Upregulation of proBDNF/p75NTR signaling in immune cells and its correlation with inflammatory markers in patients with major depression.

ProBDNF is the precursor protein of brain-derived neurotrophic factor (BDNF) expressed in the central nervous system and peripheral tissues. Previous studies showed that the blood levels of both proBDNF and p75 neurotrophic receptors (p75NTR) in major depressive disorder (MDD) were increased, but which blood cell types express proBDNF and its receptors is not known. Furthermore, the relationship between proBDNF/p75NTR and inflammatory cytokines in peripheral blood of MDD is unclear. Peripheral blood mononuclear cells (PBMCs) and serum were obtained from depressive patients (n = 32) and normal donors (n = 20). We examined the expression of proBDNF and inflammatory markers and their correlative relationship in patients with major depression. Using flow cytometry analysis, we examined which blood cells express proBDNF and its receptors. Finally, the role of proBDNF/p75NTR signal in inflammatory immune activity of PBMCs was verified in vitro experiments. Inflammatory cytokines in PBMC from MDD patients were increased and correlated with the major depression scores. The levels of IL-1ß and IL-10 were also positively correlated with the major depression scores, while the levels of TNF-α and IL-6 were negatively correlated with the major depression scores. Intriguingly, the levels of sortilin were positively correlated with IL-1ß. Q-PCR and Western blots showed proBDNF, p75NTR, and sortilin levels were significantly increased in PBMCs from MDD patients compared with that from the normal donors. Flow cytometry studies showed that proBDNF and p75NTR were present mainly in CD4+ and CD8+ T cells. The number of proBDNF and p75NTR positive CD4+ and CD8+ T cells from MDD patients was increased and subsequently reversed after therapeutic management. Exogenous proBDNF protein or p75ECD-Fc treatment of cultured PBMC affected the release of inflammatory cytokines in vitro. ProBDNF promoted the expression of inflammatory cytokines, while p75ECD-Fc inhibited the expression of inflammatory cytokines. Given there was an inflammatory response of lymphocytes to proBDNF, it is suggested that proBDNF/p75NTR signaling may upstream inflammatory cytokines in MDD. Our data suggest that proBDNF/p75NTR signaling may not only serve as biomarkers but also may be a potential therapeutic target for MDD.

proBDNF/p75NTR promotes rheumatoid arthritis and inflammatory response by activating proinflammatory cytokines.

P75 pan-neurotrophin receptor (p75NTR) is an important receptor for the role of neurotrophins in survival and death of neurons during development and after nerve injury. Our previous research found that the precursor of brain-derived neurotrophic factor (proBDNF) regulates pain as an inflammatory mediator. The current understanding of the role of proBDNF/p75NTR signaling pathway in inflammatory arthritis pain and rheumatoid arthritis (RA) is unclear. We recruited 20 RA patients, 20 healthy donors (HDs), and 10 osteoarthritis (OA) patients. Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) of proBDNF and p75NTR in synovial membrane were performed and evaluated. We next examined the mRNA and protein expression of proBDNF/p75NTR signaling pathway in peripheral blood mononuclear cells (PBMCs) and synovial tissue. ELISA and flow cytometry were assessed between the blood of RA patients and HD. To induce RA, collagen-induced arthritis (CIA) were induced in mice. We found over-synovitis of RA synovial membrane compared to OA controls in histologic sections. P75NTR and sortilin mRNA, and proBDNF protein level were significantly increased in PBMCs of RA patients compared with the HD. Consistently, ELISA showed that p75NTR, sortilin, tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and interleukin-10 (IL-10) levels in the serum of RA patients were increased compared with HD and p75NTR, sortilin were positively correlated with Disease Activity Score in 28 joints (DAS28). In addition, using flow cytometry we showed that the increased levels of proBDNF and p75NTR characterized in CD4+ and CD8+ T cells of RA patients were subsequently reversed with methotrexate (MTX) treatment. Furthermore, we found pathological changes, inflammatory pain, upregulation of the mRNA and protein expression of proBDNF/p75NTR signaling pathway, and upregulation of inflammatory cytokines in spinal cord using a well-established CIA mouse model. We showed intravenous treatment of recombinant p75ECD-Fc that biologically blocked all inflammatory responses and relieved inflammatory pain of animals with CIA. Our findings showed the involvement of proBDNF/p75NTR pathway in the RA inflammatory response and how blocking it with p75ECD-Fc may be a promising therapeutic treatment for RA.

