Mild malformations of cortical development in sleep-related hypermotor epilepsy due to KCNT1 mutations

open14siMutations in the sodium-activated potassium channel gene KCNT1 have been associated with nonlesional sleep-related hypermotor epilepsy (SHE). We report the co-occurrence of mild malformation of cortical development (mMCD) and KCNT1 mutations in four patients with SHE. Focal cortical dysplasia type I was neuropathologically diagnosed after epilepsy surgery in three unrelated MRI-negative patients, periventricular nodular heterotopia was detected in one patient by MRI. Our findings suggest that KCNT1 epileptogenicity may result not only from dysregulated excitability by controlling Na+K+ transport, but also from mMCD. Therefore, pathogenic variants in KCNT1 may encompass both lesional and nonlesional epilepsies.openRubboli G.; Plazzi G.; Picard F.; Nobili L.; Hirsch E.; Chelly J.; Prayson R.A.; Boutonnat J.; Bramerio M.; Kahane P.; Dibbens L.M.; Gardella E.; Baulac S.; Moller R.S.Rubboli, G.; Plazzi, G.; Picard, F.; Nobili, L.; Hirsch, E.; Chelly, J.; Prayson, R. A.; Boutonnat, J.; Bramerio, M.; Kahane, P.; Dibbens, L. M.; Gardella, E.; Baulac, S.; Moller, R. S

Phospho-HDAC6 Gathers Into Protein Aggregates in Parkinson’s Disease and Atypical Parkinsonisms

HDAC6 is a unique histone deacetylase that targets cytoplasmic non-histone proteins and has a specific ubiquitin-binding activity. Both of these activities are required for HDAC6-mediated formation of aggresomes, which contain misfolded proteins that will ultimately be degraded via autophagy. HDAC6 deacetylase activity is increased following phosphorylation on serine 22 (phospho-HDAC6). In human, HDAC6 localizes in neuronal Lewy bodies in Parkinson\u2019s disease (PD) and in oligodendrocytic Papp\u2013Lantos bodies in multiple system atrophy (MSA). However, the expression of phospho-HDAC6 in post-mortem human brains is currently unexplored. Here, we evaluate and compare the distribution of HDAC6 and its phosphorylated form in human brains obtained from patients affected by three forms of parkinsonism: two synucleinopathies (PD and MSA) and a tauopathy (progressive supranuclear palsy, PSP). We find that both HDAC6 and its phosphorylated form localize with pathological protein aggregates, including \u3b1-synuclein-positive Lewy bodies in PD and Papp\u2013Lantos bodies in MSA, and phospho-tau-positive neurofibrillary tangles in PSP. We further find a direct interaction of HDAC6 with \u3b1-synuclein with proximity ligation assay (PLA) in neuronal cell of PD patients. Taken together, our findings suggest that both HDAC6 and phospho-HDAC6 regulate the homeostasis of intra-neuronal proteins in parkinsonism

Immunohistochemical profile and c-kit mutations in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are low-grade sarcomas arising from the interstitial cells of Cajal, harboring mutation of c-kit. We investigated the morphological, immunohistochemical, and molecular profile of 55 GISTs to establish the prevalence of mutations, their clinical significance, and diagnostic utility. c-kit mutations were investigated by evaluating the entire coding sequence of the gene with non-radioisotopic PCR-SSCP, and characterized with fluorescent cycle sequencing. Mutations were detected in 39 tumors (71%), the majority (67%) involving exon 11. Two tumors showed exon 9 mutations (one tumor located in the small intestine and one in the stomach), whereas two cases showed a polymorphism at the splicing site of exon/intron 1 present in healthy blood donors with a 3% frequency. CD 117 was expressed in 53 tumors (96%); CD34 was positive in 42 cases (76%); 42 cases (76%) expressed both CD117 and CD34. c-kit mutations were similarly distributed in stromal tumors at low risk of aggressive behavior (78%), intermediate risk (66%), and high risk (71%). Fifteen tumors expressing CD117 showed wild-type kit gene, and on histological grounds, they were equally distributed among epithelioid and spindle cell morphology. One case neither expressed CD117 nor did it show e-kit mutation. Data suggest that both immunohistochemical and molecular evaluation may be useful in tumors likely to be classified as GISTs: molecular analysis appears valuable to support the diagnosis and to identify cases that can benefit from recent novel therapeutic tools

Search for Coxsackievirus B3 RNA in idiopathic dilated cardiomyopathy using gene amplification by polymerase chain reaction

