Dongjun Ren

Chemokines Regulate the Migration of Neural Progenitors to Sites of Neuroinflammation

Many studies have shown that transplanted or endogenous neural progenitor cells will migrate toward damaged areas of the brain. However, the mechanism underlying this effect is not clear. Here we report that, using hippocampal slice cultures, grafted neural progenitor cells (NPs) migrate toward areas of neuroinflammation and that chemokines are a major regulator of this process. Migration of NPs was observed after injecting an inflammatory stimulus into the area of the fimbria and transplanting enhanced green fluorescent protein (EGFP)-labeled NPs into the dentate gyrus of cultured hippocampal slices. Three to 7 d after transplantation, EGFP–NPs in control slices showed little tendency to migrate and had differentiated into neurons and glia. In contrast, in slices injected with inflammatory stimuli, EGFP–NPs migrated toward the site of the injection. NPs in these slices also survived less well. The inflammatory stimuli used were a combination of the cytokines tumor necrosis factor-α and interferon-γ, the bacterial toxin lipopolysaccharide, the human immunodeficiency virus-1 coat protein glycoprotein 120, or a β-amyloid-expressing adenovirus. We showed that these inflammatory stimuli increased the synthesis of numerous chemokines and cytokines by hippocampal slices. When EGFP–NPs from CC chemokine receptor CCR2 knock-out mice were transplanted into slices, they exhibited little migration toward sites of inflammation. Similarly, wild-type EGFP–NPs exhibited little migration toward inflammatory sites when transplanted into slices prepared from monocyte chemoattractant protein-1 (MCP-1) knock-out mice. These data indicate that factors secreted by sites of neuroinflammation are attractive to neural progenitors and suggest that chemokines such as MCP-1 play an important role in this process.

Chemokine receptors are expressed widely by embryonic and adult neural progenitor cells.

We investigated the expression and functions of chemokine receptors in neural progenitor cells isolated from embryonic and adult mice. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis demonstrated mRNA expression for most known chemokine receptors in neural progenitor cells grown as neurospheres from embryonic (E17) and adult (4‐week‐old) mice. The expression of CXCR4 receptors was demonstrated further in E17 neurospheres using immunohistochemistry, in situ hybridization, Northern blot analysis and fura‐2‐based Ca2+ imaging. Most neurospheres grown from E17 mice responded to stromal cell‐derived factor‐1 (SDF‐1/CXCL12) in Ca2+ imaging studies. In addition, immunohistochemical studies demonstrated that these neurospheres consisted of dividing cells that uniformly colocalized nestin and CXCR4 receptors. Differentiation of E17 neurospheres yielded astrocytes and neurons exhibiting several different phenotypes, including expression of calbindin, calretinin, gamma‐aminobutyric acid (GABA), and glutamate, and many also coexpressed CXCR4 receptors. In addition, neurospheres grown from the subventricular zone (SVZ) of 4‐week‐old mice exhibited large increases in Ca2+ in response to CXCL12 and several other chemokines. In comparison, neurospheres prepared from olfactory bulb of adult mice exhibited only small Ca2+ responses to CXCL12, whereas neurospheres prepared from hippocampus were insensitive to CXCL12, although they did respond to other chemokines. Investigations designed to investigate whether CXCL12 can act as a chemoattractant demonstrated that cells dissociated from E17 or adult SVZ neurospheres migrated toward an CXCL12 gradient and this was blocked by the CXCR4 antagonist AMD3100. These results illustrate widespread chemokine sensitivity of embryonic and adult neural progenitor cells and support the view that chemokines may be of general importance in control of progenitor cell migration in embryonic and adult brain.