Deletion of TRIM32 protects mice from anxiety- and depression-like behaviors under mild stress.

Chronic stress causes a variety of psychiatric disorders such as anxiety and depression, but its mechanism is not well understood. Tripartite motif-containing protein 32 (TRIM32) was strongly associated with autism spectrum disorder, attention deficit hyperactivity disorder, anxiety and obsessive compulsive disorder based on a study of copy number variation, and deletion of TRIM32 increased neural proliferation and reduced apoptosis. Here, we propose that TRIM32 is involved in chronic stress-induced affective behaviors. Using a chronic unpredictable mild stress mouse depression model, we studied expression of TRIM32 in brain tissue samples and observed behavioral changes in Trim32 knockout mice. The results showed that TRIM32 protein but not its mRNA was significantly reduced in hippocampus in a time-dependent manner within 8 weeks of chronic stress. These stress-induced affective behaviors and reduction of TRIM32 protein expression were significantly reversed by antidepressant fluoxetine treatment. In addition, Trim32 knockout mice showed reduced anxiety and depressive behaviors and hyperactivities compared with Trim32 wild-type mice under normal and mild stress conditions. We conclude that TRIM32 plays important roles in regulation of hyperactivities and positively regulates the development of anxiety and depression disorders induced by chronic stress.

Downregulation of DNMT3a expression by RNAi and its effect on NF-κBs expression of thymic epithelial cells.

OBJECTIVE: To understand the characteristics of DNA methyltransferase 3a (DNMT3a) in thymoma associated Myasthenia Gravis reveal its transcriptional regulator network as while as analyze the effect of DNMT3a on Rel/ nuclear factor-kappaB family (RelA/RelB) and its downstream autoimmune regulatory factor (Aire). METHODS: Tissues of 30 patients with thymoma, with or without myasthenia gravis (MG), were collected and the DNMT3a protein expression were evaluated through immunohistochemistry. We performed mRNA expression profiling microarray detection and analysis, and integrated the analysis by constructing protein-protein interaction networks and the integration with other database. We identified molecular difference between low and high DNMT3a in the thymoma by heatmap. We also performed PCR validation in thymoma tissues. The DNMT3a-shRNA plasmid was transfected into TEC cells, and these cells were treated with 5-aza-2-deoxycytidine, a blocker of DNMT3a. After the down-regulation of DNMT3a in TEC cells, the transcript and protein levels of RelA, RelB, Aire, and CHRNA3 were evaluated by western blotting. In addition, changes in gene expression profiles were screened through microarray technology. We performed differential gene analysis in the thymoma cohort by heatmap with R (v.4.3.0) software. RESULTS: In 30 matched tissue specimens, the expression of DNMT3a protein in thymoma with MG was lower than that in thymoma. Through mRNA expression profiling analysis, we constructed a co-expression network of DNMT3a and found direct interaction between IKZF1 and DNMT3a, and this co-expression relationship was overlappted with Cistrome DB database. We found up-regulation of 149 mRNAs and repression of 177 mRNAs in thymoma with MG compared with thymoma. Gene ontology and pathway analysis show the involvement of a multitude of genes in the mis-regulation of MG-related pathways. RNA interference significantly reduced the level of mRNA of DNMT3a, which proved that plasmid DNMT3a was effective. In comparison to the control group, the levels of DNMT3a, Aire, and CHRNA3 mRNA and protein in TEC cells transfected with DNMT3a-shRNA interference plasmid were significantly decreased, while the expression level of RelA and RelA/RelB was significantly increased. CONCLUSIONS: Our study reveals the DNMT3a-NF-κB pathway has a major effect on MG, and can be used as a marker for diagnosis as well as a target for MG treatment.

Alteration in gene expression profiles of thymoma: Genetic differences and potential novel targets.

