Ralf Stumm

Cxcr7 Controls Neuronal Migration by Regulating Chemokine Responsiveness

The chemokine Cxcl12 binds Cxcr4 and Cxcr7 receptors to control cell migration in multiple biological contexts, including brain development, leukocyte trafficking, and tumorigenesis. Both receptors are expressed in the CNS, but how they cooperate during migration has not been elucidated. Here, we used the migration of cortical interneurons as a model to study this process. We found that Cxcr4 and Cxcr7 are coexpressed in migrating interneurons, and that Cxcr7 is essential for chemokine signaling. Intriguingly, this process does not exclusively involve Cxcr7, but most critically the modulation of Cxcr4 function. Thus, Cxcr7 is necessary to regulate Cxcr4 protein levels, thereby adapting chemokine responsiveness in migrating cells. This demonstrates that a chemokine receptor modulates the function of another chemokine receptor by controlling the amount of protein that is made available for signaling at the cell surface.

Morphine induces terminal μ-opioid receptor desensitization by sustained phosphorylation of serine-375

Morphine is a poor inducer of μ‐opioid receptor (MOR) internalization, but a potent inducer of cellular tolerance. Here we show that, in contrast to full agonists such as [D‐Ala2‐MePhe4‐Gly‐ol]enkephalin (DAMGO), morphine stimulated a selective phosphorylation of the carboxy‐terminal residue 375 (Ser375). Ser375 phosphorylation was sufficient and required for morphine‐induced desensitization of MOR. In the presence of full agonists, morphine revealed partial agonistic properties and potently inhibited MOR phosphorylation and internalization. Upon removal of the drug, DAMGO‐desensitized receptors were rapidly dephosphorylated. In contrast, morphine‐desensitized receptors remained at the plasma membrane in a Ser375‐phosphorylated state for prolonged periods. Thus, morphine promotes terminal MOR desensitization by inducing a persistent modification of Ser375.

Heterodimerization of Substance P and μ-Opioid Receptors Regulates Receptor Trafficking and Resensitization

The μ-opioid receptor (MOR1) and the substance P receptor (NK1) coexist and functionally interact in nociceptive brain regions; however, a molecular basis for this interaction has not been established. Using coimmunoprecipitation and bioluminescence resonance energy transfer (BRET), we show that MOR1 and NK1 can form heterodimers in HEK 293 cells coexpressing the two receptors. Although NK1-MOR1 heterodimerization did not substantially change the ligand binding and signaling properties of these receptors, it dramatically altered their internalization and resensitization profile. Exposure of the NK1-MOR1 heterodimer to the MOR1-selective ligand [d-Ala2,Me-Phe4,Gly5-ol]enkephalin (DAMGO) promoted cross-phosphorylation and cointernalization of the NK1 receptor. Conversely, exposure of the NK1-MOR1 heterodimer to the NK1-selective ligand substance P (SP) promoted cross-phosphorylation and cointernalization of the MOR1 receptor. In cells expressing MOR1 alone, β-arrestin directs the receptors to clathrin-coated pits, but does not internalize with the receptor. In cells expressing NK1 alone, β-arrestin internalizes with the receptor into endosomes. Interestingly, in cells coexpressing MOR1 and NK1 both DAMGO and SP induced the recruitment of β-arrestin to the plasma membrane and cointernalization of NK1-MOR1 heterodimers with β-arrestin into the same endosomal compartment. Consequently, resensitization of MOR1-dependent receptor functions was severely delayed in coexpressing cells as compared with cells expressing MOR1 alone. Together, our findings indicate that MOR1 by virtue of its physical interaction with NK1 is sequestered via an endocytotic pathway with delayed recycling and resensitization kinetics.

CXC chemokine receptor 4 regulates neuronal migration and axonal pathfinding in the developing nervous system: implications for neuronal regeneration in the adult brain

Chemotactic cytokines (chemokines) are small secreted proteins that control leukocyte trafficking in immune organs. Chemokines which are induced in the brain during conditions of inflammation play a role in the local immune response. Recently, it has been established in the rodent brain that distinct chemokines and chemokine receptors are constitutively expressed by neurons and that these chemokines modulate neuronal functions. The CXC motif chemokine stromal cell-derived factor-1 (SDF-1), CXCL12 together with its cognate receptor CXCR4 represents the best-characterized neuronal chemokine system. Transwell migration assays with neuronal precursors, pharmacological manipulation of CXCR4 signaling in embryonic brain explants, and histochemical studies of SDF-1- or CXCR4-deficient mouse embryos provide proof that SDF-1 directs neuronal migration and axonal pathfinding in the developing nervous system. In the adult brain, SDF-1 is thought to influence neurogenesis as well as recruitment of brain resident and non-resident circulating cells toward sites of lesion. The present review summarizes patterns and functions of the SDF-1/CXCR4 system in the rodent brain with a focus on the developing and adult cerebral cortex.

