CXCR4 prevents dispersion of granule neuron precursors in the adult dentate gyrus

Neurogenesis in the adult dentate gyrus (DG) generates new granule neurons that differentiate in the inner one‐third of the granule cell layer (GCL). The migrating precursors of these neurons arise from neural stem cells (NSCs) in the subgranular zone (SGZ). Although it is established that pathological conditions, including epilepsy and stroke, cause dispersion of granule neuron precursors, little is known about the factors that regulate their normal placement. Based on the high expression of the chemokine CXCL12 in the adult GCL and its role in guiding neuronal migration in development, we addressed the function of the CXCL12 receptor CXCR4 in adult neurogenesis. Using transgenic reporter mice, we detected Cxcr4‐GFP expression in NSCs, neuronal‐committed progenitors, and immature neurons of adult and aged mice. Analyses of hippocampal NSC cultures and hippocampal tissue by immunoblot and immunohistochemistry provided evidence for CXCL12‐promoted phosphorylation/activation of CXCR4 receptors in NSCs in vivo and in vitro. Cxcr4 deletion in NSCs of the postnatal or mature DG using Cre technology reduced neurogenesis. Fifty days after Cxcr4 ablation in the mature DG, the SGZ showed a severe reduction of Sox2‐positive neural stem/early progenitor cells, NeuroD‐positive neuronal‐committed progenitors, and DCX‐positive immature neurons. Many immature neurons were ectopically placed in the hilus and inner molecular layer, and some developed an aberrant dendritic morphology. Only few misplaced cells survived permanently as ectopic neurons. Thus, CXCR4 signaling maintains the NSC pool in the DG and specifies the inner one‐third of the GCL as differentiation area for immature granule neurons. © 2013 Wiley Periodicals, Inc.     

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.     

The HIV-1 coat protein gp120 regulates CXCR4-mediated signaling in neural progenitor cells

We demonstrate that hCD4-primed gp120IIIB interacts with CXCR4 receptors expressed by postnatal mouse neural progenitor cells and elicits robust Ca²⁺ signals. The chemokine SDF-1 acted as a chemoattractant and a mitogenic stimulus for these neural progenitor cells. Although hCD4/gp120 was not able to produce chemoattraction or increase proliferaton, it completely blocked the ability of SDF-1 to produce these effects. Thus, gp120 can act both as an agonist and de facto antagonist of CXCR4-mediated signaling in neural progenitor cells. It is possible that the ability of hCD4/gp120 to block SDF-1 signaling in neural progenitors may contribute to the neuropathological effects of HIV-1.     

The role of CXCR4 signaling in the migration of transplanted oligodendrocyte progenitors into the cerebral white matter

Enhancing the ability of either endogenous or transplanted oligodendrocyte progenitors (OPs) to engage in myelination may constitute a novel therapeutic approach to demyelinating diseases of the brain. It is known that in adults neural progenitors situated in the subventricular zone of the lateral ventricle (SVZ) are capable of generating OPs which can migrate into white matter tracts such as the corpus callosum (CC). We observed that progenitor cells in the SVZ of adult mice expressed CXCR4 chemokine receptors and that the chemokine SDF-1/CXCL12 was expressed in the CC. We therefore investigated the role of chemokine signaling in regulating the migration of OPs into the CC following their transplantation into the lateral ventricle. We established OP cell cultures from Olig2-EGFP mouse brains. These cells expressed a variety of chemokine receptors, including CXCR4 receptors. Olig2-EGFP OPs differentiated into CNPase-expressing oligodendrocytes in culture. To study the migratory capacity of Olig2-EGFP OPs in vivo, we transplanted them into the lateral ventricles of mice. Donor cells migrated into the CC and differentiated into mature oligodendrocytes. This migration was enhanced in animals with Experimental Autoimmune Encephalomyelitis (EAE). Inhibition of CXCR4 receptor expression in OPs using shRNA inhibited the migration of transplanted OPs into the white matter suggesting that their directed migration is regulated by CXCR4 signaling. These findings indicate that CXCR4 mediated signaling is important in guiding the migration of transplanted OPs in the context of inflammatory demyelinating brain disease.     

