Hanneke L D M Willemen

Pre-Existing High Glucocorticoid Receptor Number Predicting Development of Posttraumatic Stress Symptoms After Military Deployment

OBJECTIVE The development of posttraumatic stress disorder (PTSD) is influenced by preexisting vulnerability factors. The authors aimed at identifying a preexisting biomarker representing a vulnerability factor for the development of PTSD. To that end, they determined whether the dexamethasone binding capacity of leukocytes, as a measure of glucocorticoid receptor (GR) number, before exposure to trauma was a predictor of development of PTSD symptoms. In addition, the authors analyzed mRNA expression for GR subtypes and GR target genes. METHOD Participants were selected from a large prospective study on deployment-related disorders, in which peripheral blood mononuclear cells (PBMCs) were obtained prior to and 1 and 6 months after military deployment. Participants included armed forces personnel with high levels of PTSD symptoms 6 months after deployment (N=34) and comparison subjects without high levels of PTSD or depressive symptoms (N=34) matched for age, rank, previous deployments, educational level, and function during deployment. RESULTS Before military deployment, the GR number in PBMCs was significantly higher in participants who developed high levels of PTSD symptoms after deployment relative to matched comparison subjects. Logistic regression analysis showed that the risk for inclusion in the PTSD group after deployment increased 7.5-fold with each GR increase of 1,000. No group differences were observed in mRNA expression of GR-α, GR-P, GR-β, glucocorticoid-induced leucine zipper (GILZ), serum and glucocorticoid-inducible kinase-1 (SGK-1), and FKBP5. The higher GR number in the PTSD group was maintained at 1 and 6 months after deployment. CONCLUSIONS These results demonstrate that a preexisting high GR number in PBMCs is a vulnerability factor for subsequent development of PTSD symptoms.

Glucocorticoid receptor pathway components predict posttraumatic stress disorder symptom development: a prospective study.

BACKGROUND Biological correlates of posttraumatic stress disorder (PTSD) have mostly been studied using cross-sectional or posttrauma prospective designs. Therefore, it remains largely unknown whether previously observed biological correlates of PTSD precede trauma exposure. We investigated whether glucocorticoid receptor (GR) pathway components assessed in leukocytes before military deployment represent preexisting vulnerability factors for development of PTSD symptoms. METHODS Four hundred forty-eight male soldiers were assessed before and 6 months after deployment to a combat zone. Participants were assigned to the PTSD or comparison group based on Self-Rating Inventory for PTSD scores after deployment. Logistic regression analysis was applied to predict development of a high level of PTSD symptoms based on predeployment GR number, messenger (m)RNA expression of GR target genes FKBP5, GILZ, and SGK1, plasma cortisol, and childhood trauma. We also investigated whether predeployment GR number and FKBP5 mRNA expression were associated with single nucleotide polymorphisms in the GR and FKBP5 genes, either alone or in interaction with childhood trauma. RESULTS Several GR pathway components predicted subsequent development of a high level of PTSD symptoms: predeployment high GR number, low FKBP5 mRNA expression, and high GILZ mRNA expression were independently associated with increased risk for a high level of PTSD symptoms. Childhood trauma also independently predicted development of a high level of PTSD symptoms. Additionally, we observed a significant interaction effect of GR haplotype BclI and childhood trauma on GR number. CONCLUSIONS Collectively, our results indicate that predeployment GR pathway components are vulnerability factors for subsequent development of a high level of PTSD symptoms.

