Biomedical subjects

Maria Teresa Ciotti

Publications and source records attributed to Maria Teresa Ciotti.

14 recordsLinked to original sources

PubMed2006

The chemokine CX3CL1 reduces migration and increases adhesion of neurons with mechanisms dependent on the beta1 integrin subunit.

Fractalkine/CX3CL1 and its specific receptor CX3CR1 are constitutively expressed in several regions of the CNS and are reported to mediate neuron-microglial interaction, synaptic transmission, and neuronal protection from toxic insults. CX3CL1 is released both by neuronal and astrocytic cells, whereas CX3CR1 is mainly expressed by microglial cells and neurons. Microglial cells efficiently migrate in response to CX3CL1, whereas no evidence is reported to date on CX3CL1-induced neuronal migration. For this reason, we have investigated in vitro the effects of CX3CL1 on basal migration of neurons and of the microglial and astrocytic populations, all these cells being obtained from the hippocampus and the cerebellum of newborn rats. We report that CX3CL1 stimulates microglial cell migration but efficiently reduces basal neuronal movement, regardless of the brain source. The effect of CX3CL1 is pertussis toxin (PTX) sensitive and PI3K dependent on hippocampal neurons, while it is PTX sensitive, PI3K dependent, and ERK dependent on cerebellar granules. Interestingly, CX3CL1 also increases neuron adhesion to the extracellular matrix component laminin, with mechanisms dependent on PTX-sensitive G proteins, and on the ERK and PI3K pathways. Both the reduction of migration and the increase of neuron adhesion require the activation of the beta(1) and alpha(6) integrin subunits with the exception of cerebellar neuron migration, which is only dependent on the beta(1) subunit. More importantly, in neurons, CX3CL1/CXCL12 cotreatment abolished the effect mediated by a single chemokine on chemotaxis and adhesion. In conclusion, our findings indicate that CX3CL1 reduces neuronal migration by increasing cell adhesion through integrin-dependent mechanisms in hippocampal and cerebellar neurons.

Animals↗

PubMed2006

Expression of NK-1 and NK-3 tachykinin receptors in pancreatic acinar cells after acute experimental pancreatitis in rats.

Activation of neurokinin (NK)-1 receptors but not of NK-3 stimulates amylase release from isolated pancreatic acini of the rat. Immunofluorescence studies show that NK-1 receptors are more strongly expressed than NK-3 receptors on pancreatic acinar cells under basal conditions. No studies have examined the expression of the two NK receptor populations in pancreatic acini during pancreatitis in rats. We therefore investigated the relationships between expression of these two tachykinin receptors and experimental acute pancreatitis induced by stimulating pancreatic amylase with caerulein (CK) in rats. Hyperstimulation of the pancreas by CK caused an increase in plasma amylase and pancreatic water content and resulted in morphological evidence of cytoplasmic vacuolization. Immunofluorescence analysis revealed a similar percentage of NK-1 receptor antibody immunoreactive acinar cells in rats with pancreatitis and in normal rat tissue but a larger percentage of NK-3 receptor immunoreactive cells in acute pancreatitis than in normal pancreas. Western blot analysis of NK-1 and NK-3 receptor protein levels after CK-induced pancreatitis showed no change in NK-1 receptors but a stronger increase in NK-3 receptor expression in pancreatic acini compared with normal rats thus confirming the immunofluorescence data. These new findings support previous evidence that substance P-mediated functions within the pancreas go beyond sensory signal transduction contributing to neurogenic inflammation, and they suggest that substance P plays a role in regulating pancreatic exocrine secretion via acinar NK-1 receptors. The significant increase in NK-3 receptors during pancreatic stimulation suggests that NK-3 receptors also intervene in the pathogenesis of mild acute pancreatitis in rats.

Animals↗

PubMed2006

NMDA receptor mediates tau-induced neurotoxicity by calpain and ERK/MAPK activation.

