Multiple sclerosis (MS) is an inflammatory disease of the CNS, characterized by demyelination, focal inflammatory infiltrates and axonal damage. Oxidative stress has been linked to MS pathology. Previous studies have suggested the involvement of NADPH oxidase 2 (Nox2), an enzyme that catalyzes the reduction of oxygen to produce reactive oxygen species, in the MS pathogenesis. The mechanisms of Nox2 activation on MS are unknown. The purpose of this study was to investigate the effect of Nox2 deletion on experimental autoimmune encephalomyelitis (EAE) onset and severity, on astrocyte activation as well as on pro-inflammatory and anti-inflammatory cytokine induction in striatum and motor cortex. Subcutaneous injection of MOG35-55 emulsified with complete Freund's adjuvant was used to evaluate the effect of Nox2 depletion on EAE-induced encephalopathy. Striatum and motor cortices were isolated and evaluated by immunoblotting and RT-PCR. Nox2 deletion resulted in clinical improvement of the disease and prevented astrocyte activation following EAE induction. Nox2 deletion prevented EAE-induced induction of pro-inflammatory cytokines and stimulated the expression of the anti-inflammatory cytokines IL-4 and IL-10. Our data suggest that Nox2 is involved on the EAE pathogenesis. IL-4 and IL-10 are likely to be involved on the protective mechanism observed following Nox2 deletion.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an effective therapeutic strategy for motor symptoms of Parkinson’s disease (PD) when L-DOPA therapy induces disabling side effects. Classical inflammatory activation of glial cells is well established in PD, contributing to the progressive neurodegenerative state; however, the role of DBS in regulating the inflammatory response remains largely unknown. To understand the involvement of astrocytes in the mechanisms of action of DBS, we evaluated the effect of STN–DBS in regulating motor symptoms, astrocyte reactivity, and cytokine expression in a 6-OHDA-induced PD rat model. To mimic in vivo DBS, we investigate the effect of high-frequency stimulation (HFS) in cultured astrocytes regulating cytokine induction and NF-κB activation. We found that STN-DBS improved motor impairment, induced astrocytic hyperplasia, and reversed increased IFN-γ and IL-10 levels in the globus pallidus (GP) of lesioned rats. Moreover, HFS activated astrocytes and prevented TNF-α-induced increase of monocyte chemoattractant protein-1 (MCP-1) and NF-κB activation in vitro. Our results indicate that DBS/HFS may act as a regulator of the inflammatory response in PD states, attenuating classical activation of astrocytes and cytokine induction, potentially through its ability to regulate NF-κB activation. These findings may help us understand the role of astrocyte signaling in HFS, highlighting its possible relationship with the effectiveness of DBS in neurodegenerative disorders.