Mesenchymal Stem Cell Transplantation Attenuates Brain Injury After Neonatal Stroke

Background and Purpose— Brain injury caused by stroke is a frequent cause of perinatal morbidity and mortality with limited therapeutic options. Mesenchymal stem cells (MSC) have been shown to improve outcome after neonatal hypoxic-ischemic brain injury mainly by secretion of growth factors stimulating repair processes. We investigated whether MSC treatment improves recovery after neonatal stroke and whether MSC overexpressing brain-derived neurotrophic factor (MSC-BDNF) further enhances recovery. Methods— We performed 1.5-hour transient middle cerebral artery occlusion in 10-day-old rats. Three days after reperfusion, pups with evidence of injury by diffusion-weighted MRI were treated intranasally with MSC, MSC-BDNF, or vehicle. To determine the effect of MSC treatment, brain damage, sensorimotor function, and cerebral cell proliferation were analyzed. Results— Intranasal delivery of MSC- and MSC-BDNF significantly reduced infarct size and gray matter loss in comparison with vehicle-treated rats without any significant difference between MSC- and MSC-BDNF–treatment. Treatment with MSC-BDNF significantly reduced white matter loss with no significant difference between MSC- and MSC-BDNF–treatment. Motor deficits were also improved by MSC treatment when compared with vehicle-treated rats. MSC-BDNF–treatment resulted in an additional significant improvement of motor deficits 14 days after middle cerebral artery occlusion, but there was no significant difference between MSC or MSC-BDNF 28 days after middle cerebral artery occlusion. Furthermore, treatment with either MSC or MSC-BDNF induced long-lasting cell proliferation in the ischemic hemisphere. Conclusions— Intranasal administration of MSC after neonatal stroke is a promising therapy for treatment of neonatal stroke. In this experimental paradigm, MSC- and BNDF-hypersecreting MSC are equally effective in reducing ischemic brain damage.

MicroRNA-124 as a novel treatment for persistent hyperalgesia

BackgroundChronic pain is often associated with microglia activation in the spinal cord. We recently showed that microglial levels of the kinase G protein–coupled receptor kinase (GRK)2 are reduced in models of chronic pain. We also found that mice with a cell-specific reduction of around 50% in GRK2 level in microglia/macrophages (LysM-GRK2+/− mice) develop prolonged inflammatory hyperalgesia concomitantly with ongoing spinal microglia/macrophage activation. The microRNA miR-124 is thought to keep microglia/macrophages in brain and spinal cord in a quiescent state. In the present study, we investigated the contribution of miR-124 to regulation of hyperalgesia and microglia/macrophage activation in GRK2-deficient mice. In addition, we investigated the effect of miR-124 on chronic inflammatory and neuropathic pain in wild-type (WT) mice.MethodsHyperalgesia was induced by intraplantar IL-1β in WT and LysM-GRK2+/− mice. We determined spinal cord microglia/macrophage miR-124 expression and levels of pro-inflammatory M1 and anti-inflammatory M2 activation markers. The effect of intrathecal miR-124 treatment on IL-1β-induced hyperalgesia and spinal M1/M2 phenotype, and on carrageenan-induced and spared nerve injury-induced chronic hyperalgesia in WT mice was analyzed.ResultsTransition from acute to persistent hyperalgesia in LysM-GRK2+/− mice is associated with reduced spinal cord microglia miR-124 levels. In our LysM-GRK2+/− mice, there was a switch towards a pro-inflammatory M1 phenotype together with increased pro-inflammatory cytokine production. Intrathecal administration of miR-124 completely prevented the transition to persistent pain in response to IL-1β in LysM-GRK2+/− mice. The miR-124 treatment also normalized expression of spinal M1/M2 markers of LysM-GRK2+/− mice. Moreover, intrathecal miR-124 treatment reversed the persistent hyperalgesia induced by carrageenan in WT mice and prevented development of mechanical allodynia in the spared nerve injury model of chronic neuropathic pain in WT mice.ConclusionsWe present the first evidence that intrathecal miR-124 treatment can be used to prevent and treat persistent inflammatory and neuropathic pain. In addition, we show for the first time that persistent hyperalgesia in GRK2-deficient mice is associated with an increased ratio of M1/M2 type markers in spinal cord microglia/macrophages, which is restored by miR-124 treatment. We propose that intrathecal miR-124 treatment might be a powerful novel treatment for pathological chronic pain with persistent microglia activation.

