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Richard J Miller

Publications and source records attributed to Richard J Miller.

At least 19 recordsLinked to original sources
PubMed2007

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.

Animals↗
PubMed2006

Molecular basis of Ca(v)2.3 calcium channels in rat nociceptive neurons.

Ca(v)2.3 calcium channels play an important role in pain transmission in peripheral sensory neurons. Six Ca(v)2.3 isoforms resulting from different combinations of three inserts (inserts I and II in the II-III loop and insert III in the carboxyl-terminal region) have been identified in different mammalian tissues. To date, however, Ca(v)2.3 isoforms unique to primary sensory neurons have not been identified. In this study, we determined Ca(v)2.3 isoforms expressed in the rat trigeminal ganglion neurons. Whole tissue reverse transcription (RT)-PCR analyses revealed that only two isoforms, Ca(v)2.3a and Ca(v)2.3e, are present in TG neurons. Using single cell RT-PCR, we found that Ca(v)2.3e is the major isoform, whereas Ca(v)2.3e expression is highly restricted to small (<16 mum) isolectin B4-negative and tyrosine kinase A-positive neurons. Ca(v)2.3e was also preferentially detected in neurons expressing the nociceptive marker, transient receptor potential vanilloid 1. Single cell RT-PCR following calcium imaging and whole-cell patch clamp recordings provided evidence of an association between an R-type calcium channel component and Ca(v)2.3e expression. Our results suggest that Ca(v)2.3e in sensory neurons may be a potential target for the treatment of pain.

Animals↗
PubMed2006

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-alpha and interferon-gamma, the bacterial toxin lipopolysaccharide, the human immunodeficiency virus-1 coat protein glycoprotein 120, or a beta-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.

Animals↗
PubMed2005

Excitatory monocyte chemoattractant protein-1 signaling is up-regulated in sensory neurons after chronic compression of the dorsal root ganglion.

Neuronal hyperexcitability in both injured and adjacent uninjured neurons is associated with states of chronic injury and pain and is likely subject to neuroinflammatory processes. Chronic inflammatory responses are largely orchestrated by chemokines. One chemokine, monocyte chemoattractant protein-1 (MCP-1), in the presence of its cognate receptor, the beta chemokine receptor 2 (CCR2), produces neural activity in dissociated neuronal cultures of neonatal dorsal root ganglion (DRG) neurons. Using a neuropathic pain model, chronic compression of the DRG (CCD), we compared anatomically separate populations of noncompressed lumbar DRG (L3/L6) with compressed lumbar DRG (L4/L5) for changes in the gene expression of CCR2. In situ hybridization revealed that CCR2 mRNA was up-regulated in neurons and nonneuronal cells present in both compressed L4/L5 and ipsilateral noncompressed L3/L6 DRGs at postoperative day 5 (POD5). The total percentages of compressed and noncompressed neurons exhibiting CCR2 mRNA transcripts in L3, L5, and L6 DRG were 33 +/- 3.5%, 49 +/- 6.2%, and 41 +/- 5.6%, respectively, and included cell bodies of small, medium, and large size. In addition, the preferred CCR2 ligand, MCP-1, was up-regulated by POD5 in both compressed L4/L5 and noncompressed L3/L6 DRG neurons. Application of MCP-1 to the cell bodies of the intact formerly compressed DRG in vitro produced potent excitatory effects not observed in control ganglia. MCP-1/CCR2 signaling is directly involved with a chronic compression injury and may contribute to associated neuronal hyperexcitability and neuropathic pain.

Animals↗
PubMed2005

Chemokinetics.

How do migrating neural progenitor cells and growing axons know where to go? In this issue of Neuron, two papers (Knaut et al. and Lieberam et al.) demonstrate that activation of Cxcr4 chemokine receptors by the chemokine SDF1/Cxcl12 can direct both of these tasks.

Animals↗
PubMed2005

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.

Animals↗
PubMed2005

Chemokines: integrators of pain and inflammation.

