Secretion-Positive LGI1 Mutations Linked to Lateral Temporal Epilepsy Impair Binding to ADAM22 and ADAM23 Receptors

Autosomal dominant lateral temporal epilepsy (ADTLE) is a focal epilepsy syndrome caused by mutations in the LGI1 gene, which encodes a secreted protein. Most ADLTE-causing mutations inhibit LGI1 protein secretion, and only a few secretion-positive missense mutations have been reported. Here we describe the effects of four disease-causing nonsynonymous LGI1 mutations, T380A, R407C, S473L, and R474Q, on protein secretion and extracellular interactions. Expression of LGI1 mutant proteins in cultured cells shows that these mutations do not inhibit protein secretion. This finding likely results from the lack of effects of these mutations on LGI1 protein folding, as suggested by 3D protein modelling. In addition, immunofluorescence and co-immunoprecipitation experiments reveal that all four mutations significantly impair interaction of LGI1 with the ADAM22 and ADAM23 receptors on the cell surface. These results support the existence of a second mechanism, alternative to inhibition of protein secretion, by which ADLTE-causing LGI1 mutations exert their loss-of-function effect extracellularly, and suggest that interactions of LGI1 with both ADAM22 and ADAM23 play an important role in the molecular mechanisms leading to ADLTE

CDX2 hox gene product in a rat model of esophageal cancer

<p>Abstract</p> <p>Background</p> <p>Barrett's mucosa is the precursor of esophageal adenocarcinoma. The molecular mechanisms behind Barrett's carcinogenesis are largely unknown. Experimental models of longstanding esophageal reflux of duodenal-gastric contents may provide important information on the biological sequence of the Barrett's oncogenesis.</p> <p>Methods</p> <p>The expression of <it>CDX2 </it>hox-gene product was assessed in a rat model of Barrett's carcinogenesis. Seventy-four rats underwent esophago-jejunostomy with gastric preservation. Excluding perisurgical deaths, the animals were sacrificed at various times after the surgical treatment (Group A: <10 weeks; Group B: 10–30 weeks; Group C: >30 weeks).</p> <p>Results</p> <p>No Cdx2 expression was detected in either squamous epithelia of the proximal esophagus or squamous cell carcinomas. <it>De novo </it>Cdx2 expression was consistently documented in the proliferative zone of the squamous epithelium close to reflux ulcers (Group A: 68%; Group B: 64%; Group C: 80%), multilayered epithelium and intestinal metaplasia (Group A: 9%; Group B: 41%; Group C: 60%), and esophageal adenocarcinomas (Group B: 36%; Group C: 35%). A trend for increasing overall Cdx2 expression was documented during the course of the experiment (<it>p </it>= 0.001).</p> <p>Conclusion</p> <p><it>De novo </it>expression of Cdx2 is an early event in the spectrum of the lesions induced by experimental gastro-esophageal reflux and should be considered as a key step in the morphogenesis of esophageal adenocarcinoma.</p

Analisi di parametri della proliferazione e del differenziamento cellulare in due modelli post-infiammatori: Metaplasia di Barrett e Rigenerazione muscolare.