BACKGROUND: This study was conducted to investigate the gene expression profiles associated with thymoma to better understand the molecular mechanism underlying the pathogenesis of thymoma. METHODS: Eight patients with thymomas (type A, AB, B1, and B2) and four controls with thymic cysts were analyzed using microarray profiling to identify changes in gene expression. RESULTS: Across all of our samples, 2319 messenger RNAs were upregulated and 2776 were downregulated in thymomas relative to thymic cysts. Gene ontology and pathway analyses revealed that a large number of genes participate in cellular functions, among which MHC class II protein complex assembly, assembly with peptide antigen, calcium activated phosphatidylcholine scrambling, and release of cytoplasmic sequestered NF-κB were dysregulated, whereas intestinal immune network for immunoglobulin A production, cytokine-cytokine receptor interaction, the calcium signaling pathway, and pathways related to autoimmune diseases were downregulated. CONCLUSIONS: Our results revealed gene expression differences between thymomas and thymic cysts, and identified key candidate genes/pathways that might be used as diagnostic markers and potential therapeutic targets to treat cancer metastasis.

Mice with Sort1 deficiency display normal cognition but elevated anxiety-like behavior.

Exposure to stressful life events plays a central role in the development of mood disorders in vulnerable individuals. However, the mechanisms that link mood disorders to stress are poorly understood. Brain-derived neurotrophic factor (BDNF) has long been implicated in positive regulation of depression and anxiety, while its precursor (proBDNF) recently showed an opposing effect on such mental illnesses. P75(NTR) and sortilin are co-receptors of proBDNF, however, the role of these receptors in mood regulation is not established. Here, we aimed to investigate the role of sortilin in regulating mood-related behaviors and its role in the proBDNF-mediated mood abnormality in mice. We found that sortilin was up-regulated in neocortex (by 78.3%) and hippocampus (by 111%) of chronically stressed mice as assessed by western blot analysis. These changes were associated with decreased mobility in the open field test and increased depression-like behavior in the forced swimming test. We also found that sortilin deficiency in mice resulted in hyperlocomotion in the open field test and increased anxiety-like behavior in both the open field and elevated plus maze tests. No depression-like behavior in the forced swimming test and no deficit in spatial cognition in the Morris water maze test were found in the Sort1-deficient mice. Moreover, the intracellular and extracellular levels of mature BDNF and proBDNF were not changed when sortilin was absent in vivo and in vitro. Finally, we found that both WT and Sort1-deficient mice injected with proBDNF in lateral ventricle displayed increased depression-like behavior in the forced swimming test but not anxiety-like behaviors in the open field and elevated plus maze tests. The present study suggests that sortilin functions as a negative regulator of mood performance and can be a therapeutic target for the treatment of mental illness.

ProBDNF Signaling Regulates Depression-Like Behaviors in Rodents under Chronic Stress.

Chronic exposure to stressful environment is a key risk factor contributing to the development of depression. However, the mechanisms involved in this process are still unclear. Brain-derived neurotropic factor (BDNF) has long been investigated for its positive role in regulation of mood, although the role of its precursor, proBDNF, in regulation of mood is not known. In this study, using an unpredictable chronic mild stress (UCMS) paradigm we found that the protein levels of proBDNF were increased in the neocortex and hippocampus of stressed mice and this UCMS-induced upregulation of proBDNF was abolished by chronic administration of fluoxetine. We then established a rat model of UCMS and found that the expression of proBDNF/p75NTR/sortilin was upregulated, whereas the expression of mature BDNF and TrkB was downregulated in both neocortex and hippocampus of chronically stressed rats. Finally, we found that the injection of anti-proBDNF antibody via intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) approaches into the UCMS rats significantly reversed the stress-induced depression-like behavior and restored the exploratory activity and spine growth. Although intramuscular injection of AAV-proBDNF did not exacerbate the UCMS-elicited rat mood-related behavioral or pathological abnormalities, i.c.v. injection of AAV-proBDNF increased the depression-like behavior in naive rats. Our findings suggest that proBDNF plays a role in the development of chronic stress-induced mood disturbances in rodents. Central (i.c.v.) or peripheral (i.p.) inhibition of proBDNF by injecting specific anti-proBDNF antibodies may provide a novel therapeutic approach for the treatment of stress-related mood disorders.