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NRXN1 is involved in synaptogenesis and have been implicated in Autism spectrum disorders. However, many rare inherited missense variants of NRXN1 have not been thoroughly evaluated. Here, functional analyses in vitro and in Drosophila of three NRXN1 missense mutations, Y282H, L893V, and I1135V identified in ASD patients in our previous study were performed. Our results showed these three mutations interfered protein degradation compared with NRXN1-WT protein. Expressing human NRXN1 in Drosophila could lead to abnormal circadian rhythm and sleep behavior, and three mutated proteins caused milder phenotypes, indicating the mutations may change the function of NRXN1 slightly. These findings highlight the functional role of rare NRXN1 missense variants identified in autism patients, and provide clues for us to better understand the pathogenesis of abnormal circadian rhythm and sleep behavior of other organisms, including humans.

Impaired sensorimotor circuits have been suggested in Attention-deficit/hyperactivity disorder (ADHD). NRXN1, highly expressed in cortex and cerebellum, was one of the candidate risk genes for ADHD, while its effects on sensorimotor circuits are unclear. In this content, we aimed to investigate the differential brain effects as functions of the cumulative genetic effects of NRXN1 variants in ADHD and healthy controls (HCs), identifying a potential pathway mapping from NRXN1, sensorimotor circuits, to ADHD. Magnetic resonance imaging, blood samples and clinical assessments were acquired from 53 male ADHD and 46 sex-matched HCs simultaneously. The effects of the cumulative genetic effects of NRXN1 variants valued by poly-variant risk score (PRS), on brain function was measured by resting-state functional connectivity (rs-FC) of cerebrocerebellar circuits. Mediation analyses were conducted to evaluate the association between NRXN1, functional abnormality, and ADHD diagnosis, as well as ADHD symptoms. The results were validated by bootstrapping and 10,000 times permutation tests. The rs-FC analyses demonstrated significant mediation models for ADHD diagnosis, and emphasized the involvement of cerebellum, middle cingulate gyrus and temporal gyrus, which are crucial parts of sensorimotor circuits. The current study suggested NRXN1 conferred risk for ADHD by regulating the function of sensorimotor circuits.

The myotendinous junction (MTJ) is a specialized interface for transmitting high forces between the muscle and tendon and yet the MTJ is a common site of strain injury with a high recurrence rate. The aim of this study was to identify previously unknown MTJ components in mature animals and humans. Samples were obtained from the superficial digital flexor (SDF) muscle-tendon interface of 20 horses, and the tissue was separated through a sequential cryosectioning approach into muscle, MTJ (muscle tissue enriched in myofiber tips attached to the tendon), and tendon fractions. RT-PCR was performed for genes known to be expressed in the three tissue fractions and t-distributed stochastic neighbor embedding (t-SNE) plots were used to select the muscle, MTJ, and tendon samples from five horses for RNA sequencing. The expression of previously known and unknown genes identified through RNA sequencing was studied by immunofluorescence on human hamstring MTJ tissue. The main finding was that RNA sequencing identified the expression of a panel of 61 genes enriched at the MTJ. Of these, 48 genes were novel for the MTJ and 13 genes had been reported to be associated with the MTJ in earlier studies. The expression of known [COL22A1 (collagen XXII), NCAM (neural cell adhesion molecule), POSTN (periostin), NES (nestin), OSTN (musclin/osteocrin)] and previously undescribed [MNS1 (meiosis-specific nuclear structural protein 1), and LCT (lactase)] MTJ genes was confirmed at the protein level by immunofluorescence on tissue sections of human MTJ. In conclusion, in muscle-tendon interface tissue enriched with myofiber tips, we identified the expression of previously unknown MTJ genes representing diverse biological processes, which may be important in the maintenance of the specialized MTJ.

Membranous lupus nephritis is a frequent cause of nephrotic syndrome in patients with systemic lupus erythematosus. It has been shown in phospholipase A2 receptor positive membranous nephropathy that known antibodies can be detected within sera, determination of the target autoantigen can have diagnostic significance, inform prognosis, and enable non-invasive monitoring of disease activity. Here we utilized mass spectrometry for antigen discovery in laser captured microdissected glomeruli from formalin-fixed paraffin embedded tissue and tissue protein G immunoprecipitation studies to interrogate immune complexes from frozen kidney biopsy tissue. We identified neural cell adhesion molecule 1 (NCAM1) to be a target antigen in some cases of membranous lupus nephritis and within rare cases of primary membranous nephropathy. The prevalence of NCAM1 association was 6.6% of cases of membranous lupus nephritis and in 2.0% of primary membranous nephropathy cases. NCAM1 was found to colocalize with IgG within glomerular immune deposits by confocal microscopy. Additionally, serum from patients with NCAM1-associated membranous nephropathy showed reactivity to NCAM1 recombinant protein on Western blotting and by indirect immunofluorescence assay, demonstrating the presence of circulating antibodies. Thus, we propose that NCAM1 is a target autoantigen in a subset of patients with membranous lupus nephritis. Future studies are needed to determine whether anti-NCAM1 antibody levels correlate with disease activity or response to therapy.

Triple-negative breast cancer (TNBCs) display lung metastasis tropism. However, the mechanisms underlying this organ-specific pattern remains to be elucidated. We sought to evaluate the utility of blocking extravasation to prevent lung metastasis. To identify potential geminin overexpression-controlled genetic drivers that promote TNBC tumor homing to lungs, we used the differential/suppression subtractive chain (D/SSC) technique. A geminin overexpression-induced lung metastasis gene signature consists of 24 genes was discovered. We validated overexpression of five of these genes (LGR5, HAS2, CDH11, NCAM2, and DSC2) in worsening lung metastasis-free survival in TNBC patients. Our data demonstrate that LGR5-induced β-catenin signaling and stemness in TNBC cells are geminin-overexpression dependent. They also demonstrate for the first-time expression of RSPO2 in mouse lung tissue only and exacerbation of its secretion in the circulation of mice that develop geminin overexpressing/LGR5+-TNBC lung metastasis. We identified a novel extravasation receptor complex, consists of CDH11, CD44v6, c-Met, and AXL on geminin overexpressing/LGR5+-TNBC lung metastatic precursors, inhibition of any of its receptors prevented geminin overexpressing/LGR5+-TNBC lung metastasis. Overall, we propose that geminin overexpression in normal mammary epithelial (HME) cells promotes the generation of TNBC metastatic precursors that home specifically to lungs by upregulating LGR5 expression and promoting stemness, intravasation, and extravasation in these precursors. Circulating levels of RSPO2 and OPN can be diagnostic biomarkers to improve risk stratification of metastatic TNBC to lungs, as well as identifying patients who may benefit from therapy targeting geminin alone or in combination with any member of the newly discovered extravasation receptor complex to minimize TNBC lung metastasis.

Objective To describe the clinical and pathological phenotype of membranous nephropathy (MN) associated with M-type-phospholipase–A2-receptor (PLA2R), thrombospondin-type-1-domain-containing-7A (THSD7A), semaphorin 3B (SEMA3B), neural-epidermal-growth-factor-like-1-protein (NELL-1), protocadherin 7 (PCDH7), exostosin 1/exostosin 2 (EXT1/EXT2) and neural cell adhesion molecule 1 (NCAM-1) as target antigens. Methods A retrospective cohort of 270 adult patients with biopsy-proven MN diagnosed between January 2015 and April 2020 was classified as PLA2R-, THSD7A-, SEMA3B-, NELL-1–, PCDH7-, EXT1/EXT2-, NCAM-1–associated or septuple-negative MN using serologic tests, immunostaining, and/or mass spectrometry. Clinical, biochemical, pathologic, and follow-up data were systematically abstracted from the medical records, including disease activity of conditions traditionally associated with MN and occurring within 5 years of MN diagnosis. Results Patients with PLA2R-associated MN were predominantly middle-aged white men without associated disease. The presence of associated disease did not affect the clinical and pathologic characteristics of PLA2R-associated MN, suggesting that they were coincidental rather than causally linked. THSD7A-, NELL-1–, PCDH7-, and NCAM-1–associated MN were rare and SEMA3B-associated MN was not discovered in our cohort. EXT1/EXT2-associated MN was primarily diagnosed in younger women with active systemic autoimmunity. A significant proportion of septuple-negative patients had associated malignancy or systemic autoimmunity. Conclusion The widely used distinction between primary and secondary MN has limitations. We propose a refined terminology that combines the target antigen and associated disease to better classify MN and guide clinical decision making.

