Down-regulation of the brain-specific cell-adhesion molecule contactin-3 in tuberous sclerosis complex during the early postnatal period

Background The genetic disorder tuberous sclerosis complex (TSC) is frequently accompanied by the development of neuropsychiatric disorders, including autism spectrum disorder and intellectual disability, with varying degrees of impairment. These co-morbidities in TSC have been linked to the structural brain abnormalities, such as cortical tubers, and recurrent epileptic seizures (in 70–80% cases). Previous transcriptomic analysis of cortical tubers revealed dysregulation of genes involved in cell adhesion in the brain, which may be associated with the neurodevelopmental deficits in TSC. In this study we aimed to investigate the expression of one of these genes – cell-adhesion molecule contactin-3. Methods Reverse transcription quantitative polymerase chain reaction for the contactin-3 gene (CNTN3) was performed in resected cortical tubers from TSC patients with drug-resistant epilepsy (n = 35, age range: 1–48 years) and compared to autopsy-derived cortical control tissue (n = 27, age range: 0–44 years), as well as by western blot analysis of contactin-3 (n = 7 vs n = 7, age range: 0–3 years for both TSC and controls) and immunohistochemistry (n = 5 TSC vs n = 4 controls). The expression of contactin-3 was further analyzed in fetal and postnatal control tissue by western blotting and in-situ hybridization, as well as in the SH-SY5Y neuroblastoma cell line differentiation model in vitro. Results CNTN3 gene expression was lower in cortical tubers from patients across a wide range of ages (fold change = − 0.5, p < 0.001) as compared to controls. Contactin-3 protein expression was lower in the age range of 0–3 years old (fold change = − 3.8, p < 0.001) as compared to the age-matched controls. In control brain tissue, contactin-3 gene and protein expression could be detected during fetal development, peaked around birth and during infancy and declined in the adult brain. CNTN3 expression was induced in the differentiated SH-SY5Y neuroblastoma cells in vitro (fold change = 6.2, p < 0.01). Conclusions Our data show a lower expression of contactin-3 in cortical tubers of TSC patients during early postnatal period as compared to controls, which may affect normal brain development and might contribute to neuropsychiatric co-morbidities observed in patients with TSC.

Analysis of the Increase of Vascular Cell Adhesion Molecule-1 (VCAM-1) Expression and the Effect of Exposure in a Hyperbaric Chamber on VCAM-1 in Human Blood Serum: A Cross-Sectional Study

Background and Objectives: Vascular cell adhesion molecule-1 (VCAM-1) was identified as a cell adhesion molecule that helps to regulate inflammation-associated vascular adhesion and the transendothelial migration of leukocytes, such as macrophages and T cells. VCAM-1 is expressed by the vascular system and can be induced by reactive oxygen species, interleukin 1 beta (IL-1β) or tumor necrosis factor alpha (TNFα), which are produced by many cell types. The newest data suggest that VCAM-1 is associated with the progression of numerous immunological disorders, such as rheumatoid arthritis, asthma, transplant rejection and cancer. The aim of this study was to analyze the increase in VCAM-1 expression and the impact of exposure in a hyperbaric chamber to VCAM-1 levels in human blood serum. Materials and Methods: The study included 92 volunteers. Blood for the tests was taken in the morning, from the basilic vein of fasting individuals, in accordance with the applicable procedure for blood collection for morphological tests. In both groups of volunteers, blood was collected before and after exposure, in heparinized tubes to obtain plasma and hemolysate, and in clot tubes to obtain serum. The level of VCAM-1 was determined using the immunoenzymatic ELISA method. Results: The study showed that the difference between the distribution of VCAM-1 before and after exposure corresponding to diving at a depth of 30 m was at the limit of statistical significance in the divers group and that, in most people, VCAM-1 was higher after exposure. Diving to a greater depth had a much more pronounced impact on changes in VCAM-1 values, as the changes observed in the VCAM-1 level as a result of diving to a depth of 60 m were statistically highly significant (p = 0.0002). The study showed an increase in VCAM-1 in relation to the baseline value, which reached as much as 80%, i.e., VCAM-1 after diving was almost twice as high in some people. There were statistically significant differences between the results obtained after exposure to diving conditions at a depth of 60 m and the values measured for the non-divers group. The leukocyte level increased statistically after exposure to 60 m. In contrast, hemoglobin levels decreased in most divers after exposure to diving at a depth of 30 m (p = 0.0098). Conclusions: Exposure in the hyperbaric chamber had an effect on serum VCAM-1 in the divers group and non-divers group. There is a correlation between the tested morphological parameters and the VCAM-1 level before and after exposure in the divers group and the non-divers group. Exposure may result in activation of the endothelium.

