Restoration of mutant bestrophin-1 expression, localisation and function in a polarised epithelial cell model

Autosomal recessive bestrophinopathy (ARB) is a retinopathy caused by mutations in the bestrophin-1 protein, which is thought to function as a Ca2+-gated Cl− channel in the basolateral surface of the retinal pigment epithelium (RPE). Using a stably transfected polarised epithelial cell model, we show that four ARB mutant bestrophin-1 proteins were mislocalised and subjected to proteasomal degradation. In contrast to the wild-type bestrophin-1, each of the four mutant proteins also failed to conduct Cl− ions in transiently transfected cells as determined by whole-cell patch clamp. We demonstrate that a combination of two clinically approved drugs, bortezomib and 4-phenylbutyrate (4PBA), successfully restored the expression and localisation of all four ARB mutant bestrophin-1 proteins. Importantly, the Cl− conductance function of each of the mutant bestrophin-1 proteins was fully restored to that of wild-type bestrophin-1 by treatment of cells with 4PBA alone. The functional rescue achieved with 4PBA is significant because it suggests that this drug, which is already approved for long-term use in infants and adults, might represent a promising therapy for the treatment of ARB and other bestrophinopathies resulting from missense mutations in BEST1

PRIM1 Deficiency Causes a Distinctive Primordial Dwarfism Syndrome

DNA replication is fundamental for cell proliferation in all organisms. Nonetheless, components of the replisome have been implicated in human disease, and here we report PRIM1 encoding the catalytic subunit of DNA primase as a novel disease gene. Using a variant classification agnostic approach, biallelic mutations in PRIM1 were identified in five individuals. PRIM1 protein levels were markedly reduced in patient cells, accompanied by replication fork asymmetry, increased interorigin distances, replication stress, and prolonged S-phase duration. Consequently, cell proliferation was markedly impaired, explaining the patients' extreme growth failure. Notably, phenotypic features distinct from those previously reported with DNA polymerase genes were evident, highlighting differing developmental requirements for this core replisome component that warrant future investigation

Reverse-Transcriptase Inhibitors in the Aicardi–Goutières Syndrome

International audienceTo the Editor:The Aicardi–Goutières syndrome is a genetic encephalopathy that is associated with childhood illness and death. The syndrome is hypothesized to be due to misidentification of self-derived nucleic acids as nonself and the subsequent induction of a type I interferon–mediated response that simulates an antiviral reaction.1 Endogenous retroelements, mobile genetic elements that can be transcribed to RNA and then to DNA by reverse transcription, constitute 40% of the human genome and represent a potential source of immunostimulatory nucleic acid in patients with this syndrome.

Type I interferon-mediated autoinflammation due to DNase II deficiency

Microbial nucleic acid recognition serves as the major stimulus to an antiviral response, implying a requirement to limit the misrepresentation of self nucleic acids as non-self and the induction of autoinflammation. By systematic screening using a panel of interferon-stimulated genes we identify two siblings and a singleton variably demonstrating severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, deforming arthropathy and increased anti-DNA antibodies. In both families we identify biallelic mutations in DNASE2, associated with a loss of DNase II endonuclease activity. We record increased interferon alpha protein levels using digital ELISA, enhanced interferon signaling by RNA-Seq analysis and constitutive upregulation of phosphorylated STAT1 and STAT3 in patient lymphocytes and monocytes. A hematological disease transcriptomic signature and increased numbers of erythroblasts are recorded in patient peripheral blood, suggesting that interferon might have a particular effect on hematopoiesis. These data define a type I interferonopathy due to DNase II deficiency in humans

The Analgesic Effect on Neuropathic Pain of Retrogradely Transported botulinum Neurotoxin A Involves Schwann Cells and Astrocytes

In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25), the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A), could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw's nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC). We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients' quality of life. © 2012 Marinelli et al

Interazione assone-glia: l'acetilcolina rilasciata lungo gli assoni regola il differenziamento delle cellule di Schwann verso il fenotipo mielinizzante.

Nel sistema nervoso, durante lo sviluppo e nell’adulto, l’interazione tra neuroni e cellule gliali e fra gli assoni in crescita e la glia mielinizzante favorisce, attraverso la produzione di specifici segnali, la modulazione della proliferazione, sopravvivenza e differenziamento di ambedue le popolazioni cellulari. Vari fattori di crescita (es.NRGs) sono stati identificati come principali modulatori di questa interazione, ma molteplici evidenze hanno indicato che anche i neurotrasmettitori (es. GABA, adenosina e ACh) possono avere un ruolo chiave in tale interazione. Negli ultimi anni la nostra attenzione si è focalizzata sul ruolo svolto dall’ACh nel modulare la crescita e il differenziamento delle cellule di Schwann, la popolazione gliale mielinizzante del SNP. Nostri precedenti dati avevano dimostrato che l’ACh, attraverso il recettore muscarinico M2, è in grado di indurre nelle cellule di Schwann, un blocco della progressione nel ciclo cellulare e una aumentata espressione delle proteine della mielina (P0, PMP22 e MBP). Per verificare se l’attivazione del recettore M2 fosse in grado di indirizzare le cellule di Schwann verso il fenotipo mielinizzante, abbiamo analizzato l’espressione di una serie di fattori responsabili della fase proliferativa e di quella differenziativa dopo attivazione del recettore M2. Cellule di Schwann, isolate dal nervo sciatico di ratti neonati (2 gg post-natale), sono state trattate in vitro con l’agonista del recettore M2, arecaidina. Analisi mediante real time PCR e western blot hanno dimostrato che fattori notoriamente coinvolti nella fase proliferativa vengono significativamente repressi (es. c-jun, NRG1/1, recettori erbB2, Notch-1, Jagged-1). Al contrario, l’espressione di fattori coinvolti nel differenziamento verso il fenotipo mielinizzante, è significativamente aumentata (Krox20/egr2; Sox-10, NRG1/3). Il trattamento con arecaidina inoltre induce un significativo cambiamento nella morfologia delle cellule che appaiono più appiattite e che sviluppano zone di adesione tra cellule adiacenti, come suggerito dalla ridistribuzione di molecole di adesione cellulare e dall’analisi al SEM. Ipotizzando che il segnale colinergico differenziante per le cellule di Schwann provenga dall’assone, allestendo colture di espianti di gangli spinali (DRG) in camere di Campenot, abbiamo dimostrato che l’ACh viene rilasciata sia nel compartimento in cui sono presenti i corpi cellulari dei neuroni, sia in quelli dove si trovano i neuriti in crescita. Inoltre abbiamo osservato che i neuroni sensoriali esprimono il mediatoforo, una proteina che risulta coinvolta nel rilascio non vescicolato dell’acetilcolina. In conclusione i nostri dati dimostrano che l’acetilcolina può essere rilasciata lungo gli assoni dei neuroni sensoriali probabilmente proprio attraverso un meccanismo di rilascio non vescicolare; questo rilascio consentirebbe alle adiacenti cellule di Schwann di rispondere allo stimolo colinergico con il blocco della proliferazione e l’attivazione della mielinizzazione, condizione necessaria per consentire la corretta formazione e conduzione di assoni di grosso calibro