CHAPTER A: CENTROSOMAL ABERRATION ANALYSIS IN A SURGICAL ESOPHAGITIS AND ESOPHAGEAL ADENOCARCINOMA ANIMAL MODEL AND IN VITRO MODEL The centrosom is the MTOC (microtubule organizing center) and is the main centre of microtubules organization. Centrosomal aberrations (CeAb), numerical and structural, have been identified in various diseases, but are mainly found in an increasing number of cancers. The CeAbs identified in various types of cancer have been identified as a potential cause of the loss of cell and tissue and as a possible source of instability (nucleation and disruption in the organization of microtubules, incorrect segregation of chromosomes resulting in aneuploidy). More recently, CeAb were found in different carcinomas in situ, making assume that these changes are also involved in tumorigenesis. My work is part of a project that involved the search for aberrations in centrosomali samples bioptici human esophageal adenocarcinoma arising on Barrett's metaplasia. Through this study it was demonstrated the presence of aberrations centrosomali in about 80% of patients and that such aberrations were very prominent and extended throughout the fabric metaplastico in 50% of cases. In light of these results were developed two experimental models, an animal model of Barrett's esophagus and a crop of primary esophageal epithelial cells. The goal was to use both models for the analysis of aberrations centrosomali so you can follow the evolution in time along the tumor progression in first and second after the treatment with two bile salts. CHAPTER B: USE OF MURINE MACROPHAGE CONDITIONED MEDIUM TO SELECT A MULTIPOTENT CELLS SUBPOPULATION AND ITS EFFECTS ON THEIR DIFFERENTIAL AND MUSCLE REGENERATION. The inflammatory processes caused by injury, disease or strenuous exercise play a key role during muscle regeneration. Many of the signals issued during these processes are exchanged between macrophages and muscle precursors, but their role is still unknown. Skeletal muscle regeneration relies onto satellite cells, a population of muscle precursors. Inflammation also plays a determinant role in the process, as upon injuring macrophages are attracted by the damaged myofibers and the activated satellite cells and act as key elements of dynamic muscle supportive stroma. Yet, it is not kwon how macrophages interact with the more profound stem cells of the satellite cell niche. This work showed that in the presence of a murine macrophage conditioned medium (mMCM) a subpopulation of stem-like cells could be selected and expanded from adult rat muscle. These cells were small, round, poorly adhesive, slow-growing and showed mesenchimal differentiation plasticity. Experiments with satellite cells mechanically isolated from suspensions of single myofibers also showed that mMCM inhibited their mesenchimal potential towards adipogenesis. In vivo, intramuscolar administrations of concentrated mMCM in a rat model of extensive surgical ablation dramatically improved muscle regeneration. Altogether, these findings suggest that macrophagic factors could be of great help in developing therapeutic protocols with myogenic stem cells.Capitolo A Il centrosoma è anche detto MTOC (microtubule organizing center), in quanto è il principale centro di organizzazione dei microtubuli. Aberrazioni centrosomali (CeAb), numeriche e strutturali, sono state individuate in diverse patologie, ma soprattutto sono riscontrate in un numero sempre maggiore di tumori. Le CeAb individuate nei diversi tipi di tumore sono state indicate come potenziale causa della perdita dell’architettura cellulare e tissutale e come possibile fonte di instabilità genetica (disfunzione nella nucleazione e organizzazione dei microtubuli, errata segregazione dei cromosomi con conseguente aneuploidia). Più recentemente, CeAb sono state riscontrate in diversi carcinomi in situ, facendo ipotizzare che tali alterazioni siano coinvolte anche nella tumorigenesi. Il mio lavoro si inserisce in un progetto che prevedeva la ricerca di aberrazioni centrosomali in campioni bioptici umani di adenocarcinoma esofageo insorto su metaplasia di Barrett. Mediante questo studio è stato possibile dimostrare la presenza di aberrazioni centrosomali in circa l’80% dei pazienti e che tali aberrazioni sono risultate molto prominenti ed estese in tutto il tessuto metaplastico nel 50% dei casi. Alla luce di questi risultati, quindi, sono stati messi a punto due modelli sperimentali, un modello animale di Esofago di Barrett e uno di colture primarie di cellule epiteliali esofagee. L’obiettivo è stato quello di utilizzare entrambi i modelli per le analisi di aberrazioni centrosomali in modo da poterne seguirne temporalmente l’evoluzione lungo la progressione tumorale nel primo caso e nel secondo da verificare se in seguito al trattamento con due Sali biliari nelle cellule epiteliali si generano aberrazioni centrosomali, in particolare rappresentate da aggregati di tubulina. CAPITOLO B: CAPACITA’ DEL MEDIUM CONDIZIONATO DA MACROFAGI MURINI DI SELEZIONARE UNA SOTTOPOPOLAZIONE DI CELLULE MULTIPOTENTI E SUOI EFFETTI SUL LORO DIFFERENZIAMENTO E SULLA RIGENERAZIONE MUSCOLARE. I processi infiammatori dovuti ad un trauma o ad una malattia muscolare o ad un importante sforzo fisico giocano un ruolo chiave nella rigenerazione muscolare. Molti dei segnali rilasciati durante questi processi vengono scambiati tra macrofagi e precursori miogenici, ma il loro ruolo è ancora poco conosciuto. La rigenerazione del muscolo scheletrico dipende dalle cellule satelliti, una popolazione di precursori miogenici. L’infiammazione gioca un ruolo determinante in questo processo, in seguito al danno i macrofagi sono attratti dalle miofibre danneggiate e la cellule satelliti vengano attivate. Non è ancora noto come i macrofagi interagiscano con le cellule staminali più profonde localizzate nelle nicchie delle cellule satellite. In questo lavoro è stato visto che in presenza di un medium condizionato da macrofagi murini (mMCM) una sottopopolazione di cellule multipotenti stem-like potrebbe essere selezionata ed espansa da muscolo di ratto adulto. Queste cellule sono piccole, rotonde, poco adesive, crescono lentamente e mostrano una plasticità di differenziamento mesenchimale. Esperimenti con cellule satelliti, isolate meccanicamente da sospensione di singole miofibre hanno mostrato che l’mMCM inibisce il loro potenziale mesenchimale verso l’adipogenesi. In vivo la somministrazione di mMCM concentrato in un modello di ratto in cui è stata praticata un’estesa ablazione chirurgica migliora enormemente la rigenerazione muscolare. Tutti questi risultati suggeriscono che i fattori macrofagici potrebbero essere di grande aiuto nello sviluppare protocolli terapeutici con cellule staminali miogeniche

CNTNAP2 mutations and autosomal dominant epilepsy with auditory features

Autosomal dominant epilepsy with auditory features (ADEAF) is clinically characterized by focal seizures with prominent auditory or aphasic auras and absence of structural brain abnormalities. Mutations in LGI1 and RELN genes account for the disorder in about 50% of ADEAF families. In a recent paper, a heterozygous intragenic deletion in the CNTNAP2 gene has been associated to ADEAF in a single family. We screened 28 ADEAF families for mutations in CNTNAP2 by next generation sequencing and copy number variation analyses and found no likely pathogenic mutations segregating with the disease. CNTNAP2 should be screened in genetically unsolved ADEAF families, but causative mutations are expected to be infrequent in this gene