Chronic (neuropathic) pain is one of the most widespread and intractable of human complaints, as well as being one of the most difficult syndromes to treat successfully with drugs or surgery. The development of new therapeutic approaches to the treatment of painful neuropathies requires a better understanding of the mechanisms that underlie the development of these chronic pain syndromes. It is clear that inflammatory responses often accompany the development of neuropathic pain, and here we discuss the idea that chemokines might be key to integrating the development of pain and inflammation and could furnish new leads in the search for effective analgesic agents for the treatment of painful neuropathies.

Chemokines↗
PubMed2005

A comparison of three techniques to obtain root coverage on mandibular incisors.

BACKGROUND: Multiple variations on the subepithelial connective tissue graft have been shown to produce good clinical results. The goal of this study was to compare three variations in the treatment of recession on mandibular incisors. METHODS: An a priori analysis was done to determine the sample size. Three groups of patients received root coverage procedures on mandibular incisors. Each group was treated with a different variation of a subepithelial graft. The first group received a connective tissue (CT) graft with a coronally positioned flap (CPF) (CPF + CT). The second group received a connective tissue graft with a double pedicle (DP) graft (DP + CT). The third group received a connective tissue graft with a tunneling (TUN) procedure and a laterally positioned (LAT) pedicle (TUN-LAT + CT). The clinical results of these procedures were compared to determine if one procedure offered an advantage over the other procedures. RESULTS: All of the procedures produced a statistically significant improvement in the clinical parameters. The groups treated with the DP + CT and TUN-LAT + CT had greater mean root coverage (95.5% and 90.5%, respectively) than the CPF + CT group (80.2%). Additionally, DP + CT produced a greater increase in keratinized tissue (3 mm) than CPF + CT (1.4 mm) or TUN-LAT + CT (1.9 mm). There was no statistically significant difference in the mean root coverage of any technique treating defects <3 mm deep (CPF + CT, 90.9%; DP + CT, 96.4%; and TUN-LAT + CT, 92.1%) or the defects >3 mm deep treated with DP + CT (95.4%) or TUN-LAT + CT (88.3%). However, the CPF + CT produced less mean root coverage (68.4%) when treating defects > or =3 mm deep. When defects treated as isolated defects were compared to cases where multiple defects were treated, the cases with multiple defects treated with the CPF + CT had less mean root coverage (77%) than cases where single defects were treated with the CPF + CT (90.3%), DP + CT (isolated, 96.7% and multiple, 95.6%), and TUN-LAT + CT (isolated, 97.2% and multiple, 87.8%). CONCLUSIONS: All three of the procedures were effective in obtaining root coverage and improved clinical parameters on mandibular incisors. Overall, the DP + CT and TUN-LAT + CT procedures had greater mean root coverage than the CPF + CT technique. Based on this study, when treating defects > or =3 mm deep, one should consider using the DP + CT or TUN-LAT + CT rather than the CPF + CT. Additionally, when treating multiple defects at a time, one should consider using the DP + CT or TUN-LAT + CT rather than the CPF + CT. In cases where an increased amount of keratinized tissue is desired, based on this study, the DP + CT may be the best procedure to use.

Adult↗
PubMed2004

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 Ca(2+) 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 proliferation, 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.

Animals↗
PubMed2004

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 Ca(2+) imaging. Most neurospheres grown from E17 mice responded to stromal cell-derived factor-1 (SDF-1/CXCL12) in Ca(2+) 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 Ca(2+) in response to CXCL12 and several other chemokines. In comparison, neurospheres prepared from olfactory bulb of adult mice exhibited only small Ca(2+) 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.

Animals↗
PubMed2004

Regulation of CXCR4 receptor dimerization by the chemokine SDF-1alpha and the HIV-1 coat protein gp120: a fluorescence resonance energy transfer (FRET) study.

