G protein coupled receptor kinase 5 modifies the nucleolar stress response activated by actinomycin D.

G protein coupled receptor kinase 5 (GRK5) is localized within the nucleus and moderates functions such as DNA transcription, in addition to its localization at the plasma membrane. In this report, we show that GRK5 modifies the nucleolar stress response activated by the DNA polymerase inhibitor, actinomycin D (ActD). We show an increased sensitivity to the apoptotic effects of ActD on cervical HeLa cells and the breast cancer cell line MDA MB 231 with reduced protein expression of GRK5. We also tested two types of breast cancer cells (MDA MB 231 and MCF7 cells) and found that the rate of response to ActD varied between them because they have innate differences in the protein expression of GRK5. We also found that GRK5 phosphorylates nucleophosmin (NPM1) at T199 before and during the early stages of ActD treatment. Phosphorylation at T199 increases the ability of NPM1 to interact with p14ARF in vitro, which may affect the protein expression levels of p14ARF. We found that the expression levels of p14ARF were lower in the cells transfected with the control shRNA, but higher in cells transfected with GRK5 shRNA. Collectively, this suggests that GRK5 modifies the nucleolar stress response associated with ActD.

G protein-coupled receptor kinase 2 modifies the cellular reaction to cisplatin through interactions with NADPH oxidase 4.

G protein-coupled receptor kinases (GRKs), in addition to their role in modulating signal transduction mechanisms associated with activated G protein-coupled receptors (GPCRs), can also interact with many non-GPCR proteins to mediate cellular responses to chemotherapeutics. The rationale for this study is based on the presumption that GRK2 modulates the responses of cancer cells to the chemotherapeutic cisplatin. In this report, we show that GRK2 modulates the responses of cancer cells to cisplatin. Cervical cancer HeLa cells stably transfected with GRK2 shRNA, to decrease GRK2 protein expression, show increased sensitivity to cisplatin. Of interest, these cells also show increased accumulation of NADPH, associating with decreased NADP buildup, at low concentrations of cisplatin tested. These changes in NADPH and NADP levels are also observed in the breast cancer MDA MB 231 cells, which has lower endogenous GRK2 protein expression levels, but not BT549, a breast cancer cell line with higher GRK2 protein expression. This effect of NADPH accumulation may be associated with a decrease in NADPH oxidase 4 (NOX4) protein expression, which is found to correlate with GRK2 protein expression in cancer cells-a relationship which mimics that observed in cardiomyocytes. Furthermore, like in cardiomyocytes, GRK2 and NOX4 interact to form complexes in cancer cells. Collectively, these results suggest that GRK2 interacts with NOX4 to modify cisplatin sensitivity in cancer cells and may also factor into the success of cisplatin-based regimens.

CXCR4 promotes differentiation of oligodendrocyte progenitors and remyelination.

Multiple sclerosis is a neurodegenerative disease characterized by episodes of autoimmune attack of oligodendrocytes leading to demyelination and progressive functional deficits. Because many patients exhibit functional recovery in between demyelinating episodes, understanding mechanisms responsible for repair of damaged myelin is critical for developing therapies that promote remyelination and prevent disease progression. The chemokine CXCL12 is a developmental molecule known to orchestrate the migration, proliferation, and differentiation of neuronal precursor cells within the developing CNS. Although studies suggest a role for CXCL12 in oligodendroglia ontogeny in vitro, no studies have investigated the role of CXCL12 in remyelination in vivo in the adult CNS. Using an experimental murine model of demyelination mediated by the copper chelator cuprizone, we evaluated the expression of CXCL12 and its receptor, CXCR4, within the demyelinating and remyelinating corpus callosum (CC). CXCL12 was significantly up-regulated within activated astrocytes and microglia in the CC during demyelination, as were numbers of CXCR4+NG2+ oligodendrocyte precursor cells (OPCs). Loss of CXCR4 signaling via either pharmacological blockade or in vivo RNA silencing led to decreased OPCs maturation and failure to remyelinate. These data indicate that CXCR4 activation, by promoting the differentiation of OPCs into oligodendrocytes, is critical for remyelination of the injured adult CNS.