Postnatal cellular contributions of the hippocampus subventricular zone to the dentate gyrus, corpus callosum, fimbria, and cerebral cortex

The rodent dentate gyrus (DG) is formed in the embryo when progenitor cells migrate from the dentate neuroepithelium to establish a germinal zone in the hilus and a secondary germinal matrix, near the fimbria, called the hippocampal subventricular zone (HSVZ). The developmental plasticity of progenitors within the HSVZ is not well understood. To delineate the migratory routes and fates of progenitors within this zone, we injected a replication-incompetent retrovirus, encoding the enhanced green fluorescent protein (EGFP), into the HSVZ of postnatal day 5 (P5) mice. Between P6 and P45, retrovirally-infected EGFP(+) of progenitors migrated into the DG, established a reservoir of progenitor cells, and differentiated into neurons and glia. By P6-7, EGFP(+) cells were observed migrating into the DG. Subsets of these EGFP(+) cells expressed Sox2 and Musashi-1, characteristic of neural stem cells. By P10, EGFP(+) cells assumed positions within the DG and expressed immature neuronal markers. By P20, many EGFP(+) cells expressed the homeobox prospero-like protein Prox1, an early and specific granule cell marker in the CNS, and extended mossy fiber projections into the CA3. A subset of non-neuronal EGFP(+) cells in the dentate gyrus acquired the morphology of astrocytes. Another subset included EGFP(+)/RIP(+) oligodendrocytes that migrated into the fimbria, corpus callosum, and cerebral cortex. Retroviral injections on P15 labeled very few cells, suggesting depletion of HSVZ progenitors by this age. These findings suggest that the early postnatal HSVZ progenitors are multipotent and migratory, and contribute to both dentate gyrus neurogenesis as well as forebrain gliogenesis.     

Expression pattern of CXCR3, CXCR4, and CCR3 chemokine receptors in the developing human brain

Chemokine receptors are essential components of the immune and central nervous systems, but little is known about their distribution during development. We evaluated the distribution of 3 chemokine receptors: CXCR3, CXCR4, and CCR3 in the human developing brain. Of these, CXCR3 was the only receptor expressed in fetal brain at 26 wk of gestation and its expression was restricted to glial cells, endothelial cells, and the choroid plexus. Neuronal staining was only seen at term in the Purkinje cells of the cerebellum. CCR3 appeared only at term in both neurons and glial cells. The expression pattern of these 2 receptors in the late gestation and term resembled that of adults. CXCR4 could not be detected in the fetal brain on neurons nor on glial cells. By examining pediatric cases, we determined that CXCR4 expression commences sometimes between 3.5 and 4.5 yr. Two of the chemokine receptors examined, CCR3 and CXCR4, can be used as co-receptor together with CD4 for HIV entry, but neither was expressed during the second trimester of pregnancy. Our findings suggest that it is unlikely that CCR3 or CXCR4 play a major role in HIV-1 transmission in the fetal brain before 37 wk of gestation.     

Identification of a sustained neurogenic zone at the dorsal surface of the adult mouse hippocampus and its regulation by the chemokine SDF‐1

We identified a previously unknown neurogenic region at the dorsal surface of the hippocampus; (the “subhippocampal zone,” SHZ) in the adult brain. Using a reporter mouse in which SHZ cells and their progeny could be traced through the expression of EGFP under the control of the CXCR4 chemokine receptor promoter we observed the presence of a pool of EGFP expressing cells migrating in direction of the dentate gyrus (DG), which is maintained throughout adulthood. This population appeared to originate from the SHZ where cells entered a caudal migratory stream (aCMS) that included the fimbria, the meninges and the DG. Deletion of CXCR4 from neural stem cells (NSCs) or neuroinflammation resulted in the appearance of neurons in the DG, which were the result of migration of NSCs from the SHZ. Some of these neurons were ectopically placed. Our observations indicate that the SHZ is a neurogenic zone in the adult brain through migration of NSCs in the aCMS. Regulation of CXCR4 signaling in these cells may be involved in repair of the DG and may also give rise to ectopic granule cells in the DG in the context of neuropathology. © 2015 Wiley Periodicals, Inc.     

