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CRISPR/Cas9-mediated therapeutic gene editing is a promising technology for durable treatment of incurable monogenic diseases such as myotonic dystrophies. Gene-editing approaches have been recently applied to in vitro and in vivo models of myotonic dystrophy type 1 (DM1) to delete the pathogenic CTG-repeat expansion located in the 3′ untranslated region of the DMPK gene. In DM1-patient-derived cells removal of the expanded repeats induced beneficial effects on major hallmarks of the disease with reduction in DMPK transcript-containing ribonuclear foci and reversal of aberrant splicing patterns. Here, we set out to excise the triplet expansion in a time-restricted and cell-specific fashion to minimize the potential occurrence of unintended events in off-target genomic loci and select for the target cell type. To this aim, we employed either a ubiquitous promoter-driven or a muscle-specific promoter-driven Cas9 nuclease and tetracycline repressor-based guide RNAs. A dual-vector approach was used to deliver the CRISPR/Cas9 components into DM1 patient-derived cells and in skeletal muscle of a DM1 mouse model. In this way, we obtained efficient and inducible gene editing both in proliferating cells and differentiated post-mitotic myocytes in vitro as well as in skeletal muscle tissue in vivo., Graphical abstract, This paper describes a gene-editing approach designed to remove the pathologic CTG expansion mutation that causes myotonic dystrophy type 1. By using muscle-specific and drug-inducible expression of the CRISPR/Cas9 complex, effective deletion of the CTG expansion was obtained in myogenic cells and in mouse skeletal muscle.

Background: Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disease caused by the abnormal expansion of CTG-triplets in the 3' untranslated region of the DMPK gene. While therapeutic approaches that neutralize the toxic DMPK transcript provide only short-term effects, CRISPR/Cas9-mediated gene editing strategies can eliminate permanently the pathogenic mutation. Methods and Results: Having previously applied CRISPR/Cas9-mediated gene therapy in DM1-patient-derived myogenic cells to eliminate the repeat expansion, we have recently employed a dual vector-mediated approach to transduce drug-inducible CRISPR/Cas9 complex components into DM1 patient-derived cells and DM1 mice carrying a mutated human DMPK transgene, either in skeletal muscle or systemically. These mice exhibit a pathologic neuromuscular phenotype and altered behavior similar to those observed in human disease. By using this strategy, we obtained efficient and inducible DMPK gene editing in the absence of off-target unintended events both in myogenic cells in vitro and in skeletal muscle tissues in vivo. Conclusions and Significance: CRISPR/Cas9-induced deletion of CTG expansion could potentially result in a durable therapeutic response in post-mitotic adult tissue, opening the way for future gene therapy application in humans. Importantly, the use of tissue-specific Cas9 and spatio-temporal control of gene editing minimizes unintended off-target activity.

Graphical abstract, Highlights • Drug design with rational methods, using structure-based methods. • Novel RNA binders based on pyrido[2,3-d]pyrimidin-7-(8H)-ones. • Myotonic Dystrophy type 1 small molecule drug discovery., Myotonic Dystrophy type 1 (DM1) is an incurable neuromuscular disorder caused by toxic DMPK transcripts that carry CUG repeat expansions in the 3′ untranslated region (3′UTR). The intrinsic complexity and lack of crystallographic data makes noncoding RNA regions challenging targets to study in the field of drug discovery. In DM1, toxic transcripts tend to stall in the nuclei forming complex inclusion bodies called foci and sequester many essential alternative splicing factors such as Muscleblind-like 1 (MBNL1). Most DM1 phenotypic features stem from the reduced availability of free MBNL1 and therefore many therapeutic efforts are focused on recovering its normal activity. For that purpose, herein we present pyrido[2,3-d]pyrimidin-7-(8H)-ones, a privileged scaffold showing remarkable biological activity against many targets involved in human disorders including cancer and viral diseases. Their combination with a flexible linker meets the requirements to stabilise DM1 toxic transcripts, and therefore, enabling the release of MBNL1. Therefore, a set of novel pyrido[2,3-d]pyrimidin-7-(8H)-ones derivatives (1a-e) were obtained using click chemistry. 1a exerted over 20% MBNL1 recovery on DM1 toxic RNA activity in primary cell biology studies using patient-derived myoblasts. 1a promising anti DM1 activity may lead to subsequent generations of ligands, highlighting a new affordable treatment against DM1.

