BAP31 regulates IRAK1-dependent neuroinflammation in microglia.

BACKGROUND: Microglia, the mononuclear immune cells of the central nervous system (CNS), are essential for the maintenance of CNS homeostasis. BAP31, a resident and ubiquitously expressed protein of the endoplasmic reticulum, serves as a sorting factor for its client proteins, mediating the subsequent export, retention, and degradation or survival. Recently, BAP31 has been defined as a regulatory molecule in the CNS, but the function of BAP31 in microglia has yet to be determined. In the present study, we investigated whether BAP31 is involved in the inflammatory response of microglia. METHODS: This study used the BV2 cell line and BAP31 conditional knockdown mice generated via the Cre/LoxP system. A BAP31 knockdown experiment was performed to elucidate the role of BAP31 in the endogenous inflammatory cytokine production by microglial BV2 cells. A mouse model of lipopolysaccharide (LPS)-induced cognitive impairment was established to evaluate the neuroprotective effect of BAP31 against neuroinflammation-induced memory deficits. Behavioral alterations were assessed with the open field test (OFT), Y maze, and Morris water maze. The activation of microglia in the hippocampus of mice was observed by immunohistochemistry. Western blot, enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining, and reverse transcription quantitative real-time polymerase chain reaction (RT-PCR) were used to clarify the mechanisms. RESULTS: BAP31 deficiency upregulates LPS-induced proinflammatory cytokines in BV2 cells and mice by upregulating the protein level of IRAK1, which in turn increases the translocation and transcriptional activity of NF-κB p65 and c-Jun, and moreover, knockdown of IRAK1 or use of an IRAK1 inhibitor reverses these functions. In the cognitive impairment animal model, the BAP31 knockdown mice displayed increased severity in memory deficiency accompanied by an increased expression of proinflammatory factors in the hippocampus. CONCLUSIONS: These findings indicate that BAP31 may modulate inflammatory cytokines and cognitive impairment induced by neuroinflammation through IRAK1, which demonstrates that BAP31 plays an essential role in microglial inflammation and prevention of memory deficits caused by neuroinflammation.

B-Cell Receptor-Associated Protein 31 Regulates the Expression of Valosin-Containing Protein Through Elf2.

BACKGROUND/AIMS: B-cell receptor-associated protein 31 (Bap31) is an evolutionarily conserved, ubiquitously expressed, polytopic integral membrane protein in the endoplasmic reticulum (ER) that is involved in the regulation of apoptosis, protein transport and degradation. Patients with Bap31 mutations exhibit symptoms similar to those exhibited by patients with central nervous system (CNS) diseases, such as deafness, dystonia, and intellectual disability. The present study aimed to investigate the function of Bap31 in CNS diseases by identifying a CNS disease-related gene regulated by Bap31 and exploring the underlying molecular mechanism. METHODS: ShRNA-Bap31 and siRNA-Bap31 were used to knockdown Bap31 in N2a cells, and real-time PCR was performed to detect the mRNA levels of genes involved in CNS diseases. Western blot analyses were used to examine the protein levels of the candidate gene (valosin-containing protein, VCP) both in vivo and in vitro. The functions of Bap31 and VCP in mediating the degradation of the hyper-unstable mutant of cystic fibrosis trans-membrane conductance regulator (CFTRΔF508) were studied. Moreover, real-time PCR, Western blot and dual luciferase reporter analyses were conducted to investigate the molecular mechanism by which Bap31 regulates the expression levels of VCP. RESULTS: VCP was identified as a candidate gene based on its differential expression in N2a cells following both shRNA- and siRNA-mediated knockdown of Bap31. Both the mRNA and protein levels of VCP were regulated by Bap31 in vivo and in vitro. In the ER-associated degradation (ERAD) pathway, Bap31 also regulated VCP expression and caused differences in the binding quantities of CFTRΔF508 and VCP. Furthermore, a transcription factor of VCP (E74-like factor 2, Elf2) was regulated by Bap31, and Elf2 mediated the changes in VCP transcription and expression in cells with altered Bap31 expression. CONCLUSION: These results indicate that Bap31 regulates the expression of VCP possibly via Elf2 and support the potential molecular function of Bap31 in CNS diseases.

[Effect of lead exposure on gene expression of Fgf3 in zebrafish embryonic development].

