Integral Membrane Protein 2B Research Articles

Exosomal miR-196a-5p contributes to esophageal squamous cell carcinoma malignant progression by inhibiting ITM2B.

Exosomes from cancer cells function as carriers to spread or transport specific microRNAs (miRNAs) to distant sites to exert their effects, but the mechanism of exosomal miRNA action in esophageal squamous cell carcinoma (ESCC) has not been fully explained. Therefore, in this study, we were interested in the impact of exosomal miR-196a-5p in ESCC progression. We found that miR-196a-5p was expressed enriched in clinical tissues, ESCC cells, and exosomes. Functionally, depletion of miR-196a-5p impeded ESCC cell growth, migration, and invasion, whereas overexpression of miR-196a-5p produced the opposite results. Moreover, enhancement of exosomal miR-196a-5p in recipient ESCC cells triggered more intense proliferation and migration. Mechanistically, we identified integral membrane protein 2B (ITM2B) as a direct target of miR-196a-5p. Silencing of ITM2B partially counteracted the inhibitory effect of miR-196a-5p inhibitors on the malignant phenotype of ESCC. Furthermore, in vivo, lower miR-196a-5p levels triggered by the introduction of antagomiR-196a-5p resulted in the generation of smaller volume and weight xenograft tumors. Thus, our results demonstrated novel mechanisms of exosomal and intracellular miR-196a-5p-mediated ESCC growth and migration and identify the interaction of miR-196a-5p with ITM2B. These works might provide new targets and basis for the development of clinical treatment options for ESCC.

Small peptide CSF fingerprint of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by abnormal protein aggregation in the motor neurons. Present and earlier proteomic studies to characterize peptides in cerebrospinal fluid (CSF) associated with motoneuron pathology did not target low molecular weight proteins and peptides. We hypothesized that specific changes in CSF peptides or low molecular weight proteins are significantly altered in ALS, and that these changes may support deciphering molecular pathophysiology and even guide approaches towards therapeutic interventions. Cerebrospinal fluid (CSF) from 50 ALS patients and 50 non-ALS controls was collected, centrifuged immediately after collection, aliquoted into polypropylene test tubes, frozen within 30-40 min after the puncture, and stored at -80°C until use. Peptides were sequenced using capillary electrophoresis or liquid chromatography/mass spectrometry (CE-MS/MS or LC-MS/MS). In the CSF of 50 patients and 50 non-ALS controls 33 peptides were found, of which 14 could be sequenced using a non-lytic single-pot proteomic detection method, CE/MS. ALS deregulated peptides vs. controls included Integral membrane protein 2B, Neurosecretory protein VGF, Osteopontin, Neuroendocrine protein 7B2 (Secretogranin-V), EGF-containing fibulin-like extracellular matrix protein 1, Xylosyltransferase 1 XT-1, Chromogranin-A, Superoxide dismutase SOD-1, Secretogranin-1 (Chromogranin B), NR2F2 Nuclear Receptor Subfamily 2 Group F Member 2 and Collagen alpha-1(VII) chain. Most striking deregulations in CSF from ALS patients were found in VGF, Osteopontin, SOD-1 and EFEMP1 peptides. No associations of disease severity, duration and region of onset with sequenced peptides were found.

Myelin Basic Protein Attenuates Furin-Mediated Bri2 Cleavage and Postpones Its Membrane Trafficking.

Myelin basic protein (MBP) is the second most abundant protein in the central nervous system and is responsible for structural maintenance of the myelin sheath covering axons. Previously, we showed that MBP has a more proactive role in the oligodendrocyte homeostasis, interacting with membrane-associated proteins, including integral membrane protein 2B (ITM2B or Bri2) that is associated with familial dementias. Here, we report that the molecular dynamics of the in silico-generated MBP-Bri2 complex revealed that MBP covers a significant portion of the Bri2 ectodomain, assumingly trapping the furin cleavage site, while the surface of the BRICHOS domain, which is responsible for the multimerization and activation of the Bri2 high-molecular-weight oligomer chaperone function, remains unmasked. These observations were supported by the co-expression of MBP with Bri2, its mature form, and disease-associated mutants, which showed that in mammalian cells, MBP indeed modulates the post-translational processing of Bri2 by restriction of the furin-catalyzed release of its C-terminal peptide. Moreover, we showed that the co-expression of MBP and Bri2 also leads to an altered cellular localization of Bri2, restricting its membrane trafficking independently of the MBP-mediated suppression of the Bri2 C-terminal peptide release. Further investigations should elucidate if these observations have physiological meaning in terms of Bri2 as a MBP chaperone activated by the MBP-dependent postponement of Bri2 membrane trafficking.