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. Indexing terms: J. Comp. Neurol. 510:207–220, 2008.

Reassessment of sst2 Somatostatin Receptor Expression in Human Normal and Neoplastic Tissues Using the Novel Rabbit Monoclonal Antibody UMB-1

OBJECTIVE The overexpression of somatostatin receptor 2 (sst2) in neuroendocrine tumors is the molecular basis for diagnostic and therapeutic application of the stable somatostatin analog octreotide. Recent evidence has shown that the immunocytochemical evaluation of sst2A status is of value for predicting response to octreotide therapy and disease prognosis. However, due to the lack of monoclonal and limited availability of specific polyclonal anti-sst2A antibodies, only very few patients can currently benefit from in vitro sst2 evaluation. METHODS In the present study, we extensively characterized the novel rabbit monoclonal anti-sst2A antibody (clone UMB-1) using tissues from sst2-deficient mice and their wild-type littermates. UMB-1 was then subjected to a comparative study of immunohistochemistry on a series of histological specimens from formalin-fixed, paraffin-embedded human tumors and adjacent normal tissues. RESULTS Immunoprecipitation experiments unequivocally demonstrated that UMB-1 selectively detected its cognate sst2A and did not cross-react with other proteins present in crude tissue homogenates. The UMB-1 monoclonal antibody, when compared with currently available polyclonal antisera, yielded several times more effective immunohistochemical staining of fixed-embedded tissues with a predominance of plasma membrane staining and very low cytoplasmic signal even without heat-based antigen retrieval. In addition, dual immunofluorescence revealed for the first time that the sst2A is present on not only gastrin-containing but also ghrelin-containing cells in human gastric mucosa. CONCLUSION Thus, the rabbit monoclonal antibody UMB-1 may prove of great value in the assessment of sst2A status in human neuroendocrine tumors during routine histopathological examination.

Clenbuterol induces growth factor mRNA, activates astrocytes, and protects rat brain tissue against ischemic damage.

The induction of growth factor synthesis in brain tissue by beta2-adrenoceptor agonists, such as clenbuterol, is a promising approach to protect brain tissue from ischemic damage. Clenbuterol (0.01-0.5 mg/kg) reduced the cortical infarct volume in Long-Evans rats as measured 7 days after permanent occlusion of the middle cerebral artery. Dosages of clenbuterol higher than 1 mg/kg showed no cerebroprotective effect due to a decrease in blood pressure and an increase in plasma glucose level. The increase in the mRNA level of nerve growth factor (NGF), basic fibroblast growth factor (basic FGF), and transforming growth factor-beta1 (TGF-beta1) mRNA in cortical and hippocampal tissue occurred earlier after middle cerebral artery occlusion and was more pronounced in animals treated with clenbuterol than in controls. In addition, glial fibrillary acidic protein (GFAP) mRNA expression was enhanced in astrocytes 6 h after ischemia in clenbuterol-treated animals. The results suggest that growth factor synthesis is enhanced in activated astrocytes and that this could be the mechanism of clenbuterol-induced cerebroprotection after ischemia.

Enhanced expression of the CXCl12/SDF-1 chemokine receptor CXCR7 after cerebral ischemia in the rat brain.

Expression patterns of the second SDF-1 receptor RDC1/CXCR7 were examined after focal ischemia in rats using in situ hybridization. CXCR7 mRNA was identified in the ventricle walls as well as neuronal, astroglial, and vascular cells. After ischemia, intact cortical regions showed a rapid, 4 days-lasting increase in neuronal CXCR7 expression. In the ischemic tissue CXCR7 expression was scarce and associated with blood vessels. Between days 2 and 10 after ischemia-onset, SDF-1 expression increased strongly in the peri-infarct and infarct region, which was accompanied by the appearance of numerous CXCR4-expressing but not CXCR7-expressing cells. These patterns suggest that SDF-1 may influence vascular, astroglial, and neuronal functions via CXCR7 and mediate cell recruitment to ischemic brain areas via CXCR4.

