Clinicopathological phenotype and outcomes of NCAM-1+ membranous lupus nephritis.

INTRODUCTION: No studies explored the long-term outcomes of neural cell adhesion molecule 1 (NCAM1) associated membranous lupus nephritis (MLN) patients. METHOD: We performed immunohistochemical studies on kidney biopsy specimens against NCAM1 in consecutive MLN patients. The clinical and histopathological characteristics and outcomes of cases of NCAM1 associated MLN patients are described and compared with NCAM1 negative patients. In addition, we detected serum circulating anti-NCAM1 antibodies through western blotting and indirect immunofluorescence assays. RESULTS: Among 361 MLN cases, 18 (5.0%) were glomerular NCAM1-positive. NCAM1 positive MLN patients were older [35 years (IQR 27-43) versus 28 (22-37); P = 0.050) and had lower systemic lupus erythematosus disease activity index [11 (IQR 8-12) versus 14 (10-18); P = 0.007], serum creatinine [60 µmol/L (IQR 50-70) versus 70 (54-114); P = 0.029], activity index [3 (IQR 2-6) versus 6 (3-9); P = 0.045] at kidney biopsy compared with NCAM1 negative patients. The percentage of positive anti-Sjogren's syndrome related antigen A antibodies in NCAM1 positive patients was significantly greater (83.3% versus 58.2%; P = 0.035) than in the NCAM1 negative patients. However, no evidence of neuropsychiatric disorders was found in these 18 patients. There were no significant differences in the treatment response and the risk of end stage renal diseases between NCAM1 positive and negative groups (P = 0.668 and P = 0.318, respectively). But the risk of death was much higher in the NCAM1 positive group than the NCAM1 negative group (27.8% vs. 8.1%, P = 0.007). Moreover, the risk of death was also much higher in the NCAM1 positive group than the matched NCAM1 negative group (Log-rank P = 0.013). Additionally, circulating anti-NCAM1 antibodies can be detected in 1/5 (20%) patients who had serum available. CONCLUSION: The prevalence of NCAM1 positivity was 5.0% in our cohort of MLN and the high mortality in these subgroup patients are needed to validate in future studies.

Development of a predictive model to distinguish prostate cancer from benign prostatic hyperplasia by integrating serum glycoproteomics and clinical variables.

BACKGROUND: Prostate Cancer (PCa) represents the second leading cause of cancer-related death in men. Prostate-specific antigen (PSA) serum testing, currently used for PCa screening, lacks the necessary sensitivity and specificity. New non-invasive diagnostic tools able to discriminate tumoral from benign conditions and aggressive (AG-PCa) from indolent forms of PCa (NAG-PCa) are required to avoid unnecessary biopsies. METHODS: In this work, 32 formerly N-glycosylated peptides were quantified by PRM (parallel reaction monitoring) in 163 serum samples (79 from PCa patients and 84 from individuals affected by benign prostatic hyperplasia (BPH)) in two technical replicates. These potential biomarker candidates were prioritized through a multi-stage biomarker discovery pipeline articulated in: discovery, LC-PRM assay development and verification phases. Because of the well-established involvement of glycoproteins in cancer development and progression, the proteomic analysis was focused on glycoproteins enriched by TiO2 (titanium dioxide) strategy. RESULTS: Machine learning algorithms have been applied to the combined matrix comprising proteomic and clinical variables, resulting in a predictive model based on six proteomic variables (RNASE1, LAMP2, LUM, MASP1, NCAM1, GPLD1) and five clinical variables (prostate dimension, proPSA, free-PSA, total-PSA, free/total-PSA) able to distinguish PCa from BPH with an area under the Receiver Operating Characteristic (ROC) curve of 0.93. This model outperformed PSA alone which, on the same sample set, was able to discriminate PCa from BPH with an AUC of 0.79. To improve the clinical managing of PCa patients, an explorative small-scale analysis (79 samples) aimed at distinguishing AG-PCa from NAG-PCa was conducted. A predictor of PCa aggressiveness based on the combination of 7 proteomic variables (FCN3, LGALS3BP, AZU1, C6, LAMB1, CHL1, POSTN) and proPSA was developed (AUC of 0.69). CONCLUSIONS: To address the impelling need of more sensitive and specific serum diagnostic tests, a predictive model combining proteomic and clinical variables was developed. A preliminary evaluation to build a new tool able to discriminate aggressive presentations of PCa from tumors with benign behavior was exploited. This predictor displayed moderate performances, but no conclusions can be drawn due to the limited number of the sample cohort. Data are available via ProteomeXchange with identifier PXD035935.

