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CXCR7 protein expression in human adult brain and differentiated neurons.

Shimizu S, Brown M, Sengupta R, Penfold ME, Meucci O - PLoS ONE (2011)

Bottom Line: Furthermore, confocal microscopy and binding assays in cultured neurons show that CXCR7 protein is mainly located into cytoplasm, while little to no protein expression is found on neuronal plasma membrane.Interestingly, specific CXCR7 ligands that inhibit CXCL12 binding to CXCR7 do not alter CXCR4-activated survival signaling (pERK/pAkt) in rat cortical neurons.Overall, these results implicate CXCR4 as the main CXCL12 signaling receptor on the surface of differentiated neurons and suggest that CXCR7 may interact with CXCR4 at the intracellular level, possibly affecting CXCR4 trafficking and/or coupling to other proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: CXCR7 and CXCR4 are receptors for the chemokine CXCL12, which is involved in essential functions of the immune and nervous systems. Although CXCR7 transcripts are widely expressed throughout the central nervous system, little is known about its protein distribution and function in the adult brain. To evaluate its potential involvement in CXCL12/CXCR4 signaling in differentiated neurons, we studied CXCR7 protein expression in human brain and cultured neurons.

Methodology/principal findings: Immunohistochemistry and RT-PCR analyses of cortex and hippocampus from control and HIV-positive subjects provided the first evidence of CXCR7 protein expression in human adult neurons, under normal and pathological conditions. Furthermore, confocal microscopy and binding assays in cultured neurons show that CXCR7 protein is mainly located into cytoplasm, while little to no protein expression is found on neuronal plasma membrane. Interestingly, specific CXCR7 ligands that inhibit CXCL12 binding to CXCR7 do not alter CXCR4-activated survival signaling (pERK/pAkt) in rat cortical neurons. Neuronal CXCR7 co-localizes to some extent with the endoplasmic reticulum marker ERp29, but not with early/late endosome markers. Additionally, large areas of overlap are detected in the intracellular pattern of CXCR7 and CXCR4 expression.

Conclusions/significance: Overall, these results implicate CXCR4 as the main CXCL12 signaling receptor on the surface of differentiated neurons and suggest that CXCR7 may interact with CXCR4 at the intracellular level, possibly affecting CXCR4 trafficking and/or coupling to other proteins.

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CXCR7 mRNA/protein expression in the brain of normal subjects and HIV                            patients.(A) CXCR7 mRNA expression in the frontal cortex of control subjects;                            human glioblastoma cells (U87MG) were used as a positive control;                            M = molecular markers;                            RT = reverse transcriptase. (B) CXCR7 protein                            expression in cortex of control patients. (C) No staining was detected                            in adjacent slices when the primary Ab was replaced by the isotype                            control. Scale bar is 0.2 mm in panels B through C and 50 µm in                            the lower left inset of panel B. (D) Double-staining with MAP2 (green)                            and CXCR7 (red) in human cortex. Scale bar: 50 µm. NC: negative                            control (shown here is a staining using CXCR7 isotype control, no MAP2                            primary, and addition of all secondaries). (E/F) CXCR7 immunostaining                            was examined in the hippocampus from control subjects (E) and HIV                            patients (F). Expression of CXCR7 protein seems predominant in cytoplasm                            in both groups. Scale bars: 10 µm.
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pone-0020680-g003: CXCR7 mRNA/protein expression in the brain of normal subjects and HIV patients.(A) CXCR7 mRNA expression in the frontal cortex of control subjects; human glioblastoma cells (U87MG) were used as a positive control; M = molecular markers; RT = reverse transcriptase. (B) CXCR7 protein expression in cortex of control patients. (C) No staining was detected in adjacent slices when the primary Ab was replaced by the isotype control. Scale bar is 0.2 mm in panels B through C and 50 µm in the lower left inset of panel B. (D) Double-staining with MAP2 (green) and CXCR7 (red) in human cortex. Scale bar: 50 µm. NC: negative control (shown here is a staining using CXCR7 isotype control, no MAP2 primary, and addition of all secondaries). (E/F) CXCR7 immunostaining was examined in the hippocampus from control subjects (E) and HIV patients (F). Expression of CXCR7 protein seems predominant in cytoplasm in both groups. Scale bars: 10 µm.

