids, its formation was studied in detail as an index of a steroid-synthesizing tissue. Pregnenolone was identified further by gas chromatography coupled to mass spectrometry, and its in vitro synthesis was inhibited by lovastatin, an inhibitor of mevalonolactone and cholesterol biosynthesis. We then examined pregnenolone synthesis in the presence of mevalonolactone as a precursor of sterol formation together with lovastatin, which reduces endogenous mevalonolactone synthesis, as well as with inhibitors of pregnenolone metabolism. The incorporation of mevalonolactone into pregnenolone and its sulfate ester was time- and concentration-dependent and blocked by aminoglutethimide, a competitive inhibitor of cytochrome P450 side-chain cleavage (P450scc) enzyme. Immunocytochemical studies with a specific antibody to P450scc revealed a primary localization of the enzyme at the retinal ganglion cell layer. A less pronounced immunostaining was also seen at cells of the inner nuclear layer. Compounds known to stimulate cyclic AMP content also stimulated pregnenolone formation by rat retinas. These results demonstrate that rat retinas synthesize steroids and, for the first time, they reveal the steroidogenic ability of neuronal cells. We propose rat retinas as an in vitro model system to study neurosteroidogenesis in the CNS.

Aminoglutethimide↗
PubMed1993

Effect of beta-N-oxalylamino-L-alanine on cerebellar cGMP level in vivo.

Beta-N-oxalylamino-L-alanine (BOAA), a non-protein amino acid present in the seeds of Lathyrus Sativus (LS), is one of several neuroactive glutamate analogs reported to stimulate excitatory receptors and, in high concentrations, cause neuronal degeneration. In the present study, the in vivo acute effects of synthetic BOAA and LS seed extract were investigated on rat cerebellar cyclic GMP following intraperitoneal (10-100 mg/kg) or oral (100 mg/kg) administration of subconvulsive doses of toxin. Furthermore, the BOAA content in LS seeds and in the cerebellum of injected rats was determined by high performance liquid chromatograph analysis. A dose- and time-dependent increase of cerebellar cyclic guanosine monophosphate (cGMP) level was observed after intraperitoneal administration of synthetic BOAA or LS extract. The neurotoxin evoked a maximum stimulation 90 min after injection within the dose range of 50-75 mg/kg, elevating cGMP from basal levels of 5.3 +/- 0.5 pmol/mg protein to 15 +/- 1.3 pmol/mg protein. Similarly, the oral intake of LS-extracted neurotoxin resulted in the elevation of cGMP content. Kynurenic acid (300 mg/kg i.p.), a non specific excitatory amino acid antagonist, was effective in blocking LS BOAA-elicited cGMP enhancement. The data suggest that in the cerebellum acute administration of low concentrations of BOAA exert in vivo activation of glutamate receptors involved in the regulation of cGMP level.

Administration, Oral↗
PubMed1993

Electroretinographic response in WAG/Rij rats after low-intensity cyclic light exposure.

In order to investigate the combined influence of age and light, the b-wave and oscillatory potentials (OPs) of the electroretinogram (ERG) were recorded in 1.5-, 7- and 12-month-old WAG/Rij rats, reared under homogenous low-intensity cyclic light exposure. Wistar albino rats of the same ages, reared under the same conditions, served as controls. The b-wave amplitude decreased, and its implicit time increased in the older age groups significantly more in WAG/Rij than in Wistar rats. Statistical analysis indicated that the b-wave amplitude is a more suitable parameter than implicit time in differentiating the ERG variations of one rat strain from the other. The added amplitude of the OPs also decreased in older age groups, but differently from the b wave. This occurred in WAG/Rij rats already at 1.5 months of age.

Aging↗
PubMed1990

Binding of flunitrazepam to differentiating neurons cultured in a chemically defined, hormone-supplemented medium.

[3H]Flunitrazepam (FNZ) binding to cortical neurons from fetal rat brain was investigated in vitro. The use of a synthetic medium specific for neurons made it possible to plot a developmental curve of 3H-FNZ binding in an almost pure neuronal culture. Detectable specific binding was present in vitro at time 0 (that is, the 16th gestational day). A progressive increase of binding, due to an increment in the number of recognition sites, was observed on the subsequent days. The affinity of the specific binding sites to 3H-FNZ was enhanced by the addition of exogenous GABA, whereas the density was not affected.

Animals↗
PubMed1988

Lateral differences in GABA binding sites in rat brain.

An asymmetric distribution of GABA binding sites was found in the cerebral cortex, hippocampus, cerebellar hemispheres, striatum, and thalamus. Higher levels of [3H]GABA binding were observed in the left-side of most brain areas and in a greater percentage of adult rats, but the opposite asymmetry was found in the thalamus. A similar left-right difference in cerebral hemispheres was also found in five day-old rats, suggesting the genetic predetermination of asymmetry.

Animals↗
PubMed1986

L-[3H]lysine binding to rat retinal membrane: I. Quantitative determination and characterization of the binding sites.

A saturable reversible binding to membranes from rat retina has been found for L-[3H]lysine. Specific binding is time, temperature and protein concentration-dependent, and shows stereospecificity. The best computer fits of the experimental data are obtained with a receptor model based on two independent binding sites, of which only one site with a Kd value of 229.4 +/- 14.23 nM and a Bmax of 2.04 +/- 0.11 pmol/mg protein could be characterized satisfactorily. Several compounds included putative neurotransmitters have moderate or no affinity for L-lysine binding sites. A different pattern of distribution of L-[3H]lysine binding sites is observed among various regions of the brain, with the highest density in the occipital cortex, and the lowest density in ponsmedulla. The existence of binding sites in rat retinal membranes for L-lysine, as well as in the areas involved in the visual pathway, suggests a role for this amino acid in the physiological mechanism of the visual function.

Animals↗
PubMed1986

L-[3H]lysine binding to rat retinal membrane: II. Effect of kainic acid, D,L-alpha-aminoadipic acid, iodoacetic acid, and modification by dark-exposure.

The rat retina and the different brain regions contain membranes sites that bind L-lysine in the nanomolar range. These binding sites undergo changes in different experimental conditions, thus: intraocular injection of kainic acid induces a reduction of the density of L-lysine binding sites, D,L-alpha-aminoadipic acid injected into the eye enhances both kinetic parameters (Bmax and Kd) of L-[3H]lysine binding sites, the intraperitoneal injection of iodoacetic acid decreases the sensitivity for its ligand binding sites, and the exposure to darkness of the rats reduces L-[3H]lysine binding in the retina, thalamus, hypothalamus and superior colliculus, but not in the occipital cortex; such a decrease appears to be characterized, at least in the retina, by a lower sensitivity of the binding sites for L-lysine after the exposure to darkness. The results show that L-lysine binding sites are located on kainic acid-sensitive cells and can be involved in the physiological mechanism of vision.

2-Aminoadipic Acid↗
PubMed1985

Lateral differences in the GABAergic system of the rat striatum.

Asymmetric differences have been found in the pre- and postsynaptic activity of the GABAergic system of the left and right striata of the rat. 3H-GABA binding shows a higher dissociation constant (KD) and a higher number of sites (Bmax) in the left striatum than in the right. Moreover, 3H-diazepam binding seems to be more extensively activated by GABA in the right striatum suggesting a more sensitive postsynaptic GABAergic activity than on the left side. However, when the presynaptic marker (GAD activity) was measured, the asymmetry was in the opposite direction. The results provide further neurochemical evidence of the functional asymmetry of the rat brain.

4-Aminobutyrate Transaminase↗