Saori Shimizu

The chemokine CXCL12 promotes survival of postmitotic neurons by regulating Rb protein.

Postmitotic neurons need to keep their cell cycle under control to survive and maintain a differentiated state. This study aims to test the hypothesis that the chemokine CXCL12 regulates neuronal survival and differentiation by promoting Rb function, as suggested by previous studies showing that CXCL12 protects neurons from apoptosis induced by Rb loss. To this end, the effect of CXCL12 on Rb expression and transcriptional activity and the role of Rb in CXCL12-induced neuronal survival were studied. CXCL12 increases Rb protein and RNA levels in rat cortical neurons. The chemokine also stimulates an exogenous Rb promoter expressed in these neurons and counteracts the inhibition of the Rb promoter induced by E2F1 overexpression. Furthermore CXCL12 stimulates Rb activity as a transcription repressor. The effects of CXCL12 are mediated by its specific receptor CXCR4, and do not require the presence of glia. Finally, shRNA studies show that Rb expression is crucial to the neuroprotective activity of CXCL12 as indicated by NMDA-neurotoxicity assays. These findings suggest that proper CXCR4 stimulation in the mature CNS can prevent impairment of the Rb-E2F pathway and support neuronal survival. This is important to maintain CNS integrity in physiological conditions and prevent neuronal injury and loss typical of many neurodegenerative and neuroinflammatory conditions.

CX3CR1 Is Expressed by Prostate Epithelial Cells and Androgens Regulate the Levels of CX3CL1/Fractalkine in the Bone Marrow: Potential Role in Prostate Cancer Bone Tropism

We have previously shown that the chemokine fractalkine promotes the adhesion of human prostate cancer cells to bone marrow endothelial cells as well as their migration toward human osteoblasts in vitro. Thus, the interaction of fractalkine with its receptor CX3CR1 could play a crucial role in vivo by directing circulating prostate cancer cells to the bone. We found that although CX3CR1 is minimally detectable in epithelial cells of normal prostate glands, it is overexpressed upon malignant transformation. Interestingly, osteoblasts, stromal and mesenchymal cells derived from human bone marrow aspirates express the cell-bound form of fractalkine, whereas the soluble form of the chemokine is detected in bone marrow supernatants. To investigate the mechanisms regulating the levels of soluble fractalkine in the bone marrow, we focused on androgens, which play a critical role in both prostate cancer progression and skeletal metastasis. Here, we show that dihydrotestosterone dramatically increases the cleavage of fractalkine from the plasma membrane of bone cells and its action is reversed by nilutamide--an antagonist of the androgen receptor--as well as the wide-spectrum inhibitor of matrix metalloproteases, GM6001. However, dihydrotestosterone was unable to induce fractalkine-cleavage from human bone marrow endothelial cells. Thus, androgens could promote the extravasation of CX3CR1-bearing cancer cells on a fractalkine concentration gradient, while leaving unaltered their ability to adhere to the bone marrow endothelium. In conclusion, our results indicate that CX3CR1, fractalkine, and the enzymes responsible for its cleavage might represent suitable targets for therapies aiming to counteract skeletal secondary tumors from prostate adenocarcinoma.

Morphine Increases Brain Levels of Ferritin Heavy Chain Leading to Inhibition of CXCR4-Mediated Survival Signaling in Neurons

This study focuses on the effect of μ-opioid receptor agonists on CXCR4 signaling in neurons and the mechanisms involved in regulation of neuronal CXCR4 by opiates. The data show that CXCR4 is negatively modulated by long-term morphine treatments both in vitro and in vivo; CXCR4 inhibition is caused by direct stimulation of μ-opioid receptors in neurons, leading to alterations of ligand-induced CXCR4 phosphorylation and upregulation of protein ferritin heavy chain (FHC), a negative intracellular regulator of CXCR4. Reduced coupling of CXCR4 to G-proteins was found in the brain of morphine-treated rats, primarily cortex and hippocampus. CXCR4-induced Gαi/Gβγ activities were suppressed after 24 h treatment of cortical neurons with morphine or the selective μ-opioid agonist DAMGO (d-Ala2-N-Me-Phe4-glycol5-enkephalin), as shown by analysis of downstream targets of CXCR4 (i.e., cAMP, Akt, and ERK1/2). These agonists also prevented CXCL12-induced phosphorylation of CXCR4, indicating a deficit of CXCR4 activation in these conditions. Indeed, morphine (or DAMGO) inhibited prosurvival signaling in neurons. These effects are not attributable to a reduction in CXCR4 expression or surface levels but rather to upregulation of FHC by opioids. The crucial role of FHC in inhibition of neuronal CXCR4 was confirmed by in vitro and in vivo RNA interference studies. Overall, these findings suggest that opiates interfere with normal CXCR4 function in the brain. By this mechanism, opiates could reduce the neuroprotective functions of CXCR4 and exacerbate neuropathology in opiate abusers who are affected by neuroinflammatory/infectious disorders, including neuroAIDS.

