USP8 protects against lipopolysaccharide-induced cognitive and motor deficits by modulating microglia phenotypes through TLR4/MyD88/NF-κB signaling pathway in mice

Ubiquitin-specific protease 8 (USP8) regulates inflammation in vitro; however, the mechanisms by which USP8 inhibits neuroinflammation and its pathophysiological functions are not completely understood. In this study, we aimed to determine whether USP8 exerts neuroprotective effects in a mouse model of lipopolysaccharide (LPS)-induced cognitive and motor impairment. We commenced intracerebroventricular USP8 administration 7 days prior to i.p. injection of LPS (750 μg/kg). All treatments and behavioral experiments were performed once per day for 7 consecutive days. Behavioral tests and pathological/biochemical assays were performed to evaluate LPS-induced hippocampal damage. USP8 attenuated LPS-induced cognitive and motor impairments in mice. Moreover, USP8 downregulated several pro-inflammatory cytokines [nitric oxide (NO), tumor necrosis factor α (TNF-α), prostaglandin E₂ (PGE₂), and interleukin-1β (IL-1β)] in the serum and brain, and the relevant protein factors [inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2)] in the brain. Furthermore, USP8 upregulated the anti-inflammatory mediators interleukin (IL)-4 and IL-10 in the serum and brain, and promoted a shift from pro-inflammatory to anti-inflammatory microglial phenotypes. The LPS-induced microglial pro-inflammatory phenotype was abolished by TLR4 inhibitor and in TLR4^−/− mice; these effects were similar to those of USP8 treatment. Mechanistically, we found that USP8 increased the expression of neuregulin receptor degradation protein-1 (Nrdp1), potently downregulated the expression of TLR4 and myeloid differentiation primary response protein 88 (MyD88) protein, and inhibited the phosphorylation of IκB kinase (IKK) β and kappa B-alpha (IκBα), thereby reducing nuclear translocation of p65 by inhibiting the activation of the nuclear factor-kappaB (NF-κB) signaling pathway in LPS-induced mice. Our results demonstrated that USP8 exerts protective effects against LPS-induced cognitive and motor deficits in mice by modulating microglial phenotypes via TLR4/MyD88/NF-κB signaling.     

Protective Effect of Melatonin on Methamphetamine-Induced Apoptosis in Glioma Cell Line

Methamphetamine (METH) is a highly addictive drug causing neurodegenerative diseases. METH has been known to be neurotoxic by inducing oxidative stress, free radical, and pro-inflammatory cytokines. Previous studies have shown that METH could induce neuron and glial cell death, especially inducing glial cell-mediated neurotoxicity that plays a critical role in stress-induced central nervous system damage. Therefore, the aim of the present study is to explore the mechanisms of METH-induced cell death in the glial cell. METH-induced glial cells death is mediated via mitochondrial damage pathway. METH activates the upregulation of the Bax, cytochrome c, cleavage caspase 9 and 3 proteins, and downregulation of Bcl-X_L protein in cascade. Pretreatment with melatonin, a neurohormone secreted by the pineal gland, effectively reduced glial cell death. Moreover, melatonin increased the Bcl-X_L/Bax ratio but reduced the level of cytochrome c, cleavage caspase 9 and 3 proteins. Therefore, these results demonstrated that melatonin could reduce the cytotoxic effect of METH by decreasing the mitochondrial death pathway activation in glial cells. This outcome suggests that melatonin might be beneficial as the neuroprotection in neurodegenerative diseases caused by METH or other pathogens.     

Heme Oxygenase-1-Inducing Activity of 4-Methoxydalbergione and 4’-Hydroxy-4-methoxydalbergione from <Emphasis Type="Italic">Dalbergia odorifera</Emphasis> and Their Anti-inflammatory and Cytoprotective Effects in Murine Hippocampal and BV2 Microglial Cell Line and Primary Rat Microglial Cells