Background Sepsis-associated encephalopathy (SAE), a diffuse cerebral dysfunction in the absence of direct CNS infection, is associated with increased rates of mortality and morbidity in patients with sepsis. Increased cytokine production and disruption of the blood-brain barrier (BBB) are implicated in the pathogenesis of SAE. The induction of pro-inflammatory mediators is driven, in part, by activation of NF-κΒ. Lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, potently activates NF-κΒ and its downstream targets, including cyclooxygenase-2 (Cox-2). Cox-2 catalyzes prostaglandin synthesis and in the brain prostaglandin, E2 is capable of inducing endothelial permeability. Depletion of polymerase δ-interacting protein 2 (Poldip2) has previously been reported to attenuate BBB disruption, possibly via regulation of NF-κΒ, in response to ischemic stroke. Here we investigated Poldip2 as a novel regulator of NF-κΒ/cyclooxygenase-2 signaling in an LPS model of SAE. Methods Intraperitoneal injections of LPS (18 mg/kg) were used to induce BBB disruption in Poldip2+/+ and Poldip2+/− mice. Changes in cerebral vascular permeability and the effect of meloxicam, a selective Cox-2 inhibitor, were assessed by Evans blue dye extravasation. Cerebral cortices of Poldip2+/+ and Poldip2+/− mice were further evaluated by immunoblotting and ELISA. To investigate the role of endothelial Poldip2, immunofluorescence microscopy and immunoblotting were performed to study the effect of siPoldip2 on LPS-mediated NF-κΒ subunit p65 translocation and Cox-2 induction in rat brain microvascular endothelial cells. Finally, FITC-dextran transwell assay was used to assess the effect of siPoldip2 on LPS-induced endothelial permeability. Results Heterozygous deletion of Poldip2 conferred protection against LPS-induced BBB permeability. Alterations in Poldip2+/+ BBB integrity were preceded by induction of Poldip2, p65, and Cox-2, which was not observed in Poldip2+/− mice. Consistent with these findings, prostaglandin E2 levels were significantly elevated in Poldip2+/+ cerebral cortices compared to Poldip2+/− cortices. Treatment with meloxicam attenuated LPS-induced BBB permeability in Poldip2+/+ mice, while having no significant effect in Poldip2+/− mice. Moreover, silencing of Poldip2 in vitro blocked LPS-induced p65 nuclear translocation, Cox-2 expression, and endothelial permeability. Conclusions These data suggest Poldip2 mediates LPS-induced BBB disruption by regulating NF-κΒ subunit p65 activation and Cox-2 and prostaglandin E2 induction. Consequently, targeted inhibition of Poldip2 may provide clinical benefit in the prevention of sepsis-induced BBB disruption.

BACKGROUND: Lung injury, a severe adverse outcome of lipopolysaccharide-induced acute respiratory distress syndrome, is attributed to excessive neutrophil recruitment and effector response. Poldip2 (polymerase δ-interacting protein 2) plays a critical role in regulating endothelial permeability and leukocyte recruitment in acute inflammation. Thus, we hypothesized that myeloid Poldip2 is involved in neutrophil recruitment to inflamed lungs. METHODS AND RESULTS: After characterizing myeloid-specific Poldip2 knockout mice, we showed that at 18  hours postlipopolysaccharide injection, bronchoalveolar lavage from myeloid Poldip2-deficient mice contained fewer inflammatory cells (8 [4–16] versus 29 [12–57]×104/mL in wild-type mice) and a smaller percentage of neutrophils (30% [28%–34%] versus 38% [33%–41%] in wild-type mice), while the main chemoattractants for neutrophils remained unaffected. In vitro, Poldip2-deficient neutrophils responded as well as wild-type neutrophils to inflammatory stimuli with respect to neutrophil extracellular trap formation, reactive oxygen species production, and induction of cytokines. However, neutrophil adherence to a tumor necrosis factor-α stimulated endothelial monolayer was inhibited by Poldip2 depletion (225 [115–272] wild-type [myePoldip2+/+] versus 133 [62–178] myeloid-specific Poldip2 knockout [myePoldip2-/-] neutrophils) as was transmigration (1.7 [1.3–2.1] versus 1.1 [1.0– 1.4] relative to baseline transmigration). To determine the underlying mechanism, we examined the surface expression of β2- integrin, its binding to soluble intercellular adhesion molecule 1, and Pyk2 phosphorylation. Surface expression of β2-integrins was not affected by Poldip2 deletion, whereas β2-integrins and Pyk2 were less activated in Poldip2-deficient neutrophils. CONCLUSIONS: These results suggest that myeloid Poldip2 is involved in β2-integrin activation during the inflammatory response, which in turn mediates neutrophil-to-endothelium adhesion in lipopolysaccharide-induced acute respiratory distress syndrome.

