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M Memo

Publications and source records attributed to M Memo.

At least 37 records · Page 2Linked to original sources
PubMed1998

Induction of tumour-suppressor phosphoprotein p53 in the apoptosis of cultured rat cerebellar neurones triggered by excitatory amino acids.

We found that primary cultures of rat cerebellar granule cells, although definitely postmitotic and terminally differentiated, express the tumour-suppressor phosphoprotein p53. In particular, granule cells both expressed significant levels of p53 mRNA and positively reacted to an anti-p53 antibody, from the first day of culturing. During neurone differentiation, p53 mRNA content did not significantly change, at least up to 12 days in vitro, while p53 immunoreactivity increased gradually. p53 expression appeared to be further modulable being upregulated after stimulation of glutamate ionotropic receptors by glutamate or kainate. Although qualitatively similar, p53 induction by glutamate and kainate differed in terms of intensity and time-course. The glutamate increase of p53 immunoreactivity appeared within 30 min after the treatment and lasted for at least 2 h. Kainate-induced increase of p53 immunoreactivity was delayed, becoming apparent within 2 h and lasting for at least 8 h. Both kainate- and glutamate-induced increases of p53 immunoreactivity were prevented by the non-competitive NMDA receptor antagonist MK 801. As shown by the electrophoretic mobility shift analysis, both glutamate and kainate induced increases of p53 DNA binding activity. Blockade of p53 induction by a specific p53 antisense oligonucleotide resulted in a partial reduction of excitotoxicity with a complete inhibition of the excitatory amino acids induced apoptosis. Our data suggest that stimulation of ionotropic glutamate receptors in neurones results in a p53-dependent apoptosis.

Animals↗
PubMed1997

Characterization of tau proteins in human neuroblastoma SH-SY5Y cell line.

Here we report three experimental paradigms in which tau proteins are differentially localized and expressed in human neuroblastoma cells SH-SY5Y. We found that in undifferentiated cells, tau proteins were predominantly localized in the nucleus. Western blot analysis of nuclear extracts revealed, among the others, a high molecular weight tau isoform and evaluation of tau mRNA levels showed a single tau isoform. After differentiation, tau immunoreactivity was detected only in cytosol and along neuritic processes. The high molecular weight tau isoform disappeared and an additional tau mRNA species was detected. Treatment of differentiated cells with doxorubicin or okadaic acid resulted in an increase of tau immunoreactivity and in a subsequent cell loss. Our results indicate that both subcellular localization and pattern of expression of tau proteins vary depending on the developmental and functional state of the cells, thus suggesting different roles in cell function.

Antibiotics, Antineoplastic↗
PubMed1997

Metabotropic glutamate receptor mRNA expression in rat spinal cord.

It has recently become clear that, analogous to the situation observed in the brain, glutamatergic neurotransmission in the spinal cord relies upon the activation of ionotropic and metabotropic glutamate receptors (mGluRs). Although electrophysiological studies have emphasized the relevance of mGluR activation in the modulation of spinal neuron responses to glutamate, a detailed study of the molecular forms of receptors belonging to the mGluR family in the spinal cord is still lacking. Using a reverse transcriptase-polymerase chain reaction technique with primers specifically recognizing each cloned mGluR subtype, we found that rat adult spinal cord specifically expresses high levels of mRNA encoding mGluR1a but not mGluR1b. The expression of mRNAs for mGluRs 5a and 5b was also found at levels roughly comparable to that observed in mGluR5-positive brain areas. mGluR2, mGluR3, mGluR4 and mGluR7 mRNAs appeared to be expressed at lower levels, while mGluR6 and mGluR8 mRNAs were not detectable.

Animals↗
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↗
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

Lewy-body dementia and responsiveness to cholinesterase inhibitors: a paradigm for heterogeneity of Alzheimer's disease?

The concept of heterogeneity of Alzheimer's disease is based on molecular, neuropathological, clinical and neuropsychological features, and also supported by the observation that Alzheimer's patients differ in their response to pharmacological interventions. Recent investigations evaluating the therapeutic potential of cholinesterase inhibitors have disclosed the existence of at least two subsets of patients with dementia, defined as 'responders' and 'nonresponders' to this therapy. In this article, Paolo Liberini and colleagues suggest that the cluster of responders to the cholinesterase inhibitors might include a significant number of subjects with a rather selective dysfunction of the cholinergic system, as in the case of Lewy-body dementia. A neuropathological demonstration of this correlation should open up new therapeutic perspectives.