Chemokine receptor expression by neural progenitor cells in neurogenic regions of mouse brain

We previously demonstrated that chemokine receptors are expressed by neural progenitors grown as cultured neurospheres. To examine the significance of these findings for neural progenitor function in vivo, we investigated whether chemokine receptors were expressed by cells having the characteristics of neural progenitors in neurogenic regions of the postnatal brain. Using in situ hybridization we demonstrated the expression of CCR1, CCR2, CCR5, CXCR3, and CXCR4 chemokine receptors by cells in the dentate gyrus (DG), subventricular zone of the lateral ventricle, and olfactory bulb. The pattern of expression for all of these receptors was similar, including regions where neural progenitors normally reside. In addition, we attempted to colocalize chemokine receptors with markers for neural progenitors. In order to do this we used nestin‐EGFP and TLX‐LacZ transgenic mice, as well as labeling for Ki67, a marker for dividing cells. In all three areas of the brain we demonstrated colocalization of chemokine receptors with these three markers in populations of cells. Expression of chemokine receptors by neural progenitors was further confirmed using CXCR4‐EGFP BAC transgenic mice. Expression of CXCR4 in the DG included cells that expressed nestin and GFAP as well as cells that appeared to be immature granule neurons expressing PSA‐NCAM, calretinin, and Prox‐1. CXCR4‐expressing cells in the DG were found in close proximity to immature granule neurons that expressed the chemokine SDF‐1/CXCL12. Cells expressing CXCR4 frequently coexpressed CCR2 receptors. These data support the hypothesis that chemokine receptors are important in regulating the migration of progenitor cells in postnatal brain. J. Comp. Neurol. 500:1007–1033, 2007.

The Chemokine Stromal Cell-Derived Factor-1 Regulates the Migration of Sensory Neuron Progenitors

Chemokines and their receptors are essential for the development and organization of the hematopoietic/lymphopoietic system and have now been shown to be expressed by different types of cells in the nervous system. In mouse embryos, we observed expression of the chemokine (CXC motif) receptor 4 (CXCR4) by neural crest cells migrating from the dorsal neural tube and in the dorsal root ganglia (DRGs). Stromal cell-derived factor-1 (SDF-1), the unique agonist for CXCR4, was expressed along the path taken by crest cells to the DRGs, suggesting that SDF-1/CXCR4 signaling is needed for their migration. CXCR4 null mice exhibited small and malformed DRGs. Delayed migration to the DRGs was suggested by ectopic cells expressing tyrosine receptor kinase A (TrkA) and TrkC, neurotrophin receptors required by DRG sensory neuron development. In vitro, the CXCR4 chemokine receptor was upregulated by migratory progenitor cells just as they exited mouse neural tube explants, and SDF-1 acted as a chemoattractant for these cells. Most CXCR4-expressing progenitors differentiated to form sensory neurons with the properties of polymodal nociceptors. Furthermore, DRGs contained a population of progenitor cells that expressed CXCR4 receptors in vitro and differentiated into neurons with a similar phenotype. Our findings indicate an important role for SDF-1/CXCR4 signaling in directing the migration of sensory neuron progenitors to the DRG and potentially in other aspects of development once the DRGs have coalesced.

STIM1-Orai1 interactions and Orai1 conformational changes revealed by live-cell FRET microscopy

Ca2+ entry through store‐operated Ca2+ release‐activated Ca2+ (CRAC) channels initiates key functions such as gene expression and exocytosis of inflammatory mediators. Activation of CRAC channels by store depletion involves the redistribution of the ER Ca2+ sensor, stromal interaction molecule 1 (STIM1), to peripheral sites where it co‐clusters with the CRAC channel subunit, Orai1. However, how STIM1 communicates with the CRAC channel and initiates the subsequent events culminating in channel opening is unclear. Here, we show that redistribution of STIM1 and Orai1 occurs in parallel with a pronounced increase in fluorescence resonance energy transfer (FRET) between STIM1 and Orai1, supporting the idea that activation of CRAC channels occurs through physical interactions with STIM1. Co‐expression of Orai1–CFP and Orai1–YFP results in a high degree of FRET in resting cells, indicating that Orai1 exists as a multimer. However, store depletion triggers molecular rearrangements in Orai1 resulting in a decline in Orai1–Orai1 FRET. The decline in Orai1–Orai1 FRET is not seen in the absence of STIM1 co‐expression and is abolished in Orai1 mutants with impaired STIM1 interaction. Both the STIM1–Orai1 interaction as well as the molecular rearrangements in Orai1 are altered by two powerful modulators of CRAC channel activity: extracellular Ca2+ and 2‐APB. These studies identify a STIM1‐dependent conformational change in Orai1 during the activation of CRAC channels and reveal that STIM1–Orai1 interaction and the downstream Orai1 conformational change can be independently modulated to fine‐tune CRAC channel activity.