Neural cell adhesion molecule 1 is a cellular target engaged plasma biomarker in demyelinating Charcot-Marie-Tooth disease.

BACKGROUND AND PURPOSE: Elevated plasma concentrations of neural cell adhesion molecule 1 (NCAM1) and p75 neurotrophin receptor (p75) in patients with peripheral neuropathy have been reported. This study aimed to determine the specificity of plasma concentration elevation of either NCAM1 or p75 in a subtype of Charcot-Marie-Tooth disease (CMT) and its correlation with pathologic nerve status and disease severity. METHODS: Blood samples were collected from 138 patients with inherited peripheral neuropathy and 51 healthy controls. Disease severity was measured using Charcot-Marie-Tooth Neuropathy Score version 2 (CMTNSv2), and plasma concentrations of NCAM1 and p75 were analyzed by enzyme-linked immunosorbent assay. Eight sural nerves from CMT patients were examined to determine the relation of histopathology and plasma NCAM1 levels. RESULTS: Plasma concentration of NCAM1, but not p75, was specifically increased in demyelinating subtypes of CMT (median = 7100 pg/mL, p < 0.001), including CMT1A, but not in axonal subtype (5964 pg/mL, p > 0.05), compared to the control (3859 pg/mL). CMT1A patients with mild or moderate severity (CMTNSv2 < 20) showed higher levels of plasma NCAM1 than healthy controls. Immunofluorescent NCAM1 staining for the sural nerves of CMT patients showed that NCAM1-positive onion bulb cells and possible demyelinating Schwann cells might be associated with the specific increase of plasma NCAM1 in demyelinating CMT. CONCLUSIONS: The plasma NCAM1 levels in demyelinating CMT might be a surrogate biomarker reflecting pathological Schwann cell status and disease progression.

Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.

Neural cell adhesion molecules 1 (NCAM1) and 2 (NCAM2) belong to the cell adhesion molecules of the immunoglobulin superfamily and have been shown to regulate formation, maturation, and maintenance of synapses. NCAM1 and NCAM2 undergo proteolysis, but the identity of all the proteases involved and how proteolysis is used to regulate their functions are not known. We report here that NCAM1 and NCAM2 are BACE1 substrates in vivo. NCAM1 and NCAM2 overexpressed in HEK cells were both cleaved by metalloproteinases or BACE1, and NCAM2 was also processed by γ-secretase. We identified the BACE1 cleavage site of NCAM1 (at Glu 671) and NCAM2 (at Glu 663) using mass spectrometry and site-directed mutagenesis. Next, we assessed BACE1-mediated processing of NCAM1 and NCAM2 in the mouse brain during aging. NCAM1 and NCAM2 were cleaved in the olfactory bulb of BACE1+/+ but not BACE1-/- mice at postnatal day 10 (P10), 4 and 12 months of age. In the hippocampus, a BACE1-specific soluble fragment of NCAM1 (sNCAM1ß) was only detected at P10. However, we observed an accumulation of full-length NCAM1 in hippocampal synaptosomes in 4-month-old BACE1-/- mice. We also found that polysialylated NCAM1 (PSA-NCAM1) levels were increased in BACE1-/- mice at P10 and demonstrated that BACE1 cleaves both NCAM1 and PSA-NCAM1 in vitro. In contrast, we did not find evidence for BACE1-dependent NCAM2 processing in the hippocampus at any age analyzed. In summary, our data demonstrate that BACE1 differentially processes NCAM1 and NCAM2 depending on the region of brain, subcellular localization, and age in vivo.

siRNA-mediated NCAM1 gene silencing suppresses oxidative stress in pre-eclampsia by inhibiting the p38MAPK signaling pathway.

Pre-eclampsia (PE), whose pathophysiology and etiology remain undefined, represents a leading consequence of fetal and maternal mortality and morbidity. Oxidative stress (OS) is recognized to involve in this disorder. In this study, we hypothesized that neural cell adhesion molecule 1 (NCAM1) gene silencing would suppress the OS in the pregnancy complicated by PE. Initially, clinical samples were collected for determination of NCAM1 expression in placental tissues and levels of OS products in blood. To assess the regulatory mechanism of NCAM1 knockdown on OS, we used small interfering RNA (siRNA) to silence NCAM1 expression in human umbilical vein endothelial cells (HUVECs). Next, cells were treated with or without hypoxia/reoxygenation to observe the level changes of OS products and p38 mitogen-activated protein kinase (p38MAPK) pathway-related genes. Finally, an evaluation of HUVEC migration and invasion abilities was conducted by wound-healing and transwell assays. Placenta of pregnancy with PE presented significantly increased NCAM1 expression in comparison to placenta of normal pregnancy. Meanwhile, enhanced OS in blood of pregnant women with PE was observed relative to women with normal pregnancy. siRNA-mediated knockdown of NCAM1 gene could inhibit the p38MAPK signaling pathway, repress OS, and promote cell migration and invasion in HUVECs, indicating that NCAM1 inhibition could reduce the influence of PE. Importantly, blocking the p38MAPK signaling pathway reversed the inhibitory role of NCAM1 gene silencing on PE. Collectively, this study defines potential role of NCAM1 gene silencing as a therapeutic target in PE through inhibiting OS and enhancing HUVEC migration and invasion by disrupting the p38MAPK signaling pathway.

