Biomedical subjects

Cristina Missale

Publications and source records attributed to Cristina Missale.

12 recordsLinked to original sources

PubMed2006

Presenilin 2 mutations alter cystatin C trafficking in mouse primary neurons.

Mutations in the presenilin genes account for the majority of familial Alzheimer disease (FAD) cases. In the present report we demonstrated that the FAD-linked presenilin 2 mutations (PS2 M239I and T122R) alter cystatin C trafficking in mouse primary neurons reducing secretion of its glycosylated form. These mutations showed a different impact on cystatin C: PS2 T122R had a much stronger effect determining a dramatic intracellular accumulation of cystatin C (native and glycosylated), followed by a reduction in the secretion of both forms. Several experimental evidences suggest that cystatin C exerts a protective role in the brain and favors stem cells proliferation. Confocal imaging showed that the effect of PS2 T122R mutation was a massive recruitment of cystatin C into the neuronal processes, in the presence of an intact cytoskeletal structure. The consequent reduction in the cystatin C extracellular levels might result in a failure of neuroregeneration. Understanding the interplay of PS2 and cystatin C in the pathogenesis of AD might highlight new therapeutic prospective.

Animals↗

PubMed2006

The NMDA/D1 receptor complex as a new target in drug development.

Dopamine and glutamate have been shown to extensively interact in the striatum, nucleus accumbens, hippocampus and prefrontal cortex, to regulate different physiological functions, including locomotor activity, positive reinforcement, attention and working memory. Although dysfunctions of dopamine transmission have long been identified as critical determinants of neurological and neuropsychiatric disorders, such as Parkinson's disease and schizophrenia, there is now increasing evidence that concurrent alterations of dopamine and glutamate function may play a central role in the pathophysiology of these diseases. Thus, defining the characteristics of dopamine-glutamate interactions may be crucial to identify alternative molecular targets for the development of novel pharmacological tools. At the postsynaptic level, interactions between the dopamine D1 and the glutamate NMDA receptors appear to be particularly relevant. Different mechanisms are involved in this interactions: 1) D1R-dependent, second messenger-mediated phosphorylation of NMDAR subunits; 2) coordinated regulation of receptor trafficking at synaptic sites; 3) formation of an heteromeric D1/NMDA receptor complex. In this paper we review the molecular mechanisms, functional implications and pharmacological significance of D1R/NMDAR interaction via direct protein-protein oligomerization.

Animals↗

PubMed2005

Loss of synaptic D1 dopamine/N-methyl-D-aspartate glutamate receptor complexes in L-DOPA-induced dyskinesia in the rat.

Glutamate-mediated mechanisms are related to the motor complications of L-DOPA therapy in Parkinson's disease (PD). In striatal postsynaptic densities (PSD), the dopamine D1 receptor (D1R) is part of an oligomeric complex with the glutamate N-methyl-D-aspartate receptor (NMDAR), determining the strength of corticostriatal transmission. We studied D1R/NMDAR complex alterations induced by L-DOPA in the 6-hydroxydopamine-lesioned rat model of PD. L-DOPA-treated hemiparkinsonian rats were determined to be dyskinetic or nondyskinetic based on behavioral testing. D1R/NMDAR assemblies containing NR1-C2 and NR2B subunits were decreased in the PSD of lesioned striatum. Short-term L-DOPA administration improved akinesia and restored the synaptic abundance of D1R, NR1-C2 and NR2B. Prolonged L-DOPA treatment also normalized synaptic D1R/NMDAR complexes in nondyskinetic rats, but remarkably reduced them in the dyskinetic group without changing their interaction. This decrease involved NR1-C2, NR1-C2', NR2A, and NR2B subunits. The composition of residual synaptic D1R/NMDAR complexes in dyskinetic rats may thus be different from that observed in lesioned rats, suggesting that expression of different motor dysfunctions might be related to the receptor profile at corticostriatal synapses. The levels of D1R/NMDAR complexes were unchanged in total striatal membrane proteins, suggesting that the decrease of these species in the PSD is likely to reflect an altered receptor trafficking. In human embryonic kidney 293 cells expressing the D1R/NMDAR, complex costimulation of both D1R and NMDAR, but not individual receptor activation, promoted internalization, suggesting that development of dyskinesias might be related to agonist-mediated down-regulation of the D1R/NMDAR complex at corticostriatal synapses.

Animals↗

PubMed2005

Group-II metabotropic glutamate receptors negatively modulate NMDA transmission at striatal cholinergic terminals: role of P/Q-type high voltage activated Ca++ channels and endogenous dopamine.