Astrocyte TNFR2 is required for CXCL12-mediated regulation of oligodendrocyte progenitor proliferation and differentiation within the adult CNS.

Multiple sclerosis (MS) is characterized by episodes of inflammatory demyelination with progressive failure of remyelination. Prior studies using murine models of MS indicate that remyelination within the adult central nervous system (CNS) requires the expression and activity of TNFR2 and CXCR4 by oligodendrocyte progenitor cells (OPCs), promoting their proliferation and differentiation into mature oligodendrocytes. Here, we extend these studies by examining the role of TNFR2 in the expression of the CXCR4 ligand, CXCL12, within the corpus callosum (CC) during cuprizone (CPZ) intoxication and by demonstrating that lentiviral-mediated gene delivery of CXCL12 to the demyelinated CC improves OPC proliferation and myelin expression during remyelination. Activated astrocytes and microglia express both TNFR1 and TNFR2 within the demyelinated CC. However, CPZ intoxicated TNFR2-/- mice exhibit loss of up-regulation of CXCL12 in astrocytes with concomitant decreases in numbers of CXCR4+ NG2+ OPCs within the CC. While CXCR4 antagonism does not affect OPC migration from subventricular zones into the CC, it decreases their proliferation and differentiation within the CC. Stereotactic delivery of lentivirus expressing CXCL12 protein into the CC of acutely demyelinated TNFR2-/- mice increases OPC proliferation and expression of myelin. In contrast, chronically demyelinated wild-type mice, which exhibit significant loss of astrocytes and OPCs, are unable to be rescued via CXCL12 lentivirus alone but instead required engraftment of CXCL12-expressing astrocytes for increased myelin expression. Our results show that TNFR2 activation induces CXCL12 expression in the demyelinated CC via autocrine signaling specifically within astrocytes, which promotes OPC proliferation and differentiation. In addition, gene delivery of critical pro-myelinating proteins might be a feasible approach for the treatment of remyelination failure in MS.

Age-related changes to tumor necrosis factor receptors affect neuron survival in the presence of beta-amyloid.

Inflammation including local accumulations of tumor necrosis factor alpha (TNF-alpha) is a part of Alzheimer's disease pathology and may exacerbate age-related neurodegeneration. Most studies on TNF-alpha and TNF neuronal receptors are conducted by using embryonic neurons. Few studies consider age-related deficits that may occur in neurons. Age-related changes in susceptibility to TNF-alpha through TNF receptor 1 (TNFR1) and receptor 2 (TNFR2) expression could increase susceptibility to beta-amyloid (1-42, Abeta42). Evidence is conflicting about which receptor mediates survival and/or apoptosis. We determined how aging affects receptor expression in cultured adult rat cortical neurons. Old neurons were more susceptible to Abeta42 toxicity than middle-aged neurons, and the addition of TNF-alpha was neuroprotective in middle-aged neurons, but exacerbated the toxicity from Abeta42 in old neurons. These pathologic and protective responses in old and middle-aged neurons, respectively, correlated with higher starting TNFR1 and TNFR2 mRNA levels in old vs. middle-aged neurons. Middle-aged neurons treated with TNF-alpha plus Abeta42 did not show an increase in either TNFR1 or TNFR2 mRNA, but old neurons showed an up-regulation in TNFR2 mRNA and not TNFR1 mRNA. Despite these mRNA changes, surface immunoreactivity of both TNFR1 and TNFR2 increased with the dose of TNF-alpha in middle-aged neurons. However, middle-aged neurons treated with TNF-alpha plus Abeta42 showed an up-regulation in both TNFR1 and TNFR2 surface expression, whereas old neurons failed to up-regulate surface expression of either receptor. These findings support the hypothesis that age-related changes in TNF-alpha surface receptor expression contribute to the neuronal loss associated with inflammation in Alzheimer's disease.