Dalbergia odorifera T. Chen (Leguminosae) grows in Central and South America, Africa, Madagascar, and Southern Asia. D. odorifera possesses many useful pharmacological properties, such as antioxidative and anti-inflammatory activities in various cell types. 4-Methoxydalbergione (MTD) and 4’-hydroxy-4-methoxydalbergione (HMTD) were isolated from the EtOH extract of D. odorifera by several chromatography methods. The chemical structures were elucidated by nuclear magnetic resonance (NMR) and mass spectrum (MS). Anti-inflammatory and cytoprotective effects were examined using BV2 microglial cells and murine hippocampus. MTD and HMTD were demonstrated to induce heme oxygenase (HO)-1 protein levels through the nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) in BV2 microglial cells, while only MTD upregulated HO-1 in HT22 cells. MTD and HMTD induced HO-1 expression through JNK MAPK pathway in BV2 cells, whereas only MTD activated the ERK and p38 pathways in HT22 cells. MTD was also shown to activated MTD and HMTD suppressed lipopolysaccharide-stimulated nitric oxide (NO) and prostaglandin E2 production by inhibiting inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in a dose-dependent manner. Furthermore, MTD and HMTD attenuated pro-inflammatory cytokine productions. These anti-inflammatory effects were found to be mediated through the nuclear factor-kappa B (NF-κB) pathway. MTD exhibited neuroprotective effects on glutamate-induced neurotoxicity by promoting HO-1 in HT22 cells. The anti-inflammatory and cytoprotective effects of MTD and HMTD were partially reversed by an HO inhibitor tin protoporphyrin IX. In addition, MTD and HMTD inhibited pro-inflammatory cytokines and NF-κB pathway in primary rat microglia. These findings suggest that MTD and HMTD have therapeutic potential against neurodegenerative diseases accompanied by microglial activation and/or oxidative cellular injury.     

Involvement of the α<Subscript>1D</Subscript>-adrenergic Receptor in Methamphetamine-Induced Hyperthermia and Neurotoxicity in Rats

Methamphetamine (METH) is a psychostimulant that damages nigrostriatal dopaminergic terminals, primarily by enhancing dopamine and glutamate release. α₁-adrenergic receptor (AR) subtype involved in METH-induced neurotoxicity in rats was investigated using selective α₁-AR antagonists. METH neurotoxicity was evaluated by (1) measuring body temperature; (2) determining tyrosine hydroxylase (TH) immunoreactivity levels; (3) examining levels of dopamine and its metabolites; and (4) assessing glial fibrillary acidic protein (GFAP) and microglial immunoreactivity in the striatum. METH caused a decrease in dopamine and TH levels and induced hyperthermia which is an exacerbating factor of METH neurotoxicity. Concurrently, METH increased GFAP expression and the number of activated microglia. Pretreatment with prazosin, a nonselective α₁-AR antagonist, completely abolished METH-induced decrease in both dopamine and TH and caused a partial reduction in hyperthermia. Prazosin also prevented METH-induced increase in both GFAP expression and the number of activated microglia. In vivo microdialysis analysis revealed that prazosin, however, does not alter the METH-induced dopamine release in the striatum. The neuroprotective effects of prazosin could be mimicked by a selective α_1D antagonist, BMY 7378, but not by selective α_1A or α_1B antagonists. These results suggest that the α_1D-AR is involved in METH-induced hyperthermia and neurotoxicity in rats.     

LRP1 deficiency in microglia blocks neuro-inflammation in the spinal dorsal horn and neuropathic pain processing

Following injury to the peripheral nervous system (PNS), microglia in the spinal dorsal horn (SDH) become activated and contribute to the development of local neuro-inflammation, which may regulate neuropathic pain processing. The molecular mechanisms that control microglial activation and its effects on neuropathic pain remain incompletely understood. We deleted the gene encoding the plasma membrane receptor, LDL Receptor-related Protein-1 (LRP1), conditionally in microglia using two distinct promoter-Cre recombinase systems in mice. LRP1 deletion in microglia blocked development of tactile allodynia, a neuropathic pain-related behavior, after partial sciatic nerve ligation (PNL). LRP1 deletion also substantially attenuated microglial activation and pro-inflammatory cytokine expression in the SDH following PNL. Because LRP1 shedding from microglial plasma membranes generates a highly pro-inflammatory soluble product, we demonstrated that factors which activate spinal cord microglia, including lipopolysaccharide (LPS) and colony-stimulating factor-1, promote LRP1 shedding. Proteinases known to mediate LRP1 shedding, including ADAM10 and ADAM17, were expressed at increased levels in the SDH after PNL. Furthermore, LRP1-deficient microglia in cell culture expressed significantly decreased levels of interleukin-1β and interleukin-6 when treated with LPS. We conclude that in the SDH, microglial LRP1 plays an important role in establishing and/or amplifying local neuro-inflammation and neuropathic pain following PNS injury. The responsible mechanism most likely involves proteolytic release of LRP1 from the plasma membrane to generate a soluble product that functions similarly to pro-inflammatory cytokines in mediating crosstalk between cells in the SDH and in regulating neuropathic pain.     