Both the chemokine SDF-1alpha and the human immunodeficiency virus-1 (HIV-1) coat protein gp120 can bind to CXCR4 chemokine receptors but with different signaling consequences. To understand the molecular basis for these differences, we tagged the rat CXCR4 receptor with enhanced cyan (ECFP) and yellow (EYFP) derivatives of the green fluorescent protein and investigated CXCR4 receptor dimerization in human embryonic kidney (HEK)-tsA201 cells using fluorescence resonance energy transfer (FRET). Elevated FRET was detected under basal conditions from EYFP-CXCR4 and ECFP-CXCR4 receptor-transfected cells indicating a high level of CXCR4 receptor dimerization. In comparison, EYFP-CXCR4 and ECFP-mu-opioid receptor-cotransfected cells displayed a much lower FRET signal. The FRET signal resulting from EYFP-CXCR4- and ECFP-CXCR4-expressing cells could be attenuated by coexpressing nontagged CXCR4 receptors suggesting competition with fluorophore-tagged receptors in the membrane. Nontagged mu-opioid, kappa-opioid, and muscarinic receptors also decreased the FRET between the tagged CXCR4 receptor pairs but to a lesser extent. Application of the CXCR4 receptor agonist SDF-1alpha (50 nM) further increased the FRET signal from tagged CXCR4 receptors, an effect that was inhibited by the CXCR4 antagonist AMD3100. SDF-1alpha had no effect when EYFP-CXCR4 and ECFP-mu-opioid receptors were coexpressed. The effect of gp120IIIB on CXCR4 FRET was dependent on the coexpression of human CD4 (hCD4) when it increased the FRET signal, and this was decreased by AMD3100 pretreatment. FRET analysis of tagged hCD4 constructs demonstrated that there was significant association of hCD4 and CXCR4, as well as hCD4 dimerization. These data suggest that CXCR4 dimerization is involved in SDF-1alpha- and gp120-induced signaling events.

Animals↗
PubMed2004

Activation of c-Jun N-terminal kinase mediates gp120IIIB- and nucleoside analogue-induced sensory neuron toxicity.

Peripheral neuropathy is the most common neurological symptom in patients with acquired immunodeficiency syndrome (AIDS). Here, we examine possible mechanisms of gp120 and nucleoside reverse transcriptase inhibitors (NRTIs) in the pathogenesis of AIDS peripheral neuropathy. Neonatal dorsal root ganglion (DRG) neurons were found to undergo apoptosis in response to chronic treatment with gp120IIIB, an effect enhanced by the co-application of hCD4, as well as upon exposure to the nucleoside reverse transcriptase inhibitor (NRTI), 2',3'-dideoxyinosine (ddI). DRG neurons were rescued from the neurotoxic effects of these agents by CEP-1347, an inhibitor of the mixed lineage kinases (MLKs), upstream activators of the c-Jun N-terminal kinase (JNK) signaling pathway. In addition, gp120- or ddI-mediated toxicity were also inhibited by neuronal expression of dominant negative versions of the MLKs. Our results suggest that both gp120 and the NRTIs cause sensory neuron apoptosis through the activation of the JNK pathway, and that CEP-1347-like compounds may serve as a therapeutic option in patients with AIDS-associated peripheral neuropathy.

Animals↗
PubMed2003

Glutamate carboxypeptidase II inhibition protects motor neurons from death in familial amyotrophic lateral sclerosis models.

Approximately 10% of cases of amyotrophic lateral sclerosis (ALS), a progressive and fatal degeneration that targets motor neurons (MNs), are inherited, and approximately 20% of these cases of familial ALS (FALS) are caused by mutations of copper/zinc superoxide dismutase type 1. Glutamate excitotoxicity has been implicated as a mechanism of MN death in both ALS and FALS. In this study, we tested whether a neuroprotective strategy involving potent and selective inhibitors of glutamate carboxypeptidase II (GCPII), which converts the abundant neuropeptide N-acetylaspartylglutamate to glutamate, could protect MNs in an in vitro and animal model of FALS. Data suggest that the GCPII inhibitors prevented MN cell death in both of these systems because of the resultant decrease in glutamate levels. GCPII inhibition may represent a new therapeutic target for the treatment of ALS.

Adenoviridae↗
PubMed2003

Direct interactions between the heterotrimeric G protein subunit G beta 5 and the G protein gamma subunit-like domain-containing regulator of G protein signaling 11: gain of function of cyan fluorescent protein-tagged G gamma 3.