Exosomal miRNA-166-5p derived from G-MDSCs promotes proliferation by targeting ITM3E in colorectal cancer.

The immune milieu of colorectal cancer is a complex phenomenon. It is imperative to investigate the crucial immune factors that promote the progression of colorectal cancer. Immune suppressor cells are granulocytic myeloid-derived suppressor cells (G-MDSCs). However, they also increased cancer growth in other ways that need to be investigated further. Using flow cytometry, we isolated G-MDSCs from colorectal cancer tissues. Ultracentrifugation was used to separate exosomes from the supernatant of G-MDSCs, and western blotting, transmission electron microscopy (TEM), and flow cytometry were used to confirm their presence. RNA sequencing was used to identify unique miRNAs and transcripts, which were subsequently confirmed by RT-qPCR (real-time quantitative real-time PCR). The CCK-8 test was used to determine the rate of proliferation. Lentiviral vectors were employed to manipulate the expression of miRNAs and genes in order to investigate their role in the development of colorectal cancer. Colorectal cancer tissues have been found to contain granulocyte-myeloid-derived suppressor cells (G-MDSCs) that secrete exosomes. These exosomes have been shown to accelerate cancer progression by promoting cell proliferation. Further research has identified microRNA-166-5p as a target from G-MDSC-derived exosomes. This downregulation leads to the inhibition of integral membrane protein 2B (ITM3E) transcription, which in turn activates the PI3K/Akt signaling pathway. This pathway promotes cell proliferation and can be inhibited using deguelin. The accelerated development of colorectal cancer has been further confirmed in mice models. The primary results of this work show that exosomes produced from G-MDSCs and the miR-166-5p/ITM3E axis have therapeutic and diagnostic promise in colorectal cancer.

Identification of glycolytic proteins as binding partners of Bri2 BRICHOS domain

Human integral membrane protein 2B (ITM2B or Bri2) is a member of the BRICHOS family, that can attenuate Aβ pathology in the brain. As a result, the identification of novel Bri2 BRICHOS client proteins has been sought to help elucidate signaling pathways and the potential identification of novel therapeutic targets. To identify Bri2 BRICHOS interacting partners, we carried out a 'protein fishing' experiment using recombinant human (rh) Bri2 BRICHOS-coated magnetic particles, in combination with proteomic analysis on cytosolic and membrane fractions of cortical homogenates from C57BL/6 J WT mouse. We identified 4 proteins from the cytosolic fractions and 44 proteins from the membrane fractions that had significant interactions (p < 0.05) with Bri2 BRICHOS domain, of which 11 proteins were previously identified as proteins that interacted with Bri2 BRICHOS domain. Enrichment analysis of the retained proteins identified glycolysis/gluconeogenesis as the most enriched pathway, with several proteins identified playing roles in carbon metabolism, amino acid synthesis. The data suggested that Bri2 BRICHOS may have a role in cellular energy demands in the brain via glycolysis and mitochondrial oxidative phosphorylation and may play a role in mitochondrial homeostasis.