NF-κB activation in cerebrospinal fluid cells from patients with meningitis

Abstract

We examined whether or not NF-κB, a factor that regulates expression of the genes that code for pro-inflammatory cytokines, is activated in cerebrospinal fluid (CSF) cells to investigate the production of pro-inflammatory cytokines by CSF cells in patients with meningitis. Western blotting demonstrated that NF-κB was more activated in CSF cells of patients with bacterial meningitis than in those of patients with aseptic meningitis. NF-κB was hardly activated in carcinomatous meningitis. The NF-κB activation in CSF cells of patients with meningitis tended to be correlated with the CSF interleukin-6 concentration. Our data suggested that CSF cells produce pro-inflammatory cytokines through NF-κB activation in meningitis, and that increased NF-κB activation in CSF cells indicate infectious meningitis rather than carcinomatous meningitis.     

SR-A Regulates the Inflammatory Activation of Astrocytes

Scavenger receptor Class A (SR-A) participates in the regulation of inflammatory processes against pathogens and in inflammatory stimulation. We have recently demonstrated the presence of SR-A in astrocytes, but its participation in their inflammatory response is unknown. Astrocytes regulate neuroinflammation through the regulation of microglial cell activation and the production of cytokines, neurotrophic factors, and reactive species. Using astrocytes from SR-A^?/? mice in culture, we assessed the participation of SR-A in their inflammatory activation, evaluating the activation of IκB/NF-κB and MAPK signaling pathways and the production of nitric oxide (NO) and IL-1β in response to SR-A ligands. In SR-A^?/? astrocytes, lipopolysaccharide (LPS) induced higher levels of NO and reduced levels of IL-1β compared to SR-A^+/+ cells. In addition, SR-A^?/? astrocytes had a reduced basal and LPS-stimulated JNK phosphorylation, and a delayed activation on IκB/NF-κB signaling pathway in response to LPS. Moreover, inhibition of the ERK pathway reduced NO production by SR-A^?/? cells, suggesting that this signaling pathway modulated LPS-induced NO production, an effect that depended on the presence of SR-A. Our results suggest that SR-A participates in the modulation of signaling pathways involved in the production of soluble molecules implicated in the neuroinflammatory response.     

Magnolol protects against trimethyltin-induced neuronal damage and glial activation in vitro and in vivo

Trimethyltin (TMT), an organotin with potent neurotoxic effects by selectively damaging to hippocampus, is used as a tool for creating an experimental model of neurodegeneration. In the present study, we investigated the protective effects of magnolol, a natural biphenolic compound, on TMT-induced neurodegeneration and glial activation in vitro and in vivo. In HT22 murine neuroblastoma cells, TMT induced necrotic/apoptotic cell death and oxidative stress, including intracellular reactive oxygen species (ROS), protein carbonylation, induction of heme oxygenase-1 (HO-1), and activation of all mitogen-activated protein kinases (MAPKs) family proteins. However, magnolol treatment significantly suppressed neuronal cell death by inhibiting TMT-mediated ROS generation and activation of JNK and p38 MAPKs. In BV-2 microglial cells, magnolol efficiently attenuated TMT-induced microglial activation via suppression of ROS generation and activation of JNK, p38 MAPKs, and nuclear factor-κB (NF-κB) signaling. In an in vivo mouse study, TMT induced massive neuronal damage and enhanced oxidative stress at day 2. We also observed a concomitant increase in glial cells and inducible nitric oxide synthase (iNOS) expression on the same day. These features of TMT toxicity were reversed by treatment of magnolol. We observed that p-JNK and p-p38 MAPK levels were increased in the mouse hippocampus at day 1 after TMT treatment and that magnolol blocked TMT-induced JNK and p38 MAPK activation. Magnolol administration prevented TMT-induced hippocampal neurodegeneration and glial activation, possibly through the regulation of TMT-mediated ROS generation and MAPK activation.     

Neurodegeneration and inflammation in Parkinson's disease

Parkinson's disease (PD) is characterized by the progressive degeneration of dopamine (DA) neurons of the substantia nigra pars compacta (SNpc) accompanied by a buildup of proteinaceous aggregates termed Lewy bodies (LB). In addition to protein aggregation and the loss of DA signaling, PD is also characterized by an active immune response. T-cell infiltration accompanies activated microglial and astrocytic accumulation in and around the SNpc. Although potentially beneficial, microglial activation is most likely responsible for furthering disease pathology and DA neuron degeneration through the release of harmful substances such as pro-inflammatory cytokines, reactive oxidative species and reactive nitrogen species. Activation of the NF-κB death pathway has been shown to occur following microglial activation related release of Cox-2, IL-1β, and Toll-like receptor activation, resulting in increased degeneration of DA neurons of the SNpc. Blockade of microglial activation can lead to DA neuron protection in animal models of PD; however, clinical application of anti-inflammatory drugs has not yielded similar benefits. Future therapeutic designs must take into account the multifactorial nature of PD, including the varied roles of the adaptive and innate immune responses.     