We used fluorescence resonance energy transfer imaging of enhanced cyan fluorescent protein (CFP)-tagged and enhanced yellow fluorescent protein (YFP)-tagged protein pairs to examine the hypothesis that G protein gamma subunit-like (GGL) domain-containing regulators of G protein signaling (RGS) can directly bind to the Gbeta5 subunit of heterotrimeric G proteins in vivo. We observed that Gbeta5 could interact with Ggamma2 and Ggamma13, after their expression in human embryonic kidney 293 cells. Interestingly, although untagged Ggamma3 did not interact with Gbeta5, CFP-tagged Ggamma3 strongly interacted with YFP-tagged Gbeta5 in FRET studies. Moreover, CFP-Ggamma3 supported Ca(2+) channel inhibition when paired with Gbeta5 or YFP-Gbeta5, indicating a "gain of function" for CFP-Ggamma3. Gbeta5 could also interact with RGS11 and its N-terminal, but not its C-terminal domain. On the other hand, RGS11 did not interact with Gbeta1. These studies demonstrate that the GGL domain-containing N terminus of RGS 11 can directly interact with Gbeta5 in vivo and supports the hypothesis that this interaction may contribute to the specificity of Gbeta5 interactions with cellular effector molecules.

Calcium Channel Blockers↗
PubMed2003

CD4 dependence of gp120IIIB-CXCR4 interaction is cell-type specific.

The HIV-1 envelope protein gp120IIIB is selective for the CXCR4 chemokine receptor and has been shown to induce apoptosis in neurons both in vivo and in vitro. We examined the ability of gp120IIIB to signal through the rat CXCR4 (rCXCR4) receptor and its dependence on the presence of the human CD4 (hCD4) protein in a number of cell systems. SDF-1alpha potently inhibited N-type Ca channels in cultured HEK293 cells expressing both the Ca channel subunits and rCXCR4 receptors. However, gp120IIIB was ineffective in producing either Ca channel inhibition or in blocking the effects of SDF-1alpha. However, when hCD4 was coexpressed with rCXCR4 and Ca channel subunits, gp120IIIB also produced Ca channel inhibition. Similarly, in PC12 cells transfected with the rCXCR4, SDF-1alpha produced mobilization of intracellular Ca, while gp120IIIB was only effective when hCD4 was coexpressed. SDF-1alpha induced endocytosis of Yellow Fluorescent Protein (YFP)-tagged rCXCR4 expressed in PC12 cells, as did gp120IIIB, an effect which was enhanced by hCD4 coexpression. When tagged rCXCR4 was expressed in F-11 cells or in rat DRG neurons, SDF-1alpha produced extensive receptor endocytosis. However, the ability of gp120IIIB to produce endocytosis was dependent on the coexpression of hCD4. Our results demonstrate that the degree of hCD4 dependence of the agonist effects of gp120IIIB at the rCXCR4 receptor is cell-type specific.

Animals↗
PubMed2003

Neurological disease: UPS stops delivering!

The ubiquitin-proteasome system (UPS) plays a vital role in directing molecules to the 26S proteasome for degradation as well as other locales in the cell. Disrupting UPS function can lead to the aggregation of mutant or misfolded proteins, which disrupts normal cellular activity in diverse ways. Here we discuss how UPS dysfunction might contribute to a variety of neurological problems.

Animals↗
PubMed2003

Electrophysiological analysis of neuronal chemokine receptors.

Several studies have shown that neurons in the central and peripheral nervous systems express a variety of chemokine receptors (CKRs). Activation of these receptors can influence neuronal signaling by regulating synaptic transmission and neuronal excitability. This article presents electrophysiological methods that are currently used to study the normal and pathophysiological role for CKRs in the nervous system. Conventional electrophysiological methods such as patch-clamp recording of isolated neurons, brain slices, and heterologous expression systems are described. In addition, single-cell reverse transcription-polymerase chain reaction is discussed as a technique that can be used in conjunction with patch-clamp recording to further investigate the molecular basis of neuronal CKR activation.

Animals↗