OBJECTIVE: To investigate the effect of lead exposure on the gene expression of fibroblast growth factor 3 (Fgf3) in zebrafish embryonic development and the mechanism of lead-induced embryonic developmental toxicity. METHODS: The embryos of zebrafish (wild types A and B) were exposed to lead acetate (PbAc) at the doses of 0, 0.1, 0.5, 2.5, and 12.5 µmol/L separately. Total RNA was extracted from each treatment group of zebrafish embryos at 8, 12, 16, 24, 36, 48, and 72 hours post fertilization (hpf). The total mRNA expression of Fgf3 was measured by real-time quantitative PCR. The spatial expression of Fgf3 in zebrafish embryos was determined by whole-mount in situ hybridization using synthesized Fgf3 RNA probe. RESULTS: The mRNA expression of Fgf3 in each group peaked at 12 hpf (P < 0.01). With the increase in PbAc concentration, the mRNA expression of Fgf3 rose. Compared with the mRNA expression level of Fgf3 in the control group, the relative mRNA expression levels of Fgf3 in the 0.1, 0.5, 2.5, and 12.5 µmol/L PbAc exposure groups were 1.02 ± 0.24, 1.05 ± 0.26, 1.22 ± 0.46, and 1.25 ± 0.38, respectively, and the 2.5 and 12.5 µmol/L PbAc exposure groups showed significantly higher Fgf3 expression than the control group (P < 0.05). The whole-mount in situ hybridization results showed that Fgf3 expression occurred mainly in the head and tail in the early stage of embryonic development and in the midbrain, fin bud, and pharyngeal arch in the middle/late stage of embryonic development; there were the most significant regions and intensities of positive hybridization signals at 12 hpf; but no significant differences were found between the control group and exposure groups in the location and intensity of Fgf3 expression CONCLUSION: Lead exposure can result in the upregulation of Fgf3 expression in zebrafish embryonic development, which might contribute to lead-induced embryonic developmental toxicity.

B-cell receptor associated protein 31 deficiency decreases the expression of adhesion molecule CD11b/CD18 and PSGL-1 in neutrophils to ameliorate acute lung injury.

Acute lung injury (ALI) and its more severe condition acute respiratory distress syndrome (ARDS) are critical life-threatening disorders characterized by an excessive influx of neutrophils into the alveolar space. Neutrophil infiltration is a multi-step process involving the sequential engagement of adhesion molecules. The adhesion molecule CD11b/CD18 acts as an important role in the recruitment of neutrophils to lung tissues in the ALI model. B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum transmembrane protein, has been reported to regulate the cellular anterograde transport of CD11b/CD18 in human neutrophils. To explore how BAP31 regulates CD11b/CD18 in mouse neutrophils, we constructed myeloid-specific BAP31 knockdown mice in this study. Biological investigations indicated that BAP31 deficiency could significantly alleviated lung injury, as evidenced by the improved histopathological morphology, reduced pulmonary wet/dry weight ratio, inhibited myeloperoxidase level and decreased neutrophil counts in the bronchoalveolar lavage fluid. Further studies clarified that BAP31 deficiency obviously down-regulated the expression of CD11b/CD18 and P-selectin glycoprotein ligand-1 (PSGL-1) by deactivating the nuclear factor kappa B (NF-κB) signaling pathway. Collectively, our results revealed that BAP31 depletion exerted a protective effect on ALI, which was possibly dependent on the attenuation of neutrophil adhesion and infiltration by blocking the expression of adhesion molecules CD11b/CD18 and PSGL-1. These findings implied the potential of BAP31 as an appealing protein to mediate the occurrence of ALI.

Recent developments of RET protein kinase inhibitors with diverse scaffolds as hinge binders.

RET is a proto-oncogene encoding a receptor tyrosine kinase. RET regulates key aspects of cellular proliferation, differentiation and survival. The activation of RET via gene fusions or point mutations is closely related to lung, thyroid and other cancers. This review summarizes the developments of a diversity of small molecule RET protein kinase inhibitors in the past 10 years. These RET inhibitors are classified according to their hinge binder chemotypes as: pyrimidines, including the pyrazolopyrimidines, pyrimidine oxazines, quinazolines, 4-aminopyrimidines and 4-aminopyridines; indolinones; 5-aminopyrazole-4-carboxamides; 3-trifluoromethylanilines; imidazopyridines, imidazopyridazines and pyrazopyridines; nicotinonitriles; pyridones and 1,2,4-triazoles. In each section, the biological activities of the inhibitors, their structure-activity relationships and possible binding modes with the RET kinase are introduced.