Neuroligin 3 (NLGN3) and neurexins (NRXNs) constitute a canonical transsynaptic cell-adhesion pair, which has been implicated in autism. In autism spectrum disorder (ASD) development of sociality can be impaired. However, the molecular mechanism underlying NLGN3-mediated social development is unclear. Here, we identify non-canonical interactions between NLGN3 and protein tyrosine phosphatase δ (PTPδ) splice variants, competing with NRXN binding. NLGN3-PTPδ complex structure revealed a splicing-dependent interaction mode and competition mechanism between PTPδ and NRXNs. Mice carrying a NLGN3 mutation that selectively impairs NLGN3-NRXN interaction show increased sociability, whereas mice where the NLGN3-PTPδ interaction is impaired exhibit impaired social behavior and enhanced motor learning, with imbalance in excitatory/inhibitory synaptic protein expressions, as reported in the Nlgn3 R451C autism model. At neuronal level, the autism-related Nlgn3 R451C mutation causes selective impairment in the non-canonical pathway. Our findings suggest that canonical and non-canonical NLGN3 pathways compete and regulate the development of sociality., Mutations of Neuroligin 3 (NLGN3) have been associated with autism spectrum disorder (ASD). Here, the authors identify a previously undescribed interaction between NLGN3 and a splice variant of protein tyrosine phosphatase δ (PTP δ) and its role in development of social behaviour in mice.

Background Rare genetic variants contribute to the etiology of both autism spectrum disorder (ASD) and schizophrenia (SCZ). Most genetic studies limit their focus to likely gene-disrupting mutations because they are relatively easier to interpret their effects on the gene product. Interpretation of missense variants is also informative to some pathophysiological mechanisms of these neurodevelopmental disorders; however, their contribution has not been elucidated because of relatively small effects. Therefore, we characterized missense variants detected in NRXN1, a well-known neurodevelopmental disease-causing gene, from individuals with ASD and SCZ. Methods To discover rare variants with large effect size and to evaluate their role in the shared etiopathophysiology of ASD and SCZ, we sequenced NRXN1 coding exons with a sample comprising 562 Japanese ASD and SCZ patients, followed by a genetic association analysis in 4273 unrelated individuals. Impact of each missense variant detected here on cell surface expression, interaction with NLGN1, and synaptogenic activity was analyzed using an in vitro functional assay and in silico three-dimensional (3D) structural modeling. Results Through mutation screening, we regarded three ultra-rare missense variants (T737M, D772G, and R856W), all of which affected the LNS4 domain of NRXN1α isoform, as disease-associated variants. Diagnosis of individuals with T737M, D772G, and R856W was 1ASD and 1SCZ, 1ASD, and 1SCZ, respectively. We observed the following phenotypic and functional burden caused by each variant. (i) D772G and R856W carriers had more serious social disabilities than T737M carriers. (ii) In vitro assay showed reduced cell surface expression of NRXN1α by D772G and R856W mutations. In vitro functional analysis showed decreased NRXN1α-NLGN1 interaction of T737M and D772G mutants. (iii) In silico 3D structural modeling indicated that T737M and D772G mutations could destabilize the rod-shaped structure of LNS2-LNS5 domains, and D772G and R856W could disturb N-glycan conformations for the transport signal. Conclusions The combined data suggest that missense variants in NRXN1 could be associated with phenotypes of neurodevelopmental disorders beyond the diagnosis of ASD and/or SCZ.

Calsyntenin-3 (Clstn3) is a postsynaptic adhesion molecule that induces presynaptic differentiation via presynaptic neurexins (Nrxns), but whether Nrxns directly bind to Clstn3 has been a matter of debate. Here, using LC–MS/MS–based protein analysis, confocal microscopy, RNAscope assays, and electrophysiological recordings, we show that β-Nrxns directly interact via their LNS domain with Clstn3 and Clstn3 cadherin domains. Expression of splice site 4 (SS4) insert–positive β-Nrxn variants, but not insert–negative variants, reversed the impaired Clstn3 synaptogenic activity observed in Nrxn-deficient neurons. Consistently, Clstn3 selectively formed complexes with SS4–positive Nrxns in vivo. Neuron-specific Clstn3 deletion caused significant reductions in number of excitatory synaptic inputs. Moreover, expression of Clstn3 cadherin domains in CA1 neurons of Clstn3 conditional knockout mice rescued structural deficits in excitatory synapses, especially within the stratum radiatum layer. Collectively, our results suggest that Clstn3 links to SS4–positive Nrxns to induce presynaptic differentiation and orchestrate excitatory synapse development in specific hippocampal neural circuits, including Schaffer collateral afferents.

Background: Cell-cell adhesion is essential in maintaining the structure and function of an organ. Several adhesion molecules have recently been identified as associated with heroin dependence in both genetic and peripheral plasma studies. Methods and Results: We reviewed literature concerning studies on adhesion molecules in opioid addictions in rodents and human, including human genetic associations in different ethnic groups, and treatment responses to methadone maintenance treatment in heroin-dependent patients. Conclusion: Some important and novel findings were summarized and discussed. Adhesion molecules in the peripheral plasma, e.g., cadherin-2 (CDH2), may be biomarkers for both methadone treatment outcome and nectin 4 may be an indicator for continued opioid use. Neural cell adhesion molecule (NCAM) in the central nervous system may regulate opioid withdrawal and analgesic responses. Future studies to uncover the mechanisms underlying the involvement of adhesion molecules in the pathological process of addictions will be an important research direction in the field.

Hypoxia in prostate tumours has been associated with disease progression and metastasis. MicroRNAs are short noncoding RNA molecules that are important in several cell processes, but their role in hypoxic signalling is still poorly understood. miR-210 has been linked with hypoxic mechanisms, but this relationship has been poorly characterised in prostate cancer. In this report, the link between hypoxia and miR-210 in prostate cancer cells is investigated. Polymerase chain reaction analysis demonstrates that miR-210 is induced by hypoxia in prostate cancer cells using in vitro cell models and an in vivo prostate tumour xenograft model. Analysis of The Cancer Genome Atlas prostate biopsy datasets shows that miR-210 is significantly correlated with Gleason grade and other clinical markers of prostate cancer progression. Neural cell adhesion molecule (NCAM) is identified as a target of miR-210, providing a biological mechanism whereby hypoxia-induced miR-210 expression can contribute to prostate cancer. This study provides evidence that miR-210 is an important regulator of cell response to hypoxic stress and proposes that its regulation of NCAM may play an important role in the pathogenesis of prostate cancer.

Glutaric acidemia type I (GA1) is caused by severe deficiency of glutaryl-CoA dehydrogenase activity, resulting in an accumulation of glutaric acid and glutarylcarnitine (C5DC) in the organism. Patients affected by GA1 are asymptomatic in the neonate period but usually manifest chronically progressive neurodegeneration apart from severe encephalopathic crises associated with acute striatum necrosis. Neurological manifestations like dyskinesia, dystonia, hypotonia, muscle stiffness, and spasticity are present. Treatment is based on protein/lysine restriction and l-carnitine supplementation. In this work, we evaluated markers of neurodegeneration and inflammation, namely BDNF (brain-derived neurotrophic factor), NCAM (neuronal adhesion molecule), PDGF-AA (platelet-derived growth factor), and cathepsin-d in plasma of six treated GA1 patients. We first found marked increases of plasma C5DC concentrations in GA1 patients, as well as increased levels of the markers BDNF and cathepsin-d as compared to those of age-matched healthy children. Furthermore, C5DC concentrations were highly correlated with the levels of cathepsin-d. These results may demonstrate that brain tissue degeneration is present in GA1 patients and that there is a relationship between increased metabolites concentrations with this process. To the best of our knowledge, this is so far the first study showing altered peripheral parameters of neurodegeneration and inflammation in GA1 patients.