Targeting CXCR7/ACKR3 as a therapeutic strategy to promote remyelination in the adult central nervous system.

Current treatment modalities for the neurodegenerative disease multiple sclerosis (MS) use disease-modifying immunosuppressive compounds but do not promote repair. Although several potential targets that may induce myelin production have been identified, there has yet to be an approved therapy that promotes remyelination in the damaged central nervous system (CNS). Remyelination of damaged axons requires the generation of new oligodendrocytes from oligodendrocyte progenitor cells (OPCs). Although OPCs are detected in MS lesions, repair of myelin is limited, contributing to progressive clinical deterioration. In the CNS, the chemokine CXCL12 promotes remyelination via CXCR4 activation on OPCs, resulting in their differentiation into myelinating oligodendrocytes. Although the CXCL12 scavenging receptor CXCR7/ACKR3 (CXCR7) is also expressed by OPCs, its role in myelin repair in the adult CNS is unknown. We show that during cuprizone-induced demyelination, in vivo CXCR7 antagonism augmented OPC proliferation, leading to increased numbers of mature oligodendrocytes within demyelinated lesions. CXCR7-mediated effects on remyelination required CXCR4 activation, as assessed via both phospho-S339-CXCR4-specific antibodies and administration of CXCR4 antagonists. These findings identify a role for CXCR7 in OPC maturation during remyelination and are the first to use a small molecule to therapeutically enhance myelin repair in the demyelinated adult CNS.

Central nervous system pathology progresses independently of KC and CXCR2 in globoid-cell leukodystrophy.

Globoid-cell Leukodystrophy (GLD; Krabbe's disease) is a rapidly progressing inherited demyelinating disease caused by a deficiency of the lysosomal enzyme Galactosylceramidase (GALC). Deficiency of GALC leads to altered catabolism of galactosylceramide and the cytotoxic lipid, galactosylsphingosine (psychosine). This leads to a rapidly progressive fatal disease with spasticity, cognitive disability and seizures. The murine model of GLD (Twitcher; GALC-/-) lacks the same enzyme and has similar clinical features. The deficiency of GALC leads to oligodendrocyte death, profound neuroinflammation, and the influx of activated macrophages into the CNS. We showed previously that keratinocyte chemoattractant factor (KC) is highly elevated in the CNS of untreated Twitcher mice and significantly decreases after receiving a relatively effective therapy (bone marrow transplantation combined with gene therapy). The action of KC is mediated through the CXCR2 receptor and is a potent chemoattractant for macrophages and microglia. KC is also involved in oligodendrocyte migration and proliferation. Based on the commonalities between the disease presentation and the functions of KC, we hypothesized that KC and/or CXCR2 contribute to the pathogenesis of GLD. Interestingly, the course of the disease is not significantly altered in KC- or CXCR2-deficient Twitcher mice. There is also no alteration in inflammation or demyelination patterns in these mice. Furthermore, transplantation of CXCR2-deficient bone marrow does not alter the progression of the disease as it does in other models of demyelination. This study highlights the role of multiple redundant cytokines and growth factors in the pathogenesis of GLD.

Mediators of oligodendrocyte differentiation during remyelination.

Myelin, a dielectric sheath that wraps large axons in the central and peripheral nervous systems, is essential for proper conductance of axon potentials. In multiple sclerosis (MS), autoimmune-mediated damage to myelin within the central nervous system (CNS) leads to progressive disability primarily due to limited endogenous repair of demyelination with associated axonal pathology. While treatments are available to limit demyelination, no treatments are available to promote myelin repair. Studies examining the molecular mechanisms that promote remyelination are therefore essential for identifying therapeutic targets to promote myelin repair and thereby limit disability in MS. Here, we present our current understanding of the critical extracellular and intracellular pathways that regulate the remyelinating capabilities of oligodendrocyte precursor cells (OPCs) within the adult CNS.

CXCR7 influences leukocyte entry into the CNS parenchyma by controlling abluminal CXCL12 abundance during autoimmunity.