GRK2: A Novel Cell-Specific Regulator of Severity and Duration of Inflammatory Pain

Chronic pain associated with inflammation is a common clinical problem, and the underlying mechanisms have only begun to be unraveled. GRK2 regulates cellular signaling by promoting G-protein-coupled receptor (GPCR) desensitization and direct interaction with downstream kinases including p38. The aim of this study was to determine the contribution of GRK2 to regulation of inflammatory pain and to unravel the underlying mechanism. GRK2+/− mice with an ∼50% reduction in GRK2 developed increased and markedly prolonged thermal hyperalgesia and mechanical allodynia after carrageenan-induced paw inflammation or after intraplantar injection of the GPCR-binding chemokine CCL3. The effect of reduced GRK2 in specific cells was investigated using Cre–Lox technology. Carrageenan- or CCL3-induced hyperalgesia was increased but not prolonged in mice with decreased GRK2 only in Nav1.8 nociceptors. In vitro, reduced neuronal GRK2 enhanced CCL3-induced TRPV1 sensitization. In vivo, CCL3-induced acute hyperalgesia in GRK2+/− mice was mediated via TRPV1. Reduced GRK2 in microglia/monocytes only was required and sufficient to transform acute carrageenan- or CCL3-induced hyperalgesia into chronic hyperalgesia. Chronic hyperalgesia in GRK2+/− mice was associated with ongoing microglial activation and increased phospho-p38 and tumor necrosis factor α (TNF-α) in the spinal cord. Inhibition of spinal cord microglial, p38, or TNF-α activity by intrathecal administration of specific inhibitors reversed ongoing hyperalgesia in GRK2+/− mice. Microglia/macrophage GRK2 expression was reduced in the lumbar ipsilateral spinal cord during neuropathic pain, underlining the pathophysiological relevance of microglial GRK2. Thus, we identified completely novel cell-specific roles of GRK2 in regulating acute and chronic inflammatory hyperalgesia.

Microglial/macrophage GRK2 determines duration of peripheral IL-1β-induced hyperalgesia: Contribution of spinal cord CX3CR1, p38 and IL-1 signaling

&NA; Chronic pain associated with inflammation is a major clinical problem, but the underlying mechanisms are incompletely understood. Recently, we reported that GRK2+/− mice with a ˜50% reduction of GRK2 develop prolonged hyperalgesia following a single intraplantar injection of the pro‐inflammatory cytokine interleukin‐1&bgr; (IL‐1&bgr;). Here we show that spinal microglia/macrophage GRK2 is reduced during chronic inflammation‐induced hyperalgesia. Next, we applied CRE‐Lox technology to create mice with low GRK2 in microglia/macrophages/granulocytes (LysM‐GRK2f/+), or sensory neurons or astrocytes. Only mice deficient in microglial/macrophage/granulocyte GRK2 display prolonged IL‐1&bgr;‐induced hyperalgesia that lasts up to 8 days. Two days after intraplantar IL‐1&bgr;, increased microglial/macrophage activity occurs in the lumbar but not thoracic spinal cord of GRK2‐deficient mice. Intrathecal pre‐treatment with minocycline, an inhibitor of microglia/macrophage activation, accelerates resolution of hyperalgesia independent of genotype and prevents transition to chronic hyperalgesia in GRK2+/− mice. Ongoing hyperalgesia in GRK2+/− mice is reversed by minocycline administration at days 1 and 2 after IL‐1&bgr; injection. Similarly, IL‐1&bgr;‐induced hyperalgesia in LysM‐GRK2f/+ mice is attenuated by intrathecal administration of anti‐CX3CR1 to abrogate fractalkine signaling, the p38 inhibitor SB239063 and the IL‐1 antagonist IL‐1ra. These data establish that chronic inflammatory hyperalgesia is associated with reduced GRK2 in microglia/macrophages and that low GRK2 in these cells is sufficient to markedly prolong hyperalgesia after a single intraplantar injection of IL‐1&bgr;. Ongoing hyperalgesia is maintained by spinal microglial/macrophage activity, fractalkine signaling, p38 activation and IL‐1 signaling. We propose that chronic inflammation decreases spinal microglial/macrophage GRK2, which prevents silencing of microglia/macrophage activity and thereby contributes to prolonged hyperalgesia.

