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Biomedical subjects

P Spano

Publications and source records attributed to P Spano.

At least 37 records · Page 2Linked to original sources
PubMed1997

Priming of cultured neurons with sabeluzole results in long-lasting inhibition of neurotoxin-induced tau expression and cell death.

Sabeluzole was described to have antiischemic, antiepileptic, and cognitive-enhancing properties, and is currently under development for Alzheimer's disease. Recently, it was reported that repeated treatments with sabeluzole protect cultured rat hippocampal neurons against NMDA- and glutamate-induced neurotoxicity. We evaluated the possibility that sabeluzole elicits neuroprotection by acting, either directly or indirectly, on tau proteins. We found that repeated treatments during development of primary cultures of cerebellar granule cells with nanomolar concentrations of sabeluzole resulted in mature cells that were resistant to the excitotoxicity induced by glutamate. Also, sabeluzole treatment specifically prevented the glutamate-induced increase of tau expression without modifying the basal pattern of expression of tau proteins, as shown by measurement of mRNA and protein levels. In human neuroblastoma cell line SH-SY5Y, differentiated by treatment with retinoic acid, doxorubicin increased tau immunoreactivity, and later induced cell death. Both effects were prevented by sabeluzole. Our data indicate that increased tau expression is a common response to different types of cells to neurotoxic agents, and that sabeluzole-induced neuroprotection is functionally associated with the prevention of the injury-mediated increase of tau expression.

Animals↗
PubMed1997

mGluR5 metabotropic glutamate receptor distribution in rat and human spinal cord: a developmental study.

By combining biochemical, molecular and immunohistochemical approaches, we have investigated the presence of metabotropic glutamate receptors (mGluRs) belonging to the subtype 5 in rat and human spinal cords and the developmental changes in their expression. A polyclonal antibody raised against the carboxy-terminal portion of mGluR5 was used to study the distribution of the receptor in rat foetal (Et15), neonatal (P8) and adult spinal cords and dorsal root ganglia (DRG). mGluR5 appeared to be predominantly expressed in regions containing the primary sensory afferents. Immunoblotting with anti-mGluR5 antibody revealed lower receptor protein levels in rat adult spinal cord when compared with P8 rat spinal cord. Reverse transcriptase-polymerase chain reaction showed both mGluR5a and mGluR5b mRNAs expression in rat spinal cord. The mGluR5a variant was found more abundant in young animals than in adults. The pattern of mGluR5 immunostaining was also studied in foetal (6-8, 10, 12 and 22 weeks of gestation) and adult human spinal cord. At all stages of human development, a strong mGluR5 immunoreactivity was observed in the dorsal roots and in the dorsal and dorsolateral funiculi with maximum levels of staining at week 12 of gestation. Foetal DRG neurons were heterogeneously labeled. mGluR5 was also diffusely detectable in the mantle layer. In adult human spinal cords, immunoreactivity was confined to laminae I and II of the dorsal horns. These results demonstrate for the first time the presence of mGluR5 in human spinal cord. The distribution of this receptor suggests a role in the development of somatosensory pathways and in the control of nociceptive neurotransmission.

Adult↗
PubMed1997

Nerve growth factor suppresses the tumoral phenotype of human prolactinomas.

We summarize here our data showing that various phenotypical characteristics distinguish prolactinoma cell lines obtained from responder and nonresponder patients, as defined by their responses to bromocriptine administration. Nonresponder cell lines have a higher degree of malignancy than responder cells and do not express D2 receptors for dopamine. Both cell lines express NGF receptors. Exposure of the most malignant nonresponder cell lines to NGF, both in vitro and when transplanted in vivo in nude mice, results in their differentiation into the responder phenotype reexpressing D2 receptors. Sequential administration of NGF and bromocriptine thus may be a promising therapy for patients refractory to bromocriptine.

Animals↗
PubMed1996

Neuroprotection by aspirin and sodium salicylate through blockade of NF-kappaB activation.

Aspirin (acetylsalicylic acid) is a commonly prescribed drug with a wide pharmacological spectrum. At concentrations compatible with amounts in plasma during chronic anti-inflammatory therapy, acetylsalicylic acid and its metabolite sodium salicylate were found to be protective against neurotoxicity elicited by the excitatory amino acid glutamate in rat primary neuronal cultures and hippocampal slices. The site of action of the drugs appeared to be downstream of glutamate receptors and to involve specific inhibition of glutamate-mediated induction of nuclear factor kappa B. These results may contribute to the emerging theme of anti-inflammatory drugs and neurodegeneration.