Striatal cholinergic nerve terminals express functional group-II metabotropic (mGlu) and NMDA glutamate receptors. To investigate whether these receptors interact to regulate ACh release, LY354740 (a group-II mGlu receptor agonist) and NMDA were co-applied in striatal synaptosomes and slices. LY354740 prevented the NMDA-evoked [3H]-choline release from synaptosomes and ACh release from slices. In synaptosomes, this modulation was prevented by omega-agatoxin IVA, suggesting that it was mediated by P/Q-type high voltage activated Ca++ channels. In slices, LY341495 (a group-II mGlu receptor antagonist) enhanced the NMDA-induced ACh release, suggesting that group-II mGlu receptor activation by endogenous glutamate inhibits NMDA transmission. Co-immunoprecipitation studies excluded direct group-II mGlu-NMDA receptor interactions. Finally, group-II mGlu negative modulation of NMDA transmission was abolished in dopamine-depleted synaptosomes and slices, suggesting that it relied on endogenous dopamine. We conclude that group-II mGlu receptors attenuate NMDA inputs at striatal cholinergic terminals via Ca++ channel modulation and dopamine-sensitive pathways.

Acetylcholine↗

PubMed2005

Identification of a mutant-like conformation of p53 in fibroblasts from sporadic Alzheimer's disease patients.

Here we show that fibroblasts from sporadic Alzheimer's disease (AD) patients specifically express an anomalous and detectable conformational state of p53 that makes these cells distinct from fibroblasts of age-matched non-AD subjects. In particular, we found that, in contrast to non-AD fibroblasts, p53 in AD fibroblasts is expressed at higher levels in resting condition, and presents a significant impairment of its DNA binding and transcriptional activity. All together, these findings figured out the presence of a mutant-like p53 phenotype. However, gene sequencing of the entire p53 gene from either AD or non-AD did not unravel point mutations. Based on immunoprecipitation studies with conformation-specific p53 antibodies (PAb1620 and PAb240), which discriminated folded versus unfolded p53 tertiary structure, we found that a significant amount of p53 assumed an unfolded tertiary structure in fibroblasts from AD patients. This conformational mutant-like p53 form was virtually undetectable in fibroblasts from non-AD patients. These data, independently from their relevance in understanding the etiopathogenesis of AD, might be useful for supporting AD diagnosis.

Aged↗

PubMed2004

Increased serum concentration of nerve growth factor in patients with microprolactinoma.

Nerve growth factor (NGF) is known to play a role as a circulating neurokine, integrating signals from the neuro-immuno-endocrine system. The ability of NGF to activate the pituitary-adrenocortical axis, together with the increase of its serum concentration in pregnancy and lactation, supports the hypothesis that NGF is secreted by the pituitary gland and plays a role as modulator of endocrine functions. Evidence obtained both in vitro and in vivo in experimental animal models suggests that lactotroph cells secrete both prolactin (PRL) and NGF. Furthermore, in previous studies we demonstrate that cell lines derived from dopamine (DA)-sensitive human prolactinomas express high levels of NGF messenger RNA and protein. On these basis, we studied serum NGF concentrations in female patients with microprolactinoma (n = 4) and in control women (n = 5). PRL and NGF were measured at the diagnosis, during the thyrotropin releasing hormone (TRH) test and after the therapy with DA D2 receptor agonist cabergoline (0.25 mg, twice a week). Results obtained suggested that hyperprolactinemia (70.3+/-8.4 ng/ml) paralleled markedly higher NGF levels (58.4+/-18.7 pg/ml) compared to controls (PRL 8.7+/-3.2 ng/ml, NGF 8.4+/-1.3 pg/ml). Serum concentrations of NGF and PRL during the TRH test were closely associated (r = 0.943, p < 0.01). Cabergoline therapy normalized PRL (7.9+/-3.6 ng/ml) and induced a significant decrease of NGF levels (12.5+/-4.9 pg/ml). In conclusions, data reported here indicated that, in human microprolactinomas, NGF is released in the bloodstream paralleling PRL-secretion and it is modulated by a neurotransmitter-regulated mechanism, since the normalization of PRL elicited by the DA D2 receptor agonist cabergoline induced a significant decrease of serum NGF as well.

Adult↗

PubMed2003

Nerve growth factor restores p53 function in pituitary tumor cell lines via trkA-mediated activation of phosphatidylinositol 3-kinase.

