Biomedical subjects

Cristina Limatola

Publications and source records attributed to Cristina Limatola.

10 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

Chemokine fractalkine/CX3CL1 negatively modulates active glutamatergic synapses in rat hippocampal neurons.

We examined the effects of the chemokine fractalkine (CX3CL1) on EPSCs evoked by electrical stimulation of Schaffer collaterals in patch-clamped CA1 pyramidal neurons from rat hippocampal slices. Acute application of CX3CL1 caused a sustained reduction of EPSC amplitude, with partial recovery after washout. CX3CL1-induced EPSC depression is postsynaptic in nature, because paired-pulse ratio was maintained, amplitude distribution of spontaneous excitatory postsynaptic currents shifted to lower values, and whole-cell current responses to AMPA were reversibly inhibited. EPSC depression by CX3CL1 is mediated by CX3CL1 receptor (CX3CR1), because CX3CL1 was unable to influence EPSC amplitude in CA1 pyramidal neurons from CX3CR1 knock-out mice. CX3CL1-induced depression of both EPSC and AMPA current was not observed in the absence of afferent fiber stimulation or AMPA receptor activation, respectively, indicating the requirement of sustained receptor activity for its development. Findings obtained from hippocampal slices, cultured hippocampal neurons, and transfected human embryonic kidney cells indicate that a Ca2+-, cAMP-, and phosphatase-dependent process is likely to modulate CX3CL1 effects because of the following: (1) CX3CL1-induced depression was antagonized by intracellular BAPTA, 8Br-cAMP, phosphatase inhibitors, and pertussis toxin (PTX); (2) CX3CL1 inhibited forskolin-induced cAMP formation sensitive to PTX; and (3) CX3CL1 inhibited forskolin-induced Ser845 GluR1 phosphorylation, which was sensitive to PTX and dependent on Ca2+ and phosphatase activity. Together, these findings indicate that CX3CL1 negatively modulates AMPA receptor function at active glutamatergic synapses through cell-signaling pathways by influencing the balance between kinase and phosphatase activity.

Animals↗

PubMed2006

Fractalkine/CX3CL1 depresses central synaptic transmission in mouse hippocampal slices.

This work reports the effect of chemokine fractalkine/CX3CL1, an endogenous small peptide highly expressed in the central nervous system, on evoked synaptic responses investigated in mouse CA1 stratum radiatum using an electrophysiological approach. We report that in acute mouse hippocampal slices, superfusion of CX3CL1 resulted in a reversible depression of the field excitatory postsynaptic potential (fEPSP) which developed within few seconds, increased for up to 10 min of application and disappeared within 30 min after the end of CX3CL1 treatment. We also show that CX3CL1-induced synaptic depression is (i) dose-dependent with IC50 and nH values of 0.7 nM and 1, respectively, (ii) not associated with a change in paired-pulse facilitation, (iii) mediated through CX3CL1 receptor (CX3CR1), being absent in CX3CR1-/- mice and inhibited in wild-type mice by a specific blocking antibody, and (iv) occluded by the induction of homosynaptic long-term depression (LTD). We conclude that CX3CL1 is a potent neuromodulator of the evoked excitatory synaptic transmission, sharing common mechanisms with LTD.

Animals↗

PubMed2006

Functional properties of neurons derived from fetal mouse neurospheres are compatible with those of neuronal precursors in vivo.

Neural stem cells can be propagated in culture as neurospheres, yielding neurons and glial cells upon differentiation. Although the neurosphere model is widely used, the functional properties of the neurosphere-derived neurons have been only partially characterized, and it is unclear whether repeated passaging alters their functional properties. In this study, we analyzed voltage- and transmitter-gated responses in neuron-like cells obtained by differentiating fetal mouse neurospheres at increasing passages in culture. We report that neurons fire overshooting action potentials in response to depolarizing currents up to passage 10 but loose this capability at later passages, as the density of voltage-gated Na(+) and K(+) currents decreases. In contrast, the immunoreactivity for the neuronal marker beta-tubulin remains unaltered up to passage 21, indicating that this marker is not representative of cell function. In almost all neurons, gamma-aminobutyric acid (GABA) evoked bicuculline-sensitive whole-cell currents, resulting from the activation of GABA(A) receptors, which appeared to be excitatory, insofar as the reversal potential of GABA-gated current was about -50 mV. Much smaller currents were elicited by the glutamatergic agonist AMPA, and only occasional responses to glycine were detected. In these functional aspects, neurosphere-derived neurons are similar to immature neurons differentiating in vivo. Therefore, at least for a limited number of passages in vitro, neurospheres provide an adequate model of in vivo neurogenesis.

Action Potentials↗

PubMed2005

Cysteine residues are critical for chemokine receptor CXCR2 functional properties.

We examined the role of cysteine (Cys) residues present in chemokine receptor CXCR2 for proper surface expression, dimerization, signaling, and chemotaxis. To address this issue, serine or leucine residues were substituted for Cys, generating nine CXCR2 mutants transiently expressed in HEK cells. Single substitution of Cys residues present in the three extracellular loops (C119L, C196L, C286S) or in the seventh-transmembrane (TM) domain (C308L) abolished CXCL8 agonist binding, while no Cys substitution abolished surface receptor expression. We have previously demonstrated that CXCR2 dimerizes under reducing conditions, due to hydrophobic interactions that involve TM3 regions, and here we show that the dimer/monomer CXCR2 ratio drastically increases when analyzed under non-reducing conditions. We report that none of the Cys-deficient CXCR2 mutants abolishes receptor dimerization, demonstrating that Cys-Cys bonds are not the exclusive determinant of CXCR2 dimerization. Furthermore, both wt- and Cys-mutated CXCR2 dimers are expressed at the cell surface, indicating that receptor dimers are efficiently transferred at the plasma membrane. We also show that every Cys substitution in CXCR2, including those that still bind CXCL8, results in an impairment of receptor activity, analyzed as cell chemotaxis and intracellular signaling, suggesting that some structural requirement is likely fulfilled by Cys presence.

Amino Acid Substitution↗

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↗

PubMed2004

Neurotrophic effects of AMPA.

AMPA-type glutamate receptors may transduce both neurotoxic and neurotrophic signals, basically depending on receptor subunit composition and on the extent of receptor activation. While a great deal of data are available on the mechanisms underlying the neurotoxic effects induced by AMPA receptor stimulation, much less is known about the molecular mechanisms responsible for their neurotrophic activities. This review describes the experimental evidences in favor of a neurotrophic effect of AMPA and compares the mechanisms identified and the signaling pathways involved with those relevant for the neurotrophic activities of other neurotrophins.

Animals↗

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

Expression of AMPA-type glutamate receptors in HEK cells and cerebellar granule neurons impairs CXCL2-mediated chemotaxis.

We find that cerebellar granule neurons (CGN) obtained from newborn rats (p3) migrate in response to both CXC chemokine ligand-2 (CXCL2) and -12 (CXCL12), while CGN from p7 rats are unresponsive to CXCL2. The expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptor 1 (GluR1) greatly impairs the chemotaxis induced by CXCL2 in CXCR2-expressing HEK cells. By immunoprecipitation, we show that CXCR2 is associated with AMPA receptors (AMPARs) in p7 CGN, and with GluR1 co-expressed in HEK cells. Taken together, these results suggest that the association between CXCR2 and AMPARs results in the inhibition of CXCL2-dependent chemotaxis, and may represent a molecular mechanism underlying the modulation of nerve cell migration.

Age Factors↗

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↗