Animals↗
PubMed1996

Interleukin-1beta and glutamate activate the NF-kappaB/Rel binding site from the regulatory region of the amyloid precursor protein gene in primary neuronal cultures.

We originally reported that members of the family of transcription factors NF-kappaB/Rel can specifically recognize two identical sequences, referred to as APPkappaB sites, which are present in the 5'-regulatory region of the APP gene. Here we show that the APPkappaB sites interact specifically with a complex which contains one of the subunits of the family, defined as p50 protein, and that they act as positive modulators of gene transcription in cells of neural origin. Additionally, the nuclear complex specifically binding to the APPkappaB sites is constitutively expressed in primary neurons from rat cerebellum and it is up-regulated in response to both the inflammatory cytokine interleukin-1beta (IL-1beta) and the excitatory amino acid glutamate. Since IL-1, whose levels are known to be induced in brain of individuals affected by Alzheimer's disease, and glutamate, are stimuli which have been regarded as major actors on the stage of neurodegenerative processes, we believe our evidence as potentially relevant for understanding the neuropathology associated with Alzheimer's disease.

Alzheimer Disease↗
PubMed1996

Nerve growth factor in the anterior pituitary: localization in mammotroph cells and cosecretion with prolactin by a dopamine-regulated mechanism.

Nerve growth factor (NGF) is well characterized for its neurotrophic actions on peripheral sensory and sympathetic neurons and on central cholinergic neurons of the basal forebrain. Recent evidence, however, has shown high levels of NGF to be present in a variety of biological fluids after inflammatory and autoimmune responses, suggesting that NGF is a mediator of immune interactions. Increased NGF serum levels have been reported in both humans and experimental animal models of psychological and physical stress, thus implicating NGF in neuroendocrine interactions as well. The possible source(s) and the regulatory mechanisms involved in the control of serum NGF levels, however, still remain to be elucidated. We now report the presence of both NGF gene transcripts and protein in the anterior pituitary. Immunofluorescence analysis indicated that hypophysial NGF is selectively localized in mammotroph cells and stored in secretory granules. NGF is cosecreted with prolactin from mammotroph cells by a neurotransmitter-dependent mechanism that can be pharmacologically regulated. Activation of the dopamine D2 receptor subtype, which physiologically controls prolactin release, resulted in a complete inhibition of vasoactive intestinal peptide-stimulated NGF secretion in vitro, whereas the specific D2 antagonist (-)-sulpiride stimulated NGF secretion in vivo, suggesting that the anterior pituitary is a possible source of circulating NGF. Given the increased NGF serum levels in stressful conditions and the newly recognized immunoregulatory function of this protein, NGF, together with prolactin, may thus be envisaged as an immunological alerting signal under neuronal control.

Animals↗
PubMed1995

Differential expression of fetal and mature tau isoforms in primary cultures of rat cerebellar granule cells during differentiation in vitro.

The molecular mechanism(s) responsible for the differential expression of various tau protein isoforms as well as their functional role in morphogenesis, neurofibrillary tangle formation and neurodegeneration have not been completely clarified. We found that the expression of tau proteins in primary cultures of cerebellar granule cells from neonatal rat brain is a developmentally regulated process affecting tau synthesis at different levels. Changes in tau RNA splicing are clearly demonstrated by PCR data showing the switching on of the mRNA containing four internal repeats by DIV 6 and the switching off of the mRNA containing three internal repeats after DIV 12. The changes in mRNA levels of the different tau isoforms during development in vitro occur in parallel with changes in tau protein expression, both qualitatively and quantitatively, as shown by Western analysis of protein extracts from granule cells at different DIV with an anti-tau polyclonal antibody. Finally, as indicated by MAP2 and tau immunocytochemistry data, the switch in tau protein expression appears to be contemporary with neurite outgrowth and cell differentiation. Our data suggest that a differential expression of various tau proteins parallels the degree of cell maturation.

Animals↗
PubMed1995

Identification and characterization of a kappa B/Rel binding site in the regulatory region of the amyloid precursor protein gene.