Background Signal regulatory protein β1 (SIRPB1) is a signal regulatory protein member of the immunoglobulin superfamily and is capable of modulating receptor tyrosine kinase-coupled signaling. Copy number variations at the SIRPB1 locus were previously reported to associate with prostate cancer aggressiveness in patients, however, the role of SIRPB1 in prostate carcinogenesis is unknown. Methods Fluorescence in situ hybridization and laser-capture microdissection coupled with quantitative polymerase chain reaction was utilized to determine SIRPB1 gene amplification and messenger RNA expression in prostate cancer specimens. The effect of knockdown of SIRPB1 by RNA interference in PC3 prostate cancer cells on cell growth in colony formation assays and cell mobility in wound-healing, transwell assays, and cell cycle analysis was determined. Overexpression of SIPRB1 in C4-2 prostate cancer cells on cell migration, invasion, colony formation and cell cycle progression and tumor take rate in xenografts was also determined. Western blot assay of potential downstream SIRPB1 pathways was also performed. Results SIRPB1 gene amplification was detected in up to 37.5% of prostate cancer specimens based on in silico analysis of several publicly available datasets. SIRPB1 gene amplification and overexpression were detected in prostate cancer specimens. The knockdown of SIRPB1 significantly suppressed cell growth in colony formation assays and cell mobility. SIRPB1 knockdown also induced cell cycle arrest during the G0 /G1 phase and enhancement of apoptosis. Conversely, overexpression of SIPRB1 in C4-2 prostate cancer cells significantly enhanced cell migration, invasion, colony formation, and cell cycle progression and increased C4-2 xenograft tumor take rate in nude mice. Finally, this study presented evidence for SIRPB1 regulation of Akt phosphorylation and showed that Akt inhibition could abolish SIRPB1 stimulation of prostate cancer cell proliferation. Conclusions These results suggest that SIRPB1 is a potential oncogene capable of activating Akt signaling to stimulate prostate cancer proliferation and could be a biomarker for patients at risk of developing aggressive prostate cancer.

Objective To characterize the role of steroid hormone and antihormone exposure on neurotrimin (NTM) expression in human leiomyoma and myometrial tissue and cells. Design Laboratory study of placebo and ulipristal acetate (UPA)–treated patient tissue. In vitro assessment of immortalized myometrial and leiomyoma cell lines after hormone and antihormone exposure. Setting Academic research center. Patient(s) Not applicable. Interventions(s) Exposure of leiomyoma cell lines to 17β-E2, medroxyprogesterone acetate (MPA), UPA, and fulvestrant. Main Outcome Measure(s) Messenger RNA expression quantified with the use of RNASeq analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Protein levels quantified by means of Western blot analysis. Immunohistochemistry (IHC) on placebo- and UPA-treated patient uterine tissue specimens. Result(s) Expression of NTM in human uterine leiomyoma specimens according to RNASeq was increased compared with myometrium (5.22 ± 0.57–fold), which was confirmed with the use of qRT-PCR (1.95 ± 0.05). Furthermore, NTM protein was elevated in leiomyoma tissue compared with matched myometrium (2.799 ± 0.575). IHC revealed increased staining intensity in leiomyoma surgical specimens compared with matched myometrium of placebo patients. Western blot analysis in immortalized leiomyoma cell lines demonstrated an up-regulation of NTM protein expression (2.4 ± 0.04). Treatment of leiomyoma cell lines with 17β-E2 yielded a 1.98 ± 0.11–fold increase in NTM protein expression; however, treatment with fulvestrant showed no significant change compared with control. Leiomyoma cell lines demonstrated a 1.91 ± 0.97–fold increase in NTM protein expression after progesterone treatment. RNASeq analysis demonstrated a reduced expression in patient leiomyoma after UPA treatment (0.75 ± 0.14). Treatment of leiomyoma cells with UPA demonstrated a reduced total NTM protein amount (0.54 ± 0.31) in patients, which was confirmed with the use of IHC (UPA10 147.2 ± 9.40, UPA20 182.8 ± 8.98). In vitro studies with UPA treatment revealed a concentration-dependent effect that supported these findings. Conclusion(s) NTM, a neural cell adhesion molecule, is increased in leiomyoma compared with myometrium in patient tissue and in vitro models after estrogen and progesterone treatment. Down-regulation of expression occurs after UPA treatment, but not after fulvestrant exposure. Clinical Trial Registration Number NCT00290251 .

Summary: Background: Synaptic proteins are increasingly studied as biomarkers for synaptic dysfunction and loss, which are early and central events in Alzheimer's disease (AD) and strongly correlate with the degree of cognitive decline. In this study, we specifically investigated the synaptic binding partners neurexin (NRXN) and neuroligin (Nlgn) proteins, to assess their biomarker's potential. Methods: we developed a parallel reaction monitoring mass spectrometric method for the simultaneous quantification of NRXNs and Nlgns in cerebrospinal fluid (CSF) of neurodegenerative diseases, focusing on AD. Specifically, NRXN-1α, NRXN-1β, NRXN-2α, NRXN-3α and Nlgn1, Nlgn2, Nlgn3 and Nlgn4 proteins were targeted. Findings: The proteins were investigated in a clinical cohort including CSF from controls (n=22), mild cognitive impairment (MCI) due to AD (n=44), MCI due to other conditions (n=46), AD (n=77) and a group of non-AD dementia (n=28). No difference in levels of NRXNs and Nlgns was found between AD (both at dementia and MCI stages) or controls or the non-AD dementia group for any of the targeted proteins. NRXN and Nlgn proteins correlated strongly with each other, but only a weak correlation with the AD core biomarkers and the synaptic biomarkers neurogranin and growth-associated protein 43, was found, possibly reflecting different pathogenic processing at the synapse. Interpretation: we conclude that NRXN and Nlgn proteins do not represent suitable biomarkers for synaptic pathology in AD. The panel developed here could aid in future investigations of the potential involvement of NRXNs and Nlgns in synaptic dysfunction in other disorders of the central nervous system. Funding: a full list of funding can be found under the acknowledgments section.

BackgroundSpinal tuberculosis (TB) may have a variable, non-specific presentation including back pain with- or without- constitutional symptoms. Further tools are needed to aid early diagnosis of this potentially severe form of TB and immunological biomarkers may show potential in this regard. The aim of this study was to investigate the utility of host serum biomarkers to distinguish spinal TB from mechanical back pain.MethodsPatients with suspected spinal TB or suspected mechanical back pain were recruited from a tertiary hospital in the Western Cape, South Africa, and provided a blood sample for biomarker analysis. Diagnosis was subsequently confirmed using bacteriological testing, advanced imaging and/or clinical evaluation, as appropriate. The concentrations of 19 host biomarkers were evaluated in serum samples using the Luminex platform. Receiver Operating Characteristic (ROC) curves and General Discriminant Analysis were used to identify biomarkers with the potential to distinguish spinal TB from mechanical back pain.ResultsTwenty-six patients with spinal TB and 17 with mechanical back pain were recruited. Seven out of 19 biomarkers were significantly different between groups, of which Fibrinogen, CRP, IFN-γ and NCAM were the individual markers with the highest discrimination utility (Area Under Curve ROC plot 0.88-0.99). A five-marker biosignature (CRP, NCAM, Ferritin, CXCL8 and GDF-15) correctly classified all study participants after leave-one-out cross-validation.ConclusionThis study identified host serum biomarkers with the potential to diagnose spinal TB, including a five-marker biosignature. These preliminary findings require validation in larger studies.

Synaptopathies are brain disorders characterized by dysfunctional synapses, which are specialized junctions between neurons that are essential for the transmission of information. Synaptic dysfunction can occur due to mutations that alter the structure and function of synaptic components or abnormal expression levels of a synaptic protein. One class of synaptic proteins that are essential to their biology are cell adhesion proteins that connect the pre- and post-synaptic compartments. Neurexins are one type of synaptic cell adhesion molecule that have, recently, gained more pathological interest. Variants in both neurexins and their common binding partners, neuroligins, have been associated with several neuropsychiatric disorders. In this review, we summarize some of the key physiological functions of the neurexin protein family and the protein networks they are involved in. Furthermore, examination of published literature has implicated neurexins in both neuropsychiatric and neurodegenerative disorders. There is a clear link between neurexins and neuropsychiatric disorders, such as autism spectrum disorder and schizophrenia. However, multiple expression studies have also shown changes in neurexin expression in several neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease. Therefore, this review highlights the potential importance of neurexins in brain disorders and the importance of doing more targeted studies on these genes and proteins.

Purpose The development of biomarkers and molecularly targeted therapies for patients with Ewing sarcoma (ES) in order to minimise morbidity and improve outcome is urgently needed. Here, we set out to isolate and characterise patient-derived ES primary cell cultures and daughter cancer stem-like cells (CSCs) to identify biomarkers of high-risk disease and candidate therapeutic targets. Methods Thirty-two patient-derived primary cultures were established from treatment-naïve tumours and primary ES-CSCs isolated from these cultures using functional methods. By RNA-sequencing we analysed the transcriptome of ES patient-derived cells (n = 24) and ES-CSCs (n = 11) to identify the most abundant and differentially expressed genes (DEGs). Expression of the top DEG(s) in ES-CSCs compared to ES cells was validated at both RNA and protein levels. The functional and prognostic potential of the most significant gene (neurexin-1) was investigated using knock-down studies and immunohistochemistry of two independent tumour cohorts. Results ES-CSCs were isolated from all primary cell cultures, consistent with the premise that ES is a CSC driven cancer. Transcriptional profiling confirmed that these cells were of mesenchymal origin, revealed novel cell surface targets for therapy that regulate cell-extracellular matrix interactions and identified candidate drivers of progression and relapse. High expression of neurexin-1 and low levels of regulators of its activity, APBA1 and NLGN4X, were associated with poor event-free and overall survival rates. Knock-down of neurexin-1 decreased viable cell numbers and spheroid formation. Conclusions Genes that regulate extracellular interactions, including neurexin-1, are candidate therapeutic targets in ES. High levels of neurexin-1 at diagnosis are associated with poor outcome and identify patients with localised disease that will relapse. These patients could benefit from more intensive or novel treatment modalities. The prognostic significance of neurexin-1 should be validated independently.