A slit-diaphragm-associated protein network for dynamic control of renal filtration

The filtration of blood in the kidney which is crucial for mammalian life is determined by the slit-diaphragm, a cell-cell junction between the foot processes of renal podocytes. The slit-diaphragm is thought to operate as final barrier or as molecular sensor of renal filtration. Using high-resolution proteomic analysis of slit-diaphragms affinity-isolated from rodent kidney, we show that the native slit-diaphragm is built from the junction-forming components Nephrin, Neph1 and Podocin and a co-assembled high-molecular weight network of proteins. The network constituents cover distinct classes of proteins including signaling-receptors, kinases/phosphatases, transporters and scaffolds. Knockout or knock-down of either the core components or the selected network constituents tyrosine kinase MER (MERTK), atrial natriuretic peptide-receptor C (ANPRC), integral membrane protein 2B (ITM2B), membrane-associated guanylate-kinase, WW and PDZ-domain-containing protein1 (MAGI1) and amyloid protein A4 resulted in target-specific impairment or disruption of the filtration process. Our results identify the slit-diaphragm as a multi-component system that is endowed with context-dependent dynamics via a co-assembled protein network.

Chemical traits of cerebral amyloid angiopathy in familial British-, Danish-, and non-Alzheimer's dementias.

Familial British dementia (FBD) and familial Danish dementia (FDD) are autosomal dominant forms of dementia caused by mutations in the integral membrane protein 2B (ITM2B, also known as BRI2) gene. Secretase processing of mutant BRI2 leads to secretion and deposition of BRI2-derived amyloidogenic peptides, ABri and ADan that resemble APP/β-amyloid (Aβ) pathology, which is characteristic of Alzheimer's disease (AD). Amyloid pathology in FBD/FDD manifests itself predominantly in the microvasculature by ABri/ADan containing cerebral amyloid angiopathy (CAA). While ABri and ADan peptide sequences differ only in a few C-terminal amino acids, CAA in FDD is characterized by co-aggregation of ADan with Aβ, while in contrast no Aβ deposition is observed in FBD. The fact that FDD patients display an earlier and more severe disease onset than FBD suggests a potential role of ADan and Aβ co-aggregation that promotes a more rapid disease progression in FDD compared to FBD. It is therefore critical to delineate the chemical signatures of amyloid aggregation in these two vascular dementias. This in turn will increase the knowledge on the pathophysiology of these diseases and the pathogenic role of heterogenous amyloid peptide interactions and deposition, respectively. Herein, we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in combination with hyperspectral, confocal microscopy based on luminescent conjugated oligothiophene probes (LCO) to delineate the structural traits and associated amyloid peptide patterns of single CAA in postmortem brain tissue of patients with FBD, FDD as well as sporadic CAA without AD (CAA+) that show pronounced CAA without parenchymal plaques. The results show that CAA in both FBD and FDD consist of N-terminally truncated- and pyroglutamate-modified amyloid peptide species (ADan and ABri), but that ADan peptides in FDD are also extensively C-terminally truncated as compared to ABri in FBD, which contributes to hydrophobicity of ADan species. Further, CAA in FDD showed co-deposition with Aβ x-42 and Aβ x-40 species. CAA+ vessels were structurally more mature than FDD/FBD CAA and contained significant amounts of pyroglutamated Aβ. When compared with FDD, Aβ in CAA+ showed more C-terminal and less N-terminally truncations. In FDD, ADan showed spatial co-localization with Aβ3pE-40 and Aβ3-40 but not with Aβx-42 species. This suggests an increased aggregation propensity of Aβ in FDD that promotes co-aggregation of both Aβ and ADan. Further, CAA maturity appears to be mainly governed by Aβ content based on the significantly higher 500/580 patterns observed in CAA+ than in FDD and FBD, respectively. Together this is the first study of its kind on comprehensive delineation of Bri2 and APP-derived amyloid peptides in single vascular plaques in both FDD/FBD and sporadic CAA that provides new insight in non-AD-related vascular amyloid pathology. Cover Image for this issue: https://doi.org/10.1111/jnc.15424.

Serum peptidome profiles immune response of COVID-19 Vaccine administration.

BackgroundCoronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused significant loss of life and property. In response to the serious pandemic, recently developed vaccines against SARS-CoV-2 have been administrated to the public. Nevertheless, the research on human immunization response against COVID-19 vaccines is insufficient. Although much information associated with vaccine efficacy, safety and immunogenicity has been reported by pharmaceutical companies based on laboratory studies and clinical trials, vaccine evaluation needs to be extended further to better understand the effect of COVID-19 vaccines on human beings.MethodsWe performed a comparative peptidome analysis on serum samples from 95 participants collected at four time points before and after receiving CoronaVac. The collected serum samples were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to profile the serum peptides, and also subjected to humoral and cellular immune response analyses to obtain typical immunogenicity information.ResultsSignificant difference in serum peptidome profiles by MALDI-TOF MS was observed after vaccination. By supervised statistical analysis, a total of 13 serum MALDI-TOF MS feature peaks were obtained on day 28 and day 42 of vaccination. The feature peaks were identified as component C1q receptor, CD59 glycoprotein, mannose-binding protein C, platelet basic protein, CD99 antigen, Leucine-rich alpha-2-glycoprotein, integral membrane protein 2B, platelet factor 4 and hemoglobin subunits. Combining with immunogenicity analysis, the study provided evidence for the humoral and cellular immune responses activated by CoronaVac. Furthermore, we found that it is possible to distinguish neutralizing antibody (NAbs)-positive from NAbs-negative individuals after complete vaccination using the serum peptidome profiles by MALDI-TOF MS together with machine learning methods, including random forest (RF), partial least squares-discriminant analysis (PLS-DA), linear support vector machine (SVM) and logistic regression (LR).ConclusionsThe study shows the promise of MALDI-TOF MS-based serum peptidome analysis for the assessment of immune responses activated by COVID-19 vaccination, and discovered a panel of serum peptides biomarkers for COVID-19 vaccination and for NAbs generation. The method developed in this study can help not only in the development of new vaccines, but also in the post-marketing evaluation of developed vaccines.

First identification of ITM2B interactome in the human retina

Integral Membrane Protein 2 B (ITM2B) is a type II ubiquitous transmembrane protein which role remains unclear. ITM2B mutations have been associated with different disorders: mutations leading to longer mutant proteins have been reported in two distinct Alzheimer-like autosomal dominant disorders with early-onset progressive dementia and cerebellar ataxia. Both disorders share neurological features including severe cerebral amyloid angiopathy, non-neuritic plaques, and fibrillary tangles as in Alzheimer disease. Our group reported a missense mutation in ITM2B, in an unusual retinal dystrophy with no dementia. This finding suggests a specific role of ITM2B in the retina. As the identification of retinal-specific ITM2B partners could bring new insights into the cellular functions of ITM2B, we performed quantitative proteomics of ITM2B interactome of the human retina. Overall, 457 ITM2B partners were identified with 8 of them involved in visual transduction. In addition, bulk Gene Ontology analyses showed that many ITM2B partners are involved in several other biological functions, such as microtubule organization, protein translation and interestingly, mitochondrial homeostasis. These data represent the first report of the ITM2B interactome in the human retina and may serve as a valuable inventory of new potential ITM2B partners for future investigations of ITM2B physiological functions and dysfunctions.

A familial Danish dementia rat shows impaired presynaptic and postsynaptic glutamatergic transmission

Familial British dementia and familial Danish dementia are neurodegenerative disorders caused by mutations in the gene integral membrane protein 2B (ITM2b) encoding BRI2, which tunes excitatory synaptic transmission at both presynaptic and postsynaptic termini. In addition, BRI2 interacts with and modulates proteolytic processing of amyloid-β precursor protein (APP), whose mutations cause familial forms of Alzheimer's disease (AD) (familial AD). To study the pathogenic mechanisms triggered by the Danish mutation, we generated rats carrying the Danish mutation in the rat Itm2b gene (Itm2bD rats). Given the BRI2/APP interaction and the widely accepted relevance of human amyloid β (Aβ), a proteolytic product of APP, to AD, Itm2bD rats were engineered to express two humanized App alleles and produce human Aβ. Here, we studied young Itm2bD rats to investigate early pathogenic changes in these diseases. We found that periadolescent Itm2bD rats not only present subtle changes in human Aβ levels along with decreased spontaneous glutamate release and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor–mediated responses but also had increased short-term synaptic facilitation in the hippocampal Schaeffer-collateral pathway. These alterations in excitatory interneuronal communication can impair learning and memory processes and were akin to those observed in adult mice producing rodent Aβ and carrying either the Danish or British mutations in the mouse Itm2b gene. Collectively, the data show that the pathogenic Danish mutation alters the physiological function of BRI2 at glutamatergic synapses across species and early in life. Future studies will determine whether this phenomenon represents an early pathogenic event in human dementia.