Delayed functional expression of neuronal chemokine receptors following focal nerve demyelination in the rat: a mechanism for the development of chronic sensitization of peripheral nociceptors

BackgroundAnimal and clinical studies have revealed that focal peripheral nerve axon demyelination is accompanied by nociceptive pain behavior. C-C and C-X-C chemokines and their receptors have been strongly implicated in demyelinating polyneuropathies and persistent pain syndromes. Herein, we studied the degree to which chronic nociceptive pain behavior is correlated with the neuronal expression of chemokines and their receptors following unilateral lysophosphatidylcholine (LPC)-induced focal demyelination of the sciatic nerve in rats.ResultsFocal nerve demyelination increased behavioral reflex responsiveness to mechanical stimuli between postoperative day (POD) 3 and POD28 in both the hindpaw ipsilateral and contralateral to the nerve injury. This behavior was accompanied by a bilateral increase in the numbers of primary sensory neurons expressing the chemokine receptors CCR2, CCR5, and CXCR4 by POD14, with no change in the pattern of CXCR3 expression. Significant increases in the numbers of neurons expressing the chemokines monocyte chemoattractant protein-1 (MCP-1/CCL2), Regulated on Activation, Normal T Expressed and Secreted (RANTES/CCL5) and interferon γ-inducing protein-10 (IP-10/CXCL10) were also evident following nerve injury, although neuronal expression pattern of stromal cell derived factor-1α (SDF1/CXCL12) did not change. Functional studies demonstrated that acutely dissociated sensory neurons derived from LPC-injured animals responded with increased [Ca2+]i following exposure to MCP-1, IP-10, SDF1 and RANTES on POD 14 and 28, but these responses were largely absent by POD35. On days 14 and 28, rats received either saline or a CCR2 receptor antagonist isomer (CCR2 RA-[R]) or its inactive enantiomer (CCR2 RA-[S]) by intraperitoneal (i.p.) injection. CCR2 RA-[R] treatment of nerve-injured rats produced stereospecific bilateral reversal of tactile hyperalgesia.ConclusionThese results suggest that the presence of chemokine signaling by both injured and adjacent, uninjured sensory neurons is correlated with the maintenance phase of a persistent pain state, suggesting that chemokine receptor antagonists may be an important therapeutic intervention for chronic pain.

The Chemokine Stromal Cell-Derived Factor-1 Regulates GABAergic Inputs to Neural Progenitors in the Postnatal Dentate Gyrus

Stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) are important regulators of the development of the dentate gyrus (DG). Both SDF-1 and CXCR4 are also highly expressed in the adult DG. We observed that CXCR4 receptors were expressed by dividing neural progenitor cells located in the subgranular zone (SGZ) as well as their derivatives including doublecortin-expressing neuroblasts and immature granule cells. SDF-1 was located in DG neurons and in endothelial cells associated with DG blood vessels. SDF-1-expressing neurons included parvalbumin-containing GABAergic interneurons known as basket cells. Using transgenic mice expressing an SDF-1-mRFP1 (monomeric red fluorescence protein 1) fusion protein we observed that SDF-1 was localized in synaptic vesicles in the terminals of basket cells together with GABA-containing vesicles. These terminals were often observed to be in close proximity to dividing nestin-expressing neural progenitors in the SGZ. Electrophysiological recordings from slices of the DG demonstrated that neural progenitors received both tonic and phasic GABAergic inputs and that SDF-1 enhanced GABAergic transmission, probably by a postsynaptic mechanism. We also demonstrated that, like GABA, SDF-1 was tonically released in the DG and that GABAergic transmission was partially dependent on coreleased SDF-1. These data demonstrate that SDF-1 plays a novel role as a neurotransmitter in the DG and regulates the strength of GABAergic inputs to the pool of dividing neural progenitors. Hence, SDF-1/CXCR4 signaling is likely to be an important regulator of adult neurogenesis in the DG.

