Biomedical subjects

M Di Luca

Publications and source records attributed to M Di Luca.

At least 55 records · Page 3Linked to original sources

PubMed1998

Dysplastic neocortex and subcortical heterotopias in methylazoxymethanol-treated rats: an intracellular study of identified pyramidal neurones.

Intracellular recordings were obtained using biocytin-filled electrodes from 78 neurones located in both dysplastic neocortex and subcortical heterotopic aggregates in a model of neuronal migration disorder induced in rats by means of a double methylazoxymethanol injection given on embryonic day 15. Both regular spiking and intrinsically bursting pyramidal neurones were found in all of the examined structures and were synaptically activated by subcortical stimulation. In a neuronal subpopulation (22%) located in the neocortex as well as in the subcortical heterotopic aggregates, the injection of depolarising current pulses elicited aberrant firing patterns, consisting of repetitive bursts of APs that gradually increased in duration and eventually merged in a long-lasting discharge. The gradual development of this 'excessive' bursting behaviour suggests a progressive run-down of the slow components of the hyperpolarising afterpotential.

Animals↗

PubMed1998

Differential level of platelet amyloid beta precursor protein isoforms: an early marker for Alzheimer disease.

OBJECTIVE: To determine whether a differential level of platelet amyloid beta precursor protein (APP) isoforms is specifically related to Alzheimer disease (AD) and whether it shows a correlation with the progression of clinical symptoms. DESIGN: After subjects were grouped according to diagnosis and severity of dementia, APP isoform levels in platelets were compared. SETTING: University medical centers. PATIENTS: Thirty-two patients who fulfilled diagnostic criteria for probable AD, 25 age-matched control subjects, and 16 patients with non-AD dementia. MAIN OUTCOME MEASURE: The levels of APP isoforms were evaluated by means of Western blot analysis and immunostaining of whole platelets. Messenger RNAs for APP transcripts were also evaluated by means of reverse transcriptase polymerase chain reaction. RESULTS: The ratio between the intensity of the 130-kd and 106- to 110-kd APP isoforms was significantly lower in the AD group (0.31 +/- 0.15, mean +/- SD) compared with both controls (0.84 +/- 0.2) and non-AD subjects (0.97 +/- 0.4). The ratio of platelet APP isoforms in patients with AD grouped by Clinical Diagnostic Rating score significantly correlated with the severity of the disease (Pearson correlation coefficient, followed by Bonferroni correction, P = .01). Reverse transcriptase polymerase chain reaction experiments showed that APP transcripts in all experimental groups were equally expressed. CONCLUSIONS: The pattern of platelet APP isoforms is specifically altered in patients with AD. In addition, the alteration of platelet APP isoforms shows a positive correlation with the progression of clinical symptoms, supporting the possibility to consider this peripheral parameter as a marker of progression of the disease. These alterations are not related to abnormalities of APP isoforms messenger RNAs in platelets.

Adult↗

PubMed1998

Altered connections between neocortical and heterotopic areas in methylazoxymethanol-treated rat.

We are currently investigating various treatments which could determine, in the rat brain, structural abnormalities mimicking those reported in human brain dysgeneses. We can induce the formation of neuronal heterotopia in the progeny of rats by means of a double injection of the cytotoxic agent methylazoxymethanol acetate (MAM) on embryonic day 15. We have now investigated the anatomical connections of these heterotopia by means of anterograde and retrograde tract tracing techniques. The induced heterotopia along the border of the lateral ventricles shared common anatomical features with the periventricular nodules in human periventricular or subcortical nodular heterotopia (PNH). The tract tracing data demonstrated the existence of reciprocal connections between the neuronal heterotopia and the ipsilateral and contralateral cortical areas, and the presence of abnormal cortico-hippocampal and cortico-cortical connections. On the basis of the connectivity patterns, it may be speculated that some cells in the heterotopia could be neurons originally committed to the cortex, that were interrupted in their migration by the MAM treatment. Given the common morphological features seen in human PNH and MAM-induced brain heterotopia, the anatomical and developmental analysis of MAM-treated rats may shed light on the mechanisms by which human brain dysgeneses develop in human patients.

Animals↗

PubMed1998

Calcium/calmodulin-dependent protein kinase II is associated with NR2A/B subunits of NMDA receptor in postsynaptic densities.