The neural cell adhesion molecule (NCAM) has previously been studied in pituitary neuroendocrine tumours (PitNETs), but its role in tumour biology and aggressiveness remains controversial, and its relationship with the tumour microenvironment remains unknown. We aimed to characterise NCAM expression in PitNETs, to correlate this with clinico-pathological features, and to assess the role of various microenvironment components on NCAM expression. NCAM and immune cells were investigated by immunohistochemistry in 16 human non-functioning-PitNETs (NF-PitNETs) and eight somatotrophinomas, including macrophages (CD68, CD163, HLA-DR), cytotoxic (CD8) and T helper (CD4) lymphocytes, regulatory T cells (FOXP3), B cells (CD20), and neutrophils (neutrophil elastase). Five normal pituitaries were included for comparison. The cytokine secretome from these PitNETs and from PitNET-derived tumour-associated fibroblasts (TAFs) were assessed on culture supernatants using a multiplex immunoassay panel. There were no significant NCAM expression differences between PitNETs and normal pituitary, and no difference between types of pituitary tumours (NF-PitNETs vs. somatotrophinomas). There was no association between NCAM expression and different clinico-pathological features, including cavernous sinus invasion and Ki-67, nor with serum hormone levels. NCAM immunoreactivity correlated negatively with PitNET-derived CXCL10 (rho = -0.417; p = .042) and CX3CL1 (rho = -0.423; p = .040) levels. NCAM immunoreactivity was negatively correlated with TAF-derived fibroblast growth factor (FGF)-2 (rho = -0.632; p = .009), but not with other TAF-derived cytokines. Within the PitNET cohort, there were no correlations between NCAM immunoreactivity and immune infiltrates or ratios, although, within NF-PitNETs, NCAM expression was higher in tumours with more FOXP3+ cells. NCAM expression does not differ between PitNETs and normal pituitary, and does not appear to relate to tumour invasiveness or proliferation. However, our data suggest a possible role for cytokines in the modulation of NCAM expression in PitNETs, particularly CXCL10, CX3CL1 and FGF-2, but not for immune cell infiltrates.

Background Irritable bowel syndrome (IBS) is characterized by abdominal pain, bowel habit alterations, and psychiatric comorbidities. Although pathophysiology remains incompletely understood, prior work demonstrates associations with brain-derived neurotrophic factor (BDNF) and catechol-O-methyltransferase (COMT). The purpose of this study was to quantify BDNF and COMT in plasma and in neuronal-enriched extracellular vesicles (nEVs), assess relationships with psychological symptoms, and gain insight on the brain-gut connection in IBS. Methods Clinical data and biorepository samples from a parent investigation were used, including scores on the Perceived Stress Scale (PSS) and Center for Epidemiological Studies Depression Scale (CES-D). Distinct subpopulations of nEVs were isolated using neural cell adhesion molecule L1CAM; levels of COMT, mature BDNF, and pro-BDNF were quantified in plasma and in nEVs using ELISA. Key results Data from 47 females (28.11 ± 6.85 years) included 18 IBS and 29 healthy control (HC) participants. IBS participants displayed reduced plasma levels of mature BDNF compared with HC (p = 0.024). Levels of COMT plasma and IBS grouping significantly predicted CES-D scores (p = 0.034). Exploratory analyses by IBS subtype and race revealed African American HC display lower levels of COMT EV than Caucasian HC (p = 0.022). Conclusions & inferences Lower levels of mature BDNF in IBS participants, preliminary patterns detected in cargo content of nEVs, and relevance of COMT and IBS status to CES-D scores, offer insight on depressive symptomatology and brain-gut dysregulation in IBS. Lower COMT levels in nEVs of African Americans highlight the relevance of race when conducting such analyses across diverse populations.

Objective. Neural cell adhesion molecule (NCAM), a glycoprotein widely distributed in the brain, has recently been shown to regulate neuroplasticity. However, the role of NCAM in vascular dementia (VaD) is still unclear. The purpose of this study is to determine whether NCAM is involved in the course of VaD. Methods. Continuous recruitment of VaD patients and control population to join this study. Doctors or nurses are responsible for collecting their clinical characteristics including age, gender, formal education, heart rate, supine systolic blood pressure, supine diastolic blood pressure, fasting glucose, high-density lipoprotein, and low-density lipoprotein. Each participant received the Montreal Cognitive Assessment (MoCA) scale after being enrolled in the group. At the same time, their peripheral blood was collected, and their serum NCAM levels were measured by enzyme-linked immunosorbent assay (ELISA). Results. 98 VaD patients and 83 age- and sex-matched controls were enrolled. There was no significant statistical difference between the VaD group and the control group in terms of the comparison of clinical characteristics ( p > 0.05 ). The MoCA score of VaD patients was significantly lower than that of the controls ( 27.9 ± 1.4 vs. 23.0 ± 2.1 points, p < 0.001 ). In addition, the circulating NCAM level of VaD patients was also significantly lower than that of controls ( 21.7 ± 3.8 vs. 17.6 ± 4.2 ng/mL, p < 0.001 ). The circulating NCAM level of VaD patients was significantly positively correlated with MoCA score ( r = 0.285 , p = 0.026 ). After adjusting for clinical characteristics, circulating NCAM levels are still an independent pathogenic factor of VaD (regression coefficient = 0.223 , p = 0.034 ). Conclusions. VaD patients have low circulating NCAM levels, which can be used as a potential predictor of VaD.

Certain behavioral characteristics of autism spectrum disorder can be found in otherwise healthy people. Individuals with difficulties in social adaptation may have subclinical autistic traits; however, effective biomarkers of these traits have not yet been established. There is a dire need for objective indices of these traits that combine behavior, brain images, and genetic information. In this study, we examined the association among a single nucleotide polymorphism of NRXN1 (rs858932; C/G), autistic traits, and brain structure in 311 healthy adults. We found that carriers of minor alleles (carriers of the G-allele) had significantly higher systemizing scores than major-allele (C-allele) homozygotes. Furthermore, the regional white matter volume in the right anterior limb of the internal capsule was significantly greater in carriers of the G-allele than in C-allele homozygotes. To the best of our knowledge, this is the first report of NRXN1 rs858932 being involved in systemizing and the brain structure of healthy adults. Our findings provide insight into the effects of genetics on autistic traits and their respective neural substrates.

Focal nodular hyperplasia (FNH) is a polyclonal tumour-like hepatic lesion characterised by parenchymal nodules, connective tissue septa without interlobular bile ducts, pronounced ductular reaction and inflammation. It may represent a response to local arterial hyperperfusion and hyperoxygenation resulting in oxidative stress. We aimed at obtaining closer insight into the pathogenesis of FNH with its characteristic morphologic features. Immunohistochemistry and immunofluorescence microscopy was performed on FNH specimens using antibodies against keratins (K) 7 and 19, neural cell adhesion molecule (NCAM), lamin B1, senescence markers (CDK inhibitor 1/p21Cip1, CDK inhibitor /p16Ink4a, senescence-associated (SA) β- galactosidase activity), proliferation markers (Ki-67, proliferating-cell nuclear antigen (PCNA)), and the abnormally phosphorylated histone γ-H2AX, indicating DNA double strand breaks; moreover SA β- galactosidase activity was determined histochemically. Ductular metaplasia of hepatocytes indicated by K7 expression in the absence of K19 plays a major role in the development of ductular reaction in FNH. Moreover, the expression of senescence markers (p21Cip1, p16Ink4a, γ-H2AX, SA β-galactosidase activity) in hepatocytes and cholangiocytes suggests that stress-induced cellular senescence contributes to fibrosis and inflammation via production of components of the senescence-associated secretory phenotype. Expression of proliferation markers (Ki-67, PCNA) was not enhanced in hepatocytes and biliary cells. Senescence and ductular metaplasia of hepatocytes may thus be involved in inflammation, fibrosis and apoptosis resistance. Hence, fibrosis, inflammation and reduced apoptotic cell death, rather than proliferation (hyperplasia) may be responsible for increased tissue mass and tumour-like appearance of FNH.