Methamphetamine-induced expression of zif268 mRNA is prevented by haloperidol in mice lacking μ-opioid receptor

We investigated the role of μ-opioid receptor (μ-OR) and dopamine receptor in the modulation of methamphetamine (METH)-induced expression of zif268 mRNA in the striatum of mice. Four groups of wild-type and μ-OR knockout mice were given a single daily intraperitoneal injection of saline (control; group 1) or METH (10 mg/kg; groups 2–4) for 7 consecutive days. On day 11 (after 4 abstinent days), groups 1 and 2 were challenged with saline, group 3 was challenged with METH (10 mg/kg), and group 4 was challenged with dopamine receptor antagonist haloperidol (0.06 mg/kg, subcutaneous injection) plus METH (10 mg/kg). Two hours after the last saline or METH injection, mouse brain tissues were taken for zif268 mRNA analysis using in situ hybridization histochemistry. In comparison to corresponding saline control group (group 1), striatal zif268 mRNA levels were unchanged in group 2 and increased in group 3 in both wild-type and μ-OR knockout mice and without genotype difference. METH challenge-enhanced expression of zif268 mRNA was completely abolished by pre-administration of haloperidol (group 4) in μ-OR knockout mice but not in wild-type mice. The results suggest a crosstalk of the two neurotransmitter systems in modulation of METH-induced IEG expression, because only in μ-OR knockout mice in which dopamine receptors were blocked were METH-induced zif268 expression abolished. METH-induced zif268 expression was not altered in μ-OR knockout mice without blockade of dopamine receptors or wild-type mice with blockade of dopamine receptors.     

Anti-Inflammatory Role of the Isoflavone Diadzein in Lipopolysaccharide-Stimulated Microglia: Implications for Parkinson’s Disease

Microglial activation and subsequent release of toxic pro-inflammatory factors are believed to play an important role in neuronal cell death associated with Parkinson’s disease (PD). Compounds that inhibit microglia activation and suppress pro-inflammatory factor release have been reported to have neuroprotective effects in animal models of PD. In this study, we tested whether diadzein, a natural isoflavone found in soybean, attenuated lipopolysaccharide (LPS)-induced release of inflammatory mediators in BV-2, a murine microglial cell line. Diadzein pretreatment was found to significantly suppress the production of the pro-inflammatory factors nitric oxide and IL-6 as well as their mRNA expression in conjunction with reductions in ROS production, p38 MAPK phosphorylation, and NF-κB activation. Furthermore, transfer of conditioned media (CM) from BV-2 cells pretreated with diadzein resulted in a significantly reduction in dopaminergic neurotoxicity compared with CM from microglia stimulated with LPS alone. Together, our results suggest that diadzein’s neuroprotective properties may be due to its ability to dampen induction of microglial activation and the subsequent release of soluble pro-inflammatory factors. This appears to be via inhibition of oxidative induction of the p38 MAP kinase-NFκB pathway, resulting in reduced expression of pro-inflammatory genes and release of their corresponding gene products.     

Activation of corticotropin-releasing factor neurons and microglia in paraventricular nucleus precipitates visceral hypersensitivity induced by colorectal distension in rats

Visceral hypersensitivity is a major contributor to irritable bowel syndrome and other disorders with visceral pain. Substantial evidence has established that glial activation and neuro-glial interaction play a key role in the establishment and maintenance of visceral hypersensitivity. We recently demonstrated that activation of spinal microglial toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor κB (NF-κB) signaling facilitated the development of visceral hypersensitivity in a rat model developed by neonatal and adult colorectal distensions (CRDs). Hypothalamic paraventricular nucleus (PVN) plays a pivotal role in the pathogenesis of chronic pain. In this study, we examined the mechanism by which microglia and neurons in PVN establish and maintain visceral hypersensitivity and the involvement of TLR4 signaling. Visceral hypersensitivity was precipitated by adult colorectal distension (CRD) only in rats that experienced neonatal CRDs. Visceral hypersensitivity was associated with an increase in the expression of c-fos, corticotropin-releasing factor (CRF) protein and mRNA in PVN, which could be prevented by intra-PVN infusion of lidocaine or small interfering RNA targeting the CRF gene. These results suggest PVN CRF neurons modulate visceral hypersensitivity. Adult CRD induced an increase in the expression of Iba-1 (a microglial marker), TLR4 protein, and its downstream effectors MyD88, NF-κB, as well as proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) only in rats that experienced neonatal CRDs. Intra-PVN infusion of minocycline, a nonselective microglial inhibitor, attenuated the hyperactivity of TLR4 signaling cascade, microglial activation, and visceral hypersensitivity. Taken together, these data suggest that neonatal CRDs induce a glial activation in PVN. Adult CRD potentiates the glial and CRF neuronal activity, and precipitates visceral hypersensitivity and pain. TLR4 signaling and proinflammatory cytokines TNF-α and IL-1β may participate in neuro-glial interaction during the pathogenesis of visceral hypersensitivity.     