NMDA receptors and Ca2+/calmodulin-dependent kinase II (CaMKII) have been reported to be highly concentrated in the postsynaptic density (PSD). Although the possibility that CaMKII in PSD might be associated with specific proteins has been put forward, the protein or proteins determining the targeting of the kinase in PSD have not yet been identified. Here we report that CaMKII binds to NR2A and NR2B subunits of NMDA receptors in PSD isolated from cortex and hippocampus. The association of NMDA receptor subunits and CaMKII was assessed by immunoprecipitating PSD proteins with antibodies specific for NR2A/B and CaMKII: CaMKII coprecipitated with NR2A/B and NR1 but not with other glutamate ionotropic receptor subunits, such as GluR1 and GluR2-3. A direct association between CaMKII and NR2A/B subunits was further confirmed by overlay experiments using either 32P-autophosphorylated CaMKII or 32P-NR2A/B and by evaluating the formation of a CaMKII-NR2A/B complex by means of the cross-linker disuccimidyl suberate. These data demonstrate an association between the NMDA receptor complex and CaMKII in the postsynaptic compartment, suggesting that this colocalization may be relevant for synaptic plasticity.

Animals↗

PubMed1997

Increased secretion of the amino-terminal fragment of amyloid precursor protein in brains of rats with a constitutive up-regulation of protein kinase C.

Protein kinase C (PKC) activation stimulates release of secreted amyloid precursor protein (APPs) in several cell lines. To ascertain the role of PKC in regulating APP metabolism in vivo, we used an animal model (methylazoxymethanol-treated rats; MAM rats) in which PKC is permanently hyperactivated in selected brain areas, i.e., cortex and hippocampus. A significant decrease in membrane-bound APP concentration was found in synaptosomes derived from cortex and hippocampus of MAM rats, where PKC is up-regulated, with a concomitant increase in APPs production in soluble fractions of the same brain areas. In contrast, in a brain area not affected by MAM treatment (i.e., cerebellum), APP secretion is similar in control and MAM rats, indicating that altered metabolism of APP is restricted to only those areas in which the PKC system is up-regulated. In addition, phorbol esters or H-7 modulate APPs release in hippocampal slices from both control and MAM rats, further supporting an in vivo role for this enzyme in regulating metabolism of mature APP.

Alkylating Agents↗

PubMed1997

Increased presynaptic protein kinase C activity and glutamate release in rats with a prenatally induced hippocampal lesion.

We have previously shown that protein kinase C (PKC) activity is up-regulated in nerve terminals of animals that have been subjected to targeted cellular ablation of cortical and hippocampal neurons by treatment with methylazoxymethanol (MAM), which results in impaired long-term potentiation (LTP) and cognitive deficit. In this study we investigated the consequences of increased membrane-bound PKC in the regulation of release of glutamate, the major excitatory transmitter involved in LTP. We show that nerve terminals of MAM-treated rats show higher PKC activity, as monitored by the in situ phosphorylation of B-50/GAP-43, in both basal and phorbol ester-stimulated conditions. In these animals, hippocampal nerve endings release a greater amount of glutamate than those of controls, both in basal conditions and when synaptosomes are stimulated with KCl or 3,4-diaminopyridine. The potentiation observed in MAM-treated rats was counteracted by the PKC blocker H-7 and the clostridial tetanus toxin. On the contrary, GABA release was not significantly up-regulated, either in basal or in depolarization-evoked conditions. Therefore our data show that the increase in synaptosomal PKC activity is paralleled by increased glutamate but not GABA release in this animal model. Whether this reflects specific up-regulation of membrane PKC activity in glutamatergic terminals or an alteration in the regulation of glutamate release remains to be determined.

Animals↗

PubMed1997

Developmental models of brain dysfunctions induced by targeted cellular ablations with methylazoxymethanol.