Genome-wide association study meta-analysis of chronic widespread pain: evidence for involvement of the 5p15.2 region

Background and objectives Chronic widespread pain (CWP) is a common disorder affecting ∼10% of the general population and has an estimated heritability of 48–52%. In the first large-scale genome-wide association study (GWAS) meta-analysis, we aimed to identify common genetic variants associated with CWP. Methods We conducted a GWAS meta-analysis in 1308 female CWP cases and 5791 controls of European descent, and replicated the effects of the genetic variants with suggestive evidence for association in 1480 CWP cases and 7989 controls. Subsequently, we studied gene expression levels of the nearest genes in two chronic inflammatory pain mouse models, and examined 92 genetic variants previously described associated with pain. Results The minor C-allele of rs13361160 on chromosome 5p15.2, located upstream of chaperonin-containing-TCP1-complex-5 gene (CCT5) and downstream of FAM173B, was found to be associated with a 30% higher risk of CWP (minor allele frequency=43%; OR=1.30, 95% CI 1.19 to 1.42, p=1.2×10−8). Combined with the replication, we observed a slightly attenuated OR of 1.17 (95% CI 1.10 to 1.24, p=4.7×10−7) with moderate heterogeneity (I2=28.4%). However, in a sensitivity analysis that only allowed studies with joint-specific pain, the combined association was genome-wide significant (OR=1.23, 95% CI 1.14 to 1.32, p=3.4×10−8, I2=0%). Expression levels of Cct5 and Fam173b in mice with inflammatory pain were higher in the lumbar spinal cord, not in the lumbar dorsal root ganglions, compared to mice without pain. None of the 92 genetic variants previously described were significantly associated with pain (p>7.7×10−4). Conclusions We identified a common genetic variant on chromosome 5p15.2 associated with joint-specific CWP in humans. This work suggests that CCT5 and FAM173B are promising targets in the regulation of pain.

Low Nociceptor GRK2 Prolongs Prostaglandin E2 Hyperalgesia via Biased cAMP Signaling to Epac/Rap1, Protein Kinase Cε, and MEK/ERK

Hyperexcitability of peripheral nociceptive pathways is often associated with inflammation and is an important mechanism underlying inflammatory pain. Here we describe a completely novel mechanism via which nociceptor G-protein-coupled receptor kinase 2 (GRK2) contributes to regulation of inflammatory hyperalgesia. We show that nociceptor GRK2 is downregulated during inflammation. In addition, we show for the first time that prostaglandin E2 (PGE2)-induced hyperalgesia is prolonged from <6 h in wild-type (WT) mice to 3 d in mice with low GRK2 in Nav1.8+ nociceptors (SNS–GRK2 +/− mice). This prolongation of PGE2 hyperalgesia in SNS–GRK2 +/− mice does not depend on changes in the sensitivity of the prostaglandin receptors because prolonged hyperalgesia also developed in response to 8-Br-cAMP. PGE2 or cAMP-induced hyperalgesia in WT mice is PKA dependent. However, PKA activity is not required for hyperalgesia in SNS–GRK2 +/− mice. SNS–GRK2 +/− mice developed prolonged hyperalgesia in response to the Exchange proteins directly activated by cAMP (Epac) activator 8-pCPT-2′-O-Me-cAMP (8-pCPT). Coimmunoprecipitation experiments showed that GRK2 binds to Epac1. In vitro, GRK2 deficiency increased 8-pCPT-induced activation of the downstream effector of Epac, Rap1, and extracellular signal-regulated kinase (ERK). In vivo, inhibition of MEK1 or PKCε prevented prolonged PGE2, 8-Br-cAMP, and 8-pCPT hyperalgesia in SNS–GRK2 +/− mice. In conclusion, we discovered GRK2 as a novel Epac1-interacting protein. A reduction in the cellular level of GRK2 enhances activation of the Epac–Rap1 pathway. In vivo, low nociceptor GRK2 leads to prolonged inflammatory hyperalgesia via biased cAMP signaling from PKA to Epac–Rap1, ERK/PKCε pathways.