DNMT3B regulates proliferation of A549 cells through the microRNA-152-3p/NCAM1 pathway.

The purpose of the present study was to examine the epigenetic mechanism by which microRNA (miR)-152-3p regulates proliferation in non-small cell lung cancer A549 cells via neural cell adhesion molecule 1 (NCAM1). Bisulfite sequencing PCR (BSP), the gold standard for methylation detection, uses bisulfite-treated DNA to determine its pattern of methylation. Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. It was conducted and demonstrated a relatively high level of methylation in the miR-152-3p promoter region. Chromatin immunoprecipitation was combined with PCR to detect the binding of DNA methyltransferase 3B (DNMT3B) protein to miR-152-3p, which tends to occur in the core region of the miR-152-3p gene in A549 cells. Luciferase assay identified NCAM1 as the target gene of miR-152-3p. MTT, colony formation and Transwell assays indicated that miR-152-3p could decrease cell proliferation and invasion and in addition to reducing the expression level of NCAM1. Overexpression of NCAM1 could attenuate the effect of miR-152-3p. DNMT3B knockdown decreased the proliferative ability of A549 cells and increased the expression of miR-152-3p, while decreased that of NCAM1. After treatment with miR-152-3p inhibitor, these effects were attenuated and the NCAM1 expression level was upregulated. The results indicated that miR-152-3p may suppress the proliferation of A549 cells by downregulating NCAM1. In addition, DNMT3B negatively regulated the expression of miR-152-3p via modulation of the methylation level in the miR-152-3p core region, thus mediating the proliferation of lung tumor cells.

Nerve bundle formation during the promotion of peripheral nerve regeneration: collagen VI-neural cell adhesion molecule 1 interaction.

The formation of nerve bundles, which is partially regulated by neural cell adhesion molecule 1 (NCAM1), is important for neural network organization during peripheral nerve regeneration. However, little is known about how the extracellular matrix (ECM) microenvironment affects this process. Here, we seeded dorsal root ganglion tissue blocks on different ECM substrates of peripheral nerve ECM-derived matrix-gel, Matrigel, laminin 521, collagen I, and collagen IV, and observed well-aligned axon bundles growing in the peripheral nerve ECM-derived environment. We confirmed that NCAM1 is necessary but not sufficient to trigger this phenomenon. A protein interaction assay identified collagen VI as an extracellular partner of NCAM1 in the regulation of axonal fasciculation. Collagen VI interacted with NCAM1 by directly binding to the FNIII domain, thereby increasing the stability of NCAM1 at the axolemma. Our in vivo experiments on a rat sciatic nerve defect model also demonstrated orderly nerve bundle regeneration with improved projection accuracy and functional recovery after treatment with 10 mg/mL Matrigel and 20 µg/mL collagen VI. These findings suggest that the collagen VI-NCAM1 pathway plays a regulatory role in nerve bundle formation. This study was approved by the Animal Ethics Committee of Guangzhou Medical University (approval No. GY2019048) on April 30, 2019.

New antigens involved in membranous nephropathy beyond phospholipase A2 receptor.

When the physiopathology of membranous nephropathy was first described, almost 30% of cases were recognized to be secondary to well-known diseases such as autoimmune diseases, tumors or infections. The remaining 70% cases were called primary membranous nephropathy as the exact mechanism or pathogenic factor involved was unknown. The discovery of the M type phospholipase A2 receptor and thrombospondin type 1 domain containing 7A as causative antigens in these "so called" primary membranous nephropathies provided new insights into the effective causes of a large proportion of these cases. Novel techniques such as laser microdissection and tandem mass spectrometry as well as immunochemistry with antibodies directed against novel proteins allowed the confirmation of new involved antigens. Finally, using confocal microscopy to localize these new antigens and immunoglobulin G and Western blot analysis of serum samples, these new antigens were detected on the glomerular membrane, and the related antibodies were detected in serum samples. The same antigens have been recognized in some cases of secondary membranous disease due to autoimmune diseases, tumors and infections. This has allowed examination of the relationship between antigens in primary membranous nephropathy and their presence in some secondary nephropathies. The aim of this study is to describe the characteristics of the new antigens discovered and their association with other diseases.

Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors.

Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.

Membranous nephropathy: a single disease or a pattern of injury resulting from different diseases.

Membranous nephropathy (MN) is defined as disease entity characterized by thickening of the glomerular basement membranes due to subepithelial (SE) deposition of immune complexes. It is typically classified into primary MN (70%) when there is no disease association, and secondary MN (30%) when there is an underlying disease association such as lupus, malignancy, infections or drugs. Phospholipase A2 receptor (PLA2R) and thrombospondin type-1 domain-containing 7A (THSD7A) are target antigens in 70% and 1-5% of primary MN, respectively. The antigens in the remaining MN were not known. Recently, multiple novel proteins/target antigens have been identified in MN. These include exostosin 1/2, neural epidermal growth-like 1 protein, semaphorin 3B, protocadherin 7 and neural cell adhesion molecule 1. Some of these antigens are present in the setting of primary MN, some in secondary MN and some in both, thus blurring the lines between primary and secondary MN. Preliminary studies show that each of the new antigen-associated MN has distinct clinical, kidney biopsy findings and outcome data. We propose that each new protein/antigen-associated MN is a specific disease that results in the common MN pattern of injury characterized by thickened glomerular basement membrane (GBM) with or without spikes or pinholes on light microscopy, granular immunoglobulin G with or without complement 3 on immunofluorescence microscopy and SE electron-dense deposits on electron microscopy. In other words, MN is truly only a pattern of injury resulting from specific diseases that cause deposition of SE immune deposits along the GBM. It is of paramount importance to ascertain the specific disease entity causing the MN pattern not only for precise diagnosis and management, but also for future studies on these newly described diseases.

Rapid Turnover of Cortical NCAM1 Regulates Synaptic Reorganization after Peripheral Nerve Injury.

Peripheral nerve injury can induce pathological conditions that lead to persistent sensitized nociception. Although there is evidence that plastic changes in the cortex contribute to this process, the underlying molecular mechanisms are unclear. Here, we find that activation of the anterior cingulate cortex (ACC) induced by peripheral nerve injury increases the turnover of specific synaptic proteins in a persistent manner. We demonstrate that neural cell adhesion molecule 1 (NCAM1) is one of the molecules involved and show that it mediates spine reorganization and contributes to the behavioral sensitization. We show striking parallels in the underlying mechanism with the maintenance of NMDA-receptor- and protein-synthesis-dependent long-term potentiation (LTP) in the ACC. Our results, therefore, demonstrate a synaptic mechanism for cortical reorganization and suggest potential avenues for neuropathic pain treatment.

Detection of natural antisense non-coding RNAs transcribed from Ncam1 in mice tissues at several developmental stages.

Natural antisense ribonucleic acids (RNAs) are transcribed from a large number of genes in various species, including humans and mice. The expression of neural cell adhesion molecule 1 (Ncam1) antisense non-coding RNAs (ncRNAs) in mice has been demonstrated by functional annotation of the mammalian genome project, but the localization of Ncam1 antisense ncRNAs has not been reported in mice tissues. In the present study, the localization of Ncam1 antisense ncRNAs was examined in tissues at several developmental stages by in situ hybridization. At days 14 and 17 of embryonic development, Ncam1 antisense ncRNAs were found in the heart, liver, lung, kidney, thymus and nerve regions including the brain (cerebral cortex, olfactory bulb, hippocampus and cerebellum) and spinal cord. In newborn mice, Ncam1 antisense ncRNAs were detected in the brain, kidney and thymus, but was not detected in other tissues. In 8-week-old mice, Ncam1 antisense ncRNAs were detected in the lung, kidney, thymus, pancreas, cornea, stomach and nerve regions including the brain. These results indicate that Ncam1 antisense ncRNAs are expressed in mice tissues. Notably, Ncam1 messenger RNAs (mRNAs), antisense ncRNAs co-localized in the Purkinje cells of the cerebellum and the levels of antisense ncRNAs appeared to be higher than those of mRNAs, suggesting that Ncam1 antisense ncRNAs may regulate the expression of Ncam1 mRNAs in the same cells.

MRNA and miRNA expression patterns associated to pathways linked to metal mixture health effects.

