FBL Promotes LPS-Induced Neuroinflammation by Activating the NF-κB Signaling Pathway.

Purpose: Neuroinflammation occurs in response to central nervous system (CNS) injury, infection, stimulation by toxins, or autoimmunity. We previously analyzed the downstream molecular changes in HT22 cells (mouse hippocampal neurons) upon lipopolysaccharide (LPS) stimulation. We detected elevated expression of Fibrillarin (FBL), a nucleolar methyltransferase, but the associated proinflammatory mechanism was not systematically elucidated. The aim of this study was to investigate the underlying mechanisms by which FBL affects neuroinflammation. Methods: RT-real-time PCR, Western blotting and immunofluorescence were used to assess the mRNA and protein expression of FBL in HT22 cells stimulated with LPS, as well as the cellular localization and fluorescence intensity of FBL. BAY-293 (a son of sevenless homolog 1 (SOS1) inhibitor), SR11302 (an activator protein-1 (AP-1) inhibitor) and KRA-533 (a KRAS agonist) were used to determine the molecular mechanisms underlying the effect of FBL. AP-1 was predicted to be the target protein of FBL by molecular docking analysis, and validation was performed with T-5224 (an AP-1 inhibitor). In addition, the downstream signaling pathways of FBL were identified by transcriptome sequencing and verified by RT-real-time PCR. Results: LPS induced FBL mRNA and protein expression in HT22 cells. In-depth mechanistic studies revealed that when we inhibited c-Fos, AP-1, and SOS1, FBL expression decreased, whereas FBL expression increased when KRAS agonists were used. In addition, the transcript levels of inflammatory genes in the NF-kB signaling pathway (including CD14, MYD88, TNF, TRADD, and NFKB1) were elevated after the overexpression of FBL. Conclusion: LPS induced the expression of FBL in HT22 cells through the RAS/MAPK signaling pathway, and FBL further activated the NF-kB signaling pathway, which promoted the expression of relevant inflammatory genes and the release of cytokines. The present study reveals the mechanism by which FBL promotes neuroinflammation and offers a potential target for the treatment of neuroinflammation.

Preso enhances mGluR1-mediated excitotoxicity by modulating the phosphorylation of mGluR1-Homer1 complex and facilitating an ER stress after traumatic brain injury.

Glutamate receptor (GluR)-mediated excitotoxicity is an important mechanism causing delayed neuronal injury after traumatic brain injury (TBI). Preso, as a core scaffolding protein of postsynaptic density (PSD), is considered an important regulator during excitotoxicity and TBI and combines with glutamate receptors to form functional units for excitatory glutamatergic neurotransmission, and elucidating the mechanisms of these functional units will provide new targets for the treatment of TBI. As a multidomain scaffolding protein, Preso directly interacts with metabotropic GluR (mGluR) and another scaffold protein, Homer. Because the mGluR-Homer complex plays a crucial role in TBI, modulation of this complex by Preso may be an important mechanism affecting the excitotoxic damage to neurons after TBI. Here, we demonstrate that Preso facilitates the interaction between metabotropic mGluR1 and Homer1 to activate mGluR1 signaling and cause excitotoxic neuronal injury and endoplasmic reticulum (ER) stress after TBI. The regulatory effect of Preso on the mGluR1-Homer1 complex is dependent on the direct association between Preso and this complex and also involves the phosphorylation of the interactive binding sites of mGluR1 and Homer1 by Preso. Further studies confirmed that Preso, as an adaptor of cyclin-dependent kinase 5 (CDK5), promotes the phosphorylation of the Homer1-binding site on mGluR1 by CDK5 and thereby enhances the interaction between mGluR1 and Homer1. Preso can also promote the formation of the mGluR1-Homer1 complex by inhibiting the phosphorylation of the Homer1 hinge region by Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα). Based on these molecular mechanisms, we designed several blocking peptides targeting the interaction between Preso and the mGluR1-Homer1 complex and found that directly disrupting the association between mGluR1 and scaffolding proteins significantly promotes the recovery of motor function after TBI.

Peripheral CCL2 induces inflammatory pain via regulation of Ih currents in small diameter DRG neurons.