Stimulation of β2-adrenergic receptors inhibits calcineurin activity in CD4+ T cells via PKA–AKAP interaction

The sympathetic nervous system (SNS) is able to modulate immune functions via adrenoceptor-dependent mechanisms. Activation of β₂-adrenergic receptors (AR) on CD4(+) T lymphocytes has been shown to inhibit Th1-cytokine production and cell proliferation. Here, we investigated the role of the calcium/calmodulin-dependent protein phosphatase calcineurin (CaN), a key element of the T cell receptor (TCR)-signaling pathway, in β₂-AR-mediated suppression of T cell function. Purified rat splenic CD4(+) T cells were stimulated with anti-CD3/anti-CD28 in presence or absence of the β₂-AR agonist terbutaline (TERB). Treatment with TERB induced a dose-dependent inhibition of cellular CaN activity, along with a reduction in IL-2 and IFN-γ production, and T cell proliferation. Co-administration of the β-AR antagonist nadolol abolished these effects. Blockade of the cAMP-dependent protein kinase A (PKA) with the inhibitor H-89 completely prevented TERB-induced CaN inhibition. However, a receptor-independent rise in the second messenger cAMP was not sufficient to suppress CaN activity. Disruption of the interaction between PKA and A-kinase anchoring protein (AKAP) by the inhibitor peptide St-Ht31 fully blocked TERB-induced CaN inhibition, demonstrating that PKA-AKAP interaction is essential for the β₂-AR-mediated CaN inhibition. Taken together, this study provides evidence for a link between the β₂-AR and TCR signaling pathways since expression of IL-2 and IFN-γ in activated T cells largely depends on dephosphorylation of the transcription factor NFAT by CaN, and identifies a novel intracellular mechanism that can lead to downregulation of T cell function after SNS activation.

Cell-specific roles of GRK2 in onset and severity of hypoxic-ischemic brain damage in neonatal mice

The ubiquitously expressed kinase GRK2 protects against cellular overstimulation by desensitizing G protein-coupled receptors and regulating intracellular signaling. Recently, we described that hypoxia-ischemia (HI)-induced brain damage was accelerated and increased in GRK2(+/-) neonatal mice. Using Cre-Lox technology we now investigated the role of decreased GRK2 in only microglia/macrophages or forebrain neurons in development of HI brain injury. Low GRK2 in microglia/macrophages (LysM-GRK2(f/+) mice) was sufficient to accelerate onset of HI damage, without affecting the severity of brain injury at 24h post-HI as compared to LysM-GRK2(+/+) littermates. Consistently, the ipsilateral hemisphere of GRK2(+/-) mice contained microglia with a more rounded phenotype compared to WT mice at 3h post-HI. Inhibition of microglial/macrophage activity by minocycline treatment prevented the early onset of HI injury in GRK2(+/-) mice. In vitro, primary GRK2(+/-) microglia stimulated with LPS produced more TNF-alpha than WT microglia via a p38-dependent pathway. In vivo, HI-induced cerebral p38 activation and TNF-alpha production were increased in GRK2(+/-) mice or in LysM-GRK2(f/+) mice. Our findings indicate that low GRK2 in microglia/macrophages accelerates brain damage via a GRK2/p38/TNF-alpha-dependent pathway. Reduced GRK2 only in forebrain neurons (CamKIIalpha-GRK2(f/+) mice) significantly increased severity of HI brain damage without affecting the onset of brain damage. In conclusion, our data indicate that low GRK2 in microglia/macrophages facilitates activation of these cells which may contribute to the earlier onset of cerebral HI injury associated with increased p38 phosphorylation and TNF-alpha production. The level of GRK2 in neurons is crucial for determining the ultimate severity of HI damage in the newborn brain.