Interactions between Chemokines REGULATION OF FRACTALKINE/CX3CL1 HOMEOSTASIS BY SDF/CXCL12 IN CORTICAL NEURONS

The soluble form of the chemokine fractalkine/CX3CL1 regulates microglia activation in the central nervous system (CNS), ultimately affecting neuronal survival. This study aims to determine whether CXCL12, another chemokine constitutively expressed in the CNS (known as stromal cell-derived factor 1; SDF-1), regulates cleavage of fractalkine from neurons. To this end, ELISA was used to measure protein levels of soluble fractalkine in the medium of rat neuronal cultures exposed to SDF-1. Gene arrays, quantitative RT-PCR, and Western blot were used to measure overall fractalkine expression in neurons. The data show that the rate of fractalkine shedding in healthy cultures positively correlates with in vitro differentiation and survival. In analogy to non-neuronal cells, metalloproteinases (ADAM10/17) are involved in cleavage of neuronal fractalkine as indicated by studies with pharmacologic inhibitors. Moreover, treatment of the neuronal cultures with SDF-1 stimulates expression of the inducible metalloproteinase ADAM17 and increases soluble fractalkine content in culture medium. The effect of SDF-1 is blocked by an inhibitor of both ADAM10 and -17, but only partially affected by a more specific inhibitor of ADAM10. In addition, SDF-1 also up-regulates expression of the fractalkine gene. Conversely, exposure of neurons to an excitotoxic stimulus (i.e. NMDA) inhibits α-secretase activity and markedly diminishes soluble fractalkine levels, leading to cell death. These results, along with previous findings on the neuroprotective role of both SDF-1 and fractalkine, suggest that this novel interaction between the two chemokines may contribute to in vivo regulation of neuronal survival by modulating microglial neurotoxic properties.

CXCR7 Protein Expression in Human Adult Brain and Differentiated Neurons

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.

Regulation of neuronal P53 activity by CXCR4

Abnormal activation of CXCR 4 during inflammatory/infectious states may lead to neuronal dysfunction or damage. The major goal of this study was to determine the coupling of CXCR 4 to p53-dependent survival pathways in primary neurons. Neurons were stimulated with the HIV envelope protein gp120(IIIB) or the endogenous CXCR 4 agonist, SDF-1 alpha. We found that gp120 stimulates p53 activity and induces expression of the p53 pro-apoptotic target Apaf-1 in cultured neurons. Inhibition of CXCR 4 by AMD 3100 abrogates the effect of gp120 on both p53 and Apaf-1. Moreover, gp120 neurotoxicity is markedly reduced by the p53-inhibitor, pifithrin-alpha. The viral protein also regulates p53 phosphorylation and expression of other p53-responsive genes, such as MDM 2 and p21. Conversely, SDF-1 alpha, which can promote neuronal survival, increases p53 acetylation and p21 expression in neurons. Thus, the stimulation of different p53 targets could be instrumental in determining the outcome of CXCR 4 activation on neuronal survival in neuro-inflammatory disorders.

Modulation of dendritic cell maturation and function by the Tax protein of human T cell leukemia virus type 1

Human T cell leukemia virus type 1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by the generation of an intense CTL cell response directed against the viral transactivator protein Tax. In addition, patients diagnosed with HAM/TSP exhibit rapid activation and maturation of dendritic cells (DC), likely contributing to the robust, Tax‐specific CTL response. In this study, extracellular Tax has been shown to induce maturation and functional alterations in human monocyte‐derived DC, critical observations being confirmed in freshly isolated myeloid DC. Tax was shown to promote the production of proinflammatory cytokines and chemokines involved in the DC activation process in a dose‐ and time‐dependent manner. Furthermore, Tax induced the expression of DC activation (CD40, CD80, and CD86) and maturation (CD83) markers and enhanced the T cell proliferation capability of DC. Heat inactivation of Tax resulted in abrogation of these effects, indicating a requirement for the native structure of Tax, which was found to bind efficiently to the DC membrane and was internalized within a few hours, suggesting that extracellular Tax may possess an intracellular mechanism of action subsequent to entry. Finally, inhibitors of cellular signaling pathways, NF‐κB, protein kinase, tyrosine kinase, and phospholipase C, were shown to inhibit Tax‐mediated DC activation. This is the first study reporting the immunomodulatory effects of extracellular Tax in the DC compartment. These results suggest that DC, once exposed to Tax by uptake from the extracellular environment, can undergo activation, providing constant antigen presentation and costimulation to T cells, leading to the intense T cell proliferation and inflammatory responses underlying HAM/TSP.

Disruption of neuronal CXCR4 function by opioids: preliminary evidence of ferritin heavy chain as a potential etiological agent in neuroAIDS.

The chemokine CXCL12 and its receptor, CXCR4, regulate neuronal migration, differentiation, and survival. Alterations of CXCL12/CXCR4 signaling are implicated in different neuropathologies, including the neurological complications of HIV infection. Opiates are important co-factors for progression to neuroAIDS and can disrupt the CXCL12/CXCR4 axis in vitro and in vivo. This paper will review recently identified mechanisms of opiate-induced CXCR4 impairment in neurons and introduce results from pilot studies in human brain tissue, which highlight the role of the protein ferritin heavy chain in HIV neuropathology in patients with history of drug abuse.

Bilaminar co-culture of primary rat cortical neurons and glia.

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons. Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection. A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions(1). At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods(1-3). Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology(4), cellular and molecular biology(5-8), biochemistry(5), imaging and microscopy(4,6,7,9,10). The primary neurons extend axons and dendrites to form functional synapses(11), a process which is not observed in neuronal cell lines, although some cell lines do extend processes. A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously(4,12,13). Here we detail a modified protocol suited for cortical neurons. As approximately 20x10(6) cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Multispectral imaging and automated laser capture microdissection of human cortical neurons: A quantitative study of CXCR4 expression

Quantifying protein and RNA expression within specific cell populations in vivo is an essential step in unraveling the complex mechanisms of neurological disease. The challenges associated with studying human brain tissue are commonly compounded by variations in postmortem interval, formalin fixation time, and tissue processing methods among others. The result is a sample population that is inherently heterogeneous, implying the need for reliable protocols that are sensitive to low levels of antigen while minimizing background and nonspecific staining. Here, we describe a single immunohistochemistry protocol on formalin-fixed, paraffin-embedded human cortex which can be adapted to (1) quantify the relative protein expression of the chemokine receptor, CXCR4, using multispectral image or (2) isolate neuronal RNA through automated laser capture microdissection.