Chemical neurotransmission in the brain typically occurs through synapses, which are structurally and functionally defined as sites of close apposition between an axon terminal and a postsynaptic domain. Ultrastructural examinations of axon terminals established by monoamine neurons in the brain often failed to identify a similar tight pre- and postsynaptic coupling, giving rise to the concept of “diffuse” or “volume” transmission. Whether this results from intrinsic properties of such modulatory neurons remains undefined. Using an efficient co-culture model, we find that dopaminergic neurons establish an axonal arbor that is distinctive compared to glutamatergic or GABAergic neurons in both size and propensity of terminals to avoid direct contact with target neurons. Furthermore, while most dopaminergic varicosities express key proteins involved in exocytosis such as synaptotagmin 1, only ~20% of these are synaptic. The active zone protein bassoon was found to be enriched in a subset of dopaminergic terminals that are in proximity to a target cell. Irrespective of their structure, a majority of dopaminergic terminals were found to be active. Finally, we found that the presynaptic protein Nrxn-1αSS4- and the postsynaptic protein NL-1AB, two major components involved in excitatory synapse formation, play a critical role in the formation of synapses by dopamine neurons. Taken together, our findings support the idea that dopamine neurons in the brain are endowed with a distinctive developmental program that leads them to adopt a fundamentally different mode of connectivity, compared to glutamatergic and GABAergic neurons involved in fast point-to-point signaling.SIGNIFICANCE STATEMENTMidbrain dopamine (DA) neurons regulate circuits controlling movement, motivation, and learning. The axonal connectivity of DA neurons is intriguing due to its hyperdense nature, with a particularly large number of release sites, most of which not adopting a classical synaptic structure. In this study, we provide new evidence highlighting the unique ability of DA neurons to establish a large and heterogeneous axonal arbor with terminals that, in striking contrast with glutamate and GABA neurons, actively avoid contact with the target cells. The majority of synaptic and non-synaptic terminals express proteins for exocytosis and are active. Finally, our finding suggests that, NL-1A+B and Nrxn-1αSS4-, play a critical role in the formation of synapses by DA neurons.

Whereas remarkable advances have uncovered mechanisms that drive nervous system assembly, the processes responsible for the lifelong maintenance of nervous system architecture remain poorly understood. Subsequent to its establishment during embryogenesis, neuronal architecture is maintained throughout life in the face of the animal’s growth, maturation processes, the addition of new neurons, body movements, and aging. The Caenorhabditis elegans protein SAX-7, homologous to the vertebrate L1 protein family of neural adhesion molecules, is required for maintaining the organization of neuronal ganglia and fascicles after their successful initial embryonic development. To dissect the function of sax-7 in neuronal maintenance, we generated a null allele and sax-7S-isoform-specific alleles. We find that the null sax-7(qv30) is, in some contexts, more severe than previously described mutant alleles and that the loss of sax-7S largely phenocopies the null, consistent with sax-7S being the key isoform in neuronal maintenance. Using a sfGFP::SAX-7S knock-in, we observe sax-7S to be predominantly expressed across the nervous system, from embryogenesis to adulthood. Yet, its role in maintaining neuronal organization is ensured by postdevelopmentally acting SAX-7S, as larval transgenic sax-7S(+) expression alone is sufficient to profoundly rescue the null mutants’ neuronal maintenance defects. Moreover, the majority of the protein SAX-7 appears to be cleaved, and we show that these cleaved SAX-7S fragments together, not individually, can fully support neuronal maintenance. These findings contribute to our understanding of the role of the conserved protein SAX-7/L1CAM in long-term neuronal maintenance and may help decipher processes that go awry in some neurodegenerative conditions.

Objectives Salivary gland regeneration is closely related to the parasympathetic nerve; however, the mechanism behind this relationship is still unclear. The aim of this study was to evaluate the relationship between the parasympathetic nerve and morphological differences during salivary gland regeneration. Materials and Methods We used a duct ligation/deligation‐induced submandibular gland regeneration model of Sprague‐Dawley (SD) rats. The regenerated submandibular gland with or without chorda lingual (CL) innervation was detected by haematoxylin–eosin staining, real‐time PCR (RT‐PCR), immunohistochemistry and Western blotting. We counted the number of Ki67‐positive cells to reveal the proliferation process that occurs during gland regeneration. Finally, we examined the expression of the following markers: aquaporin 5, cytokeratin 7, neural cell adhesion molecule (NCAM) and polysialyltransferases. Results Intact parasympathetic innervation promoted submandibular gland regeneration. The process of gland regeneration was significantly repressed by cutting off the CL nerve. During gland regeneration, Ki67‐positive cells were mainly found in the ductal structures. Moreover, the expression of NCAM and polysialyltransferases‐1 (PST) expression in the innervation group was significantly increased during early regeneration and decreased in the late stages. In the denervated submandibular glands, the expression of NCAM decreased during regeneration. Conclusions Our findings revealed that the regeneration of submandibular glands with intact parasympathetic innervation was associated with duct cell proliferation and the increased expression of PST and NCAM., Intact parasympathetic nerve innervation promoted submandibular gland regeneration. The process of gland regeneration was significantly repressed by cutting off the parasympathetic nerve. During regeneration, the proliferating cells were mainly found in the ductal structures with parasympathetic innervation. On the contrary, almost no proliferative ductal cells were found in the denervation group. This differential pattern of cell proliferation is most associated with the increased expression of PST and NCAM.

The most distal portion of the ventricular conduction system (VCS) contains cardiac Purkinje cells (PCs), which are essential for synchronous activation of the ventricular myocardium. Contactin-2 (CNTN2), a member of the immunoglobulin superfamily of cell adhesion molecules (IgSF-CAMs), was previously identified as a marker of the VCS. Through differential transcriptional profiling, we discovered two additional highly enriched IgSF-CAMs in the VCS: NCAM-1 and ALCAM. Immunofluorescence staining showed dynamic expression patterns for each IgSF-CAM during embryonic and early postnatal stages, but ultimately all three proteins became highly enriched in mature PCs. Mice deficient in NCAM-1, but not CNTN2 or ALCAM, exhibited defects in PC gene expression and VCS patterning, as well as cardiac conduction disease. Moreover, using ST8sia2 and ST8sia4 knockout mice, we show that inhibition of post-translational modification of NCAM-1 by polysialic acid leads to disrupted trafficking of sarcolemmal intercalated disc proteins to junctional membranes and abnormal expansion of the extracellular space between apposing PCs. Taken together, our data provide insights into the complex developmental biology of the ventricular conduction system., Summary: The cell adhesion molecule NCAM-1 and its post-translational modification by polysialylation are required for normal formation and function of the specialized ventricular conduction system.

Made available in DSpace on 2020-12-12T01:47:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) This study proposed to determine global microRNA (miRNA) expression and miRNA-regulated pathways in Intestinal Neuronal Dysplasia type B (IND-B). Fifty patients (0–15 years old) with IND-B were included in the study. Peripheral blood samples were collected from all 50 patients and from 10 healthy asymptomatic children (controls). Rectal biopsies were collected from 29/50 patients; biopsy tissues were needle microdissected to isolate the different intestinal layers, for molecular analysis. Global miRNA expression was determined using TaqMan arrays. Correlation analysis between miRNA expression in plasma and biopsy samples as well as among tissues derived from the distinct intestinal layers was performed. Computational approaches were used for miRNA target prediction/identification of miRNA-regulated genes and enriched pathways biologically relevant to IND-B pathogenesis. miRNAs were statistically significantly deregulated (FC ≥ 2 and p ≤ 0.05) in submucosal and muscular layers: over-expressed (miR-146a and miR-146b) and under-expressed (miR-99a, miR-100, miR-130a, miR-133b, miR-145, miR-365, miR-374-5p, miR-451). Notably, let-7a-5p was highly over-expressed in patient plasma compared to healthy controls (FC = 17.4). In addition, miR-451 was significantly under-expressed in both plasma and all biopsy tissues from the same patients. Enriched pathways (p < 0.01) were axon guidance, nerve growth factor signalling, NCAM signalling for neurite out-growth, neuronal system and apoptosis. miRNA expression is deregulated in the submucosa and muscular layers of the rectum and detected in plasma from patients with IND-B. Biologically enriched pathways regulated by the identified miRNAs may play a role in IND-B disease pathogenesis, due to the activity related to the neurons of the enteric nervous system. UNESP - São Paulo State University Faculty of Medicine Department of Surgery and Orthopedics UNESP - São Paulo State University Faculty of Medicine Experimental Research Unity (UNIPEX) Institute of Livestock and Biotechnology Laboratory of Molecular Physiology Toribio Rodriguez de Mendoza National University UNESP - São Paulo State University Faculty of Medicine Department of Pathology UNESP - São Paulo State University Faculty of Medicine Department of Surgery and Orthopedics UNESP - São Paulo State University Faculty of Medicine Experimental Research Unity (UNIPEX) UNESP - São Paulo State University Faculty of Medicine Department of Pathology FAPESP: 2015/03664-2

