Stimulation of chemokine CXC receptor 4 induces synaptic depression of evoked parallel fibers inputs onto Purkinje neurons in mouse cerebellum

In the present work, we studied the effects of the stimulation of the chemokine CXC receptor 4 (CXCR4) by the stromal-derived cell growth factor-1alpha (SDF-1alpha) on the evoked excitatory postsynaptic current. This was generated in Purkinje neurons (PN) from mouse cerebellar slices by the stimulation of parallel fibers. It was found that the amplitude of EPSC was reversibly reduced by SDF-1alpha application. This effect was dose-dependent (IC(50)=0.34 nM) and was abolished by the anti-CXCR4 monoclonal antibody (mAb) 12G5. This SDF-1alpha-induced synaptic depression was caused by a decrease of evoked glutamate release, rather than a decrease in the postsynaptic glutamate receptor (GluR) sensitivity, as the mean amplitude of the spontaneous EPSCs was not influenced by chemokine application. Moreover, NMDA receptors (NMDARs) are involved in EPSC depression being inhibited by the NMDAR blocker 2-amino-5-phosphonopentanoic acid (AP-5). The mechanisms by which SDF-1alpha modulates neurotransmission in the cerebellar cortex are discussed.     

Modulation of network-driven, GABA-mediated giant depolarizing potentials by SDF-1alpha in the developing hippocampus

Chemokine stromal cell-derived factor-1 (SDF-1, or CXCL12) plays an important role in brain development and functioning. Whole-cell patch clamp recordings were conducted on CA3 neurons in hippocampal slices prepared from neonatal rats between postnatal days 2 and 6 to study the modulatory effects of SDF-1alpha on network-driven, gamma-aminobutyric-acid-mediated giant depolarizing potentials (GDPs), a hallmark of the developing hippocampus. We found that SDF-1alpha, the only natural ligand for chemokine CXC motif receptor 4 (CXCR4), decreased GDP firing without significant effects on neuronal passive membrane properties in neonatal hippocampal neurons. The SDF-1alpha-mediated decrease in GDP firing was blocked by T140, a CXCR4 receptor antagonist, suggesting that SDF-1alpha modulates GDP firing via CXCR4. We also showed that endogenous SDF-1 exerts a tonic inhibitory action on GDPs in the developing hippocampus. As SDF-1/CXCR4 are highly expressed in the developing brain and GDPs are involved in activity-dependent synapse formation and functioning, the inhibitory action of SDF-1alpha on GDPs may reflect a potential mechanism for chemokine regulation of neural development in early neonatal life.     

Stromal cell-derived factor 1alpha mediates neural progenitor cell motility after focal cerebral ischemia.

In the adult rodent, stroke induces an increase in endogenous neural progenitor cell (NPC) proliferation in the subventricular zone (SVZ) and neuroblasts migrate towards the ischemic boundary. We investigated the role of stromal cell-derived factor 1alpha (SDF-1alpha) in mediating NPC migration after stroke. We found that cultured NPCs harvested from the normal adult SVZ, when they were overlaid onto stroke brain slices, exhibited significantly (P<0.01) increased migration (67.2+/-25.2 microm) compared with the migration on normal brain slices (29.5+/-29.5 microm). Immunohistochemistry showed that CXCR 4, a receptor of SDF-1alpha, is expressed in the NPCs and migrating neuroblasts in stroke brain. Blocking SDF-1alpha by a neutralizing antibody against CXCR 4 significantly attenuated stroke-enhanced NPC migration. ELISA analysis revealed that SDF-1alpha levels significantly increased (P<0.01) in the stroke hemisphere (43.6+/-6.5 pg/mg) when compared with the normal brain (25.2+/-1.9 pg/mg). Blind-well chamber assays showed that SDF-1alpha enhanced NPC migration in a dose-dependent manner with maximum migration at a dose of 500 ng/mL. In addition, SDF-1alpha induced directionally selective migration. These findings show that SDF-1alpha generated in the stroke hemisphere may guide NPC migration towards the ischemic boundary via binding to its receptor CXCR 4 in the NPC. Thus, our data indicate that SDF-1alpha/CXCR 4 is important for mediating specific migration of NPCs to the site of ischemic damaged neurons.     