Abnormal brain development represents one of the major causes of neurological disorders in humans, and determining the factors responsible for generating specific brain malformations represents a formidable task for developmental neurobiology. The knowledge of the precise neurogenetic time table and the use of toxins, like methylazoxymethanol, able to interfere with neuroepithelial cells entering their last mitotic cycle, have allowed for targeted neuronal ablations in specific brain areas of the central nervous system (CNS) when administered at different gestational or postnatal days in various animal species. Of particular relevance are the studies in which ablations of neuronal populations of cortex, hippocampus, and cerebellum have been made. The results obtained show that these early ablations induce a number of neuroanatomic, neurochemical, and electrophysiological changes that give us the possibility to unravel the biochemical strategies utilized by surviving neurons to adapt to the perturbated environment. Most striking are the findings that target deprivation does not affect the survival of afferent neurons in the CNS (except for neurons of the lateral geniculate nucleus), in sharp contrast to the notion of target dependence for peripheral nervous system neurons. Animals showing selective ablations in the Ammon's horn of the hippocampus allow us to understand the complex biochemical pathways leading to changes in activity-dependent synaptic plasticity, and the data underscore the fundamental role of diverse Ca(2+)-dependent protein kinases, and their substrates, in modulating pre- and postsynaptic events during induction and maintenance of long-term potentiation (LTP). Because LTP represents a useful model to study molecular substrates of learning and memory, this animal model might be of relevance in understanding cognitive brain dysfunctions.

Animals↗

PubMed1996

Differential translocation of protein kinase C isozymes in rats characterized by a chronic lack of LTP induction and cognitive impairment.

The translocation of protein kinase C isozymes was investigated in an animal model of cognitive deficit and lack of induction of long-term potentiation (LTP). In MAM rats, presynaptic alpha, beta, epsilon PKC showed enhanced translocation, while postsynaptic gamma PKC displayed decreased translocation when compared to control levels. This imbalance of PKC isozyme translocation between the pre- and post-synaptic compartment might therefore represent a possible molecular cause for the lack of synaptic plasticity observed in these animals.

Animals↗

PubMed1996

Determination of the endogenous phosphorylation state of B-50/GAP-43 and neurogranin in different brain regions by electrospray mass spectrometry.

Electrospray mass spectrometry coupled to liquid chromatography was utilized to measure two PKC neuronal substrates, B-50/GAP-43 and neurogranin, in single rat brain areas. Aliquots of perchloric acid extracts were directly injected and mass spectra recorded. At elution times of 14.2 and 27.0 min two molecular species of MW 7450 and 23 602 Da were observed. These values are in excellent agreement for the expected MW for rat neurogranin and B-50/GAP-43. The presence of molecular species shifted by 80 mass units in both cases indicates that these proteins are present in phosphorylated forms in cortical and hippocampal extracts.

Animals↗

PubMed1996

Abnormal pattern of platelet APP isoforms in Alzheimer disease and Down syndrome.

OBJECTIVE: To determine if changes in levels of amyloid precursor protein (APP) isoforms in periphery are associated with Alzheimer disease and Down syndrome. DESIGN: After subjects were grouped according to diagnosis, APP isoform levels in platelets were compared. SETTING: University medical center. SUBJECTS: Ten patients who fulfilled diagnostic criteria for probable Alzheimer disease, 22 healthy volunteers, and 7 elderly (mean age, 42.7 years) and 7 young (mean age, 19.0 years) patients with Down syndrome. MAIN OUTCOME MEASURES: The levels of APP isoforms were evaluated by means of Western blot analysis and immunostaining of whole platelets. RESULTS: The ratio between the 130- and the 106- to 110-kd APP isoforms was markedly lower in patients with Alzheimer disease and in elderly patients with Down syndrome than in control subjects. In young patients with Down syndrome, the ratio did not significantly differ from that in control subjects. CONCLUSIONS: A consistent alteration in platelet APP isoforms has been found in Alzheimer disease and Down syndrome. Further studies will determine whether this alteration could provide a peripheral biochemical marker of the disorder and whether it could intervene in the pathogenesis of Alzheimer disease.

Aged↗

PubMed1995

Selective in vitro blockade of neuroepithelial cells proliferation by methylazoxymethanol, a molecule capable of inducing long lasting functional impairments.

In order to characterize the antiproliferative effect of methylazoxymethanol neuroepithelial cells derived from the rat striata primordia at embryonic day 14 have been exposed to graded doses of this compound. It was found that methylazoxymethanol application to striatal neuroblasts elicits a blockade of cell proliferation at a dose which does not interfere with cell survival. By using synchronized cells and short term exposures to this compound, we found that the antiproliferative effect of methylazoxymethanol is strikingly correlated to the number of cells actively dividing in culture, thus indicating that the cells targeted by methylazoxymethanol must be in an active mitotic phase. To test for the selectivity of action of Methylazoxymethanol for dividing neuroblasts either cultures composed of mature proliferating astrocytes or muscle cells have been subjected to the same treatment. It has been observed that astrocytes proliferation was not affected by the dose of methylazoxymethanol shown to be effective on neuroepithelial cells. Finally we demonstrated that methylazoxymethanol is able only transiently to interfere with smooth muscle cell division, further supporting its selectivity of action within the developing CNS.