CXCL12‐mediated feedback from granule neurons regulates generation and positioning of new neurons in the dentate gyrus

Adult hippocampal neurogenesis is implicated in learning and memory processing. It is tightly controlled at several levels including progenitor proliferation as well as migration, differentiation and integration of new neurons. Hippocampal progenitors and immature neurons reside in the subgranular zone (SGZ) and are equipped with the CXCL12‐receptor CXCR4 which contributes to defining the SGZ as neurogenic niche. The atypical CXCL12‐receptor CXCR7 functions primarily by sequestering extracellular CXCL12 but whether CXCR7 is involved in adult neurogenesis has not been assessed. We report that granule neurons (GN) upregulate CXCL12 and CXCR7 during dentate gyrus maturation in the second postnatal week. To test whether GN‐derived CXCL12 regulates neurogenesis and if neuronal CXCR7 receptors influence this process, we conditionally deleted Cxcl12 and Cxcr7 from the granule cell layer. Cxcl12 deletion resulted in lower numbers, increased dispersion and abnormal dendritic growth of immature GN and reduced neurogenesis. Cxcr7 ablation caused an increase in progenitor proliferation and progenitor numbers and reduced dispersion of immature GN. Thus, we provide a new mechanism where CXCL12‐signals from GN prevent dispersion and support maturation of newborn GN. CXCR7 receptors of GN modulate the CXCL12‐mediated feedback from GN to the neurogenic niche.     

Cux2 activity defines a subpopulation of perinatal neurogenic progenitors in the hippocampus

The hippocampus arises from the medial region of the subventricular (SVZ) within the telencephalon. It is one of two regions in the postnatal brain that harbors neural progenitors (NPs) capable of giving rise to new neurons. Neurogenesis in the hippocampus is restricted to the subgranular zone (SGZ) of the dentate gyrus (DG) where it contributes to the generation of granule cell layer (gcl) neurons. It is thought that SGZ progenitors are heterogeneous, differing in their morphology, expression profiles, and developmental potential, however it is currently unknown whether they display differences in their developmental origins and cell fate‐restriction in the DG. Here we demonstrate that Cux2 is a marker for SGZ progenitors and nascent granule cell neurons in the perinatal brain. Cux2 was expressed in the presumptive hippocampal forming region of the embryonic forebrain from E14.5 onwards. At fetal stages, Cux2 was expressed in early‐forming Prox1⁺ granule cell neurons as well as the SVZ of the DG germinal matrix. In the postnatal brain, Cux2 was expressed in several types of progenitors in the SGZ of the DG, including Nestin/Sox2 double‐positive radial glia, Sox2⁺ cells that lacked a radial glial process, DCX⁺ neuroblasts, and Calretinin‐expressing nascent neurons. Another domain characterized by a low level of Cux2 expression emerged in Calbindin⁺ neurons of the developing DG blades. We used Cux2‐Cre mice in genetic fate‐mapping studies and showed almost exclusive labeling of Calbindin‐positive gcl neurons, but not in any progenitor cell types or astroglia. This suggests that Cux2⁺ progenitors directly differentiate into gcl neurons and do not self‐renew. Interestingly, developmental profiling of cell fate revealed an outside‐in formation of gcl neurons in the DG, likely reflecting the activity of Cux2 in the germinative matrices during DG formation and maturation. However, DG morphogenesis proceeded largely normally in hypomorphic Cux2 mutants lacking Cux2 expression. Taken together we conclude that Cux2 expression reflects hippocampal neurogenesis and identifies non‐self‐renewing NPs in the SGZ. © 2014 The Authors Hippocampus Published by Wiley Periodicals, Inc.     