Two groups of prolactinomas were identified, one slowly proliferating and responsive to bromocriptine and one fast proliferating and bromocriptine resistant. Nerve growth factor (NGF) inhibits proliferation of bromocriptine-resistant cells by mechanisms that are still unclear. The tumor suppressor p53 is one of the key regulators of cell proliferation and in most tumors, but not pituitary adenomas, it is inactivated by genomic mutations. Here we investigated whether in prolactinoma cell lines NGF influences cell cycle-related pathways involving p53. By using conformation-specific antibodies and immunocytochemistry we found that in bromocriptine-resistant cells p53 adopts a mutant conformation that precludes its nuclear translocation and transcriptional activity. NGF administration to these cells refolds p53 into wild-type tertiary structure, promotes its nuclear translocation, and restores its DNA-binding activity as demonstrated by the transcriptional activation of p21Cip1/WAF1 and the resulting down-regulation of different cyclins and cyclin-dependent kinase 2. Inactivation of trkA, but not of p75NTR, and wortmannin prevented NGF-induced p53 nuclear translocation. Thus, in prolactinoma cells p53 is inactivated by conformational mutation and cytoplasmic segregation. This defect is reversible because NGF reconstitutes active p53 in these cells. This effect of NGF is exclusively mediated by trkA through activation of phosphatidylinositol-3-kinase and may be related to its growth-inhibitory action.

Animals↗

PubMed2003

CaMKII-dependent phosphorylation regulates SAP97/NR2A interaction.

Synapse-associated protein 97 (SAP97), a member of membrane-associated guanylate kinase protein family, has been implicated in the processes of targeting ionotropic glutamate receptors at postsynaptic sites. Here we show that SAP97 is enriched at the postsynaptic density where it co-localizes with both ionotropic glutamate receptors and downstream signaling proteins such as Ca2+/calmodulin-dependent protein kinase II (CaMKII). SAP97 and alphaCaMKII display a high co-localization pattern in hippocampal neurons as well as in transfected COS-7 cells. Metabolic labeling of hippocampal cultures reveals that N-methyl-D-aspartic acid (NMDA) receptor activation induces CaMKII-dependent phosphorylation of SAP97; co-incubation with the CaMKII-specific inhibitor KN-93 reduces SAP97 phosphorylation to basal levels. Our results show that SAP97 directly interacts with the NR2A subunit of NMDA receptor both in an in vitro "pull-out" assay and in co-immunoprecipitation experiments from homogenates and synaptosomes purified from hippocampal rat tissue. Interestingly, in the postsynaptic density fraction, SAP97 fails to co-precipitate with NR2A. We show here that SAP97 is directly associated with NR2A through its PDZ1 domain, and CaMKII-dependent phosphorylation of SAP97-Ser-232 disrupts NR2A interaction both in an in vitro pull-out assay and in transfected COS-7 cells. Moreover, expression of SAP97(S232D) mutant has effects similar to those observed upon constitutively activating CaMKII. Our findings suggest that SAP97/NR2A interaction is regulated by CaMKII-dependent phosphorylation and provide a novel mechanism for the regulation of synaptic targeting of NMDA receptor subunits.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Regulation of dopamine D1 receptor trafficking and desensitization by oligomerization with glutamate N-methyl-D-aspartate receptors.

Activation of dopamine D1 receptors is critical for the generation of glutamate-induced long-term potentiation at corticostriatal synapses. In this study, we report that, in striatal neurons, D1 receptors are co-localized with N-methyl-d-aspartate (NMDA) receptors in the postsynaptic density and that they co-immunoprecipitate with NMDA receptor subunits from postsynaptic density preparations. Using modified bioluminescence resonance energy transfer, we demonstrate that D1 and NMDA receptor clustering reflects the existence of direct interactions. The tagged D1 receptor and NR1 subunit cotransfected in COS-7 cells generated a significant bioluminescence resonance energy transfer signal that was insensitive to agonist stimulation and that did not change in the presence of the NR2B subunit, suggesting that the D1 receptor constitutively and selectively interacts with the NR1 subunit of the NMDA channel. Oligomerization with the NR1 subunit substantially modified D1 receptor trafficking. In individually transfected HEK293 cells, NR1 was localized in the endoplasmic reticulum, whereas the D1 receptor was targeted to the plasma membrane. In cotransfected cells, both the D1 receptor and NR1 subunit were retained in cytoplasmic compartments. In the presence of the NR2B subunit, the NR1-D1 receptor complex was translocated to the plasma membrane. These data suggest that D1 and NMDA receptors are assembled within intracellular compartments as constitutive heteromeric complexes that are delivered to functional sites. Coexpression with NR1 and NR2B subunits also abolished agonist-induced D1 receptor cytoplasmic sequestration, indicating that oligomerization with the NMDA receptor could represent a novel regulatory mechanism modulating D1 receptor desensitization and cellular trafficking.

Animals↗

PubMed2002

Nerve growth factor regulates dopamine D(2) receptor expression in prolactinoma cell lines via p75(NGFR)-mediated activation of nuclear factor-kappaB.