The altered function and/or structure of tau protein is postulated to cause cell death in tauopathies and Alzheimer's disease. However, the mechanisms by which tau induces neuronal death remain unclear. Here we show that overexpression of human tau and of some of its N-terminal fragments in primary neuronal cultures leads to an N-methyl-D-aspartate receptor (NMDAR)-mediated and caspase-independent cell death. Death signaling likely originates from stimulation of extrasynaptic NR2B-subunit-containing NMDARs because it is accompanied by dephosphorylation of cAMP-response-element-binding protein (CREB) and it is inhibited by ifenprodil. Interestingly, activation of NMDAR leads to a crucial, sustained, and delayed phosphorylation of extracellular-regulated kinases 1 and 2, whose inhibition largely prevents tau-induced neuronal death. Moreover, NMDAR involvement causes the fatal activation of calpain, which, in turn, degrades tau protein into a 17-kDa peptide and possibly other highly toxic N-terminal peptides. Some of these peptides are hypothesized, on the basis of our in vitro experiments, to initiate a negative loop, ultimately leading to cell death. Thus, inhibition of calpain largely prevents tau degradation and cell death. Our findings unravel a cellular mechanism linking tau toxicity to NMDAR activation and might be relevant to Alzheimer's disease and tauopathies where NMDAR-mediated toxicity is postulated to play a pivotal role.

Alzheimer Disease↗

PubMed2005

Chemokine CX3CL1 protects rat hippocampal neurons against glutamate-mediated excitotoxicity.

Excitotoxicity is a cell death caused by excessive exposure to glutamate (Glu), contributing to neuronal degeneration in many acute and chronic CNS diseases. We explored the role of fractalkine/CX3CL1 on survival of hippocampal neurons exposed to excitotoxic doses of Glu. We found that: CX3CL1 reduces excitotoxicity when co-applied with Glu, through the activation of the ERK1/2 and PI3K/Akt pathways, or administered up to 8 h after Glu insult; CX3CL1 reduces the Glu-activated whole-cell current through mechanisms dependent on intracellular Ca2+; CX3CL1 is released from hippocampal cells after excitotoxic insult, likely providing an endogenous protective mechanism against excitotoxic cell death.

Animals↗

PubMed2005

Interaction of Tau with Fe65 links tau to APP.

The beta-amyloid precursor protein APP and the microtubule-associated protein Tau play a crucial role in the pathogenesis of Alzheimer's disease (AD). However, the possible molecular events linking these two proteins are still unknown. Here, we show that Fe65, one of the ligands of the APP cytodomain, is associated with Tau in vivo and in vitro, as demonstrated by co-immunoprecipitation, co-localization, and FRET experiments. Deletion studies indicated that the N-terminal domain of Tau and the PTB1 domain of Fe65 are required for this association. This interaction is regulated by the phosphorylation of Tau at selected sites, by glycogen synthase kinase-3beta (GSK3beta) and cyclin-dependent kinase 5 (Cdk5), and requires an intact microtubule network. Furthermore, laser scanner microscopy and co-immunoprecipitation experiments provide preliminary evidence of possible complex(es) involving Tau, Fe65, APP. These findings open new perspectives for the study of the possible crosstalk between these proteins in the pathogenesis of AD.

Alzheimer Disease↗

PubMed2005

Role of the autophagic-lysosomal system on low potassium-induced apoptosis in cultured cerebellar granule cells.