Pathologic Proteolytic Processing of N-Cadherin as a Marker of Human Fibrotic Disease

Prior research has implicated the involvement of cell adhesion molecule N-cadherin in tissue fibrosis and remodeling. We hypothesize that anomalies in N-cadherin protein processing are involved in pathological fibrosis. Diseased tissues associated with fibrosis of the heart, lung, and liver were probed for the precursor form of N-cadherin, pro-N-cadherin (PNC), by immunohistochemistry and compared to healthy tissues. Myofibroblast cell lines were analyzed for cell surface pro-N-cadherin by flow cytometry and immunofluorescent microscopy. Soluble PNC products were immunoprecipitated from patient plasmas and an enzyme-linked immunoassay was developed for quantification. All fibrotic tissues examined show aberrant PNC localization. Cell surface PNC is expressed in myofibroblast cell lines isolated from cardiomyopathy and idiopathic pulmonary fibrosis but not on myofibroblasts isolated from healthy tissues. PNC is elevated in the plasma of patients with cardiomyopathy (p ≤ 0.0001), idiopathic pulmonary fibrosis (p ≤ 0.05), and nonalcoholic fatty liver disease with cirrhosis (p ≤ 0.05). Finally, we have humanized a murine antibody and demonstrate that it significantly inhibits migration of PNC expressing myofibroblasts. Collectively, the aberrant localization of PNC is observed in all fibrotic tissues examined in our study and our data suggest a role for cell surface PNC in the pathogenesis of fibrosis.

A fragment of cell adhesion molecule L1 reduces amyloid-β plaques in a mouse model of Alzheimer’s disease

Deposition of amyloid-β (Aβ) in the brain is one of the important histopathological features of Alzheimer’s disease (AD). Previously, we reported a correlation between cell adhesion molecule L1 (L1) expression and the occurrence of AD, but its relationship was unclear. Here, we report that the expression of L1 and a 70 kDa cleavage product of L1 (L1-70) was reduced in the hippocampus of AD (APPswe) mice. Interestingly, upregulation of L1-70 expression in the hippocampus of 18-month-old APPswe mice, by parabiosis involving the joining of the circulatory system of an 18-month-old APPswe mouse with a 2-month-old wild-type C57BL/6 mouse, reduced amyloid plaque deposition. Furthermore, the reduction was accompanied by the appearance of a high number of activated microglia. Mechanistically, we observed that L1-70 could combine with topoisomerase 1 (Top1) to form a complex, L1-70/Top1, that was able to regulate expression of macrophage migration inhibitory factor (MIF), resulting in the activation of microglia and reduction of Aβ plaques. Also, transforming growth factor β1 (TGFβ-1) transferred from the blood of young wild-type C57BL/6 mice to the aged AD mice, was identified as a circulating factor that induces full-length L1 and L1-70 expression. All together, these findings suggest that L1-70 contributes to the clearance of Aβ in AD, thereby adding a novel perspective in understanding AD pathogenesis.

Integration of miRNA-lncRNA-mRNA profiles in liver tissue from EpCAM knockout mice

The epithelial cell adhesion molecule (EpCAM) is highly expressed in the liver during development and diseases. However, its role in the development and pathology of liver remains to be explored. The liver tissues of EpCAM-/- and wildtype (WT) mice at P0 stage were used for RNA sequencing. The differently expressed miRNAs, lncRNAs and mRNAs were selected and confirmed by qPCR. The expression of metabolism-related gene SET domain bifurcated 2 (Setdb2) was significantly increased in the liver of EpCAM-/- mice; the triglyceride (TG) and total cholesterol (TC) levels in the liver were also markedly decreased in EpCAM-/- mice. The microRNA (miRNA)-long noncoding RNA (lncRNA)-mRNA regulatory networks indicated that EpCAM may play important roles in glucose and lipid metabolism of the liver during development and in disease. The comprehensive miRNA, lncRNA and mRNA expression profiles in the developing liver of EpCAM-/- mice established here might help to elucidate functions and mechanisms of EpCAM during development and in diseases of the liver.

The Role of Dielectrophoresis for Cancer Diagnosis and Prognosis

Liquid biopsy is emerging as a potential diagnostic tool for prostate cancer (PC) prognosis and diagnosis. Unfortunately, most circulating tumor cells (CTC) technologies, such as AdnaTest or Cellsearch®, critically rely on the epithelial cell adhesion molecule (EpCAM) marker, limiting the possibility of detecting cancer stem-like cells (CSCs) and mesenchymal-like cells (EMT-CTCs) that are present during PC progression. In this context, dielectrophoresis (DEP) is an epCAM independent, label-free enrichment system that separates rare cells simply on the basis of their specific electrical properties. As compared to other technologies, DEP may represent a superior technique in terms of running costs, cell yield and specificity. However, because of its higher complexity, it still requires further technical as well as clinical development. DEP can be improved by the use of microfluid, nanostructured materials and fluoro-imaging to increase its potential applications. In the context of cancer, the usefulness of DEP lies in its capacity to detect CTCs in the bloodstream in their epithelial, mesenchymal, or epithelial–mesenchymal phenotype forms, which should be taken into account when choosing CTC enrichment and analysis methods for PC prognosis and diagnosis.