The C-C motif chemokine ligand 2 (CCL2) has been implicated in chronic pain, but its exact mechanism of peripheral sensitization is unknown. In this study, we aimed to clarify the mechanism of CCL2 regulation of ion channels. Our behavioral experiments revealed that ZD7288, a blocker of Ih current, can inhibit CFA and CCL2-mediated mechanical and thermal nociceptive sensitization. Furthermore, patch clamp studies demonstrated that CFA-induced peripheral sensitization primarily affects the excitability of small-diameter DRG neurons. Further studies revealed that inflammatory pain caused by CFA or incubation of DRG with CCL2 mainly affected Ih currents in small-diameter DRG neurons, which were blocked by co-incubation CCR2 antagonist INCB3344 or adenylate cyclase inhibitor SQ22536. Immunohistochemical staining showed that both intraplantar injection of CFA as well as DRG injection of CCL2 resulted in significant upregulation of CCR2+/HCN2+ expression. In conclusion, we suggest in the inflammatory pain state, CCL2 can act on small-diameter DRG neurons, leading to upregulation of HCN2 expression and consequently Ih, which in turn leads to neuronal hyperexcitability.

Quantitative proteomic and phosphoproteomic analyses of the hippocampus reveal the involvement of NMDAR1 signaling in repetitive mild traumatic brain injury.

The cumulative damage caused by repetitive mild traumatic brain injury can cause long-term neurodegeneration leading to cognitive impairment. This cognitive impairment is thought to result specifically from damage to the hippocampus. In this study, we detected cognitive impairment in mice 6 weeks after repetitive mild traumatic brain injury using the novel object recognition test and the Morris water maze test. Immunofluorescence staining showed that p-tau expression was increased in the hippocampus after repetitive mild traumatic brain injury. Golgi staining showed a significant decrease in the total density of neuronal dendritic spines in the hippocampus, as well as in the density of mature dendritic spines. To investigate the specific molecular mechanisms underlying cognitive impairment due to hippocampal damage, we performed proteomic and phosphoproteomic analyses of the hippocampus with and without repetitive mild traumatic brain injury. The differentially expressed proteins were mainly enriched in inflammation, immunity, and coagulation, suggesting that non-neuronal cells are involved in the pathological changes that occur in the hippocampus in the chronic stage after repetitive mild traumatic brain injury. In contrast, differentially expressed phosphorylated proteins were mainly enriched in pathways related to neuronal function and structure, which is more consistent with neurodegeneration. We identified N-methyl-D-aspartate receptor 1 as a hub molecule involved in the response to repetitive mild traumatic brain injury , and western blotting showed that, while N-methyl-D-aspartate receptor 1 expression was not altered in the hippocampus after repetitive mild traumatic brain injury, its phosphorylation level was significantly increased, which is consistent with the omics results. Administration of GRP78608, an N-methyl-D-aspartate receptor 1 antagonist, to the hippocampus markedly improved repetitive mild traumatic brain injury-induced cognitive impairment. In conclusion, our findings suggest that N-methyl-D-aspartate receptor 1 signaling in the hippocampus is involved in cognitive impairment in the chronic stage after repetitive mild traumatic brain injury and may be a potential target for intervention and treatment.

Cingulate cGMP-dependent protein kinase I facilitates chronic pain and pain-related anxiety and depression.

ABSTRACT: Patients with chronic pain often experience exaggerated pain response and aversive emotion, such as anxiety and depression. Central plasticity in the anterior cingulate cortex (ACC) is assumed to be a critical interface for pain perception and emotion, which has been reported to involve activation of NMDA receptors. Numerous studies have documented the key significance of cGMP-dependent protein kinase I (PKG-I) as a crucial downstream target for the NMDA receptor-NO-cGMP signaling cascade in regulating neuronal plasticity and pain hypersensitivity in specific regions of pain pathway, ie, dorsal root ganglion or spinal dorsal horn. Despite this, whether and how PKG-I in the ACC contributes to cingulate plasticity and comorbidity of chronic pain and aversive emotion has remained elusive. Here, we uncovered a crucial role of cingulate PKG-I in chronic pain and comorbid anxiety and depression. Chronic pain caused by tissue inflammation or nerve injury led to upregulation of PKG-I expression at both mRNA and protein levels in the ACC. Knockdown of ACC-PKG-I relieved pain hypersensitivity as well as pain-associated anxiety and depression. Further mechanistic analysis revealed that PKG-I might act to phosphorylate TRPC3 and TRPC6, leading to enhancement of calcium influx and neuronal hyperexcitability as well as synaptic potentiation, which results in the exaggerated pain response and comorbid anxiety and depression. We believe this study sheds new light on the functional capability of ACC-PKG-I in modulating chronic pain as well as pain-associated anxiety and depression. Hence, cingulate PKG-I may represent a new therapeutic target against chronic pain and pain-related anxiety and depression.