Apoptotic and autophagic cell death have been implicated, on the basis of morphological and biochemical criteria, in neuronal loss occurring in neurodegenerative diseases and it has been shown that they may overlap. We have studied the relationship between apoptosis and autophagic cell death in cerebellar granule cells (CGCs) undergoing apoptosis following serum and potassium deprivation. We found that apoptosis is accompanied by an early and marked proliferation of autophagosomal-lysosomal compartments as detected by electron microscopy and immunofluorescence analysis. Autophagy is blocked by hrIGF-1 and forskolin, two well-known inhibitors of CGC apoptosis, as well as by adenovirus-mediated overexpression of Bcl-2. 3-Methyladenine (3-MA) an inhibitor of autophagy, not only arrests this event but it also blocks apoptosis. The neuroprotective effect of 3-MA is accompanied by block of cytochrome c (cyt c) release in the cytosol and by inhibition of caspase-3 activation which, in turn, appears to be mediated by cathepsin B, as CA074-Me, a selective inhibitor of this enzyme, fully blocks the processing of pro-caspase-3. Immunofluorescence analysis demonstrated that cathepsin B, normally confined inside the lysosomal-endosomal compartment, is released during apoptosis into the cytosol where this enzyme may act as an execution protease. Collectively, these observations indicate that autophagy precedes and is causally connected with the subsequent onset of programmed death.

Adenine↗

PubMed2005

AMPA receptors are modulated by tachykinins in rat cerebellum neurons.

The peptides of the tachykinin family are widely distributed within the mammalian peripheral and central nervous systems and play a well-recognized role as neuromodulators, although their direct action on cerebellum granule cells have not yet been demonstrated. We have examined the effect of the best known members of the family, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors from rat cerebellar granule cells in culture to assess the ability of these peptides to regulate the glutamatergic input. Both NKA and NKB, but not SP, produce a significant enhancement of ionic current through AMPA receptors activated by the agonist kainate in 53.5 and 46% of patched neurons, respectively. This effect was not observable in the presence of MEN 10,627 and Trp(7)betaAla(8), NKA and NKB competitive antagonist receptors, respectively, indicating that the current modulations were mediated by the respective receptors. NKB also produces a significant enhancement of ionic current through the AMPA receptors activated directly by its agonist AMPA and cyclothiazide, an allosteric modulator that selectively suppresses desensitization of AMPA receptors. The presence of NK3 receptors was demonstrated in these neurons by RT-PCR amplification of total RNA extracted from cerebellar granule cells, using NK3-specific primer pairs. Immunocytochemistry experiments, using a specific polyclonal antibody directed against NK3, also confirmed the presence of NK3 receptors and their co-localization with the GLUR2 AMPA subunit in about 54% of cerebellar granule neurons. This study adds the tachykinins to the list of neuromodulators capable of exerting a excitatory action on cerebellar granule cells.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2004

Dual control of neurogenesis by PC3 through cell cycle inhibition and induction of Math1.

Growing evidence indicates that cell cycle arrest and neurogenesis are highly coordinated and interactive processes, governed by cell cycle genes and neural transcription factors. The gene PC3 (Tis21/BTG2) is expressed in the neuroblast throughout the neural tube and inhibits cell cycle progression at the G1 checkpoint by repressing cyclin D1 transcription. We generated inducible mouse models in which the expression of PC3 was upregulated in neuronal precursors of the neural tube and of the cerebellum. These mice exhibited a marked increase in the production of postmitotic neurons and impairment of cerebellar development. Cerebellar granule precursors of PC3 transgenic mice displayed inhibition of cyclin D1 expression and a strong increase in the expression of Math1, a transcription factor required for their differentiation. Furthermore, PC3, encoded by a recombinant adenovirus, also induced Math1 in postmitotic granule cells in vitro and stimulated the Math1 promoter activity. In contrast, PC3 expression was unaffected in the cerebellar primordium of Math1 null mice, suggesting that PC3 acts upstream to Math1. As a whole, our data suggest that cell cycle exit of cerebellar granule cell precursors and the onset of cerebellar neurogenesis are coordinated by PC3 through transcriptional control of cyclin D1 and Math1, respectively.

Animals↗

PubMed2004

Oxidative stress induces p53-mediated apoptosis in glia: p53 transcription-independent way to die.