Atomic Level Dissection of the Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) Homophilic Binding Interface: Implications for Endothelial Cell Barrier Function

Objective: PECAM-1 (platelet endothelial cell adhesion molecule 1) is a 130 kDa member of the immunoglobulin (Ig) gene superfamily that is expressed on the surfaces of platelets and leukocytes and concentrated at the intercellular junctions of confluent endothelial cell monolayers. PECAM-1 Ig domains 1 and 2 (IgD1 and IgD2) engage in homophilic interactions that support a host of vascular functions, including support of leukocyte transendothelial migration and the maintenance of endothelial junctional integrity. The recently solved crystal structure of PECAM-1 IgD1 and IgD2 revealed a number of intermolecular interfaces predicted to play important roles in stabilizing PECAM-1/PECAM-1 homophilic interactions and in formation and maintenance of endothelial cell-cell contacts. We sought to determine whether the protein interfaces implicated in the crystal structure reflect physiologically important interactions. Approach and Results: We assessed the impact of single amino acid substitutions at the interfaces between opposing PECAM-1 molecules on homophilic binding and endothelial cell function. Substitution of key residues within the IgD1-IgD1 and IgD1-IgD2 interfaces but not those within the smaller IgD2-IgD2 interface, markedly disrupted PECAM-1 homophilic binding and its downstream effector functions, including the ability of PECAM-1 to localize at endothelial cell-cell borders, mediate the formation of endothelial tubes, and restore endothelial barrier integrity. Conclusions: Taken together, these results validate the recently described PECAM-1 IgD1/IgD2 crystal structure by demonstrating that specific residues visualized within the IgD1-IgD1 and IgD1-IgD2 interfaces of opposing molecules in the crystal are required for functionally important homophilic interactions. This information can now be exploited to modulate functions of PECAM-1 in vivo.

The neural cell adhesion molecule NrCAM regulates development of hypothalamic tanycytes

Hypothalamic tanycytes are neural stem and progenitor cells, but little is known of how they are regulated. Here we provide evidence that the cell adhesion molecule, NrCAM, regulates tanycytes in the adult niche. NrCAM is strongly expressed in adult mouse tanycytes. Immunohistochemical and in situ hybridization analysis revealed that NrCAM loss of function leads to both a reduced number of tanycytes and reduced expression of tanycyte-specific cell markers, along with a small reduction in tyrosine hydroxylase-positive arcuate neurons. Similar analyses of NrCAM mutants at E16 identify few changes in gene expression or cell composition, indicating that NrCAM regulates tanycytes, rather than early embryonic hypothalamic development. Neurosphere and organotypic assays support the idea that NrCAM governs cellular homeostasis. Single-cell RNA sequencing (scRNA-Seq) shows that tanycyte-specific genes, including a number that are implicated in thyroid hormone metabolism, show reduced expression in the mutant mouse. However, the mild tanycyte depletion and loss of markers observed in NrCAM-deficient mice were associated with only a subtle metabolic phenotype.

Challenging the Current Paradigm of Liquid Biopsy Through Dielectrophoresis (DEP) In Prostate Cancer

Liquid biopsy via isolation of circulating tumour cells (CTCs) represents a promising diagnostic tool capable of supplementing state-of-the-art for prostate cancer (PC) prognosis. Unfortunately, most of CTC technologies, such as AdnaTest or Cellsearch, critically rely on the Epithelial-Cell-Adhesion-Molecule (EpCAM) marker, limiting the possibility of detecting stem-like cells (CSCs) and mesenchymal-like cells (EMT-CTCs) that are present during PC progression. In this tontext, dielectrophoresis (DEP) is an epCAM independent, label-free, enrichment system, separating rare cells simply on the basis of their specific electrical properties. As compared to other technollgies, DEP represents a superior technique in terms of running costs, cells yield and specificity, but due to its higher complexity, requires still further technical as well as clinical development. Interestingly, DEP can be improved by the use of microfluid, nanostructured materials and fluoroimaging in order to increase its potential applications. In the context of PC, the utility of DEP can be translated in its capacity to detect CTC in the bloodstream in their epithelial, mesenchymal, or epithelial-mesenchymal phenotypes, which should be taken into account when choosing CTC enrichment and analysis methods for PC prognosis and early diagnosis.