Animals↗

PubMed1995

Changes in protein kinase C and its presynaptic substrate B-50/GAP-43 after intrauterine exposure to methylazoxy-methanol, a treatment inducing cortical and hippocampal damage and cognitive deficit in rats.

The involvement of protein kinase C (PKC)-dependent processes in adaptive and plastic changes underlying neuronal plasticity was tested in an in vivo animal model characterized by targeted cellular ablation of cortical and hippocampal neurons, cognitive impairment and lack of induction of long-term potentiation. [3H]Phorbol ester binding performed on brain slices revealed a 67.4 and 35.0% increase in membrane-bound protein kinase C in the cortex and hippocampus respectively of rats treated with methylazoxy-methanol acetate compared with saline-treated control rats, and there was no modification in the expression of mRNAs of different protein kinase C isozymes. In situ phosphorylation experiments performed with 32Pi-labelled synaptosomes from the affected areas demonstrated that the phosphorylation of the nervous tissue-specific presynaptic membrane-associated protein kinase C substrate B-50/GAP-43 was increased by 51.4 and 44.8% in cortex and hippocampus respectively. Western blot analysis of protein kinase C in synaptosomal cytosol and membrane fractions prepared from cortex and hippocampus showed an increased proportion of protein kinase C in the membrane compartment in treated animals, but no change in the total synaptosomal protein kinase C activity. Our data are consistent with increased activity of presynaptic protein kinase C and predict a sustained increase in glutamate release in methylazoxy-methanol-treated rats.

Animals↗

PubMed1994

Synaptic protein phosphorylation changes in animals exposed to neurotoxicants during development.

Protein phosphorylation represents a key process by which neuronal function is regulated by first messengers interacting with extracellular membrane receptors. Protein kinases transfer the phosphate group from ATP to neuron specific proteins and phosphatases, catalyzing the removal of the phosphate group, shut off the signal by restoring the reactive form of the protein. These phosphorylation processes seem to be particularly important in long-term changes which follow sustained activation of neurons. Particular importance has been given to the Calcium/phospholipid-dependent protein kinase (PKC) as the molecular mechanism in synaptic plasticity associated with learning and memory. We have studied the changes of PKC activity in an animal model of impaired cognitive functions as a consequence of an exposure during embryonic life to an antimitotic agent, methylazoxy-methanol acetate (MAM). Treatment at gestational day (GD) 15 results in offspring showing a dose-dependent reduction in the size of cortex and hippocampus. When adult, these animals show impairments in several tests for learning and memory. In hippocampal slice preparations from MAM-treated rats, Long-Term Potentiation could not be induced in the CA1 region, the area affected by the treatment. However, in the hippocampal dentate gyrus, an area not affected by the treatment, LTP could be induced. Moreover, these animals show area-specific changes in the phosphorylation state of the protein B-50/GAP-43, a well characterized neuron specific substrate for PKC. By changing the time of MAM exposure, i.e. at GD19, a different pattern of brain damage occurs and this results both in a different pattern in behavior and B-50 phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Protein kinase C-dependent phosphorylation in prenatally induced microencephaly.

We describe here integrated studies conducted in an animal model of brain malformation induced by prenatal treatment with a potent antimitotic agent, methylazoxymethanol acetate (MAM). When given at gestational day 15, MAM induces a marked and dose-dependent hypoplasia of cortex and hippocampus. The alteration of specific neurotransmitter systems in these brain areas reflect the specificity of the damage induced by MAM administration at this particular stage of brain development. These animals, when adult, show impairments in learning and memory performance, without gross alterations of spontaneous behavior. The impairment in cognitive functions is correlated with changes, both in cortex and hippocampus, of the phosphorylation state of the neuron-specific protein B-50, a substrate of Protein Kinase C, known to play a key role in synaptic plasticity. Moreover, Long-Term Potentiation (LTP), a cellular model for studying synaptic plasticity associated with learning and memory, is impaired in the hippocampal subfields affected by MAM treatment. All these results--obtained with anatomical, behavioral, neurochemical and electrophysiological studies-point to the usefulness of this animal model to understand the long-lasting consequences of the interference of neurotoxic compounds with the developing CNS.