Mentions: Following this initial validation, we started the analysis of the human tissue samples from the NNTC to determine expression of CXCR7 RNA (Figure 3A) and protein (Figure 3B–F) in both control and HIV patients. Brain samples (frontal cortex and hippocampus) from control subjects and HIV+ patients included a total of 9 subjects of different race, age, and gender as indicated in Table 1. The data in normal brain tissue confirmed our previous observations with the tissue arrays. As shown in Figure 3, both CXCR7 RNA and protein are found in the human brain. CXCR7-expressing cells were identified as neurons based on morphology/localization (Figure 3B) as well as co-staining with the neuronal marker MAP2 (Figure 3D). CXCR7-positive cells were observed in both cortex (Figure 3A–D) and hippocampus (Figure 3E/F) of control and HIV patients. Our analysis primarily focused on MAP2 positive cells in the frontal cortex, since the major goal of this study was to evaluate expression of the CXCR7 protein in mature cortical neurons. These IHC studies showed that the vast majority of MAP2 positive cells are also positive to CXCR7. Specifically, within the population of cells that stained positively for MAP2, the percentage of cells also expressing CXCR7 was 95% and 91%, respectively, in control and HIV tissue (total number of cells analyzed = 125 cells; these included CXCR7+/MAP2+, CXCR7+/MAP2-, and CXCR7-/MAP2+). These data suggest that both excitatory and inhibitory mature neurons express the CXCR7 protein and are in agreement with recent studies in the developing brain showing expression of cxcr7 transcripts in migrating interneurons as well as immature excitatory neurons [38]. Furthermore, in these human brain samples only a minority of CXCR7 positive cells (i.e. 11% and 11.4% respectively control/HIV) did not stain for MAP2. As MAP2 is a marker for mature neurons, this smaller group of cells could include resident/infiltrating immune cells (including glia) and/or neural precursors. Further studies are necessary to fully characterize this CXCR7 positive/MAP2 negative cell population (i.e. to establish cell types, differentiation stages, and leukocytes infiltration) and its potential changes during disease progression in specific brain areas. Analysis of a larger group of samples is underway to achieve this goal and to identify potential differences in the expression of CXCR7 between HIV-positive patients without neurological problems and HIV positive patients affected by neurocognitive impairment. The present study demonstrates that CXCR7 protein is widely expressed in mature neurons of the human frontal cortex and hippocampus under both normal and diseased (i.e. HIV) conditions, thus providing the fundamental basis of future investigations focused on its specific role in brain pathology.


CXCR7 protein expression in human adult brain and differentiated neurons.

Shimizu S, Brown M, Sengupta R, Penfold ME, Meucci O - PLoS ONE (2011)