Alzheimer Disease↗
PubMed1996

Activation of multiple metabotropic glutamate receptor subtypes prevents NMDA-induced excitotoxicity in rat hippocampal slices.

Metabotropic glutamate receptors (mGluRs) belong to a relative large receptor family consisting of multiple members with important roles in a number of brain functions. We report here that activation of mGluRs prevents the neurotoxic effect induced by N-methyl-D-aspartate (NMDA) in slices from the rat hippocampus. Neuroprotection was elicited when slices were simultaneously exposed to both the selective mGluR agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (tACPD) and NMDA. Persisting stimulation of mGluRs after the toxic exposure did not improve the survival of pyramidal or granular cells. The neuroprotection elicited by tACPD toxic exposure did not improve the survival of pyramidal or granular cells. The neuroprotection elicited by tACPD was also evoked by its active isomer, (1S, 3R)-ACPD, and was prevented by the selective mGluR antagonist (+)-alpha-methyl-4-carboxyphenyl-glycine (500 microM), confirming that mGluR activation is involved in the mechanism of action of tACPD. The effect of 100 microM tACPD was reproduced by 100 microM quisqualate, an agonist of mGluR2 and mGluR3 subtypes. No neuroprotection was induced by L-2-amino-4-phosphonobutyrate, a selective agonist for mGluR4, mGluR6, mGluR7 and mGluR8, at 500 microM. Since the NMDA-mediated cell death in hippocampal slices is considered relevant to ischaemia-induced brain injury, these results indicate that mGluRs may be important safety devices used by neurons to decrease their sensitivity to excitotoxic stimuli and increase their chance of survival.

Animals↗
PubMed1996

Metabotropic and ionotropic transducers of glutamate signal inversely control cytoplasmic Ca2+ concentration and excitotoxicity in cultured cerebellar granule cells: pivotal role of protein kinase C.

We investigated the functional role of metabotropic glutamate receptors (mGluRs) in modulating glutamate-affected neuronal intracellular calcium concentration ([Ca2+]i) and cell viability in rat cerebellar granule cells. The mGluR agonist trans-1-amino-cyclopentane-1,3-dicarboxylic acid (tACPD) induced a transient increase in [Ca2+]i, which seemed to be developmentally regulated and maximal at 4 days in vitro. In addition, tACPD significantly prevented the [Ca2+]i rise produced by glutamate or by N-methyl-D-aspartate. The mGluR antagonists L-2-amino-3-phosphonopropionic and (+)-alpha-methyl-4-carboxyphenylglycine blocked the effects of tACPD but intrinsically, they magnified the glutamate-mediated [Ca2+]i elevation. The tACPD-mediated decrease in [Ca2+]i rise occurred under experimental conditions superimposable on those producing neuroprotection in glutamate-exposed cultures. tACPD affected neither [Ca2+]i elevation due to KCI nor that evoked by the calcium ionophore A 23187. The inhibitory effect of tACPD was also unaffected by K+ channel blockade produced by tetraethylammonium. The tACPD effects were fully mimicked by quisqualate and (RS)-3,5-dihydroxyphenylglycine, whereas they were only partially reproduced by (2S,1'S,2'S)-2-carboxycyclopropyl-glycine. L-2-Amino-4-phosphonobutyrate was inactive in preventing glutamate-mediated [Ca2+]i rise and neurotoxicity. The tACPD inhibitory responses seemed to be highly sensitive to protein kinase C blockade by bisindolylmaleimide or staurosporine, whereas they were weakly affected by the cAMP analogue dibutyryl cAMP. The protein kinase C activator 4beta-phorbol-12,13-dibutyrate reproduced mGluR-mediated inhibition of both glutamate-induced [Ca2+]i rise and neurotoxicity. In summary, these data suggest that activation of mGluR1-5 subtypes reduce glutamate-mediated (Ca2+]i rise through a mechanism involving protein kinase C activation. Such an effect results in neuroprotection.

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↗