NGF-activated protein tyrosine phosphatase 1B mediates the phosphorylation and degradation of I-κ-Bα coupled to NF-κ-B activation,thereby controlling dendrite morphology

NGF diminishes dendrite complexity in cultured hippocampal neurons by decreasing the number of primary and secondary dendrites, while increasing the length of those that remain. The transduction pathway used by NGF to provoke dendrite elongation involves the activation of NF-κ-B and the expression of the homologues of Enhancer-of-split 1 gene. Here, we define important steps that link NGF with NF-κ-B activation, through the activity of protein tyrosine phosphatase 1B (PTP1B). Binding of NGF to p75^NTR stimulates PTP1B activity, which can be blocked by either pharmacological inhibition of the phosphatase or by transfecting neurons with a dn PTP1B isoform, whereby NGF is no longer able to stimulate dendrite growth. Indeed, overexpressing PTP1B alone provoked dendrite growth and further studies revealed a role for the src kinase downstream of PTP1B. Again, loss of src activity largely cancelled out the capacity of NGF to promote dendrite growth, whereas overexpression of v-src in neurons was sufficient to promote dendrite growth. Finally, the NGF/p75^NTR/PTP1B/src kinase pathway led to the tyrosine phosphorylation of I-κ-Bα prior to its degradation, an event that is necessary for NF-κ-B activation. Indeed, the dendrite growth response to NGF was lost when neurons were transfected with a mutant form of I-κ-Bα that lacks tyr42. Thus, our data suggest that PTP1B fulfils a central role in the NGF signalling that controls dendrite patterning in hippocampal neurons.     

Inhibition of nuclear factor-kappa B sensitises anterior pituitary cells to tumour necrosis factor-α- and lipopolysaccharide-induced apoptosis

Nuclear factor-kappa B (NF-κB), an important pro-inflammatory factor, is a crucial regulator of cell survival. Both lipopolysaccharide (LPS) and tumour necrosis factor (TNF)-α activate NF-κB signalling. Oestrogens were shown to suppress NF-κB activation. Oestrogens exert a sensitising action to pro-apoptotic stimuli such as LPS and TNF-α in anterior pituitary cells. In the present study, we show by western blotting that 17β-oestradiol (E(2)) decreases TNF-α-induced NF-κB/p65 and p50 nuclear translocation in primary cultures of anterior pituitary cells from ovariectomised (OVX) rats. Also, the in vivo administration of E(2) decreases LPS-induced NF-κB/p65 and p50 nuclear translocation. To investigate whether the inhibition of NF-κB pathway sensitises anterior pituitary cells to pro-apoptotic stimuli, we used an inhibitor of NF-κB activity, BAY 11-7082 (BAY). BAY, at a concentration that fails to induce apoptosis, has permissive action on TNF-α-induced apoptosis of lactotrophs and somatotrophs from OVX rats, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Pharmacological inhibition of NF-κB signalling enhances E(2)-sensitising effect to TNF-α-induced apoptosis in lactotrophs but not in somatotrophs. In vivo administration of BAY allowed LPS-induced apoptosis in anterior pituitary cells from OVX rats (determined by fluorescence activated cell sorting). Furthermore, LPS-induced expression of Bcl-xL in pituitaries of OVX rats is decreased by E(2) administration. Our results show that inhibition of the NF-κB signalling pathway sensitises anterior pituitary cells to the pro-apoptotic action of LPS and TNF-α. Because E(2) inhibits LPS- and TNF-α-activated NF-κB nuclear translocation, the present study suggests that E(2) sensitises anterior pituitary cells to TNF-α- and LPS-induced apoptosis by inhibiting NF-κB activity.