The synthesis and characterization of Bri2 BRICHOS coated magnetic particles and their application to protein fishing: Identification of novel binding proteins

Human integral membrane protein 2B (ITM2B or Bri2) is a member of the BRICHOS family, proteins that efficiently prevent Aβ42 aggregation via a unique mechanism. The identification of novel Bri2 BRICHOS client proteins could help elucidate signaling pathways and determine novel targets to prevent or cure amyloid diseases. To identify Bri2 BRICHOS interacting partners, we carried out a ‘protein fishing’ experiment using recombinant human (rh) Bri2 BRICHOS-coated magnetic particles, which exhibit essentially identical ability to inhibit Aβ42 fibril formation as free rh Bri2 BRICHOS, in combination with proteomic analysis on homogenates of SH-SY5Y cells. We identified 70 proteins that had more significant interactions with rh Bri2 BRICHOS relative to the corresponding control particles. Three previously identified Bri2 BRICHOS interacting proteins were also identified in our ‘fishing’ experiments. The binding affinity of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the top ‘hit’, was calculated and was identified as a strong interacting partner. Enrichment analysis of the retained proteins identified three biological pathways: Rho GTPase, heat stress response and pyruvate, cysteine and methionine metabolism.

Danish and British dementia ITM2b/BRI2 mutations reduce BRI2 protein stability and impair glutamatergic synaptic transmission

Mutations in integral membrane protein 2B (ITM2b/BRI2) gene cause familial British and Danish dementia (FBD and FDD), autosomal dominant disorders characterized by progressive cognitive deterioration. Two pathogenic mechanisms, which may not be mutually exclusive, have been proposed for FDD and FBD: 1) loss of BRI2 function; 2) accumulation of amyloidogenic mutant BRI2-derived peptides, but the mechanistic details remain unclear. We have previously reported a physiological role of BRI2 in excitatory synaptic transmission at both presynaptic termini and postsynaptic termini. To test whether pathogenic ITM2b mutations affect these physiological BRI2 functions, we analyzed glutamatergic transmission in FDD and FBD knock-in mice, which carry pathogenic FDD and FBD mutations into the mouse endogenous Itm2b gene. We show that in both mutant lines, spontaneous glutamate release and AMPAR-mediated responses are decreased, while short-term synaptic facilitation is increased, effects similar to those observed in Itm2bKO mice. In vivo and in vitro studies show that both pathogenic mutations alter maturation of BRI2 resulting in reduced levels of functional mature BRI2 protein at synapses. Collectively, the data show that FDD and FBD mutations cause a reduction of BRI2 levels and function at synapses, which results in reduced glutamatergic transmission. Notably, other genes mutated in Familial dementia, such as APP, PSEN1/PSEN2, are implicated in glutamatergic synaptic transmission, a function that is altered by pathogenic mutations. Thus, defects in excitatory neurotransmitter release may represent a general and convergent mechanism leading to neurodegeneration. Targeting these dysfunction may offer a unique disease modifying method of therapeutic intervention in neurodegenerative disorders.

Analyses Mutations in GSN, CST3, TTR, and ITM2B Genes in Chinese Patients With Alzheimer's Disease.