Animals↗

PubMed1993

Selective alteration in B-50/GAP-43 phosphorylation in brain areas of animals characterized by cognitive impairment.

When methylazoxymethanol acetate is administered to pregnant rats at gestational day 19, the offspring are greatly impaired in the learning of a two-way active avoidance task and these behavioral changes are paralleled by a change in the phosphorylation of the protein B-50/GAP-43 in hippocampus but not in cortex. The expression of the protein is not altered, indicating that the phosphorylation of B-50 is a sensitive marker of alterations in synaptic plasticity associated with impairments of learning abilities in rats.

Animals↗

PubMed1993

The impaired long-term potentiation in the CA1 field of the hippocampus of cognitive deficient microencephalic rats is restored by D-serine.

Rat embryos exposed on gestational day 15 to methyl-azoxymethanol acetate develop a microencephaly characterized primarily by a hypoplasia of the neocortex and CA fields of the hippocampus that in adulthood is associated with disturbances in learning. In brain slices prepared from microencephalic rats, we have examined the field excitatory postsynaptic potentials and population spike in the CA1 field of the hippocampus evoked by stimulation of the stratum radiatum. These parameters did not differ from those obtained in slices from control rats. High frequency stimulation of the stratum radiatum afferent fibres, which readily induced long-term potentiation of the field excitatory postsynaptic potentials and population spike in the CA1 field of the hippocampus of control rats, failed to induce long-term potentiation in that of microencephalic rats. High frequency stimulation of the perforant path readily elicited long-term potentiation in the dentate gyrus of both control and microencephalic rats. Picrotoxin had no apparent effect on field excitatory postsynaptic potentials and population spike in the CA1 field of the microencephalic rats, indicating that little GABAergic inhibition was present in slices from these rats. D-2-Amino-phosphonovalerate suppressed the field potentials in slices from microencephalic rats by more than 50%, suggesting that N-methyl-D-aspartate receptors contributed markedly to the synaptic responses evoked by single stimuli. D-Serine, but not picrotoxin, restored long-term potentiation in the CA1 field of the microencephalic rats. The D-serine effect was prevented by pretreating the slices with either 7-chloro-kynurenate or D-2-amino-phosphonovalerate. The failure to induce long-term potentiation, if also found in vivo, may be among the factors related to the learning deficits displayed by these rats.

2-Amino-5-phosphonovalerate↗

PubMed1992

Cellular expression of somatostatin in MAM-induced microencephaly in the rat.

Methylazoxymethanol acetate (MAM) is a mitotic inhibitor that has been used to selectively destroy neuroblasts at specific times during gestation. The administration of MAM results in a dose-dependent microencephaly. Following MAM treatment at 15 days of gestation, we have noted an increase in the level of SS immunoreactivity in the neocortex, as determined by radioimmunoassay. Northern blot analysis for preproSS mRNA revealed an increase in MAM-treated cortex. The cellular distribution of SS has been determined using in situ hybridization and immunocytochemistry. There was a 30% increase in the density of SS-immunoreactive neurons in the cortex of the MAM-treated animals. These data suggest that SS neurons in the cortex are spared following MAM treatment at GD 15.

Animals↗

PubMed1992

Measurement of relative amounts of phospho- and dephospho-B-50(GAP-43) peptides by fast atom bombardment-mass spectrometry.

The biological role of phosphoproteins depends upon their degree of phosphorylation in vivo. Methods currently available to measure the degree of phosphorylation of a protein involve indirect procedures to detect the 32P-phosphate incorporation. We report here a direct method to measure relative amounts of phospho- and dephospho-forms of peptides based upon a mass spectrometric technique. The intensities of the molecular ions corresponding to the two forms of the peptides are proportional to their relative amounts. This is demonstrated for a peptide fragment of the protein B-50(GAP-43) and for kemptide, respectively substrates for protein kinases C and A, and demonstrates the applicability of fast atom bombardment-mass spectrometry to quantitate peptides bearing post-translational modifications.

Amino Acid Sequence↗