Several observations support the hypothesis that pathogenetic mechanisms of beta amyloid formation in Alzheimer's disease may involve alterations in amyloid precursor protein (APP) gene expression. In this regard, molecular dissection of the APP gene transcriptional regulation is of primary importance. We report evidence that members of the family of transcription factors NF kappa B/Rel can specifically recognize two identical sequences located in the 5'-regulatory region of APP. These sequences, which we refer to as APP kappa B sites, interact preferentially with p50-containing members of the family. In particular, p50 homodimers and p50/p65 and p50/c-Rel heterodimers act as transcriptional activators at the APP kappa B site. Finally, the nuclear complex specifically binding to the APP kappa B sites proves to be an integral part of neurons and lymphocytes.

Alzheimer Disease↗
PubMed1995

Inhibition of glutamate-induced neurotoxicity by a tau antisense oligonucleotide in primary culture of rat cerebellar granule cells.

Short-term exposure of primary cultures of cerebellar granule cells from neonatal rat brain to high concentrations of glutamate resulted in a significant increase of both immunoreactivity to and mRNA levels of tau protein. Time-course experiments revealed the increases of tau immunoreactivity and mRNA levels to be maximal 2 h after the glutamate pulse. To investigate the relationship between newly synthesized tau protein and glutamate-induced neurotoxicity, neurons were preincubated with a specific tau antisense oligonucleotide. This treatment resulted in (i) inhibition of the glutamate-induced increase of tau immunoreactivity and (ii) a decrease in the sensitivity of the neurons to neurotoxic concentrations of glutamate. These data indicate that induction of the cytoskeleton-associated tau protein participates in the cascade of events promoted by glutamate leading to neurodegeneration.

Animals↗
PubMed1995

Opposing regulation of amyloid precursor protein by ionotropic and metabotropic glutamate receptors.

The effects of the ionotropic glutamate receptor (iGluR) selective agonist N-methyl-D-aspartate (NMDA) on amyloid precursor protein (APP) levels were investigated in primary cultures of rat cerebellar granule cells. Both immunocytochemistry and immunoblotting techniques showed increased APP levels 4 h after a 15 min pulse with NMDA. This effect was completely prevented by incubating the neurones in the presence of the selective metabotropic GluR (mGluR) agonist 1S,3R-ACPD. This phenomenon was related, in terms of doses and time, with the observed 1S,3R-ACPD-mediated protection on NMDA-induced granule cell death. Our findings indicate that APP metabolism is differentially regulated by the stimulation of various GluR subtypes. The GluR-mediated changes in APP content might participate in the control of neuronal viability.

Amyloid beta-Protein Precursor↗
PubMed1995

N-methyl-D-aspartate neurotoxicity in hippocampal slices: protection by aniracetam.

Aniracetam, a drug known to elicit cognition enhancing properties in both animals and humans, was found to counteract the neurotoxicity induced by excitatory amino acids in primary cultures of cerebellar neurons. We report here that aniracetam prevents the neurotoxic effect induced by N-methyl-D-aspartate (NMDA) in rat hippocampal slices. Time-course experiments showed that the aniracetam-induced neuroprotection does not require preincubation of the slices with the drug. Maximal effective concentration of aniracetam was 10 microM. Since the NMDA-mediated cell death in hippocampal slices is considered a valuable experimental model of ischemia, these results suggest a possible novel therapeutic application for aniracetam.

Animals↗
PubMed1995

Nerve growth factor promotes the differentiation of pituitary mammotroph cells in vitro.

It is well known that the differentiation of somatotroph cells is dependent on GRF. The extracellular signals that direct the initial proliferation and differentiation of mammotroph cells during pituitary development, however, have remained elusive. In the present study we first report that somatomammotroph and mammotroph cells present in early postnatal rat pituitary cultures express the receptor for nerve growth factor (NGF) and contain NGF. In addition, we demonstrate that endogenous NGF supports the proliferation and terminal differentiation of mammotroph cells during postnatal in vitro maturation. Early postnatal rat pituitary cells were cultured for 1-12 days, double stained with antibodies to PRL and GH, and analyzed by fluorescence-activated cell sorting. Mature mammotroph cells were generated in culture as shown by 1) the increase in PRL production, 2) the spreading of cells secreting only PRL in relation to those producing both PRL and GH, and 3) the expression of D-2 receptors for dopamine. Exposure of cell cultures to NGF produced a remarkable increase in the rate of appearance of mammotroph cells and an increase in their abundance from 20 +/- 0.5% to 44 +/- 1%. Deprivation of secreted NGF by means of a specific antibody completely prevented the generation of mature mamotrophs. The fluorescence-activated cell sorting analysis of cells grown in the presence of the antibody to NGF showed, in fact, that although the proportions of somatotrophs and somatomammotrophs were unchanged, that of mammotrophs was much lower than that in untreated cultures. We, thus, conclude that NGF is critical for generation of the mammotroph phenotype during pituitary maturation.