CXCR7 mRNA/protein expression in the brain of normal subjects and HIV                            patients.(A) CXCR7 mRNA expression in the frontal cortex of control subjects;                            human glioblastoma cells (U87MG) were used as a positive control;                            M = molecular markers;                            RT = reverse transcriptase. (B) CXCR7 protein                            expression in cortex of control patients. (C) No staining was detected                            in adjacent slices when the primary Ab was replaced by the isotype                            control. Scale bar is 0.2 mm in panels B through C and 50 µm in                            the lower left inset of panel B. (D) Double-staining with MAP2 (green)                            and CXCR7 (red) in human cortex. Scale bar: 50 µm. NC: negative                            control (shown here is a staining using CXCR7 isotype control, no MAP2                            primary, and addition of all secondaries). (E/F) CXCR7 immunostaining                            was examined in the hippocampus from control subjects (E) and HIV                            patients (F). Expression of CXCR7 protein seems predominant in cytoplasm                            in both groups. Scale bars: 10 µm.
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pone-0020680-g003: CXCR7 mRNA/protein expression in the brain of normal subjects and HIV patients.(A) CXCR7 mRNA expression in the frontal cortex of control subjects; human glioblastoma cells (U87MG) were used as a positive control; M = molecular markers; RT = reverse transcriptase. (B) CXCR7 protein expression in cortex of control patients. (C) No staining was detected in adjacent slices when the primary Ab was replaced by the isotype control. Scale bar is 0.2 mm in panels B through C and 50 µm in the lower left inset of panel B. (D) Double-staining with MAP2 (green) and CXCR7 (red) in human cortex. Scale bar: 50 µm. NC: negative control (shown here is a staining using CXCR7 isotype control, no MAP2 primary, and addition of all secondaries). (E/F) CXCR7 immunostaining was examined in the hippocampus from control subjects (E) and HIV patients (F). Expression of CXCR7 protein seems predominant in cytoplasm in both groups. Scale bars: 10 µm.
Mentions: Following this initial validation, we started the analysis of the human tissue samples from the NNTC to determine expression of CXCR7 RNA (Figure 3A) and protein (Figure 3B–F) in both control and HIV patients. Brain samples (frontal cortex and hippocampus) from control subjects and HIV+ patients included a total of 9 subjects of different race, age, and gender as indicated in Table 1. The data in normal brain tissue confirmed our previous observations with the tissue arrays. As shown in Figure 3, both CXCR7 RNA and protein are found in the human brain. CXCR7-expressing cells were identified as neurons based on morphology/localization (Figure 3B) as well as co-staining with the neuronal marker MAP2 (Figure 3D). CXCR7-positive cells were observed in both cortex (Figure 3A–D) and hippocampus (Figure 3E/F) of control and HIV patients. Our analysis primarily focused on MAP2 positive cells in the frontal cortex, since the major goal of this study was to evaluate expression of the CXCR7 protein in mature cortical neurons. These IHC studies showed that the vast majority of MAP2 positive cells are also positive to CXCR7. Specifically, within the population of cells that stained positively for MAP2, the percentage of cells also expressing CXCR7 was 95% and 91%, respectively, in control and HIV tissue (total number of cells analyzed = 125 cells; these included CXCR7+/MAP2+, CXCR7+/MAP2-, and CXCR7-/MAP2+). These data suggest that both excitatory and inhibitory mature neurons express the CXCR7 protein and are in agreement with recent studies in the developing brain showing expression of cxcr7 transcripts in migrating interneurons as well as immature excitatory neurons [38]. Furthermore, in these human brain samples only a minority of CXCR7 positive cells (i.e. 11% and 11.4% respectively control/HIV) did not stain for MAP2. As MAP2 is a marker for mature neurons, this smaller group of cells could include resident/infiltrating immune cells (including glia) and/or neural precursors. Further studies are necessary to fully characterize this CXCR7 positive/MAP2 negative cell population (i.e. to establish cell types, differentiation stages, and leukocytes infiltration) and its potential changes during disease progression in specific brain areas. Analysis of a larger group of samples is underway to achieve this goal and to identify potential differences in the expression of CXCR7 between HIV-positive patients without neurological problems and HIV positive patients affected by neurocognitive impairment. The present study demonstrates that CXCR7 protein is widely expressed in mature neurons of the human frontal cortex and hippocampus under both normal and diseased (i.e. HIV) conditions, thus providing the fundamental basis of future investigations focused on its specific role in brain pathology.

Bottom Line: Furthermore, confocal microscopy and binding assays in cultured neurons show that CXCR7 protein is mainly located into cytoplasm, while little to no protein expression is found on neuronal plasma membrane.Interestingly, specific CXCR7 ligands that inhibit CXCL12 binding to CXCR7 do not alter CXCR4-activated survival signaling (pERK/pAkt) in rat cortical neurons.Overall, these results implicate CXCR4 as the main CXCL12 signaling receptor on the surface of differentiated neurons and suggest that CXCR7 may interact with CXCR4 at the intracellular level, possibly affecting CXCR4 trafficking and/or coupling to other proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: CXCR7 and CXCR4 are receptors for the chemokine CXCL12, which is involved in essential functions of the immune and nervous systems. Although CXCR7 transcripts are widely expressed throughout the central nervous system, little is known about its protein distribution and function in the adult brain. To evaluate its potential involvement in CXCL12/CXCR4 signaling in differentiated neurons, we studied CXCR7 protein expression in human brain and cultured neurons.

Methodology/principal findings: Immunohistochemistry and RT-PCR analyses of cortex and hippocampus from control and HIV-positive subjects provided the first evidence of CXCR7 protein expression in human adult neurons, under normal and pathological conditions. Furthermore, confocal microscopy and binding assays in cultured neurons show that CXCR7 protein is mainly located into cytoplasm, while little to no protein expression is found on neuronal plasma membrane. Interestingly, specific CXCR7 ligands that inhibit CXCL12 binding to CXCR7 do not alter CXCR4-activated survival signaling (pERK/pAkt) in rat cortical neurons. Neuronal CXCR7 co-localizes to some extent with the endoplasmic reticulum marker ERp29, but not with early/late endosome markers. Additionally, large areas of overlap are detected in the intracellular pattern of CXCR7 and CXCR4 expression.

Conclusions/significance: Overall, these results implicate CXCR4 as the main CXCL12 signaling receptor on the surface of differentiated neurons and suggest that CXCR7 may interact with CXCR4 at the intracellular level, possibly affecting CXCR4 trafficking and/or coupling to other proteins.

Show MeSH
Related in: MedlinePlus