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy affecting many different body tissues, predominantly skeletal and cardiac muscles and the central nervous system. The expansion of CTG repeats in the DM1 protein-kinase (DMPK) gene is the genetic cause of the disease. The pathogenetic mechanisms are mainly mediated by the production of a toxic expanded CUG transcript from the DMPK gene. With the availability of new knowledge, disease models, and technical tools, much progress has been made in the discovery of altered pathways and in the potential of therapeutic intervention, making the path to the clinic a closer reality. In this review, we describe and discuss the molecular therapeutic strategies for DM1, which are designed to directly target the CTG genomic tract, the expanded CUG transcript or downstream signaling molecules.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by abnormally expanded stretches of CTG DNA triplets in the DMPK gene, leading to mutated-transcript RNA-toxicity. MicroRNAs (miRNAs) are short non-coding RNAs that, after maturation, are loaded onto the RISC effector complex that destabilizes target mRNAs and represses their translation. In DM1 muscle biopsies not only the expression, but also the intracellular localization of specific miRNAs is disrupted, leading to the dysregulation of the relevant mRNA targets. To investigate the functional alterations of the miRNA/target interactions in DM1, we analyzed by RNA-sequencing the RISC-associated RNAs in skeletal muscle biopsies derived from DM1 patients and matched controls. The mRNAs found deregulated in DM1 biopsies were involved in pathways and functions relevant for the disease, such as energetic metabolism, calcium signaling, muscle contraction and p53-dependent apoptosis. Bioinformatic analysis of the miRNA/mRNA interactions based on the RISC enrichment profiles, identified 24 miRNA/mRNA correlations. Following validation in 21 independent samples, we focused on the couple miR-29c/ASB2 because of the role of miR-29c in fibrosis (a feature of late-stage DM1 patients) and of ASB2 in the regulation of muscle mass. Luciferase reporter assay confirmed the direct interaction between miR-29c and ASB2. Moreover, decreased miR-29c and increased ASB2 levels were verified also in immortalized myogenic cells and primary fibroblasts, derived from biopsies of DM1 patients and controls. CRISPR/Cas9-mediated deletion of CTG expansions rescued normal miR-29c and ASB2 levels, indicating a direct link between the mutant repeats and the miRNA/target expression. In conclusion, functionally relevant miRNA/mRNA interactions were identified in skeletal muscles of DM1 patients, highlighting the dysfunction of miR-29c and ASB2.

RIFLESSIONE SULLA LINGUA E MODELLO VALENZIALEAtti dei corsi di formazione per insegnanti sulla grammatica valenziale: “C’è grammatica e grammatica…”.Università degli Studi di Padova ottobre/febbraio 2017/2018 - ottobre/giugno 2018-2019. A cura di Elena Maria Duso e Walter Paschetto. Contributi di Elena Maria Duso, Maria G. Lo Duca, Donatella Lovison, Cristiana De Santis, Elena Martinelli, Claudia Provenzano, Laura Vanelli, Michele Prandi, Diana Vedovato e Vera Zanette, Federica di Maria, Chiara Giannone, Paola Iannacci e Paola Marinetto, Maria Rizzato.

RIFLESSIONE SULLA LINGUA E MODELLO VALENZIALE Atti dei corsi di formazione per insegnanti sulla grammatica valenziale: “C’è grammatica e grammatica…”. Università degli Studi di Padova ottobre/febbraio 2017/2018 - ottobre/giugno 2018-2019. A cura di Elena Maria Duso e Walter Paschetto. Contributi di Elena Maria Duso, Maria G. Lo Duca, Donatella Lovison, Cristiana De Santis, Elena Martinelli, Claudia Provenzano, Laura Vanelli, Michele Prandi, Diana Vedovato e Vera Zanette, Federica di Maria, Chiara Giannone, Paola Iannacci e Paola Marinetto, Maria Rizzato.