Metals are a threat to human health by increasing disease risk. Experimental data have linked altered miRNA expression with exposure to some metals. MiRNAs comprise a large family of non-coding single-stranded molecules that primarily function to negatively regulate gene expression post-transcriptionally. Although several human populations are exposed to low concentrations of As, Cd and Pb as a mixture, most toxicology research focuses on the individual effects that these metals exert. Thus, this study aims to evaluate global miRNA and mRNA expression changes induced by a metal mixture containing NaAsO2, CdCl2, Pb(C2H3O2)2·3H2O and to predict possible metal-associated disease development under these conditions. Our results show that this metal mixture results in a miRNA expression profile that may be responsible for the mRNA expression changes observed under experimental conditions in which coding proteins are involved in cellular processes, including cell death, growth and proliferation related to the metal-associated inflammatory response and cancer.

Expression and Significance of MicroRNA-595 in Inflammatory Bowel Disease / 胃肠病学

Background:Dysregulation of microRNAs is associated with intestinal mucosal barrier injury,intestinal inflammation and intestinal dysfunction. Abnormal expression of microRNAs occurs in patients with inflammatory bowel disease(IBD). Aims:To investigate the expression and significance of microRNA-595( miR-595)in IBD. Methods:A total of 100 patients with IBD at Nanjing General Hospital of Nanjing Military Command of PLA from July 2012 to July 2014 were enrolled,in which 63 cases were ulcerative colitis(UC)and 37 cases were Crohn’s disease(CD). According to disease activity,patients were divided into active UC(aUC)group,remissive UC(rUC)group,active CD(aCD)group and remissive CD(rCD)group. A total of 42 healthy subjects were served as normal control(NC)group. Specimens of serum and intestinal tissue were collected. Expression of miR-595 in serum and intestinal tissue was determined by fluorescence quantitative PCR. Luciferase report gene plasmid containing the 3’UTR of neural cell adhesion molecule 1(NCAM1)or fibroblast growth factor receptor 2(FGFR2)and plasmid containing miR-595 were co-transfected into human colon cancer cell line HCT116 to detect the effect of miR-595 on transcriptional activities of NCAM1 and FGFR2. Results:Expression of miR-595 in serum and intestinal tissue in UC and CD groups was significantly higher than that in NC group(P < 0. 05), and that in aUC and aCD groups was significantly higher than that in rUC and rCD groups,respectively(P < 0. 05). MiR-595 could down-regulate the transcriptional activities of NCAM1 and FGFR2 through directly binding to the 3’UTR of NCAM1 and FGFR2. Conclusions:Expression of miR-595 in serum and intestinal tissue is increased in patients with IBD and correlates with disease activity. MiR-595 inhibits the expressions of tight junction protein NCAM1 and FGFR2,thereby inducing injury of intestinal mucosal barrier and promoting intestinal inflammation. MiR-595 can serve as a serum biomarker for diagnosis of IBD and disease activity evaluation.

Correlation of neural cell adhesion molecule 1 and pathogenesis of cognitive dysfunction in epileptic rats / 中华神经医学杂志

Objective To study the correlation of neural cell adhesion molecule 1 (NCAM1) with pathogenesis of cognitive dysfunction in epileptic rats and explore its effect on cognitive dysfunction in epileptic rats.Methods A total of 120 Wistar rats were randomly assigned to control group (n=20) and experimental group (n=100); and rats in the experimental group were subdivided into 5 groups (n=20),the epilepsy models were induced by pilocarpine,and rats in these 5 groups received daily treatments for 30 days with either saline,carbamazine,oxcarbazine,aniracetam or donepezil (nootopic).Spatial learning and memory abilities were assessed with Water Maze test.Hippocampus tissue was assessed for NCAM1 mRNAs by RT-PCR and proteins by immunochemistry.Results The mean escape latency of rats in the Morris water maze test:carbamazepine treatment group ＞oxcarbazepine treatment group ＞aniracetam treatment group ＞epilepsy group ＞donepezi treatment group; significant differences were noted between each two groups (F=91.920,P=0.000).Immunohistochemical and RT-PCR results:donepezi treatment rgroup ＞aniracetam treatment group ＞epileptic group ＞oxcarbazepine treatment group ＞carbamazepine treatment group＞ control group; there were differences between each 2 groups (Immunohistochemical result:F=324.510,P=0.000; RT-PCR result:F=81.160,P=0.000).Conclusion NCAM1 expression in the hippocampus rises within 30 days of epileptic attack,who participates in the pathogenesis of cognitive dysfunction; carbamazepine can aggravate the cognitive dysfunction and donepezil can obviously improve the cognitive function of epilepsy.