Cristina Limatola

CXC chemokines interleukin-8 (IL-8) and growth-related gene product α (GROα) modulate Purkinje neuron activity in mouse cerebellum

Abstract We give here evidence that Purkinje neurons (PNs) of mouse cerebellar slices studied with patch clamp technique combined with laser confocal microscopy, respond to human IL-8 and GROα by (i) a cytosolic Ca2+ transient compatible with inositol (1,4,5) trisphosphate (InsP3) formation; (ii) an enhancement of the neurotransmitter release; and (iii) an impairment of the long-term depression of synaptic strength (LTD). It was also found the expression of IL-8 receptor type 2 in PN and granule cells by immunofluorescence, immunoblotting and RT-PCR analysis. Considered together these findings suggest that IL-8 and GROα may play a neuromodulatory role on mouse cerebellum.

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.

SDF‐1α‐mediated modulation of synaptic transmission in rat cerebellum

The functional expression of the seven‐transmembrane domain G protein‐coupled chemokine receptor CXCR‐4/fusin in rat nerve cell was demonstrated by staining with a polyclonal anti‐CXCR‐4 Ab, and by evaluating the calcium responses to the physiological agonist stromal‐derived cell factor‐1α (SDF‐1α) in both cerebellar granule cells in culture and Purkinje neurons (PNs) in cerebellar slices. Cerebellar glial, granule and Purkinje cells showed a pronounced staining for CXCR‐4. Furthermore, cultured granule cells exhibited Ca2+ transients elicited by the application of SDF‐1α, both in cell bodies and in neuronal processes. Whole‐cell patch‐clamped PNs in cerebellar slices responded to SDF‐1α application by a slow inward current followed by an increase of both intracellular Ca2+ level and spontaneous synaptic activity. In particular, the SDF‐1α‐induced slow inward current was considerably reduced by ionotropic glutamate receptor blockers, but developed fully in a medium in which synaptic transmission was inhibited, indicating that this current might be, at least in part, mediated by extrasynaptic glutamate, possibly released from the surrounding glial and/or nerve cells. Taken together, these findings indicate a functional involvement of CXCR‐4 in the modulation of synaptic transmission, adding another member to the repertoire of the chemokine receptors exerting a neuromodulatory role in the cerebellum.

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 β) 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.

Modulating neurotoxicity through CX3CL1/CX3CR1 signaling.

Since the initial cloning of fractalkine/CX3CL1, it was proposed that the only known member of the CX3C or δ subfamily of chemotactic cytokines could play some significant role in the nervous system, due to its high expression on neurons. The pivotal description of the localization of the unique CX3CL1 receptor, CX3CR1, on microglial cells, firmed up by the generation of cx3cr1GFP/GFP mice, opened the road to the hypothesis of some specific key interactions between microglia and neurons mediated by this pair. This expectation has been indeed supported by recent exciting evidence indicating that CX3CL1-mediated microglia-neuron interaction modulates basic physiological activities during development, adulthood and aging, including: synaptic pruning; promoting survival of neurons and neural precursors; modulating synaptic transmission and plasticity; enhancing synapse and network maturation; and facilitating the establishment of neuropathic pain circuits. Beyond playing such fascinating roles in physiological conditions, CX3CL1 signaling has been implicated in different neuropathologies. Early papers demonstrated that the levels of CX3CL1 may be modulated by various toxic stimuli in vitro and that CX3CL1 signaling is positively or negatively regulated in EAE and MS, in HIV infection and LPS challenge, in epilepsy, in brain tumors, and in other neuropathologies. In this review we focus on the experimental evidence of CX3CL1 involvement in neuroprotection and survey the common molecular and cellular mechanisms described in different brain diseases.

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.

Activity of Adenosine Receptors Type 1 Is Required for CX3CL1-Mediated Neuroprotection and Neuromodulation in Hippocampal Neurons