The neurotrophins Ngf, Bdnf, NT-3, NT4–5 have key roles in development, survival, and plasticity of neuronal cells. Their action involves broad gene expression changes at the level of transcription and translation. MicroRNAs (miRs)—small RNA molecules that control gene expression post-transcriptionally—are increasingly implicated in regulating development and plasticity of neural cells. Using PC12 cells as a model system, we show that Ngf modulates changes in expression of a variety of microRNAs, including miRs known to be modulated by neurotrophins—such as the miR-212/132 cluster—and several others, such as miR-21, miR-29c, miR-30c, miR-93, miR-103, miR-207, miR-691, and miR-709. Pathway analysis indicates that Ngf-modulated miRs may regulate many protein components of signaling pathways involved in neuronal development and disease. In particular, we show that miR-21 enhances neurotrophin signaling and controls neuronal differentiation induced by Ngf. Notably, in a situation mimicking neurodegeneration—differentiated neurons deprived of Ngf—this microRNA is able to preserve the neurite network and to support viability of the neurons. These findings uncover a broad role of microRNAs in regulating neurotrophin signaling and suggest that aberrant expression of one or more Ngf-modulated miRs may be involved in neurodegenerative diseases. Electronic supplementary material The online version of this article (doi:10.1007/s12017-014-8292-z) contains supplementary material, which is available to authorized users.

TTF-1/Nkx2.1 is a homeodomain-containing transcription factor required for the proper development of ventral forebrain, including some structures of the hypothalamus. TTF-1/Nkx2.1 remains expressed in the hypothalamus after birth and it plays a crucial role during sexual development. To identify putative TTF-1/Nkx2.1 target genes in GnRH neurons, we have studied the gene expression profile of the GT1-7 cells exogenously expressing TTF-1/Nkx2.1 coding gene. Our transcriptome analysis confirms that TTF-1/Nkx2.1 is involved in neuron morphogenesis and differentiation. Many of the newly identified TTF-1/Nkx2.1 target genes have a direct involvement with the central regulation of sexual maturity. In particular, we have identified Sparc as a gene directly regulated by TTF-1/Nkx2.1 at the promoter level. To further support the role of TTF-1 in GnRH neurons, we show that Sparc is involved in the regulation of the GnRH secretion in GT1-7 cells.

Benign hereditary chorea (BHC) is an autosomal dominant disorder of early onset characterised by non progressive choreic movements with normal cognitive function occasionally associated with hypothyroidism and respiratory problems. Numerous pieces of evidence link BHC with TITF-1/NKX2.1 gene mutations. We studied a patient with a familial benign hereditary chorea and normal thyroid and respiratory function. Sequence analysis of TITF-1 revealed the presence of a heterozygous C>T substitution at nucleotide 532, predicted to change an arginine (CGA) with a stop codon (TGA) at position 178 (R178X). A functional analysis shows that the mutated TTF-1 is not binding DNA, nor activating the canonical thyroid target gene promoter or interfering with the ability of wild type TTF-1 to activate transcription. In addition, the mutated protein is predominantly cytoplasmic, rather than nuclear as in the case of the wild type TTF-1. Thus, we have identified a new mutation in the TTF-1 coding gene in a patient with benign hereditary chorea. The results show that the mutation leads to a haploinsufficiency of TITF-1 and opens the question of genotype/phenotype correlation.

The thyroid transcription factor 1 (TTF-1) is encoded, on chromosome 14813, by the gene termed TITF-1/NKX2.1. Mutations in this gene have been associated with chorea, hypothyroidism, and lung disease, all included in the "brain-thyroid-lung syndrome." We here describe two cases of novel missense mutations [NM_003317.3:c.516G>T and c.623G>C resulting in p.(GIn172His) and p.(Trp208Ser), respectively] in TITF-1/NKX2-1 in non-consanguineous patients. We provide a functional study of the role of the two mutations on the TTF-1 ability to bind DNA and to trans-activate both thyroid and lung specific gene promoters. Our results confirm the difficulty to correlate the TTF-1 activity with the clinical phenotype of affected patients and highlight the need to increase the limited knowledge we have on the activity of TTF-1 in neuronal cells. (C) 2015 Elsevier B.V. All rights reserved.