Amyloid protein deposition is a common mechanism of hereditary amyloidosis (HA) and Alzheimer’s disease (AD). Mutations of gelsolin (GSN), cystatin C (CST3), transthyretin (TTR), and integral membrane protein 2B (ITM2B) genes can lead to HA. But the relationship is unclear between these genes and AD. Genes targeted sequencing (GTS), including GSN, CST3, TTR, and ITM2B, was performed in a total of 636 patients with clinical AD and 365 normal controls from China. As a result, according to American College of Medical Genetics and Genomics (ACMG) guidelines, two novel likely pathogenic frame-shift mutations (GSN:c.1036delA:p.K346fs and GSN:c.8_35del:p.P3fs) were detected in five patients with AD, whose initial symptom was memory decline, accompanied with psychological and behavioral abnormalities later. Interestingly, the patient with K346fs mutation, presented cerebral β-amyloid protein deposition, had an early onset (48 years) and experienced rapid progression, while the other four patients with P3fs mutation had a late onset [(Mean ± SD): 69.50 ± 5.20 years] and a long course of illness [(Mean ± SD): 9.24 ± 4.86 years]. Besides, we also discovered 17 variants of uncertain significance (VUS) in these four genes. To our knowledge, we are the first to report AD phenotype with GSN mutations in patients with AD in the Chinese cohort. Although mutations in the GSN gene are rare, it may explain a small portion of clinically diagnosed AD.

G-MDSCs-Derived Exosomal miRNA-143-3p Promotes Proliferation via Targeting of ITM2B in Lung Cancer.

BackgroundThe immune environment of lung cancer is complex, and the critical immune factors that promote lung cancer progression need to be explored. Granulocytic myeloid-derived suppressor cells (G-MDSCs) are regarded as immune suppressing cells. However, they also promote tumor progression through other ways, which needs to be explored further. Therefore, we sought to study the regulatory mechanisms underlying the cancer promoting function of G-MDSCs in lung cancer.MethodsG-MDSCs were isolated from lung cancer tissues using flow cytometry. Exosomes were separated from the G-MDSCs supernatant by ultracentrifugation and verified using flow cytometry, Western blot, and transmission electron microscopy (TEM). RNA sequencing was used to identify the differential miRNAs and genes. Real-time quantitative real-time PCR (RT-qPCR) confirmed these results. The proliferation rate was assessed using the CCK-8 assay. Lentiviral vectors were used to alter the expression of the miRNAs and genes to analyze their effects on lung cancer progression.ResultsG-MDSCs secreted more exosomes in the lung cancer tissues, which promoted cancer progression by accelerating proliferation. Micro RNA-143-3p (miR-143-3p) increased in G-MDSCs derived exosomes and downregulated integral membrane protein 2B (ITM2B) by targeting the 3ʹ-untranslated region (UTR) region. Overexpression of miR-143-3p enhanced proliferation by inhibiting transcription of ITM2B to activate the PI3K/Akt signaling pathway, which can be blocked by deguelin. This phenomenon was further confirmed by accelerated tumor growth and worse prognosis in mice.ConclusionThe key findings of this study highlight the potential of the G-MDSC-derived exosomes and the miR-143-3p/ITM2B axis as therapeutic targets and clinical indicators of lung cancer.

Interaction Between ITM2B and GLUT9 Links Urate Transport to Neurodegenerative Disorders.

Hyperuricemia plays a critical causative role in gout. In contrast, hyperuricemia has a protective effect in neurodegenerative disorders, including Alzheimer’s Disease. Genetic variation in the SLC2A9 gene, encoding the urate transporter GLUT9, exerts the largest single-gene effect on serum uric acid (SUA). We report here the identification of two GLUT9-interacting proteins, integral membrane protein 2B (ITM2B) and transmembrane protein 85 (TMEM85), isolated from a human kidney cDNA library using the dual-membrane yeast two-hybrid system. ITM2B is a ubiquitously expressed, N-glycosylated transmembrane regulatory protein, involved in familial dementias and retinal dystrophy; the function of TMEM85 is less defined. Using coimmunoprecipitation, we confirmed the physical interaction between ITM2B or TMEM85 and N-terminal GLUT9 isoforms (GLUT9a and GLUT9b) in transfected HEK 293T cells and Xenopus oocytes, wherein ITM2B but not TMEM85 inhibited GLUT9-mediated urate uptake. Additionally, co-expression of ITM2B with GLUT9 in oocytes inhibited N-glycosylation of GLUT9a more than GLUT9b and stimulated urate efflux by both isoforms. However, urate uptake by N-glycosylation and N-terminal deletion GLUT9 mutants was efficiently inhibited by ITM2B, indicating that neither N-glycosylation nor the N terminus is necessary for functional interaction of GLUT9 with ITM2B. Notably, ITM2B variants linked to familial Danish dementia and retinal dystrophy significantly attenuated the inhibition of GLUT9–mediated urate influx. We propose ITM2B as a potential regulatory link between urate homeostasis and neurodegenerative disorders.