Background: Polymerase δ-interacting protein 2 (Poldip2) is a multifunctional protein that regulates vascular extracellular matrix composition and matrix metalloproteinase (MMP) activity. The blood-brain barrier (BBB) is a dynamic system assembled by endothelial cells, basal lamina, and perivascular astrocytes, raising the possibility that Poldip2 may be involved in maintaining its structure. We investigated the role of Poldip2 in the late BBB permeability induced by cerebral ischemia. Methods: Transient middle cerebral artery occlusion (tMCAO) was induced in Poldip2 +/+ and Poldip2 +/- mice. The volume of the ischemic lesion was measured in triphenyltetrazolium chloride-stained sections. BBB breakdown was evaluated by Evans blue dye extravasation. Poldip2 protein expression was evaluated by western blotting. RT-PCR, zymography, and ELISAs were used to measure mRNA levels, activity, and protein levels of cytokines and MMPs. Cultured astrocytes were transfected with Poldip2 siRNA, and mRNA levels of cytokines were evaluated as well as IΚBα protein degradation. Results: Cerebral ischemia induced the expression of Poldip2. Compared to Poldip2 +/+ mice, Poldip2 +/- animals exhibited decreased Evans blue dye extravasation and improved survival 24 h following stroke. Poldip2 expression was upregulated in astrocytes exposed to oxygen and glucose deprivation (OGD) and siRNA-mediated downregulation of Poldip2 abrogated OGD-induced IL-6 and TNF-α expression. In addition, siRNA against Poldip2 inhibited TNF-α-induced IΚBα degradation. TNF-α, IL-6, MCP-1, VEGF, and MMP expression induced by cerebral ischemia was abrogated in Poldip2 +/- mice. The protective effect of Poldip2 depletion on the increased permeability of the BBB was partially reversed by systemic administration of TNF-α. Conclusions: Poldip2 is upregulated following ischemic stroke and mediates the breakdown of the BBB by increasing cerebral cytokine production and MMP activation. Therefore, Poldip2 appears to be a promising novel target for the development of therapeutic strategies to prevent the development of cerebral edema in the ischemic brain.

Stroke is a multiphasic process involving a direct ischemic brain injury which is then exacerbated by the influx of immune cells into the brain tissue. Activation of brain endothelial cells leads to the expression of adhesion molecules such vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells, further increasing leukocyte recruitment. Polymerase δ-interacting protein 2 (Poldip2) promotes brain vascular inflammation and leukocyte recruitment via unknown mechanisms. This study aimed to define the role of Poldip2 in mediating vascular inflammation and leukocyte recruitment following cerebral ischemia. Cerebral ischemia was induced in Poldip2+/+ and Poldip2+/− mice and brains were isolated and processed for flow cytometry or RT-PCR. Cultured rat brain microvascular endothelial cells were used to investigate the effect of Poldip2 depletion on focal adhesion kinase (FAK)-mediated VCAM-1 induction. Poldip2 depletion in vivo attenuated the infiltration of myeloid cells, inflammatory monocytes/macrophages and decreased the induction of adhesion molecules. Focusing on VCAM-1, we demonstrated mechanistically that FAK activation was a critical intermediary in Poldip2-mediated VCAM-1 induction. In conclusion, Poldip2 is an important mediator of endothelial dysfunction and leukocyte recruitment. Thus, Poldip2 could be a therapeutic target to improve morbidity following ischemic stroke.

Tissue morphogenesis requires dynamic intercellular contacts that are subsequently stabilized as tissues mature. The mechanisms governing these competing adhesive properties are not fully understood. Using gain- and loss-of-function approaches, we tested the role of p120-catenin (p120) and VE-cadherin (VE-cad) endocytosis in vascular development using mouse mutants that exhibit increased (VE-cadGGG/GGG) or decreased (VE-cadDEE/DEE) internalization. VE-cadGGG/GGG mutant mice exhibited reduced VE-cad-p120 binding, reduced VE-cad levels, microvascular hemorrhaging, and decreased survival. By contrast, VE-cadDEE/DEE mutants exhibited normal vascular permeability but displayed microvascular patterning defects. Interestingly, VE-cadDEE/DEE mutant mice did not require endothelial p120, demonstrating that p120 is dispensable in the context of a stabilized cadherin. In vitro, VE-cadDEE mutant cells displayed defects in polarization and cell migration that were rescued by uncoupling VE-cadDEE from actin. These results indicate that cadherin endocytosis coordinates cell polarity and migration cues through actin remodeling. Collectively, our results indicate that regulated cadherin endocytosis is essential for both dynamic cell movements and establishment of stable tissue architecture.