Background The aim of this study was to identify subgroups with good or bad prognosis in patients with pulmonary large cell neuroendocrine carcinoma (LCNEC) based on immunostaining patterns with neuroendocrine markers and compare them with small cell lung carcinoma (SCLC). Methods From January 2001 to December 2017, of all patients with resected LCNEC and SCLC, we selected patients whose pathological tumor sizes were ≤30 mm in diameter (defined as small‐sized tumors) and who underwent complete resection with lymphadenectomy. We classified patients with small‐sized LCNEC (sLCNEC) into two subgroups based on immunostaining patterns with three neuroendocrine markers (chromogranin A, synaptophysin, and NCAM) and compared them to small‐sized SCLC (sSCLC). Results A total of 48 patients with sLCNEC and 39 patients with sSCLC were enrolled. Of 48 patients with sLCNEC, 21 were categorized as the small‐sized triple‐positive group (sTP), whose patients were positive for the three neuroendocrine markers, and 27 patients were categorized as the small‐sized nontriple‐positive group (sNTP), whose patients were not positive for all three neuroendocrine markers. The percentage of lymph node metastasis was significantly lower in sNTP than in sTP and sSCLC. There was no significant difference in overall survival, but recurrence‐free survival (RFS) and tumor‐specific survival (TSS) were significantly poorer in sTP and sSCLC than in sNTP. Multivariate analysis revealed sTP and sSCLC were independent prognostic factors for poorer RFS and TSS than those of sNTP. Conclusions The sNTP subgroup had a good prognosis and the sTP subgroup a poor prognosis. There were some similarities in clinicopathological features between sTP and sSCLC.

Objective Resistance training (RT) can improve whole muscle strength without increasing muscle fiber size or contractility. Neural adaptations, which lead to greater neural activation of muscle, may mediate some of these improvements, particularly in older adults, where motor neuron denervation is common. The purpose of this study was to explore the relationship of neural adaptations, as reflected by neural cell adhesion molecule (NCAM) expression, to improvements in (1) whole muscle strength and (2) muscle fiber size following RT in older adults with knee osteoarthritis. We performed whole muscle strength measurements and immunohistochemical analysis of fiber size, type, and NCAM expression before and after a 14-week RT program. Results RT increased whole-muscle strength as measured by 1-repetition maximum (1-RM) leg press (P = 0.01), leg extension (P = 0.03), and knee extensor peak torque (P = 0.050), but did not alter NCAM expression. Greater NCAM expression in myosin heavy chain (MHC) II fibers was associated with greater whole muscle strength gains (knee extensor peak torque r = 0.93; P Trial registration NCT01190046

Schizophrenia, Schizoaffective, and Bipolar disorders share behavioral and phenomenological traits, intermediate phenotypes, and some associated genetic loci with pleiotropic effects. Volumetric abnormalities in brain structures are among the intermediate phenotypes consistently reported associated with these disorders. In order to examine the genetic underpinnings of these structural brain modifications, we performed genome-wide association analyses (GWAS) on 60 quantitative structural brain MRI phenotypes in a sample of 777 subjects (483 cases and 294 controls pooled together). Genotyping was performed with the Illumina PsychChip microarray, followed by imputation to the 1000 genomes multiethnic reference panel. Enlargement of the Temporal Horns of Lateral Ventricles (THLV) is associated with an intronic SNP of the gene NRXN1 (rs12467877, P = 6.76E–10), which accounts for 4.5% of the variance in size. Enlarged THLV is associated with psychosis in this sample, and with reduction of the hippocampus and enlargement of the choroid plexus and caudate. Eight other suggestively significant associations (P

Introduction Muscle fiber denervation increases with age, yet studies at the tissue level are sparse due to the challenging nature of establishing the relative role of regeneration and denervation. Methods Muscle biopsies were obtained from the vastus lateralis of 70 healthy men (aged 72 ± 6 years; range, 65-94). Messenger RNA (mRNA) levels of acetylcholine receptors (AchR) were measured, and sections were stained for embryonic myosin, neonatal myosin (MHCn ), and neural cell adhesion molecule (NCAM). Results Embryonic myosin+ fibers were rare, while MHCn + and NCAM+ fibers were observed in all samples. Age (range, 65-94 years) was negatively associated with AchRγ mRNA. Discussion Muscle from healthy older individuals expressed developmental myosins to varying degrees but more than has been previously reported for young individuals. Along with the AchR correlations, we propose that these findings support the presence of neuromuscular junction destabilization, denervation, and reinnervation in aging human skeletal muscle.

A large body of the literature has demonstrated that the polysialic acid (polySia) modification of the neural cell adhesion molecule (NCAM) is a key regulator of cellular interactions during brain development, maintenance and plasticity. To properly fulfill these functions, polySia concentration has to be carefully controlled. This is done by the regulation of the expression of the two polySia-synthesizing enzymes ST8SiaII and ST8SiaIV. From this point of view we and others have demonstrated that downregulation of ST8SiaIV during oligodendrocyte differentiation is a prerequisite for efficient myelin formation and maintenance. Here, we addressed the question whether the prevention of polySia downregulation in neurons affects brain and particularly myelin development and functioning. For this purpose, we developed transgenic (tg) mouse lines overexpressing the polysialyltransferase ST8SiaIV in neurons. tg expression of ST8SiaIV prevented the postnatal downregulation of polySia, and most of the polySias in the forebrain and brain stem of adult tg mice were associated with NCAM-140 and NCAM-180 isoforms. Structural examination of the brain revealed no overt abnormalities of axons and myelin. In addition, ultrastructural and western blot analyses indicated normal myelin development. However, behavioral studies revealed reduced rearing activity, a measure for exploratory behavior, while parameters of motor activity were not affected in tg mice. Taken together, these results suggest that a persisting presence of polySia in neurons has no major effect on brain structure, myelination and myelin maintenance, but causes mild behavioral changes.

Formation of protein complexes across synapses is a critical process in neurodevelopment, having direct implications on brain function and animal behavior. Here, we present the understanding, importance, and potential impact of a newly found regulator of such a key interaction. A systematic search of the literature was conducted on PubMed (Medline), Embase, and Central-Cochrane Database. Membrane-associated mucin domain-containing glycosylphosphatidylinositol anchor proteins (MDGAs) were recently discovered to regulate synaptic development and transmission via suppression of neurexins–neuroligins trans-synaptic complex formation. MDGAs also regulate axonal migration and outgrowth. In the context of their physiological role, we begin to consider the potential links to the etiology of certain neurodevelopmental disorders. We present the gene expression and protein structure of MDGAs and discuss recent progress in our understanding of the neurobiological role of MDGAs to explore its potential as a therapeutic target. MDGAs play a key role in neuron migration, axon guidance and synapse development, as well as in regulating brain excitation and inhibition balance.

Purpose: To assess the contribution of rare variants in the genetic background toward variability of neurodevelopmental phenotypes in individuals with rare copy-number variants (CNVs) and gene-disruptive variants. Methods: We analyzed quantitative clinical information, exome sequencing, and microarray data from 757 probands and 233 parents and siblings who carry disease-associated variants. Results: The number of rare likely deleterious variants in functionally intolerant genes (“other hits”) correlated with expression of neurodevelopmental phenotypes in probands with 16p12.1 deletion (n=23, p=0.004) and in autism probands carrying gene-disruptive variants (n=184, p=0.03) compared with their carrier family members. Probands with 16p12.1 deletion and a strong family history presented more severe clinical features (p=0.04) and higher burden of other hits compared with those with mild/no family history (p=0.001). The number of other hits also correlated with severity of cognitive impairment in probands carrying pathogenic CNVs (n=53) or de novo pathogenic variants in disease genes (n=290), and negatively correlated with head size among 80 probands with 16p11.2 deletion. These co-occurring hits involved known disease-associated genes such as SETD5, AUTS2, and NRXN1, and were enriched for cellular and developmental processes. Conclusion: Accurate genetic diagnosis of complex disorders will require complete evaluation of the genetic background even after a candidate disease-associated variant is identified.