The Familial dementia gene ITM2b/BRI2 facilitates glutamate transmission via both presynaptic and postsynaptic mechanisms

Mutations in the Integral membrane protein 2B (ITM2b/BRI2) gene, which codes for a protein called BRI2, cause familial British and Danish dementia (FBD and FDD). Loss of BRI2 function and/or accumulation of amyloidogenic mutant BRI2-derived peptides have been proposed to mediate FDD and FBD pathogenesis by impairing synaptic Long-term potentiation (LTP). However, the precise site and nature of the synaptic dysfunction remain unknown. Here we use a genetic approach to inactivate Itm2b in either presynaptic (CA3), postsynaptic (CA1) or both (CA3 + CA1) neurons of the hippocampal Schaeffer-collateral pathway in both female and male mice. We show that after CA3 + CA1 Itm2b inactivation, spontaneous glutamate release and AMPAR-mediated responses are decreased, while short-term synaptic facilitation is increased. Moreover, AMPAR-mediated responses are decreased after postsynaptic but not presynaptic deletion of Itm2b. In contrast, the probability of spontaneous glutamate release is decreased, while short-term synaptic facilitation is increased, primarily after presynaptic deletion of Itm2b. Collectively, these results indicate a dual physiological role of Itm2b in the regulation of excitatory synaptic transmission at both presynaptic termini and postsynaptic termini and suggest that presynaptic and postsynaptic dysfunctions may be a pathogenic event leading to dementia and neurodegeneration in FDD and FBD.

Blood–brain and blood–cerebrospinal fluid passage of BRICHOS domains from two molecular chaperones in mice

Targeting toxicity associated with β-amyloid (Aβ) misfolding and aggregation is a promising therapeutic strategy for preventing or managing Alzheimer's disease. The BRICHOS domains from human prosurfactant protein C (proSP-C) and integral membrane protein 2B (Bri2) efficiently reduce neurotoxicity associated with Aβ42 fibril formation both in vitro and in vivo In this study, we evaluated the serum half-lives and permeability into the brain and cerebrospinal fluid (CSF) of recombinant human (rh) proSP-C and Bri2 BRICHOS domains injected intravenously into WT mice. We found that rh proSP-C BRICHOS has a longer blood serum half-life compared with rh Bri2 BRICHOS and passed into the CSF but not into the brain parenchyma. As judged by Western blotting, immunohistochemistry, and ELISA, rh Bri2 BRICHOS passed into both the CSF and brain. Intracellular immunostaining for rh Bri2 BRICHOS was observed in the choroid plexus epithelium as well as in the cerebral cortex. Our results indicate that intravenously administered rh proSP-C and Bri2 BRICHOS domains have different pharmacokinetic properties and blood-brain/blood-CSF permeability in mice. The finding that rh Bri2 BRICHOS can reach the brain parenchyma after peripheral administration may be harnessed in the search for new therapeutic strategies for managing Alzheimer's disease.

Identification of plasma RGS18 and PPBP mRNAs as potential biomarkers for gastric cancer using transcriptome arrays.

Coding and noncoding RNAs serve a crucial role in tumorigenesis. Circulating RNAs have been recognized as a novel category of biomarkers for a variety of physiological and pathological conditions. To identify plasma RNA biomarkers for gastric cancer (GC), a genome-wide transcriptome analysis using GeneChip® Human Transcriptome Array, which contains probe sets covering exons of ~67500 coding and noncoding transcripts of annotated genes, was performed to screen for the RNAs that exhibited differential expression in the plasma samples of patients with GC and controls. The expression levels of 6 candidate RNAs, including regulator of G-protein signaling 18 (RGS18), integral membrane protein 2B, pro-platelet basic protein (PPBP), nucleosome assembly protein1-like 1, n324674 and ENST00000442382 were assessed in the plasma samples of 81 patients with GC and 77 healthy participants using reverse transcription-quantitative polymerase chain reaction. Furthermore, the expression levels of RGS18 and PPBP mRNAs were indicated to be significantly differentially expressed (P<0.0001) in an independent panel of plasma samples of 36 patients with GC compared with 34 healthy participants. The potential association of RGS18 and PPBP mRNA expression levels with clinicopathological features was subsequently analyzed. Receiver operating characteristic analysis indicated that the combination of these 2 mRNAs with an area under curve <0.812 was an improved indicator for gastric cancer compared with respective individual levels. The results of the present study indicate that RGS18 and PPBP mRNA expression was significantly downregulated in the plasma of patients with GC, and the combination of these 2 mRNAs may be a useful diagnostic or prognostic marker for GC.