Identification of INSRR as an immune-related gene in the tumor microenvironment of glioblastoma by integrated bioinformatics analysis.

Gliomas are the most common malignant tumors in the central nervous system. The tumor microenvironment (TME) plays a crucial role in tumor proliferation, invasion, angiogenesis, and immune escape. However, little is known about TME in gliomas. The purpose of this study was to explore the biomarkers associated with TME in glioblastoma (GBM) to predict immunotherapy effectiveness and prognosis in patients. Based on RNA-seq transcriptome data and clinical features of 1222 samples (113 normal samples and 1109 tumor samples) in The Cancer Genome Atlas (TCGA) database, the ImmuneScore, StromalScore, and ESTIMATEScore were calculated by ESTIMATE algorithm. The differentially expressed genes (DEGs) and differentially mutated genes (DMGs) were determined in the TCGA GBM cohort. Furthermore, gene set enrichment analysis (GSEA) was used to investigate the enrichment pathways of INSRR genes with abnormal expression. The proportion of tumor-infiltrating immune cells (TIICs) was evaluated by CIBERSORT. Frequent mutations of TP53, EGFR, and PTEN occurred in high and low immune scores. The cross-analysis of DEGs and DMGs revealed that INSRR was an immune-related biomarker in the TCGA GBM cohort. According to GSEA, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway with INSRR abnormal expression were IgA-produced intestinal immune network and Alzheimer's disease, oxidative phosphorylation, and Parkinson's disease, respectively. Additionally, INSRR expression was correlated with dendritic cells activated, dendritic cells resting, T cells CD8, and T cell gamma delta. INSRR is associated with the immune microenvironment in GBM and is used as a biomarker to predict immune invasion.

Pulsed Electromagnetic Fields Protect Against Brain Ischemia by Modulating the Astrocytic Cholinergic Anti-inflammatory Pathway.

Neuroinflammation is one of the most important pathological processes following brain ischemia. Pulsed electromagnetic fields (PEMFs) protect against brain ischemia, but their role in regulating neuroinflammation remains unclear. In the present study, we investigated the biological effects of PEMF exposure on brain ischemia-induced neuroinflammation through the astrocytic cholinergic anti-inflammatory pathway. PEMF exposure reduced the activation of astrocytes and neuroinflammation following brain ischemia by directly modulating astrocytic injury and inflammatory cytokine release. Inhibition of nicotinic acetylcholine receptor alpha 7 subunit (α7nAChR) by a specific antagonist reversed the regulatory effects of PEMF on astrocytes. Furthermore, negative regulation of signal transducer and activator of transcription 3 (STAT3) by α7nAChR was found to be an important downstream mechanism through which PEMF regulates astrocyte-related neuroinflammation. PEMF suppressed STAT3 phosphorylation and nuclear translocation by activating α7nAChR. These results demonstrate that PEMF exerts anti-inflammatory effects in the context of brain ischemia by modulating astrocytic α7nAChR/STAT3 signaling.

Identification of a Fibroblast-Related Prognostic Model in Glioma Based on Bioinformatics Methods.