Chemokine receptors and mechanisms of cell death in HIV neuropathogenesis

Several chemokine receptors are used as coreceptors for HIV-1 entry in the central nervous system (CNS). CCR5 is the major coreceptor together with CD4 for HIV-1 infection of microglia, the major target cells for HIV-1 infection in the CNS. CXCR4 and CCR3 are also expressed on microglia and can mediate infection by certain HIV-1 isolates but at lower efficiency than CCR5. Additional chemokine coreceptors are expressed in the brain, but their role in HIV-1 neuropathogenesis has not been defined. The expression of CXCR4, and possibly other chemokine receptors, on subpopulations of neurons and glial cells may render neurons vulnerable to mechanisms of CNS injury induced by the HIV-1 gp120 Env protein. HIV-1 viruses which use CXCR4 and emerge during the late stages of HIV-1 infection may impact disease progression in the CNS by inducing apoptosis of neurons and other cell types. The neurodegenerative mechanisms may involve infection of microglia by certain CXCR4 tropic viruses in addition to cellular dysfunction and apoptosis induced by HIV-1 gp120 binding to CXCR4. Understanding the role of CXCR4 and other chemokine receptors in HIV-1 neuropathogenesis will help to advance the development of new therapeutic strategies for the prevention and treatment of neurologic disorders associated with HIV-1 infection.     

Chemokine receptor CXCR4 signaling modulates the growth factor-induced cell cycle of self-renewing and multipotent neural progenitor cells

CXC chemokine receptor CXCR4 is expressed in vitro in both human and rodent adult neural progenitor cells (NPCs). It has been suggested that the CXCL12-CXCR4 axis potentially enhances the proliferation of NPCs. However, whether CXCR4 is expressed in the neural stem cells (NSCs), a subset of self-renewing and multipotent NPCs, and whether CXCR4 signaling is directly required for their proliferation are not clear. In this study, we report that CXCR4 is expressed in a subpopulation of NPCs in the early embryonic ventricular zone. In studies of a CXCR4(eGFP) bacterial artificial chromosomal (BAC) transgenic mouse line, we further isolated NPCs from E12.5 transgenic telencephalon and GFP(+) cells demonstrated self-renewal and multipotency in neurosphere assays in vitro. Consistent with these observations, we enriched GFP(+)/CXCR4(+) cells by fluorescence activated cell sorting (FACS) with either CXCR4 antibody 12G5 or GFP. Furthermore, we observed that CXCL12 alone did not activate the self-renewal of NPCs or increase the proliferation of NPCs that are induced by bFGF/EGF. However, we found that blocking CXCR4 receptor with antagonist AMD3100 impaired the bFGF/EGF-induced expansion of GFP(+) NPCs through modulating their cell cycling. In addition, AMD3100 treatment of pregnant mice reduced the generation of neurospheres from E12.5 embryos. Our data suggest that CXCR4 is a potential cell surface marker for early embryonic NSCs and modulates growth-factor signaling.     

Chemokine receptors and virus entry in the central nervous system

Several members of the chemokine receptor family are used as coreceptors together with CD4 for HIV and SIV entry in the central nervous system (CNS). CCR5 is the major coreceptor for HIV-1 infection of macrophages and microglia, the major target cells for HIV-1 infection in the CNS. CXCR4 and CCR3 are also expressed on microglia and can mediate infection by certain HIV-1 isolates but at lower efficiency than CCR5. Additional chemokine receptors that can function as HIV-1 and SIV coreceptors for a subset of viruses are expressed in the brain (i.e. Apj, CX3CR1, STRL33/BONZO, and gpr1), but their role in CNS infection has not been defined. The expression of CXCR4, and possibly other chemokine receptors, on subpopulations of neurons and glial cells may contribute to mechanisms of CNS injury that are independent of viral infection. Understanding the role of chemokine receptors and their chemokine ligands in HIV-1 and SIV infection of the CNS will elucidate mechanisms of viral tropism and pathogenesis and advance the development of new therapeutic strategies.     