Abstract 527: Identification of novel target mRNAs of microRNA-143 in glioblastoma cells

Abstract The purpose of this study is to assess the biological role of microRNA-143 (miR-143) in Glioblastoma multiforme (GBM). In the United States, the incidence of GBM is about 17% of all primary brain tumors and about 60-75% of all Astrocytomas. Many patients recur even after therapy with a median survival rate of 15 months. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally. Deregulated miRNAs have been identified in cancer including GBM. In this study, we aim to thoroughly uncover the role of miR-143 by using GBM cell lines, mouse models, and patient samples. Previous qPCR data from FFPE samples of Puerto Rican GBM patients showed a higher relative expression of miR-143 in GBM patients compared to control individuals and paired surrounding non-cancerous tissue suggesting an oncogenic role. Moreover, in vitro data of transient transfections of a miR-143 oligonucleotide inhibitor in GBM cells showed reduced cell proliferation (clonogenic assay), increased apoptosis and cell cycle arrest in the G0/G1-S phase (flow cytometry and western blot analysis). Furthermore, we want to identify novel target mRNAs of miR-143 in GBM cells that may explain these results. Using in silico analysis tools and a qPCR analysis we identified 5 candidate genes as potential miR-143 targets. Western blot analysis of the proteins encoded by these genes using miR-143 overexpressing cells showed that the Integral membrane protein 2B (ITM2B) and Zinc transporter 8 (SLC30A8) protein levels were reduced and when miR-143 was transiently inhibited their protein levels where increased. These results suggest that miR-143 targets these mRNAs. Ongoing luciferase reporter assays will determine direct binding of miR-143 to the 3'UTR of these mRNAs in GBM cells. Citation Format: Eunice Lozada-Delgado, Fatma Valiyeva, Maria Marcos, Pablo Vivas. Identification of novel target mRNAs of microRNA-143 in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 527.

BRICHOS domain of Bri2 inhibits islet amyloid polypeptide (IAPP) fibril formation and toxicity in human beta cells

Aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils in islets of Langerhans is associated with type 2 diabetes, and formation of toxic IAPP species is believed to contribute to the loss of insulin-producing beta cells. The BRICHOS domain of integral membrane protein 2B (Bri2), a transmembrane protein expressed in several peripheral tissues and in the brain, has recently been shown to prevent fibril formation and toxicity of Aβ42, an amyloid-forming peptide in Alzheimer disease. In this study, we demonstrate expression of Bri2 in human islets and in the human beta-cell line EndoC-βH1. Bri2 colocalizes with IAPP intracellularly and is present in amyloid deposits in patients with type 2 diabetes. The BRICHOS domain of Bri2 effectively inhibits fibril formation in vitro and instead redirects IAPP into formation of amorphous aggregates. Reduction of endogenous Bri2 in EndoC-βH1 cells with siRNA increases sensitivity to metabolic stress leading to cell death while a concomitant overexpression of Bri2 BRICHOS is protective. Also, coexpression of IAPP and Bri2 BRICHOS in lateral ventral neurons of Drosophila melanogaster results in an increased cell survival. IAPP is considered to be the most amyloidogenic peptide known, and described findings identify Bri2, or in particular its BRICHOS domain, as an important potential endogenous inhibitor of IAPP aggregation and toxicity, with the potential to be a possible target for the treatment of type 2 diabetes.