Oxidative stress has been implicated in the pathogenesis of stroke, traumatic brain injuries, and neurodegenerative diseases affecting both neuronal and glial cells in the central nervous system (CNS). The tumor suppressor protein p53 plays a pivotal function in neuronal apoptosis triggered by oxidative stress. We investigated the role of p53 and related molecular mechanisms that support oxidative stress-induced apoptosis in glia. For this purpose, we exposed C6 glioma cells and primary cultures of rat cortical astrocytes to an H(2)O(2)-induced oxidative stress protocol followed by a recovery period. We evaluated the effects of pifithrin-alpha (PF-alpha), which has been reported to protect neurons from ischemic insult by specifically inhibiting p53 DNA-binding activity. Strikingly, PF-alpha was unable to prevent oxidative stress-induced astrocyte apoptosis. We demonstrate that p53 is able to mediate an apoptotic response by direct signaling at mitochondria, despite its transcriptional activity. The z-VAD-fmk-sensitive apoptotic response requires a caspase-dependent MDM-2 degradation, leading to p53 mitochondrial targeting accompanied by cytochrome c release and nucleosomal fragmentation.

Amino Acid Chloromethyl Ketones↗

PubMed2004

Gene expression profiles of apoptotic neurons.

The multigenic program underlying neuronal apoptosis is mostly unknown. To study the program, we used genome-scale screening by oligonucleotide microarrays during serum and potassium deprivation-induced apoptosis of cerebellar granule neurons. From the 8740 genes interrogated by the arrays, 423 genes were found to be regulated at both the transcriptional and the posttranscriptional level and segregated into distinct clusters. Semantic clustering based on gene ontologies showed coordinated expression of genes with common biological functions and metabolic pathways. Among the genes implicated in apoptotic cerebellar granule neurons, 70 were in common with those differentially expressed in cortical neurons exposed to amyloid beta-protein, indicating the existence of common mechanisms responsible for neuronal cell death. Our results offer a genomic view of the changes that accompany neuronal apoptosis and yield new insights into the underlying molecular basis.

Amyloid beta-Peptides↗

PubMed2003

Neuronal fibrillogenesis: amyloid fibrils from primary neuronal cultures impair long-term memory in the crab Chasmagnathus.

Amyloid beta protein (Abeta) fibrillogenesis is considered one of the crucial steps of Alzheimer's disease (AD) pathogenesis. The effect of endogenous neuronal amyloid fibrils on memory processes is unknown. To investigate this issue, we first characterised the Abeta fibrillar aggregates secreted by cerebellar granule cells and then we evaluated the effect of neuronal fibrils on an invertebrate model of memory. An increase of fibril formation, assessed by Thioflavin T (ThT) fluorescence, was observed in the conditioned medium of apoptotic neurons during 48 h of the apoptotic process. Moreover, the depolarisation-stimulated secretion of cerebellar granule cells contains monomers of endogenous Abeta, which undergo cell-free fibrillogenesis over several days of incubation. The pattern of single endogenous fibrils, examined by electron microscopy, was similar to that of synthetic Abeta while a tighter and more complex interfibrillar organization was observed in endogenous fibrils. The biological effect of neuronal fibrils was studied in a long-term memory (LTM) paradigm, namely the context-signal learning of the crab Chasmagnathus. Pre-training injection of neuronal fibril extract (protein concentration, 1 microg/ml) induced amnesia in a dose-dependent manner. On the contrary, no effect on retention was observed with the administration of two orders higher doses (100 microg/ml) of synthetic Abeta1-40. These results indicate that only naturally secreted fibrils, but not synthetic Abeta, clearly interfere with memory process.

Amyloid beta-Peptides↗

PubMed2003

Ligand-independent CXCR2 dimerization.