BACKGROUND: Glioma is the most common primary tumor of the central nervous system with a high lethality rate. This study aims to mine fibroblast-related genes with prognostic value and construct a corresponding prognostic model. METHODS: A glioma-related TCGA (The Cancer Genome Atlas) cohort and a CGGA (Chinese Glioma Genome Atlas) cohort were incorporated into this study. Variance expression profiling was executed via the "limma" R package. The "clusterProfiler" R package was applied to perform a GO (Gene Ontology) analysis. The Kaplan-Meier (K-M) curve, LASSO regression analysis, and Cox analyses were implemented to determine the prognostic genes. A fibroblast-related risk model was created and affirmed by independent cohorts. We derived enriched pathways between the fibroblast-related high- and low-risk subgroups using gene set variation analysis (GSEA). The immune infiltration cell and the stromal cell were calculated using the microenvironment cell populations-counter (MCP-counter) method, and the immunotherapy response was assessed with the SubMap algorithm. The chemotherapy sensitivity was estimated using the "pRRophetic" R package. RESULTS: A total of 93 differentially expressed fibroblast-related genes (DEFRGs) were uncovered in glioma. Seven prognostic genes were filtered out to create a fibroblast-related gene signature in the TCGA-glioma cohort training set. We then affirmed the fibroblast-related risk model via TCGA-glioma cohort and CGGA-glioma cohort testing sets. The Cox regression analysis proved that the fibroblast-related risk score was an independent prognostic predictor in prediction of the overall survival of glioma patients. The fibroblast-related gene signature revealed by the GSEA was applicable to the immune-relevant pathways. The MCP-counter algorithm results pointed to significant distinctions in the tumor microenvironment between fibroblast-related high- and low-risk subgroups. The SubMap analysis proved that the fibroblast-related risk score could predict the clinical sensitivity of immunotherapy. The chemotherapy sensitivity analysis indicated that low-risk patients were more sensitive to multiple chemotherapeutic drugs. CONCLUSION: Our study identified prognostic fibroblast-related genes and generated a novel risk signature that could evaluate the prognosis of glioma and offer a theoretical basis for clinical glioma therapy.

Bioactivity-Guided Synthesis Accelerates the Discovery of Evodiamine Derivatives as Potent Insecticide Candidates.

Pests threaten worldwide food security by decreasing crop yields and damaging their quality. Natural product-based molecular design and structural optimization have been one of the most effective ways to innovate pesticides for integrated insect management. To continue our previous studies on the discovery of insecticidal lead, a series of evodiamine derivatives were designed, synthesized, and evaluated for their insecticidal activities. The bioassay results demonstrated that compounds Ian and Iao exhibited 90 and 80% insecticidal activities against Mythimna separata at 2.5 mg/L, respectively, which were superior to evodiamine (10% at 10 mg/L), matrine (45% at 600 mg/L), and rotenone (30% at 200 mg/L). Compounds Ian-Iap showed 90% insecticidal activities against Plutella xylostella at 1.0 mg/L, far more potent than those of evodiamine, matrine, and rotenone. Compound Ian displayed 60% insecticidal activity against Helicoverpa armigera at 5.0 mg/L, while evodiamine, matrine, and rotenone showed very poor activities. The study on the insecticidal mechanism of action by a calcium imaging experiment indicated that the insect ryanodine receptors (RyRs) could be the potential target of Ian. Furthermore, the molecular docking indicated that Ian anchored in the binding site of the RyR of P. xylostella. The above results manifested the potential of evodiamine derivatives as potent insecticide candidates.

Nociceptor-localized cGMP-dependent protein kinase I is a critical generator for central sensitization and neuropathic pain.

Patients with neuropathic pain often experience exaggerated pain and anxiety. Central sensitization has been linked with the maintenance of neuropathic pain and may become an autonomous pain generator. Conversely, emerging evidence accumulated that central sensitization is initiated and maintained by ongoing nociceptive primary afferent inputs. However, it remains elusive what mechanisms underlie this phenomenon and which peripheral candidate contributes to central sensitization that accounts for pain hypersensitivity and pain-related anxiety. Previous studies have implicated peripherally localized cGMP-dependent protein kinase I (PKG-I) in plasticity of nociceptors and spinal synaptic transmission as well as inflammatory hyperalgesia. However, whether peripheral PKG-I contributes to cortical plasticity and hence maintains nerve injury-induced pain hypersensitivity and anxiety is unknown. Here, we demonstrated significant upregulation of PKG-I in ipsilateral L3 dorsal root ganglia (DRG), no change in L4 DRG, and downregulation in L5 DRG upon spared nerve injury. Genetic ablation of PKG-I specifically in nociceptors or post-treatment with intervertebral foramen injection of PKG-I antagonist, KT5823, attenuated the development and maintenance of spared nerve injury-induced bilateral pain hypersensitivity and anxiety. Mechanistic analysis revealed that activation of PKG-I in nociceptors is responsible for synaptic potentiation in the anterior cingulate cortex upon peripheral neuropathy through presynaptic mechanisms involving brain-derived neurotropic factor signaling. Our results revealed that PKG-I expressed in nociceptors is a key determinant for cingulate synaptic plasticity after nerve injury, which contributes to the maintenance of pain hypersensitivity and anxiety. Thereby, this study presents a strong basis for opening up a novel therapeutic target, PKG-I, in nociceptors for treatment of comorbidity of neuropathic pain and anxiety with least side effects.