Characterization and visualization of [125I] stromal cell-derived factor-1alpha binding to CXCR4 receptors in rat brain and human neuroblastoma cells

Stromal cell-Derived Factor-1 (SDF-1alpha), binds to the seven-transmembrane G protein-coupled CXCR4 receptor and modulates cell migration, differentiation, and proliferation. CXCR4 has been reported to be expressed in various tissues including brain. Moreover, CXCR4 has recently been shown to be one of the coreceptors for HIV-1 infection which could be implicated in HIV encephalitis. In the present study, the binding properties and autoradiographic distribution of [125I]SDF-1alpha binding to CXCR4 were characterized in the adult rat brain. SDF-1alpha binding and CXCR4 coupling system were also studied in human neuroblastoma cell line SK-N-SH. The binding of [125I]SDF-1alpha on rat brain sections was specific, time-dependent and reversible. The highest densities of CXCR4 were detected in the choroid plexus of the lateral and the dorsal third ventricle. Lower densities of [125I]SDF-1alpha binding sites were observed in various brain regions including cerebral cortex, anterior olfactory nuclei, hippocampal formation, thalamic nuclei, blood vessels and pituitary gland. In the choroid plexus, the IC(50) and K(d) of [125I]SDF-1alpha binding were respectively 0.6 nM and 0. 36 nM. Similar IC(50) values were obtained in other brain structures. A CXCR4 antagonist, bicyclam, competed with SDF-1alpha binding (30% inhibition at 10(-6) M). In SK-N-SH cells, [125I]SDF-1alpha bound to CXCR4 with a K(d) of 5.0 nM and a maximal binding capacity of 460 fmol/mg of protein. SDF-1alpha induced a rapid and transient intracellular calcium increase in SK-N-SH cells. These findings suggest that CXCR4 is highly expressed in some brain structures and have a regulatory role in the nervous system. The significance of this expression in the brain parenchyma and more specifically in the choroid plexus remains to be clarified in the normal as well as in the infected brain.     

Intracellular CXCR4 signaling, neuronal apoptosis and neuropathogenic mechanisms of HIV-1-associated dementia.

The mechanism(s) by which HIV-1 affects neural injury in HIV-1-associated dementia (HAD) remains unknown. To ascertain the role that cellular and viral macrophage products play in HAD neurotoxicity, we explored one potential route for neuronal demise, CXCR4. CXCR4, expressed on lymphocytes and neurons, is both a part of neural development and a co-receptor for HIV-1. Its ligand, stromal cell-derived factor-1alpha (SDF-1alpha), affects neuronal viability. GTP binding protein (G-protein) linked signaling after neuronal exposure to SDF-1alpha, virus-infected monocyte-derived macrophage (MDM) secretory products, and virus was determined. In both human and rat neurons, CXCR4 was expressed at high levels. SDF-1alpha/beta was detected predominantly in astrocytes and at low levels in MDM. SDF-1beta/beta was expressed in HAD brain tissue and upregulated in astrocytes exposed to virus infected and/or immune activated MDM conditioned media (fluids). HIV-1-infected MDM secretions, virus and SDF-1beta induced a G inhibitory (Gi) protein-linked decrease in cyclic AMP (cAMP) and increase inositol 1,4, 5-trisphosphate (IP3) and intracellular calcium. Such effects were partially blocked by antibodies to CXCR4 or removal of virus from MDM fluids. Changes in G-protein-coupled signaling correlated, but were not directly linked, to increased neuronal synaptic transmission, Caspase 3 activation and apoptosis. These data, taken together, suggest that CXCR4-mediated signal transduction may be a potential mechanism for neuronal dysfunction during HAD.     

Electrophysiologic properties of red nucleus neurons in the rat brain slices]

Intracellular recordings from the red nucleus (RN) neurons were made in experiments on the rat brain slices. Passive membrane properties (input resistance and membrane time constant) of the RN neurons were evaluated. Phenomena of potential-dependent rebound depolarization and time-dependent inward rectification were revealed by means of passing hyperpolarizing current pulses through the recorded cells. Injections of depolarizing currents caused repetitive firing of neurons with frequencies directly depending on the intensity of injected currents. Repetitive firing was also characterized by a fast frequency adaptation during injections of currents of different intensities. Stimulation of a region of slices presumably corresponding to the decussation of brachium conjunctivum evoked mainly monosynaptic EPSPs with a "fast"-rise time in the RN neurons, which suggests activation of the synaptic input from the cerebellar nucleus interpositus. Stimulation of the same region sometimes evoked EPSP-IPSP mixtures or pure IPSPs in the RN neurons.     

Regulation of calcium currents by chemokines and their receptors

We investigated the modulation of voltage dependent Ca(2+) currents by chemokine receptors in heterologous expression systems and neurons. Fractalkine, SDF-1alpha, RANTES and MDC inhibited the I(Ba) in CX3CR1-, CXCR4-, CCR5- and CCR4-expressing G1A1 cells, respectively. The I(Ba) inhibition was voltage-dependent, exhibited prepulse facilitation, and was blocked by N-ethylmaleimide and pertussis toxin pretreatment, indicating that it was mediated by Gi/Go. Some chemokines also inhibited the I(Ba) in subpopulations of dorsal root ganglion neurons and area postrema/nucleus tractus solitarius neurons. These data provide evidence that chemokines can potentially modulate neuronal signaling through the inhibition of neuronal Ca(2+) currents.     

Stromal-derived factor 1 signalling regulates radial and tangential migration in the developing cerebral cortex

Stromal-derived factor 1 (SDF-1), a known chemoattractant, and its receptor CXCR4 are widely expressed in the developing and adult cerebral cortex. Recent studies have highlighted potential roles for SDF-1 during early cortical development. In view of the current findings, our histological analysis has revealed a distinct pattern of SDF-1 expression in the developing cerebral cortex at a time when cell proliferation and migration are at peak. To determine the role of chemokine signalling during early cortical development, embryonic rat brain slices were exposed to a medium containing secreted SDF-1 to perturb the endogenous levels of chemokine. Alternatively, brain slices were treated with 40 muM of T140 or AMD3100, known antagonists of CXCR4. Using these experimental approaches, we demonstrate that chemokine signalling is imperative for the maintenance of the early cortical plate. In addition, we provide evidence that both neurogenesis and radial migration are concomitantly regulated by this signalling system. Conversely, interneurons, although not dependent on SDF-1 signalling to transgress the telencephalic boundary, require the chemokine to maintain their tangential migration. Collectively, our results demonstrate that SDF-1 with its distinct pattern of expression is essential and uniquely positioned to regulate key developmental events that underlie the formation of the cerebral cortex.     

Stromal-cell-derived factor 1alpha /CXCL12 modulates high-threshold calcium currents in rat substantia nigra

Dopaminergic neurons of the substantia nigra constitutively express the CXCR4 receptor for the chemokine stromal-cell-derived factor 1α (CXCL12) but, to date, no direct effect of CXCR4 activation by CXCL12 on membrane conductance of dopaminergic neurons has been demonstrated. We tested the effects of CXCL12 on whole-cell currents of dopaminergic neurons recorded in patch clamp in substantia nigra slices and showed that CXCL12 (0.01–10 n m ) increased the amplitude of total high-voltage-activated (HVA) Ca currents through CXCR4 activation. This effect was reversibly reduced by ϖ-conotoxin-GVIA, suggesting that CXCL12 acted on N-type Ca currents, known to be involved in dopamine (DA) release. We therefore investigated the effects of CXCL12 on DA release from cultured dopaminergic neurons from the rat mesencephalon. In basal conditions, CXCL12 alone had no effect on DA release. When neurons were depolarized with KCl (20 m m ), and thus when HVA Ca currents were activated, low CXCL12 concentrations (1–50 n m ) increased DA release via CXCR4 stimulation. These data strongly suggest that the chemokine CXCL12 can act directly as a neuromodulator of dopaminergic neuronal electrical activity through the modulation of HVA currents.     

Regulation of neuronal P53 activity by CXCR 4

Abnormal activation of CXCR 4 during inflammatory/infectious states may lead to neuronal dysfunction or damage. The major goal of this study was to determine the coupling of CXCR 4 to p53-dependent survival pathways in primary neurons. Neurons were stimulated with the HIV envelope protein gp120(IIIB) or the endogenous CXCR 4 agonist, SDF-1 alpha. We found that gp120 stimulates p53 activity and induces expression of the p53 pro-apoptotic target Apaf-1 in cultured neurons. Inhibition of CXCR 4 by AMD 3100 abrogates the effect of gp120 on both p53 and Apaf-1. Moreover, gp120 neurotoxicity is markedly reduced by the p53-inhibitor, pifithrin-alpha. The viral protein also regulates p53 phosphorylation and expression of other p53-responsive genes, such as MDM 2 and p21. Conversely, SDF-1 alpha, which can promote neuronal survival, increases p53 acetylation and p21 expression in neurons. Thus, the stimulation of different p53 targets could be instrumental in determining the outcome of CXCR 4 activation on neuronal survival in neuro-inflammatory disorders.     

Exposure of nicotine to ventral tegmental area slices induces glutamatergic synaptic plasticity on dopamine neurons

Nicotine promotes glutamatergic synaptic plasticity in dopaminergic (DA) neurons in the ventral tegmental area (VTA), which is thought to be an important mechanism underlying nicotine reward. However, it is unclear whether exposure of nicotine alone to VTA slice is sufficient to increase glutamatergic synaptic strength on DA neurons and which nicotinic acetylcholine receptor (nAChR) subtype mediates this effect. Here, we report that the incubation of rat VTA slices with 500 nM nicotine induces glutamatergic synaptic plasticity in DA neurons. We measure the ratio of AMPA and NMDA receptor‐mediated currents (AMPA/NMDA) and compare these ratios between nicotine‐treated and ‐untreated slices. Our results demonstrate that the incubation of VTA slices with 500 nM nicotine for 1 h (but not for 10 min) significantly increases the AMPA/NMDA ratio when compared with controls. Preincubation with 10 nM of the α7‐nAChR antagonist, methyllycaconitine (MLA) but not 1 μM α4‐containing nAChR antagonist, dihydro‐β‐erythroidine (DHβE) prevents nicotinic effect, suggesting that α7‐nAChRs are mainly mediated this nicotinic effect. This finding is further supported by the disappearance of this nicotinic effect in nAChR α7 knockout (KO) mice. Furthermore, nicotine reduced paired‐pulse ratio (PPR) of evoked excitatory postsynaptic potential (eEPSP) in the VTA slices prepared from wild‐type (WT) mice but not α7 KO mice. Collectively, these findings suggest that exposure of smoking‐relevant concentrations of nicotine to VTA slices is sufficient to increase glutamatergic synaptic strength on DA neurons and that α7‐nAChRs likely mediate this nicotinic effect through increasing presynaptic release of glutamate. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

Stromal cell-derived factor 1-mediated CXCR4 signaling in rat and human cortical neural progenitor cells

Stromal cell-derived factor 1 (SDF-1) and the chemokine receptor CXCR4 are highly expressed in the nervous system. Knockout studies have suggested that both SDF-1 and CXCR4 play essential roles in cerebellar, hippocampal, and neocortical neural cell migration during embryogenesis. To extend these observations, CXCR4 signaling events in rat and human neural progenitor cells (NPCs) were examined. Our results show that CXCR4 is expressed in abundance on rat and human NPCs. Moreover, SDF-1alpha induced increased NPCs levels of inositol 1,4,5-triphosphate, extracellular signal-regulated kinases 1/2, Akt, c-Jun N-terminal kinase, and intracellular calcium whereas it diminished cyclic adenosine monophosphate. Finally, SDF-1alpha can induce human NPC chemotaxis in vitro, suggesting that CXCR4 plays a functional role in NPC migration. Both T140, a CXCR4 antagonist, and pertussis toxin (PTX), an inactivator of G protein-coupled receptors, abrogated these events. Ultimately, this study suggested that SDF-1alpha can influence NPC function through CXCR4 and that CXCR4 is functional on NPC.     