Homo- and hetero-oligomerization have been reported for several G protein-coupled receptors (GPCRs). The CXCR2 is a GPCR that is activated, among the others, by the chemokines CXCL8 (interleukin-8) and CXCL2 (growth-related gene product beta) to induce cell chemotaxis. We have investigated the oligomerization of CXCR2 receptors expressed in human embryonic kidney cells and generated a series of truncated mutants to determine whether they could negatively regulate the wild-type (wt) receptor functions. CXCR2 receptor oligomerization was also studied by coimmunoprecipitation of green fluorescent protein- and V5-tagged CXCR2. Truncated CXCR2 receptors retained their ability to form oligomers only if the region between the amino acids Ala-106 and Lys-163 was present. In contrast, all of the deletion mutants analyzed were able to form heterodimers with the wt CXCR2 receptor, albeit with different efficiency, competing for wt/wt dimer formation. The truncated CXCR2 mutants were not functional and, when coexpressed with wt CXCR2, interfered with receptor functions, impairing cell signaling and chemotaxis. When CXCR2 was expressed with the AMPA-type glutamate receptor GluR1, CXCR2 dimerization was again impaired in a dose-dependent way, and receptor functions were prejudiced. In contrast, CXCR1, a chemokine receptor that shares many similarities with CXCR2, did not dimerize alone or with CXCR2 and when coexpressed with CXCR2 did not impair receptor signaling and chemotaxis. The formation of CXCR2 dimers was also confirmed in cerebellar neuron cells. Taken together, we conclude from these studies that CXCR2 functions as a dimer and that truncated receptors negatively modulate receptor activities competing for the formation of wt/wt dimers.

Alanine↗

PubMed2003

A tachykinin-like factor increases glutamate toxicity in rat cerebellar granule cells.

Tachykinins (TKs), which include substance P, neurokinin A and neurokinin B, constitute a group of neuropeptides widely expressed in the CNS where they play several functions connected with neural modulation often in synergy with glutamate excitatory transmission. The aim of this study was to assess whether TKs modulate glutamate response of in vitro cultured cerebellar granule neurons and whether GSA (glutamate-sensitizing activity), a peptide released by these neurons, belongs to the TKs family. Treatment with substance P and other neurokinin 1 receptor (NK1) agonists does not affect the response of cerebellar granule neurons to glutamate toxicity. On the contrary, agonists neurokinin 2 receptor (NK2) and neurokinin 3 receptor (NK3) agonists increase, in a dose and time dependent fashion, the response of the same neurons to glutamate. MEN 10,627, a selective NK2 receptor antagonist, and (Trp(7),betaAla(8)) NKA (4-10), a selective NK3 receptor antagonist inhibit not only the sensitizing action to glutamate of their respective agonists. These antagonists almost equally reduce the glutamate-sensitizing activity of GSA. Such activity is also abolished in the presence of a polyclonal antibody directed against neurokinin B (NKB). These findings indicate that TKs increase glutamate sensitivity in cerebellar granule neurons and that the GSA previously detected in conditioned media of the same cultured neurons belongs to the TK family although its primary structure as compared to known TKs remains to be established.

Animals↗

PubMed2003

Signalling pathways involved in the chemotactic activity of CXCL12 in cultured rat cerebellar neurons and CHP100 neuroepithelioma cells.

We compared the signal transduction pathways activated by stromal cell-derived factor-1 (CXCL12) chemokine in two different cell systems: primary cultures of rat cerebellar granule neurons (CGN) and human neuroepithelioma CHP100 cells. Both cell types express functional CXC chemokine receptor 4 (CXCR4), which is coupled both to extracellular signal-regulated kinase (ERK) and Akt phosphorylation pathways. The activation of ERK shows different dependency on the phosphatidylinositol 3-kinase (PI3-K) pathway and different sensitivity to pertussis toxin (PTX) treatment, indicative of coupling to different G proteins in the two cell systems considered. We demonstrate that the inhibition of either the ERK kinase or the PI3-K pathways blocks the CXCL12 induced-chemotaxis in CHP100 cells; while only PI3-K activity is stringently necessary for CGN migration.

Animals↗