A new textured polyphosphazene biomaterial with improved blood coagulation and microbial infection responses.

A new poly[bis(octafluoropentoxy) phosphazene] (OFP) was synthesized for the purpose of blood contacting medical devices. OFP was further either developed into crosslinkable polyphosphazene (X-OFP) or blended with polyurethane (PU) as the mixture (OFP/PU) for improvement of mechanical property of polyphosphazene polymers. All the materials were fabricated as smooth films or further textured with submicron pillars for the assay of antimicrobial and antithrombotic properties. Results showed that crosslinkable OFP (X-OFP) and blends of OFP/PU successfully improved the mechanical strength of OFP and fewer defects of pillars were found on the textured polyphosphazene surfaces. The antithrombotic experiments showed that polyphosphazene OFP materials reduced human Factor XII activation and platelet adhesion, thereby being resistant to plasma coagulation and thrombosis. The bacterial adhesion and biofilm experiments demonstrated that OFP materials inhibited staphylococcal bacterial adhesion and biofilm formation. The surface texturing further reduced the platelet adhesion and bacterial adhesion, and inhibited biofilm formation up to 23 days. The data suggested that textured OFP materials may provide a practical approach to improve the biocompatibility of current biomaterials in the application of blood contacting medical devices with significant reduction in risk of pathogenic infection and thrombosis. STATEMENT OF SIGNIFICANCE: The thromboembolic events and microbial infection have been the significant barriers for the long term use of biomaterials in blood-contacting medical devices. The development of new materials with multiple functions including anti-thrombosis and antibacterial surfaces is a high research priority. This study synthesized new biostable and biocompatible polyphosphazene polymers, poly[bis(octafluoropentoxy)phosphazene] (OFP) and crosslinkable OFP, and successfully improved the mechanical strength of polyphosphazenes. Polymers were fabricated into textured films with submicron pillars on the surfaces. The antimicrobial and antithrombotic assays demonstrated that new materials combined with surface physical modification have significant reduction in risk of pathogenic infection and thrombosis, and improve the biocompatibility of current biomaterials in the application of blood-contacting medical devices. It would be interest to biomaterials and bioengineering related communities.

Nanodisco balls: control over surface versus core loading of diagnostically active nanocrystals into polymer nanoparticles.

Nanoparticles of complex architectures can have unique properties. Self-assembly of spherical nanocrystals is a high yielding route to such systems. In this study, we report the self-assembly of a polymer and nanocrystals into aggregates, where the location of the nanocrystals can be controlled to be either at the surface or in the core. These nanospheres, when surface decorated with nanocrystals, resemble disco balls, thus the term nanodisco balls. We studied the mechanism of this surface loading phenomenon and found it to be Ca(2+) dependent. We also investigated whether excess phospholipids could prevent nanocrystal adherence. We found surface loading to occur with a variety of nanocrystal types including iron oxide nanoparticles, quantum dots, and nanophosphors, as well as sizes (10-30 nm) and shapes. Additionally, surface loading occurred over a range of polymer molecular weights (∼30-3000 kDa) and phospholipid carbon tail length. We also show that nanocrystals remain diagnostically active after loading onto the polymer nanospheres, i.e., providing contrast in the case of magnetic resonance imaging for iron oxide nanoparticles and fluorescence for quantum dots. Last, we demonstrated that a fluorescently labeled protein model drug can be delivered by surface loaded nanospheres. We present a platform for contrast media delivery, with the unusual feature that the payload can be controllably localized to the core or the surface.

Organic-inorganic hybrid photonic hydrogels as a colorful platform for visual detection of SCN-.

This communication demonstrates a facile method to detect SCN(-) by the naked eye through color change based on responsive organic-inorganic hybrid photonic hydrogels.