Background The classification of intrahepatic cholangiocarcinomas (ICCs) has been reported in several studies, however, it remains controversial. Materials and methods Between January 2003 and December 2015, 94 patients underwent hepatectomy for ICC. The ICCs of 63 of these patients were classified as predominantly small bile duct type or bile ductular type ICC and were included in this analysis. Results Thirty-seven patients (58.7%) were classified into the small bile duct ICC group, and 26 (41.3%) into the bile ductular ICC group. A multivariate analysis identified intrahepatic metastasis [hazard ratio (HR)=2.53, p=0.011], small bile duct ICC (HR=2.05, p=0.046) and portal vein invasion (HR 2.05, p=0.047) as independent prognostic factors for poorer survival. Conclusion It is important to correctly distinguish between small bile duct and bile ductular ICC types because these two types clearly have different clinicopathological and prognostic features.

The prognostic importance of the neuroendocrine (NE) markers involving neural cell adhesion molecule (NCAM) has been unclear enough to be adopted for WHO classification in patients with pancreatic neuroendocrine neoplasms (Pan-NENs). This study aimed to elucidate whether the three NE markers such as chromogranin A, synaptophysin, and NCAM decide prognoses for patients with well-differentiated tumors. Between April 2002 and October 2018, 217 patients were included in this study. Tissue samples from tumors of Pan-NENs were immunochemically stained for the aforementioned NE markers. Diffuse and intense staining was defined as positive, while faint or focal staining and non-staining were considered negative. The median age of patients was 55 years. The median observation period was 1415 days. In multivariate analysis of progression-free survival (PFS), liver metastasis, Ki-67 index, and triple-positive staining of NE markers were risk factors. The 5-year PFS rate of patients with and without triple-positive NE markers was 56.3% and 23.8%, respectively (P

Denervation and mitochondrial impairment are implicated in age-related skeletal muscle atrophy and may play a role in physical frailty. We recently showed that denervation modulates muscle mitochondrial function in octogenarian men, but this has not been examined in elderly women. On this basis, we tested the hypothesis that denervation plays a modulating role in mitochondrial impairment in skeletal muscle from prefrail or frail elderly (FE) women. Mitochondrial respiratory capacity and reactive oxygen species emission were examined in permeabilized myofibers obtained from vastus lateralis muscle biopsies from FE and young inactive women. Muscle respiratory capacity was reduced in proportion to a reduction in a mitochondrial marker protein in FE, and mitochondrial reactive oxygen species emission was elevated in FE versus young inactive group. Consistent with a significant accumulation of neural cell adhesion molecule-positive muscle fibers in FE (indicative of denervation), a 50% reduction in reactive oxygen species production after pharmacologically inhibiting the denervation-mediated reactive oxygen species response in FE women suggests a significant modulation of mitochondrial function by denervation. In conclusion, our data support the hypothesis that denervation plays a modulating role in skeletal muscle mitochondrial function in FE women, suggesting therapeutic strategies in advanced age should focus on the causes and treatment of denervation.

Neurons in the murine olfactory epithelium (OE) differ by the olfactory receptor they express as well as other molecular phenotypes that are regionally restricted. These patterns can be precisely regenerated following epithelial injury, suggesting that spatial cues within the tissue can direct neuronal diversification. Nonetheless, the permanency and mechanism of this spatial patterning remain subject to debate. Via transplantation of stem and progenitor cells from dorsal OE into ventral OE, we demonstrate that, in mice of both sexes, nonautonomous spatial cues can direct the spatially circumscribed differentiation of olfactory sensory neurons. The vast majority of dorsal transplant-derived neurons express the ventral marker OCAM (NCAM2) and lose expression of NQO1 to match their new location. Single-cell analysis also demonstrates that OSNs adopt a fate defined by their new position following progenitor cell transplant, such that a ventral olfactory receptor is expressed after stem and progenitor cell engraftment. Thus, spatially constrained differentiation of olfactory sensory neurons is plastic, and any bias toward an epigenetic memory of place can be overcome.SIGNIFICANCE STATEMENTSpatially restricted differentiation of olfactory sensory neurons is both key to normal olfactory function and a challenging example of biological specificity. That the stem cells of the olfactory epithelium reproduce the organization of the olfactory periphery to a very close approximation during lesion-induced regeneration begs the question of whether stem cell-autonomous genomic architecture or environmental cues are responsible. The plasticity demonstrated after transfer to a novel location suggests that cues external to the transplanted stem and progenitor cells confer neuronal identity. Thus, a necessary prerequisite is satisfied for using engraftment of olfactory stem and progenitor cells as a cellular therapeutic intervention to reinvigorate neurogenesis whose exhaustion contributes to the waning of olfaction with age.

The axis of the vertebrate neural tube is patterned, in part, by a ventral to dorsal gradient of Shh signaling. In the ventral spinal cord, Shh induces concentration-dependent expression of transcription factors, subdividing neural progenitors into distinct domains that subsequently produce distinct neuronal and glial subtypes. In particular, progenitors of the pMN domain express the bHLH transcription factor Olig2 and produce motor neurons followed by oligodendrocytes, the myelinating glial cell type of the central nervous system. In addition to its role in patterning ventral progenitors, Shh signaling must be maintained through development to specify pMN progenitors for oligodendrocyte fate. Using a forward genetic screen in zebrafish for mutations that disrupt the development of oligodendrocytes, we identified a new mutant allele of boc, which encodes a type I transmembrane protein that functions as a coreceptor for Shh. Embryos homozygous for the bocco25 allele, which creates a missense mutation in a Fibronectin type III domain that binds Shh, have normally patterned spinal cords but fail to maintain pMN progenitors, resulting in a deficit of oligodendrocytes. Using a sensitive fluorescent detection method for in situ RNA hybridization, we found that spinal cord cells express boc in a graded fashion that is inverse to the gradient of Shh signaling activity and that boc function is necessary to maintain pMN progenitors by shaping the Shh signaling gradient.

Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation predisposing to schizophrenia. Previous studies showed that engineered heterozygous NRXN1 deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multi-center effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. We show that in neurons that were trans-differentiated from induced pluripotent stem cells derived from three NRXN1-deletion patients, the same impairment in neurotransmitter release was observed as in engineered NRXN1-deficient neurons. This impairment manifested as a decrease in spontaneous synaptic events and in evoked synaptic responses, and an alteration in synaptic paired-pulse depression. Nrxn1-deficient mouse neurons generated from embryonic stem cells by the same method as human neurons did not exhibit impaired neurotransmitter release, suggesting a human-specific phenotype. NRXN1 deletions produced a reproducible increase in the levels of CASK, an intracellular NRXN1-binding protein, and were associated with characteristic gene expression changes. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons regardless of genetic background, enabling future drug discovery efforts.

Neural cell adhesion molecule (NCAM) is involved in cell multi-directional differentiation, but its role in osteoblast differentiation is still poorly understood. In the present study, we investigated whether and how NCAM regulates osteoblastic differentiation. We found that NCAM silencing inhibited osteoblast differentiation in pre-osteoblastic MC3T3-E1 cells. The function of NCAM was further confirmed in NCAM-deficient mesenchymal stem cells (MSCs), which also had a phenotype with reduced osteoblastic potential. Moreover, NCAM silencing induced decrease of Wnt/β-catenin and Akt activation. The Wnt inhibitor blocked osteoblast differentiation, and the Wnt activator recovered osteoblast differentiation in NCAM-silenced MC3T3-E1 cells. We lastly demonstrated that osteoblast differentiation of MC3T3-E1 cells was inhibited by the PI3K-Akt inhibitor. In conclusion, these results demonstrate that NCAM silencing inhibited osteoblastic differentiation through inactivation of Wnt/β-catenin and PI3K-Akt signaling pathways.