Patterns of SDF-1α and SDF-1γ mRNAs, migration pathways, and phenotypes of CXCR4-expressing neurons in the developing rat telencephalon

Cortical GABAergic neurons originate in the ventral telencephalon, invade the cortex via tangential migration, and integrate into the cortical plate by surface‐directed and ventricle‐directed migration. In mice lacking CXCR4 or SDF‐1, GABAergic neurons fail to complete their migration. It is presently unknown which parts of the migration of CXCR4‐expressing GABAergic neurons are driven by SDF‐1. Here we compared patterns of SDF‐1 isoforms and CXCR4 in the developing rat telencephalon. In the ventral telencephalon, radial glia, striatal, and migratory GABAergic neurons expressed CXCR4. Tangentially migrating CXCR4‐expressing neurons populated the marginal zone and started to invade the lateral intermediate zone at embryonic day (E)14. Until E17 the spread of CXCR4‐expressing neurons in the dorsomedial direction was accompanied by progressive upregulation of SDF‐1α in the dorsomedial intermediate/subventricular zone. In the meninges, SDF‐1α and SDF‐1γ were expressed persistently. During invasion of the cortical plate the orientation of CXCR4‐immunoreactive neurons changed gradually from tangential (E17/E18) to radial (postnatal day [P] 0), which was paralleled by downregulation of SDF‐1α in the intermediate/subventricular zone. At E17, CXCR4‐immunoreactive cells were colabeled with markers for ventral forebrain‐derived neurons (Dlx) but not markers for glutamatergic (Tbr) or subplate (calretinin) neurons. Postnatally, calretinin‐ and somatostatin‐expressing but not parvalbumin‐expressing GABAergic neurons or pyramidal cells contained CXCR4. Pyramidal cells and few large blood vessels expressed SDF‐1α, while microvessels contained SDF‐1γ transcripts. In summary, SDF‐1α is expressed along cortical but not subcortical migration routes of GABAergic neurons. We propose that regulated expression of SDF‐1 in the intermediate/subventricular zone influences lateromedial tangential migration of CXCR4‐expressing GABAergic neurons. J. Comp. Neurol. 502:382–399, 2007.

Overexpression of Stromal Cell–Derived Factor 1 and Its Receptor CXCR4 Induces Autocrine/Paracrine Cell Proliferation in Human Pituitary Adenomas

Purpose: Hypothalamic or locally produced growth factors and cytokines control pituitary development, functioning, and cell division. We evaluated the expression of the chemokine stromal cell–derived factor 1 (SDF1) and its receptor CXCR4 in human pituitary adenomas and normal pituitary tissues and their role in cell proliferation. Experimental Design: The expression of SDF1 and CXCR4 in 65 human pituitary adenomas and 4 human normal pituitaries was determined by reverse transcription-PCR, immunohistochemistry, and confocal immunofluorescence. The proliferative effect of SDF1 was evaluated in eight fibroblast-free human pituitary adenoma cell cultures. Results: CXCR4 mRNA was expressed in 92% of growth hormone (GH)-secreting pituitary adenomas (GHoma) and 81% of nonfunctioning pituitary adenomas (NFPA), whereas SDF1 was identified in 63% and 78% of GHomas and NFPAs, respectively. Immunostaining for CXCR4 and SDF1 showed a strong homogenous labeling in all tumoral cells in both GHomas and NFPAs. In normal tissues, CXCR4 and SDF1 were expressed only in a subset of anterior pituitary cells, with a lower expression of SDF1 compared with its cognate receptor. CXCR4 and SDF1 were not confined to a specific cell population in the anterior pituitary but colocalized with discrete subpopulations of GH-, prolactin-, and adrenocorticorticotropic hormone–secreting cells. Conversely, most of the SDF1-containing cells expressed CXCR4. In six of eight pituitary adenoma primary cultures, SDF1 induced a statistically significant increase in DNA synthesis that was prevented by the treatment with the CXCR4 antagonist AMD3100 or somatostatin. Conclusions: CXCR4 and SDF1 are overexpressed in human pituitary adenomas and CXCR4 activation may contribute to pituitary cell proliferation and, possibly, to adenoma development `in humans.