Loss of CXCL12, a leukocyte localizing cue, from abluminal surfaces of the blood-brain barrier occurs in multiple sclerosis (MS) lesions. However, the mechanisms and consequences of reduced abluminal CXCL12 abundance remain unclear. Here, we show that activation of CXCR7, which scavenges CXCL12, is essential for leukocyte entry via endothelial barriers into the central nervous system (CNS) parenchyma during experimental autoimmune encephalomyelitis (EAE), a model for MS. CXCR7 expression on endothelial barriers increased during EAE at sites of inflammatory infiltration. Treatment with a CXCR7 antagonist ameliorated EAE, reduced leukocyte infiltration into the CNS parenchyma and parenchymal VCAM-1 expression, and increased abluminal levels of CXCL12. Interleukin 17 and interleukin 1ß increased, whereas interferon-γ decreased, CXCR7 expression on and CXCL12 internalization in primary brain endothelial cells in vitro. These findings identify molecular requirements for the transvascular entry of leukocytes into the CNS and suggest that CXCR7 blockade may have therapeutic utility for the treatment of MS.

Age-related differences in NFkappaB translocation and Bcl-2/Bax ratio caused by TNFalpha and Abeta42 promote survival in middle-age neurons and death in old neurons.

Alzheimer's disease is associated with an age-related accumulation of Abeta and inflammation. The inflammatory mediator, TNFalpha activates a signaling cascade involving NFkappaB translocation to the nucleus and a beneficial or detrimental transcriptional response, depending on the age of the neurons and the type of stress applied. Relative to treatment with Abeta42 alone, previously we found that TNFalpha plus Abeta42, applied to old rat neurons (24 month) is toxic, while the same treatment of middle-age neurons (10 month) is protective. In contrast to improved survival of middle-age rat cortical neurons, neurons from old rats are killed by TNFalpha plus Abeta42 despite greater p50 nuclear translocation. In middle-age neurons, blocking TNFR1 does not affect NFkappaB translocation, whereas blocking TNFR2 results in an increase in NFkappaB translocation. For old neurons, blocking either receptor, does not change NFkappaB translocation, but improves cell survival. To account for these effects on cell viability in response to TNF+Abeta, measures of the Bcl-2/Bax ratio positively correlate with survival. In the setting of old neurons, these results suggest that overactivated nuclear translocation of NFkappaB and lower Bcl-2 levels promote death that is reduced by inhibition of either TNFR1 or R2.

Age-related changes in neuronal glucose uptake in response to glutamate and beta-amyloid.

Energy supplies that may decline with age are crucial for cells to maintain ionic homeostasis and prevent neuron death. We examined baseline glucose transporter expression and rate of glucose uptake in cultured hippocampal neurons from embryonic, middle-age (12-month-old), and old (24-month-old) rats and exposed the neurons to glutamate, beta-amyloid, and mitochondrial inhibitors. Without stress, the rate of glucose uptake was similar in middle-age and old neurons, and the rate of glucose uptake in embryonic neurons was threefold greater than that in middle-age and old neurons. Glucose uptake increased in the presence of mitochondrial inhibitors (FCCP and oligomycin) for embryonic and middle-age neurons. The old neurons failed to increase glucose uptake. In the presence of glutamate, FCCP, and oligomycin, embryonic neurons showed a decrease in glucose uptake and the middle-age and old neurons showed no change in glucose uptake. Middle-age neurons took up significantly more glucose than old neurons when under mitochondrial and glutamate stress. In the presence of beta-amyloid, only embryonic neurons increased glucose uptake; middle-age and old neurons did not. Fluorescence imaging of immunoreactive glut3 in response to beta-amyloid demonstrated a 16-49% increase in glut3 immunoreactivity at the plasma membrane for the three ages. The results suggest that old neurons were not able to upregulate glucose uptake to ensure cell survival. Neuron aging does not indicate a defect in normal glut3 function; rather, our results suggest that mechanisms regulating glucose uptake under stress fail to react in time to ensure cell survival.