Homozygous Dab1 yotari mutant mice, Dab1yot (yot/yot) mice, have an autosomal recessive mutation of Dab1 and show reeler-like phenotype including histological abnormality of the cerebellum, hippocampus, and cerebral cortex. We here show abnormal hippocampal development of yot/yot mice where granule cells and pyramidal cells fail to form orderly rows but are dispersed diffusely in vague multiplicative layers. Possibly due to the positioning failure of granule cells and pyramidal cells and insufficient synaptogenesis, axons of the granule cells did not extend purposefully to connect with neighboring regions in yot/yot mice. We found that both hippocampal granule cells and pyramidal cells of yot/yot mice expressed proteins reactive with the anti-Dab1 antibody. We found that Y198- phosphorylated Dab1 of yot/yot mice was greatly decreased. Accordingly the downstream molecule, Akt was hardly phosphorylated. Especially, synapse formation was defective and the distribution of neurons was scattered in hippocampus of yot/yot mice. Some of neural cell adhesion molecules and hippocampus associated transcription factors of the neurons were expressed aberrantly, suggesting that the Reelin-Dab1 signaling pathway seemed to be importantly involved in not only neural migration as having been shown previously but also neural maturation and/or synaptogenesis of the mice. It is interesting to clarify whether the defective neural maturation is a direct consequence of the dysfunctional Dab1, or alternatively secondarily due to the Reelin-Dab1 intracellular signaling pathways.

Muscular regeneration is a complex biological process that occurs during acute injury and chronic degeneration, implicating several cell types. One of the earliest events of muscle regeneration is the inflammatory response, followed by the activation and differentiation of muscle progenitor cells. However, the process of novel neuromuscular junction formation during muscle regeneration is still largely unexplored. Here, we identify by single-cell RNA sequencing and isolate a subset of vessel-associated cells able to improve myogenic differentiation. We termed them 'guide' cells because of their remarkable ability to improve myogenesis without fusing with the newly formed fibers. In vitro, these cells showed a marked mobility and ability to contact the forming myotubes. We found that these cells are characterized by CD44 and CD34 surface markers and the expression of Ng2 and Ncam2. In addition, in a murine model of acute muscle injury and regeneration, injection of guide cells correlated with increased numbers of newly formed neuromuscular junctions. Thus, we propose that guide cells modulate de novo generation of neuromuscular junctions in regenerating myofibers. Further studies are necessary to investigate the origin of those cells and the extent to which they are required for terminal specification of regenerating myofibers. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:22 issue:4 ispartof: location:Switzerland status: published

Background Myosatellite cells are myogenic stem cells that can transform to provide nuclei for existing muscles or generate new muscle fibers as documented after extended exercise programs. Objectives The authors investigated whether the simultaneous application of High-Intensity Focused Electromagnetic (HIFEM) and Synchrode radiofrequency (RF) affects the levels of satellite cells similarly as the prolonged exercise does to achieve muscle growth. Methods Three 30-minute simultaneous HIFEM and Synchrode RF treatments (once a week) were administered over the abdominal area of 5 Large White swine aged approximately 6 months. All animals were anesthetized during the treatments and biopsy acquisition. Biopsies of muscle tissue were collected at baseline, 4 days, 2 weeks, and 1 month post-treatment. After binding the specific antibodies, the NCAM/CD56 levels, a marker of activated satellite cells, were quantified employing the immunofluorescence microscopy technique with a UV lamp. Results Examined slices showed a continuous increase in satellite cell levels throughout the study. Four days after the treatment, we observed a 26.1% increase in satellite cells, which increased to 30.2% at 2-week follow-up. Additional histological analysis revealed an increase in the cross-sectional area of muscle fibers and the signs of newly formed fibers of small diameters at 2 weeks after the treatment. No damage to muscle tissue and no adverse effects related to the treatment were observed. Conclusions The findings indicate that the simultaneous application of HIFEM and novel Synchrode RF treatment can initiate differentiation of satellite cells to support the growth of existing muscles and, presumably, even the formation of new myofibers.

Thyroid cancer (TC) is the most common endocrine malignant disease with a rising morbidity year by year. Accumulating studies have shown that microRNAs (miRNAs) play a regulatory role in the progression of various tumors, but the molecular regulatory mechanism of miR-196a-2 in TC is still unknown. qRT-PCR was employed to measure the expression of miR-196a-2 and NRXN1 mRNA in TC cells, while western blot was used to detect the protein expression of NRXN1. CCK-8, colony formation and flow cytometry assays were used to measure cell proliferation and apoptosis of TC cells. Dual-luciferase reporter gene assay was used to predict and verify the targeted binding relationship between miR-196a-2 and NRXN1. Our study results manifested that miR-196a-2 was dramatically overexpressed in cells of TC, while NRXN1 was lowly expressed. miR-196a-2 could promote cell proliferation and inhibit cell apoptosis of TC. Additionally, miR-196a-2 could also target and inhibit the expression of NRXN1. Silencing NRXN1 could reverse the inhibitory effect of miR-196a-2 downregulation on cell proliferation of TC, as well as the promoting effect on cell apoptosis. In a conclusion, we found that miR-196a-2 could promote cell proliferation and inhibit cell apoptosis of TC by targeting NRXN1. Therefore, miR-196a-2/NRXN1 is potential to be a molecular therapeutic target for TC.

Autism spectrum disorder (ASD) is characterized by a complex polygenic background, but with the unique feature of a subset of cases (~15%‐30%) presenting a rare large‐effect variant. However, clinical interpretation in these cases is often complicated by incomplete penetrance, variable expressivity and different neurodevelopmental trajectories. NRXN1 intragenic deletions represent the prototype of such ASD‐associated susceptibility variants. From chromosomal microarrays analysis of 104 ASD individuals, we identified an inherited NRXN1 deletion in a trio family. We carried out whole‐exome sequencing and deep sequencing of mitochondrial DNA (mtDNA) in this family, to evaluate the burden of rare variants which may contribute to the phenotypic outcome in NRXN1 deletion carriers. We identified an increased burden of exonic rare variants in the ASD child compared to the unaffected NRXN1 deletion‐transmitting mother, which remains significant if we restrict the analysis to potentially deleterious rare variants only (P = 6.07 × 10−5). We also detected significant interaction enrichment among genes with damaging variants in the proband, suggesting that additional rare variants in interacting genes collectively contribute to cross the liability threshold for ASD. Finally, the proband's mtDNA presented five low‐level heteroplasmic mtDNA variants that were absent in the mother, and two maternally inherited variants with increased heteroplasmic load. This study underlines the importance of a comprehensive assessment of the genomic background in carriers of large‐effect variants, as penetrance modulation by additional interacting rare variants to might represent a widespread mechanism in neurodevelopmental disorders.

Presenilins (PS) form the active subunit of the gamma-secretase complex, which mediates the proteolytic clearance of a broad variety of type-I plasma membrane proteins. Loss-of-function mutations in PSEN1/2 genes are the leading cause of familial Alzheimer's disease (fAD). However, the PS/gamma-secretase substrates relevant for the neuronal deficits associated with a loss of PS function are not completely known. The members of the neurexin (Nrxn) family of presynaptic plasma membrane proteins are candidates to mediate aspects of the synaptic and memory deficits associated with a loss of PS function. Previous work has shown that fAD-linked PS mutants or inactivation of PS by genetic and pharmacological approaches failed to clear Nrxn C-terminal fragments (NrxnCTF), leading to its abnormal accumulation at presynaptic terminals. Here, we generated transgenic mice that selectively recreate the presynaptic accumulation of NrxnCTF in adult forebrain neurons, leaving unaltered the function of PS/gamma-secretase complex towards other substrates. Behavioral characterization identified selective impairments in NrxnCTF mice, including decreased fear-conditioning memory. Electrophysiological recordings in medial prefrontal cortex-basolateral amygdala (mPFC-BLA) of behaving mice showed normal synaptic transmission and uncovered specific defects in synaptic facilitation. These data functionally link the accumulation of NrxnCTF with defects in associative memory and short-term synaptic plasticity, pointing at impaired clearance of NrxnCTF as a new mediator in AD., This work was funded by a grant from Ministerio de Ciencia, Innovación y Universidades (RTI2018-101886-B-100) to FGS and AM-M, Junta de Andalucía (PY18-823) to JMG-D, and co-funded by ERDF. ACS-H was the recipient of a fellowship from Junta de Andalucía (P11-CVI-7599). FAA was the recipient of a fellowship from Ministerio de Economía, Industria y Competitividad (BES-2016-076579). CM-C was the recipient of a Garantía Juvenil contract from Universidad de Sevilla.

Significance This study addresses a fundamental question in neuroscience, namely how does the presynaptic component of the synapse precisely align with the postsynaptic component? This is essential for the proper transmission of signals across the synapse. This paper focuses on a set of transsynaptic, epilepsy-related proteins that are essential for this alignment. We show that the LGI1–ADAM22–MAGUK complex is a key player in the nanoarchitecture of the synapse, such that the release site is directly apposed to the nanocluster of glutamate receptors., Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1–ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv1 channels, and LRRTM4–Neurexin adhesion molecules. Adam22ΔC5/ΔC5 knock-in mice devoid of the ADAM22–MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1–ADAM22–MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.