In questo articolo vengono presentati i primi risultati della ricerca GRASS (Riflessione Grammaticale a Scuola: il Soggetto Sintattico), che ha coinvolto 444 tra alunni e studenti dalla scuola primaria al primo anno di università e 16 insegnanti. Muovendo da alcune riflessioni generali sull’insegnamento della grammatica nella scuola italiana, la cui criticità sta, fra l’altro, nella scarsa considerazione degli usi linguistici reali nello sviluppo della riflessione metalinguistica, l’articolo tratta nello specifico alcuni aspetti emersi dalla ricerca. Innanzitutto, la confusione tra funzione grammaticale e referenza, ben testimoniata dalle espressioni incongrue e idiosincratiche usate dagli informanti per esplicitare i soggetti individuati in un testo. Poi, i giudizi di grammaticalità rispetto a frasi dell’italiano contenenti casi problematici di accordo verbo-soggetto. Infine, l’analisi delle definizioni esplicite di soggetto fornite da alunni e studenti, che forniscono indicazioni sulla sottostante nozione di soggetto così come viene appresa nel corso della scolarizzazione. Il saggio si conclude con alcune riflessioni su come ricerche di questo tipo possano avere ricadute in termini di formazione degli insegnanti e di pratiche didattiche. The paper discusses some preliminary results of the research project GRASS - Grammar Reflection at School: Syntactic Subject, which involved 444 junior and senior students from lower to higher education grades and 16 teachers. Starting from some general remarks on the current state of grammar teaching in the Italian school system, particularly critical as far as real speech, discourse and metalinguistic awareness are concerned, the paper highlights and discusses three main critical issues: a)the confusion between (con)textual reference and grammatical function, well attested by thediverse and incongruous ways used by the respondents to identify and represent the subject of sentences from a text; b) the pupils’ and students’ grammaticality judgements on verb and subject agreement, especially in cases of semantic/syntactic mismatch; c) the relation between the respondents’ explicit definitions of grammatical subject and the emerging underlying notions that characterize the different stages of Italian education as far as primary vs. secondary schools are concerned. Finally, some conclusions are drawn on the possible outcomes that this type of research can have in terms of teachers’ training and teaching practices.

The paper discusses some preliminary results of the research project GRASS - Grammar Reflection at School: Syntactic Subject, which involved 444 junior and senior students from lower to higher education grades and 16 teachers. Starting from some general remarks on the current state of grammar teaching in the Italian school system, particularly critical as far as real speech, discourse and metalinguistic awareness are concerned, the paper highlights and discusses three main critical issues: a)the confusion between (con)textual reference and grammatical function, well attested by thediverse and incongruous ways used by the respondents to identify and represent the subject of sentences from a text; b) the pupils’ and students’ grammaticality judgements on verb and subject agreement, especially in cases of semantic/syntactic mismatch; c) the relation between the respondents’ explicit definitions of grammatical subject and the emerging underlying notions that characterize the different stages of Italian education as far as primary vs. secondary schools are concerned. Finally, some conclusions are drawn on the possible outcomes that this type of research can have in terms of teachers’ training and teaching practices. In questo articolo vengono presentati i primi risultati della ricerca GRASS (Riflessione Grammaticale a Scuola: il Soggetto Sintattico), che ha coinvolto 444 tra alunni e studenti dalla scuola primaria al primo anno di università e 16 insegnanti. Muovendo da alcune riflessioni generali sull’insegnamento della grammatica nella scuola italiana, la cui criticità sta, fra l’altro, nella scarsa considerazione degli usi linguistici reali nello sviluppo della riflessione metalinguistica, l’articolo tratta nello specifico alcuni aspetti emersi dalla ricerca. Innanzitutto, la confusione tra funzione grammaticale e referenza, ben testimoniata dalle espressioni incongrue e idiosincratiche usate dagli informanti per esplicitare i soggetti individuati in un testo. Poi, i giudizi di grammaticalità rispetto a frasi dell’italiano contenenti casi problematici di accordo verbo-soggetto. Infine, l’analisi delle definizioni esplicite di soggetto fornite da alunni e studenti, che forniscono indicazioni sulla sottostante nozione di soggetto così come viene appresa nel corso della scolarizzazione. Il saggio si conclude con alcune riflessioni su come ricerche di questo tipo possano avere ricadute in termini di formazione degli insegnanti e di pratiche didattiche.

A number of microRNAs have been shown to regulate skeletal muscle development and differentiation. MicroRNA-222 is downregulated during myogenic differentiation and its overexpression leads to alteration of muscle differentiation process and specialized structures. By using RNA-induced silencing complex (RISC) pulldown followed by RNA sequencing, combined with in silico microRNA target prediction, we have identified two new targets of microRNA-222 involved in the regulation of myogenic differentiation, Ahnak and Rbm24. Specifically, the RNA-binding protein Rbm24 is a major regulator of muscle-specific alternative splicing and its downregulation by microRNA-222 results in defective exon inclusion impairing the production of muscle-specific isoforms of Coro6, