HIV-1 gp120 enhances giant depolarizing potentials via chemokine receptor CXCR4 in neonatal rat hippocampus

In the immature hippocampus, the giant depolarizing potentials (GDPs) are recurrent network-driven synaptic events generated by gamma-aminobutyric acid (GABA), which in neonatal life is depolarizing and excitatory. The GDPs enable a high degree of synchrony in immature neurons and participate in activity-dependent growth and synapse formation. To understand how human immunodeficiency virus type one (HIV-1) infection in the immature brain impairs brain growth and development, we studied the effects of HIV-1 envelope glycoprotein, gp120, a viral toxin shed in abundance by infected cells, on spontaneous occurring GDPs recorded in the CA3 pyramidal cells in neonatal (P2-P6) Sprague-Dawley rat hippocampal slices using whole-cell patch technique. Bath application of gp120 produced a sustained enhancement of GDP frequency in a concentration-dependent manner without affecting passive membrane properties, suggesting that the site of action is most likely on neural network, other than on the recorded neurons. The gp120-induced enhancement of GDPs was blocked by T140, a highly specific antagonist for the chemokine receptor, CXCR4, indicating the involvement of CXCR4 in the gp120-induced increase of GDPs. Bath application of stromal cell-derived factor-1alpha (SDF-1alpha), the only CXCR4 ligand, mimicked the effects of gp120 on GDPs, supporting the engagement of CXCR4 receptors in the gp120-induced increase of GDP occurrence. Further studies revealed the involvement of protein kinase A/C in the gp120-induced enhancement of GDPs. These results demonstrate that gp120 enhances GDPs in the neonatal rat hippocampus. This enhancement may cause an excessive increase in intracellular calcium and resultant neuronal injury, leading to retardation of the brain and behavioural development as seen in paediatric AIDS patients.     

Complex effects of stromal cell-derived factor-1 alpha on melanin-concentrating hormone neuron excitability

Stromal cell-derived factor 1alpha (SDF-1alpha), a chemoattractant for leucocytes and neurons, and its receptor, CXCR4 are expressed in subsets of neurons of specific brain areas. In rat lateral hypothalamic area (LHA) we show, using immunocytochemistry, that CXCR4 is localized within melanin-concentrating hormone (MCH)-expressing neurons, mainly involved in feeding behaviour regulation. We investigated whether SDF-1alpha may control MCH neuronal activity. Patch-clamp recordings in rat LHA slices revealed multiple effects of SDF-1alpha on the membrane potential of MCH neurons, indirect through glutamate/GABA release and direct through GIRK current activation. Moreover, SDF-1alpha at 0.1-1 nM decreased peak and discharge frequency of action potential evoked by current pulses. These effects were further confirmed in voltage-clamp experiments, SDF-1alpha depressing both potassium and sodium currents. At 10 nM, however, SDF-1alpha increased peak and discharge frequency of action potential evoked by current pulses. Using a specific CXCR4 antagonist, we demonstrated that only the depressing effect on AP discharge was mediated through CXCR4 while the opposite effect was indirect. Together, our studies reveal for the first time a direct effect of SDF-1alpha on voltage-dependent membrane currents of neurons in brain slices and suggest that this chemokine may regulate MCH neuron activity.     

Dexamethasone downregulates chemokine receptor CXCR4 and exerts neuroprotection against hypoxia/ischemia-induced brain injury in neonatal rats

OBJECTIVE: Hypoxia/ischemia (H/I) induces rapid and massive brain damage in neonatal rat brain, resulting in long-term consequences on structural and functional maturation of the central nervous system. Inflammatory mediators contribute to these permanent pathological changes, which are sensitive to corticoid treatments. Since the chemokine receptor CXCR4, specific for the SDF-1 alpha/CXCL12 ligand, regulates both apoptotic and neuroregeneration processes, this receptor was quantified 2 days following H/I in neonatal rat brain in relation with dexamethasone (DEX) treatment. METHODS: Seven-day-old male rats were exposed to a 90-min hypoxia following unilateral carotid ligation (H/I) and were sacrificed 48 h later. Glucocorticoid-pretreated animals were injected subcutaneously 5 h prior to hypoxia with 0.5 microg/g DEX. Glial fibrillary acidic protein and cresyl violet staining were used for assessing the extent of brain lesion subdivided into necrotic and penumbra-like areas. The density of CXCR4 receptors was determined by quantitative autoradiography using [(125)I]SDF-1 alpha as a ligand. RESULTS: The H/I resulted in a massive lesion ipsilateral to the carotid ligation, which was extended to cortical, striatal, hippocampal and thalamic areas, while the contralateral hemisphere remained apparently unaffected. DEX decreased the lesion size by reducing mainly the necrotic area. H/I induced a marked increase in CXCR4 receptor binding in the penumbra-like areas. DEX pretreatment decreased CXCR4 receptor density in the penumbra and attenuated astrocytosis. Furthermore, DEX strongly lowered mortality rate and reduced functional recovery time right after hypoxia. CONCLUSION: The rapid enhancement in CXCR4 chemokine receptor binding in the affected brain areas suggests that SDF-1 alpha/CXCR4 may play a role in the hypoxia-induced inflammatory reaction in the neonatal brain. Attenuation of CXCR4 expression and astrogliosis could contribute to the neuroprotective effect of DEX pretreatment via influencing the inflammatory cascade induced by H/I in the neonatal brain.     

Regional and cellular localization of the CXCl12/SDF-1 chemokine receptor CXCR7 in the developing and adult rat brain

The chemokine stromal cell-derived factor-1 (SDF-1) regulates neuronal development via the chemokine receptor CXCR4. In the adult brain the SDF-1/CXCR4 system was implicated in neurogenesis, neuromodulation, brain inflammation, tumor growth, and HIV encephalopathy. Until the recent identification of RDC1/CXCR7 as the second SDF-1 receptor, CXCR4 was considered to be the only receptor for SDF-1. Here we provide the first map of CXCR7 mRNA expression in the embryonic and adult rat brain. At embryonic stages, CXCR7 and CXCR4 were codistributed in the germinative zone of the ganglionic eminences, caudate putamen, and along the routes of GABAergic precursors migrating toward the cortex. In the cortex, CXCR7 was identified in GABAergic precursors and in some reelin-expressing Cajal-Retzius cells. Unlike CXCR4, CXCR7 was abundant in neurons forming the cortical plate and sparse in the developing dentate gyrus and cerebellar external germinal layer. In the adult brain, CXCR7 was expressed by blood vessels, pyramidal cells in CA3, and mature dentate gyrus granule cells, which is reminiscent of the SDF-1 pattern. CXCR7 and CXCR4 overlapped in the wall of the four ventricles. Further neuronal structures expressing CXCR7 comprised the olfactory bulb, accumbens shell, supraoptic and ventromedial hypothalamic nuclei, medial thalamus, and brain stem motor nuclei. Also, GLAST-expressing astrocytes showed signals for CXCR7. Thus, CXCR4 and CXCR7 may cooperate or act independently in SDF-1-dependent neuronal development. In mature neurons and blood vessels CXCR7 appears to be the preponderant SDF-1-receptor.     

HIV-1, chemokines and neurogenesis

HIV-1 infection of the brain results in a large number of behavioural defecits accompanied by diverse neuropathological signs. However,it is not clear how the virus produces these effects or exactly how the neuropathology and behavioural defecits are related. In this article we discuss the possibility that HIV-1 infection may negatively impact the process of neurogenesis in the adult brain and that this may contribute to HIV-1 related effects on the nervous system. We have previously demonstrated that the development of the dentate gyrus during embryogenesis requires signaling by the chemokine SDF-1 via its receptor CXCR4. We demonstrated that neural progenitor cells that give rise to dentate granule neurons express CXCR4 and other chemokine receptors and migrate into the nascent dentate gyrus along SDF-1 gradients. Animals deficient in CXCR4 receptors exhibit a malformed dentate gyrus in which the migration of neural progenitors is stalled. In the adult, neurogenesis continues in the dentate gyrus. Adult neural progenitor cells existing in the subgranlar zone, that produce granule neurons, express CXCR4 and other chemokine receptors, and granule neurons express SDF-1 suggesting that SDF-1/CXCR4 signaling is also important in adult neurogenesis. Because the cellular receptors for HIV-1 include chemokine receptors such as CXCR4 and CCR5 it is possible that the virus may interfere with SDF-1/CXCR4 signaling in the brain including disruption of the formation of new granule neurons in the adult brain.     

Dual role of GABA in the neonatal rat hippocampus

The effects of modulators of GABA-A receptors on neuronal network activity were studied in the neonatal (postnatal days 0-5) rat hippocampus in vitro. Under control conditions, the physiological pattern of activity of the neonatal hippocampal network was characterized by spontaneous network-driven giant depolarizing potentials (GDPs). The GABA-A receptor agonist isoguvacine (1-2 microM) and the allosteric modulator diazepam (2 microM) induced biphasic responses: initially the frequency of GDPs increased 3 to 4 fold followed by blockade of GDPs and desynchronization of the network activity. The GABA-A receptor antagonists bicuculline (10 microM) and picrotoxin (100 microM) blocked GDPs and induced glutamate (AMPA and NMDA)-receptor-mediated interictal- and ictal-like activities in the hippocampal slices and the intact hippocampus. These data suggest that at early postnatal ages GABA can exert a dual - both excitatory and inhibitory - action on the network activity. Copyright Copyright 1999 S. Karger AG, Basel     

Regulation of cell cycle proteins by chemokine receptors: A novel pathway in human immunodeficiency virus neuropathogenesis?

In order to test the hypothesis that alteration of cell cycle proteins are involved in the neuronal damage caused by human immunodeficiency virus (HIV), the authors have been studying the effect of chemokines on the CDK/Rb/E2F-1 pathway--which is involved in neuronal apoptosis and differentiation. First, they have asked whether CXCR4, the specific receptor for the chemokine SDF-1 and X4-using gp120s, can regulate Rb and E2F-1 activity in cultures of differentiated rat neurons. Although CCR3 and CCR5 are known to mediate infection of microglia by HIV-1, recent evidence indicate that CXCR4 also play important roles in HIV-induced neuronal injury, and dual-tropic isolates that use CXCR4 to infect macrophages have recently been reported. The authors have focused on two specific brain areas in which CXCR4 is physiologically relevant, i.e., the cerebellum and the hippocampus. So far, the data indicate that changes in the nuclear and cytosolic levels of Rb, which result in the functional loss of this protein, are associated with apoptosis in these neurons, and that SDF-1alpha and gp120IIIB affect this pathway. A summary of the findings are presented.     

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.     

SDF-1alpha is expressed in astrocytes and neurons in the AIDS dementia complex: an in vivo and in vitro study

Recent in vitro studies suggest that the alpha chemokine stromal-derived factor-1alpha (SDF-1alpha) and its receptor CXCR-4 may contribute to neuronal apoptosis in HIV infection of the brain. The cellular and regional expression of this chemokine and its relationship to the AIDS dementia complex (ADC), however, have remained undetermined. Using immunohistochemistry and semiquantitative RT-PCR, we examined the expression of SDF-1alpha in the frontal cortex (FC), the adjacent deep white matter (DWM). and the basal ganglia (BG) of 17 patients with ADC and 5 normal controls, and the FC and temporal cortex of 6 patients with Alzheimer disease (AD). Additionally, SDF-1alpha expression was studied in 3 different neuronal cultures: differentiated SK-N-MC cells, primary human fetal neuronal, and mouse hippocampal cultures. SDF-1alpha staining was predominantly localized to astrocytes in all 3 groups in the gray matter of the FC and the BG, often in the vicinity of cortical and basal ganglia neurons, but was generally absent in the DWM. Further, the number of positive neurons was significantly greater in the BG of AIDS subjects with advanced brain disease compared to subjects with lesser disease (p = 0.029). All cultures showed prominent SDF-1alpha staining of neurons within the cytoplasm and in neurites, whereas preferential expression in GABA-ergic neurons was found in hippocampal cultures. This is the first study to show that SDF-1alpha is constitutively expressed in astrocytes of the deep and cortical gray matter as well as in neurons of the human brain. Its increased expression in basal ganglia neurons of patients with advanced HIV CNS disease suggests it may also contribute to pathogenesis.     

Neuroanatomical distribution of CXCR4 in adult rat brain and its localization in cholinergic and dopaminergic neurons

Accumulating evidence supports a role of chemokines and their receptors in brain function. Up to now scarce evidence has been given of the neuroanatomical distribution of chemokine receptors. Although it is widely accepted that chemokine receptors are present on glial cells, especially in pathological conditions, it remains unclear whether they are constitutively present in normal rat brain and whether neurons have the potential to express such chemokine receptors. CXCR4, a G protein-coupled receptor for the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) was reported to have possible implications in brain development and AIDS-related dementia. By dual immunohistochemistry on brain sections, we clearly demonstrate that CXCR4 is constitutively expressed in adult rat brain, in glial cells (astrocytes, microglia but not oligodendrocytes) as well as in neurons. Neuronal expression of CXCR4 is mainly found in cerebral cortex, caudate putamen, globus pallidus, substantia innominata, supraoptic and paraventricular hypothalamic nuclei, ventromedial thalamic nucleus and substantia nigra. Using confocal microscopy, a differential distribution of CXCR4 in neuronal perikarya and dendrites can be observed according to the brain structure. Furthermore, this work demonstrates for the first time the coexistence of a chemokine receptor with classical neurotransmitters. A localization of CXCR4 is thus observed in neuronal cell bodies expressing choline acetyltransferase-immunoreactivity in the caudate putamen and substantia innominata, as well as in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. In conclusion, the constitutive neuronal CXCR4 expression suggests that SDF-1/CXCL12 could be involved in neuronal communication and possibly linked up with cholinergic and dopaminergic neurotransmission and related disorders.     

Highly regionalized distribution of stromal cell-derived factor-1/CXCL12 in adult rat brain: constitutive expression in cholinergic, dopaminergic and vasopressinergic neurons

The stromal cell-derived factor-1 (SDF-1)/CXCL12 and its receptor CXCR4 are key modulators of immune functions. In the nervous system, SDF-1/CXCL12 is crucial for neuronal guidance in developing brain, intercellular communication and the neuropathogenesis of acquired immunodeficiency syndrome. However, cerebral functions of SDF-1/CXCL12 in adult brain are poorly understood. The understanding of its role in the adult brain needs a detailed neuroanatomical mapping of SDF-1/CXCL12. By dual immunohistochemistry we demonstrate that this chemokine is constitutively expressed not only in astrocytes and microglia but also in neurons, in discrete neuroanatomical regions. Indeed, neuronal expression of SDF-1/CXCL12 is mainly found in cerebral cortex, substantia innominata, globus pallidus, hippocampus, paraventricular and supraoptic hypothalamic nuclei, lateral hypothalamus, substantia nigra and oculomotor nuclei. Moreover, we provide the first evidence that SDF-1/CXCL12 is constitutively expressed in cholinergic neurons in the medial septum and substantia innominata and in dopaminergic neurons in substantia nigra pars compacta and the ventral tegmental area. Interestingly we also show, for the first time, a selective co-localization of SDF-1/CXCL12 with vasopressin-expressing neurons in the supraoptic and paraventricular hypothalamic nuclei. In addition, in the lateral hypothalamic area, SDF-1/CXCL12 was found to be located on melanin concentrating hormone-expressing neurons. Altogether, these original data suggest that SDF-1/CXCL12 could be a modulatory neuropeptide regulating both central cholinergic and dopaminergic systems. In addition, a key role for SDF-1/CXCL12 in neuroendocrine regulation of vasopressin-expressing neurons represents an exciting new field of research.     

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.     

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.     

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.     

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.     

Mice deficient in the chemokine receptor CXCR4 exhibit impaired limb innervation and myogenesis

The chemokine CXCL12/SDF-1 and its receptor CXCR4 regulate the development and the function of the hematopoietic system and control morphogenesis of distinct brain areas. Here, we demonstrate that inactivation of CXCR4 results in a massive loss of spinal cord motoneurons and dorsal root ganglion neurons and, subsequently, in a reduced innervation of the developing mouse fore- and hindlimbs. However, only the death of sensory neurons seems to be a direct consequence of receptor inactivation as suggested by the observations that DRG neurons, but not motoneurons, of wild-type animals express CXCR4 and respond to CXCL12 with an increase in cell survival. In contrast, the increased death of motoneurons in CXCR4-deficient animals seems to result from impaired limb myogenesis and a subsequent loss of muscle-derived neurotrophic support. In summary, our findings unravel a previously unrecognized complex role of CXCL12/CXCR4 in the control of limb neuromuscular development.     

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.