A polymerase chain reaction (PCR) amplification assay was developed to detect Coxsackievirus B3 ribonucleic acid (RNA) in blood and myocardial tissue of explanted hearts from 40 patients who underwent cardiac transplantation and in 1 normal heart. Twenty-one patients were affected by idiopathic dilated cardiomyopathy of different duration and 19 by coronary artery disease. Coxsackievirus B3 in vitro infected Vero cells and cells infected by related human enteroviruses (Coxsackievirus B2, B4, and poliovirus 1) were used as reaction controls. PCR was performed using 4 pairs of primers homologous to Coxsackievirus B3 sequences. Three sets were located in regions of the genome conserved at nucleotide level between several enterovirus species (replicase gene, 5′ noncoding region), while one was located in a Coxsackievirus B3-specific region (VP1 gene). Total RNA was prepared by acid guanidinium isothyocyanate extraction from tissue stored frozen at -80 °C. One microgram of total RNA was retrotranscribed with either antisense primer or with random hexanucleotide primers and then subjected to 40 cycles of amplification. PCR products were separated by electrophoresis on a 10% polyacrilamide gel, electrotransferred to a nylon membrane and then hybridized to oligonucleotide probes specific for the coxsackievirus B3 genome radiolabeled with radioactive isotope of phosphorous. All pairs of primers yielded specific amplification products when tested on Coxsackievirus B3-infected Vero cells, with a sensitivity of 1 infected cell out of 105 to 106 cells starting from 1 μg total RNA. Primer sets for regions of Coxsackievirus B3 genome highly conserved between related enteroviral species gave positive amplification also when challenged with RNA from cells infected by Coxsackievirus B2, B4 and poliovirus 1. The VP1 gene primer set produced positive amplification only with RNA of Coxsackievirus B3-infected cells. Coxsackievirus B3-specific amplification products were distinguished from those of related enteroviruses by hybridization with specific oligoprobes. However, Coxsackievirus B2, B4 and poliovirus 1-specific PCR products showed positive hybridization if probed with Coxsackievirus B3 genomic probes. All total RNAs from blood and myocardial samples examined by our PCR assay failed to reveal any amplification product that could be related to Coxsackievirus B3 or to enteroviruses in general, after gel electrophoresis and low stringency Southern blot hybridization with the Coxsackievirus B3 specific oligoprobes or the Coxsackievirus B3 genomic probes. The negative results obtained in our series question the hypothesized widespread persistence of enteroviral RNA in hearts with idiopathic dilated cardiomyopathy. © 1992

Gene expression, cytoskeletal changes and extracellular matrix synthesis in human osteoblasts treated with cyclosporin A.

Cyclosporin A (CyA) is an immunosuppressive agent used to prevent allograft rejection, but unfortunately it causes adverse effects such as bone diseases, osteoporosis and osteomalacia. These pathologies involve an imbalance between synthesis, degradation and mineralization of extracellular matrix. CyA can modify extracellular matrix components such as glycosaminoglycans (GAG) and collagen fibers. In addition, normal cell activity is dependent on cell morphology and substrate cell attachment. We treated normal human osteoblasts with CyA and analyzed: (i) gene expression by a microarray method; (ii) extracellular GAG and collagen after (3)H-glucosamine and Western blot analysis; and (iii) cytoskeletal changes, using actin and tubulin fluorescent antibodies. CyA increased intra- and extracellular GAG and extracellular GAG classes such as hyaluronic acid, chondroitin sulphate, and dermatan sulphate; there was no noteworthy effect on heparan sulphate and the ratio of non-sulphated to sulphated GAG. In osteoblast cultures the drug reduced cytoskeletal actin, while tubulin did not change. In vivo the osteoblasts showed morphological changes with different extracellular matrix synthesis. Microarray analysis indicated the inhibition of gene pathways related to Wnt signaling molecules, and the cytoskeletal and focal adhesion cascade. In in vitro human osteoblasts CyA modified gene expression related to cytoskeletal pattern organization and cell morphology. Since in bone pathologies osteoblasts show different morphology related to cell size, these data suggest that in vivo osteoblast different functions could be dependent on alteration of osteoblast differentiation

Gene expression, cytoskeletal changes and extracellular matrix synthesis in human osteoblasts treated with cyclosporin A.

Cyclosporin A (CyA) is an immunosuppressive agent used to prevent allograft rejection, but unfortunately it causes adverse effects such as bone diseases, osteoporosis and osteomalacia. These pathologies involve an imbalance between synthesis, degradation and mineralization of extracellular matrix. CyA can modify extracellular matrix components such as glycosaminoglycans (GAG) and collagen fibers. In addition, normal cell activity is dependent on cell morphology and substrate cell attachment. We treated normal human osteoblasts with CyA and analyzed: (i) gene expression by a microarray method; (ii) extracellular GAG and collagen after (3)H-glucosamine and Western blot analysis; and (iii) cytoskeletal changes, using actin and tubulin fluorescent antibodies. CyA increased intra- and extracellular GAG and extracellular GAG classes such as hyaluronic acid, chondroitin sulphate, and dermatan sulphate; there was no noteworthy effect on heparan sulphate and the ratio of non-sulphated to sulphated GAG. In osteoblast cultures the drug reduced cytoskeletal actin, while tubulin did not change. In vivo the osteoblasts showed morphological changes with different extracellular matrix synthesis. Microarray analysis indicated the inhibition of gene pathways related to Wnt signaling molecules, and the cytoskeletal and focal adhesion cascade. In in vitro human osteoblasts CyA modified gene expression related to cytoskeletal pattern organization and cell morphology. Since in bone pathologies osteoblasts show different morphology related to cell size, these data suggest that in vivo osteoblast different functions could be dependent on alteration of osteoblast differentiation