Animals↗
PubMed1994

Epidermal growth factor promotes uncoupling from adenylyl cyclase of the rat D2S receptor expressed in GH4C1 cells.

In anterior pituitary cells or when transfected into host cell lines, the D2 dopamine receptor inhibits adenylyl cyclase and activates potassium channels. The GH-3 pituitary tumor cell line, which lacks functional D2 receptors, responds to epidermal growth factor (EGF) by expressing a D2 receptor that, paradoxically, couples to potassium channel activation but poorly inhibits adenylyl cyclase; this was correlated with a pronounced increase in alpha subunit of the G protein Gi3. In this study we have investigated the effects of EGF on the transduction mechanisms of D2 receptors in GH4C1 cells transfected and permanently overexpressing the rat short D2 receptor. Activation of D2 receptors in these cells resulted in both inhibition of adenylyl cyclase and opening of potassium channels and inhibition of prolactin release by both cyclic AMP-dependent and independent mechanisms. Exposure of the transfected GH4C1 cells to EGF caused a dramatic decrease in the coupling efficiency of the D2 receptor to inhibit cyclic AMP-dependent responses, leaving its activity toward potassium channels unchanged. The EGF treatment led to the concomitant increase in the membrane content of Gi3 protein. These results suggest that the transmembrane signaling specificity of G protein-coupled receptors can be modulated by the relative amounts of different G proteins at the cell membrane.

Adenylyl Cyclase Inhibitors↗
PubMed1994

Antisense strategy unravels a dopamine receptor distinct from the D2 subtype, uncoupled with adenylyl cyclase, inhibiting prolactin release from rat pituitary cells.

The antisense strategy was used to unravel the functional contribution of the mRNAs encoding dopamine (DA) receptors to the multiple transduction mechanisms operated by DA in rat pituitary cells. An antisense oligonucleotide was designed to recognize seven nucleotides upstream and 11 nucleotides downstream from the initiation translation codon of the mRNA that encodes the DA D2 receptor. Addition of the antisense oligonucleotide for 7 days to primary culture of rat pituitary cells resulted in a decreased expression of DA D2 receptor as shown by (a) the virtual disappearance of [3H]spiroperidol binding sites and (b) the marked reduction in the levels of both the long and the short splice variant of the D2 receptor mRNAs. After this treatment, the DA D2 receptor agonist bromocriptine lost its capability both to inhibit adenylyl cyclase activity and to reduce prolactin mRNA levels. On the contrary, the inhibition of prolactin release induced by bromocriptine was affected minimally by the antisense oligonucleotide treatment. These data indicate that (a) translation of the mRNA encoding DA D2 receptors results in receptors that are negatively coupled with adenylyl cyclase and functionally linked to inhibition of prolactin synthesis; and (b) the release of prolactin might be regulated, at least in part, by a DA receptor that is encoded by mRNA species distinct from those encoding the D2 receptor.

Adenylyl Cyclase Inhibitors↗
PubMed1994

Nerve growth factor directs differentiation of the bipotential cell line GH-3 into the mammotroph phenotype.

GH-3 is an established cell line which, for the production of both PRL and GH, may be related to the bipotential somatomammotroph from which both somatotroph and mammotroph cells derive. In the present study we first report that GH-3 cells express both the gp140trk and the gp75 components of the nerve growth factor (NGF) receptor and that NGF dictates a nonneuronal type of differentiation of this cell line of ectodermal origin. After exposure to NGF, GH-3 cells markedly decreased their proliferation rate. This effect, which was maximal (50% inhibition) 3 days after beginning the treatment and was maintained during the following days of exposure, was paralleled by a change in the hormone production. The secretion of PRL was increased 6-fold, but that of GH was remarkably inhibited. Moreover, GH-3 cells expressed the mammotroph-specific D-2 receptor protein in response to NGF, as shown by binding with the D-2 receptor ligand N-(p-aminophenetyl)spiperone coupled to fluorescein. The present data thus show that NGF induces the differentiation of GH-3 cells into one of their physiological counterparts, the mammotroph cell, and together with the finding that NGF receptors are expressed in the anterior pituitary suggest a physiological role for the neurotrophic factor in pituitary ontogenesis.

Cell Differentiation↗