Two groups of prolactinoma cell lines were identified. One group (responder) expresses both D(2) dopamine receptors and an autocrine loop mediated by nerve growth factor (NGF) and one group (nonresponder) lacks both D(2) receptors and NGF production. D(2) receptor expression in these cell lines is dependent on NGF. Indeed, NGF inactivation in responder cells decreases D(2) receptor density, while NGF treatment induces D(2) receptor expression in nonresponders. Here we show that inactivation of p75(NGFR), but not of trkA, resulted in D(2) receptor loss in responder cells and prevented D(2) receptor expression induced by NGF in the nonresponder. Analysis of nuclear factor-kappaB (NF-kappaB) nuclear accumulation and binding to corresponding DNA consensus sequences indicated that in NGF-secreting responder cells, but not in nonresponders, NF-kappaB is constitutively activated. Moreover, NGF treatment of nonresponder cells induced both nuclear translocation and DNA binding activity of NF-kappaB complexes containing p50, p65/RelA, and cRel subunits, an effect prevented by anti-p75(NGFR) antibodies. Disruption of NF-kappaB nuclear translocation by SN50 remarkably impaired D(2) receptor expression in responder cells and prevented D(2) gene expression induced by NGF in nonresponders. These data indicate that in prolactinoma cells the effect of NGF on D(2) receptor expression is mediated by p75(NGFR) in a trkA-independent way and that NGF stimulation of p75(NGFR) activates NF-kappaB, which is required for D(2) gene expression. We thus suggest that NF-kappaB is a key transcriptional regulator of the D(2) gene and that this mechanism may not be confined to pituitary tumors, but could also extend to other dopaminergic systems.

Animals↗

PubMed2002

Nerve growth factor and retinoic acid interactions in the control of small cell lung cancer proliferation.

OBJECTIVE: Nerve growth factor (NGF) has antiproliferative and differentiating effects in neuroendocrine tumors. In cell lines derived from small cell lung cancer (SCLC), NGF treatment stimulates NGF receptor expression, activates NGF secretion, inhibits proliferation and abrogates invasion. Since these effects are lost upon NGF withdrawal, it is relevant to identify other differentiation factors that may co-operate with the NGF system to control SCLC growth and differentiation. DESIGN: Retinoic acid (RA), which has been shown to inhibit cell transformation and proliferation, modulates the expression of NGF receptors and the sensitivity to NGF in different cell models. In the present study, we have investigated whether NGF and RA may interact to control the proliferation of SCLC cell lines. METHODS: SCLC cells were exposed to 50 ng/ml NGF or 1 microM all-trans RA for different times. Cell proliferation was measured by the [(3)H]thymidine incorporation test and NGF receptor expression was evaluated by immunofluorescence. RESULTS: We found that RA increased the expression of both trkA and p75 NGF receptors in NCI-N-592 and GLC8 cell lines and prevented the loss of both NGF production and NGF receptor expression occurring when NGF treatment was discontinued. As a result, RA, which did not inhibit the proliferation of untreated cells, abolished NGF withdrawal-related increase in cell proliferation both in vitro and in vivo, thus making permanent the antiproliferative effects of NGF. CONCLUSIONS: These data suggest that combined treatments with NGF and RA or mimicking drugs may represent a strategy to be further investigated for the treatment of SCLC.

Carcinoma, Small Cell↗

PubMed2002

Nerve growth factor induces the re-expression of functional androgen receptors and p75(NGFR) in the androgen-insensitive prostate cancer cell line DU145.

BACKGROUND: One of the paracrine/autocrine factors regulating prostate growth and differentiation is nerve growth factor (NGF). The role of NGF and its receptors in the prostate, however, remains controversial. We have shown that NGF treatment of human prostate cancer cell lines reduced their tumorigenicity, both in vitro and in vivo. OBJECTIVE: To investigate the involvement of NGF as a differentiation factor in prostate cancer cells. DESIGN: We exposed the androgen-independent/androgen receptor (AR)-negative prostate cancer cell line DU145 to NGF to study whether this neurotrophin could revert DU145 cells to a less malignant phenotype. METHODS: DU145 cells were treated with NGF, then ARs and NGF receptor p75(NGFR) expression and telomerase activity were studied. Finally, we investigated whether re-expression of ARs could restore the androgen sensitivity in this cell line. RESULTS AND CONCLUSIONS: NGF treatment induced a reversion of DU145 cells to a less malignant phenotype, characterized by the re-expression of ARs and p75(NGFR) NGF receptors. Re-expression of ARs restored the androgen sensitivity, as suggested by the fact that exposure to dihydrotestosterone stimulated the growth of NGF-treated DU145 cells. This effect was blocked by androgen antagonist drugs, such as hydroxyflutamide and cyproterone acetate, which also induced apoptotic death of NGF-treated cells. The hypothesis that a differentiation pathway is activated by exogenous NGF in DU145 cells is also supported by findings indicating that NGF-treated DU145 cells expressed a low telomerase activity, as a result of a decrease in human telomerase reverse transcriptase transcription.

Androgen Antagonists↗