CCR2 chemokine receptor signaling mediates pain in experimental osteoarthritis

Osteoarthritis is one of the leading causes of chronic pain, but almost nothing is known about the mechanisms and molecules that mediate osteoarthritis-associated joint pain. Consequently, treatment options remain inadequate and joint replacement is often inevitable. Here, we use a surgical mouse model that captures the long-term progression of knee osteoarthritis to longitudinally assess pain-related behaviors and concomitant changes in the innervating dorsal root ganglia (DRG). We demonstrate that monocyte chemoattractant protein (MCP)-1 (CCL2) and its high-affinity receptor, chemokine (C-C motif) receptor 2 (CCR2), are central to the development of pain associated with knee osteoarthritis. After destabilization of the medial meniscus, mice developed early-onset secondary mechanical allodynia that was maintained for 16 wk. MCP-1 and CCR2 mRNA, protein, and signaling activity were temporarily up-regulated in the innervating DRG at 8 wk after surgery. This result correlated with the presentation of movement-provoked pain behaviors, which were maintained up to 16 wk. Mice that lack Ccr2 also developed mechanical allodynia, but this started to resolve from 8 wk onwards. Despite severe allodynia and structural knee joint damage equal to wild-type mice, Ccr2-null mice did not develop movement-provoked pain behaviors at 8 wk. In wild-type mice, macrophages infiltrated the DRG by 8 wk and this was maintained through 16 wk after surgery. In contrast, macrophage infiltration was not observed in Ccr2-null mice. These observations suggest a key role for the MCP-1/CCR2 pathway in establishing osteoarthritis pain.

Visualization of Chemokine Receptor Activation in Transgenic Mice Reveals Peripheral Activation of CCR2 Receptors in States of Neuropathic Pain

CCR2 chemokine receptor signaling has been implicated in the generation of diverse types of neuropathology, including neuropathic pain. For example, ccr2 knock-out mice are resistant to the establishment of neuropathic pain, and mice overexpressing its ligand, monocyte chemoattractant protein-1 (MCP1; also known as CCL2), show enhanced pain sensitivity. However, whether CCR2 receptor activation occurs in the central or peripheral nervous system in states of neuropathic pain has not been clear. We developed a novel method for visualizing CCR2 receptor activation in vivo by generating bitransgenic reporter mice in which the chemokine receptor CCR2 and its ligand MCP1 were labeled by the fluorescent proteins enhanced green fluorescent protein and monomeric red fluorescent protein-1, respectively. CCR2 receptor activation under conditions such as acute inflammation and experimental autoimmune encephalomyelitis could be faithfully visualized by using these mice. We examined the status of CCR2 receptor activation in a demyelination injury model of neuropathic pain and found that MCP1-induced CCR2 receptor activation mainly occurred in the peripheral nervous system, including the injured peripheral nerve and dorsal root ganglia. These data explain the rapid antinociceptive effects of peripherally administered CCR2 antagonists under these circumstances, suggesting that CCR2 antagonists may ameliorate pain by inhibiting CCR2 receptor activation in the periphery. The method developed here for visualizing CCR2 receptor activation in vivo may be extended to G-protein-coupled receptors (GPCRs) in general and will be valuable for studying intercellular GPCR-mediated communication in vivo.

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.