The effects of QX-314 on medullary respiratory neurones

The synaptic and current-evoked responses of respiratory neurones located in the nucleus of the tractus solitarius, the para- and retroambigual regions and the nucleus ambiguus, were examined after voltage-dependent sodium currents were blocked by intracellular application of the quaternary lidocaine derivative QX-314. (1) QX-314 abolished orthodromically and antidromically evoked action potential discharge. Only antidromic action potentials recovered during negative DC current injection. (2) QX-314 did not alter the amplitude or duration of small and short excitatory and inhibitory postsynaptic potentials evoked by vagus or superior laryngeal nerve stimulation. Larger and longer waves of spontaneous membrane depolarizations, however, were slightly diminished. (3) The repetitive discharge evoked by depolarizing current pulses was blocked by QX-314. Positive current pulses produced less membrane depolarization than under control and often evoked only a single action potential at the beginning of the pulse, indicating that QX-314 interferes with the processes responsible for repetitive firing. (4) When fast spike discharges were completely blocked, positive current pulses occasionally evoked depolarizing 'spikes' and potentials which were followed by a hyperpolarization. We conclude that a noninactivating sodium inward current and calcium currents contribute to the electroresponsiveness of respiratory neurones.     

Stromal derived growth factor-1alpha as a beacon for stem cell homing in development and injury

This review extrapolates the functions of SDF-1alpha and its receptor, CXCR4, as regulators of hematopoietic stem cells and discusses their potential roles in the development and regeneration of tissues. The discussion focuses on the repair of neural tissues while parallels are made with bone marrow hematopoietic stem cells. Overall, the organization links the basic biology of SDF-1alpha and CXCR4 to topics in medicine and show how any disease processes involving the SDF-1alpha-CXCR4 system could be central points in medicine. Discussions focused on potential therapies for SDF-1 and CXCR4 in clinical disorders. Breast and prostate cancers are selected as examples of solid tumors while leukemia is discussed as an example of hematological malignancies. Diffuse macular edema is discussed as potential therapy for a non-malignant disease.     

Dendrite-selective redistribution of the chemokine receptor CXCR4 following agonist stimulation

The chemokine SDF-1 is a secreted protein that plays a critical role in several aspects of neuron development through interaction with its unique receptor CXCR4. A key mechanism that controls neuron responsiveness to extracellular signals during neuronal growth is receptor endocytosis. Since we previously reported that SDF-1 regulates axon development without affecting the other neurites, we asked whether this could correlate with a compartment-selective trafficking of CXCR4. We thus studied CXCR4 behavior upon SDF-1 exposure in rat hippocampus slices and in transfected neuron cultures. A massive agonist-induced redistribution of CXCR4 in endosomes was observed in dendrites whereas no modification was evidenced in axons. Our data suggest that CXCR4 trafficking may play a role in mediating selective effects of SDF-1 on distinct neuronal membrane subdomains.     

SDF-1alpha-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-1alpha (SDF-1alpha) 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-1alpha, both in cell bodies and in neuronal processes. Whole-cell patch-clamped PNs in cerebellar slices responded to SDF-1alpha application by a slow inward current followed by an increase of both intracellular Ca2+ level and spontaneous synaptic activity. In particular, the SDF-1alpha-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.     

High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells

Neural precursor cells (NPCs) are self-renewing, multipotent progenitors that give rise to neurons, astrocytes and oligodendrocytes in the central nervous system (CNS). Fetal NPCs have attracted attention for their potential use in studying normal CNS development. Several studies of rodent neural progenitors have suggested that chemokines and their receptors are involved in directing NPC migration during CNS development. In this study, we established a consistent system to culture human NPCs and examined the expression of chemokine receptors on these cells. NPCs were found to express the markers nestin and CD133 and to differentiate into neurons, astrocytes and oligodendrocytes at the clonal level. Flow cytometry and RNase protection assay (RPA) indicated that NPCs express high levels of CXCR4 and low levels of several other chemokine receptors. When examined using a chemotaxis assay, NPCs were able to respond to CXCL12/SDF-1alpha, a ligand of CXCR4. Treatment with anti-CXCR4 antibody or HIV-1 gp120 abolished the migratory response of NPCs towards CXCL12/SDF-1alpha. These findings suggest that CXCR4 may play a significant role in directing NPC migration during CNS development.