The chemokine fractalkine (CX3CL1) is constitutively expressed by central neurons, regulating microglial responses including chemotaxis, activation, and toxicity. Through the activation of its own specific receptor, CX3CR1, CX3CL1 exerts both neuroprotection against glutamate (Glu) toxicity and neuromodulation of the glutamatergic synaptic transmission in hippocampal neurons. Using cultured hippocampal neuronal cell preparations, obtained from CX3CR1−/− (CX3CR1GFP/GFP) mice, we report that these same effects are mimicked by exposing neurons to a medium conditioned with CX3CL1-treated mouse microglial cell line BV2 (BV2-st medium). Furthermore, CX3CL1-induced neuroprotection from Glu toxicity is mediated through the adenosine receptor 1 (AR1), being blocked by neuronal cell preparations treatment with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a specific inhibitor of AR1, and mimicked by both adenosine and the specific AR1 agonist 2-chloro-N6-cyclopentyladenosine. Similarly, experiments from whole-cell patch-clamped hippocampal neurons in culture, obtained from CX3CR1+/+ mice, show that CX3CL1-induced depression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- (AMPA-) type Glu receptor-mediated current (AMPA-current), is associated with AR1 activity being blocked by DPCPX and mimicked by adenosine. Furthermore, BV2-st medium induced a similar AMPA-current depression in CX3CR1GFP/GFP hippocampal neurons and this depression was again blocked by DPCPX. We also report that CX3CL1 induced a significant release of adenosine from microglial BV2 cells, as measured by HPLC analysis. We demonstrate that (i) CX3CL1, along with AR1, are critical players for counteracting Glu-mediated neurotoxicity in the brain and (ii) AR1 mediates neuromodulatory action of CX3CL1 on hippocampal neurons.

CX3CR1 Deficiency Alters Hippocampal-Dependent Plasticity Phenomena Blunting the Effects of Enriched Environment

In recent years several evidence demonstrated that some features of hippocampal biology, like neurogenesis, synaptic transmission, learning, and memory performances are deeply modulated by social, motor, and sensorial experiences. Fractalkine/CX3CL1 is a transmembrane chemokine abundantly expressed in the brain by neurons, where it modulates glutamatergic transmission and long-term plasticity processes regulating the intercellular communication between glia and neurons, being its specific receptor CX3CR1 expressed by microglia. In this paper we investigated the role of CX3CL1/CX3CR1 signaling on experience-dependent hippocampal plasticity processes. At this aim wt and CX3CR1GFP/GFP mice were exposed to long-lasting-enriched environment (EE) and the effects on hippocampal functions were studied by electrophysiological recordings of long-term potentiation of synaptic activity, behavioral tests of learning and memory in the Morris water maze paradigm and analysis of neurogenesis in the subgranular zone of the dentate gyrus (DG). We found that CX3CR1 deficiency increases hippocampal plasticity and spatial memory, blunting the potentiating effects of EE. In contrast, exposure to EE increased the number and migration of neural progenitors in the DG of both wt and CX3CR1GFP/GFP mice. These data indicate that CX3CL1/CX3CR1-mediated signaling is crucial for a normal experience-dependent modulation of hippocampal functions.

Anomalous levels of Cl− transporters cause a decrease of GABAergic inhibition in human peritumoral epileptic cortex

Purpose:  Several factors contribute to epileptogenesis in patients with brain tumors, including reduced γ‐aminobutyric acid (GABA)ergic inhibition. In particular, changes in Cl− homeostasis in peritumoral microenvironment, together with alterations of metabolism, are key processes leading to epileptogenesis in patients afflicted by glioma. It has been recently proposed that alterations of Cl− homeostasis could be involved in tumor cell migration and metastasis formation. In neurons, the regulation of intracellular Cl− concentration ([Cl−]i) is mediated by NKCC1 and KCC2 transporters: NKCC1 increases while KCC2 decreases [Cl−]i. Experiments were thus designed to investigate whether, in human epileptic peritumoral cortex, alterations in the balance of NKCC1 and KCC2 activity may decrease the hyperpolarizing effects of GABA, thereby contributing to epileptogenesis in human brain tumors.

Modulation of the neurotransmitter release in rat cerebellar neurons by GROβ

WE report here that, in cultured cerebellar granule cells, the CXC chemokine GRO β stimulates the signaling pathway of the extracellular signal-regulated kinases, and enhances both evoked and spontaneous post-synaptic currents in patch clamped Purkinje neurons from rat cerebellar slices. The GRO β-induced enhancement of the excitatory post-synaptic currents evoked by stimulating the parallel fibres is blocked by the inhibitor of the extracellular signal-regulated kinases pathway PD98059, which also reduces both basal frequency of spontaneous post-synaptic currents and mean amplitude of evoked excitatory post-synaptic currents. Our results suggest that GRO β modulates neurotransmitter release in the cerebellum through the activation of the extra-cellular signal-regulated kinases pathway.