Synthesis and biological evaluation of selective histone deacetylase 6 inhibitors as multifunctional agents against Alzheimer's disease.

Histone deacetylase 6 (HDAC6) is a potential target for Alzheimer's disease (AD). In this study, a series of novel phenothiazine-, memantine-, and 1,2,3,4-tetrahydro-γ-carboline-based HDAC6 inhibitors with a variety of linker moieties were designed and synthesized. As a hydrochloride salt, the phenothiazine-based hydroxamic acid W5 with a pyridyl-containing linker motif was identified as a high potent and selective HDAC6 inhibitor. It inhibited HDAC6 with an IC50 of 2.54 nM and was more than 290- to 3300-fold selective over other HDAC isoforms. In SH-SY5Y cells, W5 dose-dependently increased the acetylated α-tubulin levels and reduced the hyperphosphorylated tau proteins at Ser396. As an effective metal chelator, W5 inhibited Cu2+-induced Aß1-42 aggregation and disaggregated Cu2+-Aß1-42 oligomers, and showed protective effects on the SH-SY5Y cells against Aß1-42- as well as Cu2+-Aß1-42 induced cell damages, serving as a potential ligand to target AD metal dyshomeostasis. Moreover, W5 promoted the differentiated neuronal neurite outgrowth, increased the mRNA expression of the recognized neurogenesis markers, GAP43, N-myc, and MAP-2. Therefore, W5 might be a good lead for the development of novel HDAC6 inhibitors targeting multi-facets of AD.

Regulation of Superoxide by BAP31 through Its Effect on p22phox and Keap1/Nrf2/HO-1 Signaling Pathway in Microglia.

Reactive oxygen species (ROS) production by activation of microglia is considered to be a major cause of neuronal dysfunction, which can lead to damage and death through direct oxidative damage to neuronal macromolecules or derangement of neuronal redox signaling circuits. BAP31, an integral ER membrane protein, has been defined as a regulatory molecule in the CNS. Our latest studies have found that BAP31 deficiency leads to activation of microglia. In this study, we discovered that BAP31 deficiency upregulated LPS-induced superoxide anion production in BV2 cells and mice by upregulating the expression level of p22phox and by inhibiting the activation of Nrf2-HO-1 signaling. Knockdown of p22phox/keap1 or use of an NADPH oxidase inhibitor (apocynin) reversed the production of superoxide anion and inflammatory cytokines, which then reduced neuronal damage and death in vitro and in vivo. These results suggest that BAP31 deficiency contributes to microglia-related superoxide anion production and neuroinflammation through p22phox and keap1. Furthermore, the excess superoxide anion cooperated with inflammatory cytokines to induce the damage and death of neurons. Thus, we determined that BAP31 is an important regulator in superoxide anion production and neuroinflammation, and the downstream regulators or agonists of BAP31 could therefore be considered as potential therapeutic targets in microglial-related superoxide anion production and neuroinflammation.

B-Cell Receptor-Associated Protein 31 Negatively Regulates the Expression of Monoamine Oxidase A Via R1.

B-cell receptor-associated protein 31 (Bap31) is a three trans-membrane protein of the endoplasmic reticulum (ER). Patients who have loss of function of Bap31 suffered from X-linked syndrome, such as motor and intellectual disabilities, dystonia, and sensorineural deafness. However, the underlying mechanism of Bap31 on X-linked syndrome remains unclear. Here, we found that a total of 21 proteins (9 up-regulated and 12 down-regulated proteins) related with X-linked syndrome were screened from shRNA-Bap31 transfected cells with the isobaric tags for relative and absolute quantification (iTRAQ) technique. One gene with the greatest change trend, monoamine oxidase A (MAOA), was identified. MAOA expression was up-regulated by Bap31 knockdown. However, Bap31 did not affect the ubiquitination degradation of MAOA protein. Of note, Bap31 selectively regulated the expression of cell division cycle associated 7-like (R1/RAM2/CDCA7L/JPO2, a transcriptional repressor of MAOA) and the binding activity of R1 with MAOA promoter, thereby affecting MAOA expression. This study demonstrates the molecular mechanisms of Bap31 in MAOA via R1 and supports the potential function of Bap31 on X-linked syndrome.