LGI1 tumor tissue expression and serum autoantibodies in patients with primary malignant glioma

Objectives: The Leucine-rich glioma inactivated 1 (LGI1) protein is thought to be implicated in malignant progression of glioma tumors, and mutations in the encoding gene, LGI1, cause autosomal dominant lateral temporal epilepsy, a genetic focal epilepsy syndrome. The aim of this study was to investigate the possible involvement of LGI1 in high-grade glioma-associated epilepsy by analyzing its expression in tumor specimens of patients with and without epilepsy and by searching for LGI1 autoantibodies in the sera these patients. Patients and methods: We examined tumor tissue samples from 24 patients with high-grade gliomas (12 with and 12 without epilepsy) by immunoblot and detected variable amounts of LGI1 in tumor tissues from 9/24 (37%) patients. Results: LGI1 was detected in 7/12 (58%) patients with epilepsy and in 2/12 (16%) patients without epilepsy (p = 0.0894; Fisher's exact test). Moreover, testing blood sera of five patients for antibodies against LGI1 revealed LGI1 autoantibodies in two patients, both suffering from epilepsy and expressing LGI1 in tumor tissue. Conclusion: Our findings suggest that there may be a preferential expression of LGI1 in high-grade glioma tumors of patients with epilepsy. We also unveil the presence of serum LGI1 autoantibodies in some patients with high-grade gliomas, where they might play an epileptogenic role

Suggestive linkage of familial mesial temporal lobe epilepsy to chromosome 3q26

Purpose: To describe the clinical findings in a family with a benign form of mesial temporal lobe epilepsy and to identify the causative genetic factors. Methods: All participants were personally interviewed and underwent neurologic examination. The affected subjects underwent EEG and most of them neuroradiological examinations (MRI). All family members were genotyped with the HumanCytoSNP-12 v1.0 beadchip and linkage analysis was performed with Merlin and Simwalk2 programs. Exome sequencing was performed on HiSeq2000, after exome capture with SureSelect 50Mb kit v2.0. Results: The family had 6 members with temporal lobe epilepsy. Age at seizure onset ranged from 8 to 13 years. Five patients had epigastric auras often associated to oro-alimentary automatic activity, 3 patients presented loss of contact, and 2 experienced secondary generalizations. Febrile seizures occurred in 2 family members, 1 of whom also had temporal lobe epilepsy. EEG showed focal slow waves and epileptic abnormalities on temporal regions in 1 patient and was normal in the other affected individuals. MRI was normal in all temporal lobe epilepsy patients. We performed single nucleotide polymorphism-array linkage analysis of the family and found suggestive evidence of linkage (LOD score = 2.106) to a region on chromosome 3q26. Haplotype reconstruction supported the linkage data and showed that the majority of unaffected family members carried the haplotype at risk. Whole exome sequencing failed to identify pathogenic mutations in genes of the candidate region. Conclusions: Our data suggest the existence of a novel locus for benign familial mesial temporal lobe epilepsy on chromosome 3q26. Our failure to identify pathogenic mutations in genes of this region may be due to limitations of the exome sequencing technology. (C) 2013 Elsevier B.V. All rights reserved

LGI1 mutations impair interaction with ADAM22 and ADAM23.

<p>HEK293T cells were transiently co-transfected with (A) HA-ADAM22 or (B) HA-ADAM23 and LGI1 wild-type or mutants. Inputs (6.5% of total proteins) and HA-immunoprecipitations were separated in SDS-PAGE, transferred and immunoblotted using the indicated antibody. Co-immunoprecipitations showed that wild type LGI1 interacts with HA-ADAM22 (A) and HA-ADAM23 (B), while the interaction is abolished in the presence of T380A mutant. LGI1 mutants R407C, S473L and R474Q displayed a reduced interaction compared to the wild-type. Each of these experiments was performed twice with similar results. In the experiment shown in panel B, HA-ADAM23 failed to transfect HEK293T cells together with LGI1-S473L.</p

Percentage of cells with LGI1 (wild type or mutated) bound to either ADAM22 or ADAM23 receptor on the cell membrane.

<p>Percentage of cells with LGI1 (wild type or mutated) bound to either ADAM22 or ADAM23 receptor on the cell membrane.</p

Genetic features of study mutations.

<p>Genetic features of study mutations.</p

Secretion of wild type LGI1 co-expressed with LGI1 mutant proteins.

<p>Western blot of concentrated media of HEK293T cells co-transfected with GFP-tagged wild type (wt) LGI1 and one of the indicated Flag-tagged LGI1 constructs: wt, R407C (sec+), or I122K (sec-). Bands were revealed with an anti-LGI1 antibody. IgG gel loading control added to cell culture medium prior concentration is shown below. Vector, empty vector; MW, molecular weight markers.</p