Moderate traumatic brain injury promotes proliferation of quiescent neural progenitors in the adult hippocampus

Recent evidence shows that traumatic brain injury (TBI) regulates proliferation of neural stem/progenitor cells in the dentate gyrus (DG) of adult hippocampus. There are distinct classes of neural stem/progenitor cells in the adult DG, including quiescent neural progenitors (QNPs), which carry stem cell properties, and their progeny, amplifying neural progenitors (ANPs). The response of each class of progenitors to TBI is not clear. We here used a transgenic reporter Nestin-GFP mouse line, in which QNP and ANP cells are easily visualized and quantified, to determine the targets of the TBI in the DG. We examined changes in proliferation of QNPs and ANPs in the acute phase following TBI and found that QNPs were induced by TBI insult to enter the cell cycle whereas proliferation of ANPs was not significantly affected. These results indicate that different subtypes of neural stem/progenitor cells respond differently to TBI insult. Stem cell activation by the TBI may reflect the induction of innate repair and plasticity mechanisms by the injured brain.     

Developmental expression patterns of CCR5 and CXCR4 in the rhesus macaque brain.

Emerging data indicate that chemokine receptors on neurons and glia in the central nervous system (CNS) play a role in normal CNS development, intercellular communication, and the neuropathogenesis of AIDS. To further understand chemokine receptors in the brain and explore their potential role in HIV neuropathogenesis, particularly in pediatrics, we examined the regional and cellular distribution of CCR5 and CXCR4 in normal fetal, neonatal, and adult rhesus macaques. CCR5 and CXCR4 were detected by immunohistochemistry and immunofluorescence within the cytoplasm of subpopulations of neurons in the neocortex, hippocampus, basal nuclei, thalamus, brain stem, and cerebellum and by flow cytometry on the surface of neurons and glia. Interestingly, expression of CCR5 and CXCR4 increased significantly (p<0.05) from birth to 9 months of age. We further characterize this dynamic developmental pattern of CCR5 and CXCR4 expression in resident cells of the CNS.     

Regulation of chemokine receptor expression in human microglia and astrocytes

It has been proposed that the positioning of mobile cells within a tissue is determined by their overall profile of chemokine receptors. This study examines the profiles of chemokine receptors expressed on resting and activated adult human microglial cells, astrocytes and a microglial cell line, CHME3. Microglia express highest levels of CXCR1, CXCR3 and CCR3. Astrocytes also have moderate levels of CXCR1 and CXCR3, and some CCR3, while both cell types also expressed CCR4, CCR5, CCR6, CXCR2, CXCR4 and CXCR5 at lower levels. Activation of the cells with the inflammatory cytokine tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) increased the expression of some but not all receptors over a period of 24 h. Microglia showed moderate enhancement of receptor expression, while astrocytes responded particularly strongly to TNFalpha with enhanced CXCR3, CCR3 and CXCR1. However, the migratory and proliferative responses of the microglia and astrocytes to the same chemokine were different, with microglia migrating and astrocytes proliferating in response to CXCL10. The data indicates a mechanism by which activated microglia and astrocytes become selectively more sensitive to inflammatory chemokines during CNS disease, and the paper discusses which of the many chemokines present in CNS would have priority of action on microglia and astrocytes.     

Astrocytes express functional chemokine receptors

Multiple lines of evidence are presented characterizing the functional expression of chemokine receptors CXCR4, CCR1, CCR5, and CX3CR1 on astrocytes. Most of these receptors are expressed at low levels and may only be detectable on a subset of cells during disease or following cytokine induction. The expression of CXCR2, CCR2, CCR3, CCR10, CCR11, and several orphan receptors associated with HIV-1 infection has also been proposed. The appearance of several chemokine receptors implies a wider role for chemokines in the regulation of central nervous system functions. Available evidence indicates that selected chemokines induce further chemokine synthesis in astrocytes providing a mechanism to amplify inflammatory responses in the central nervous system.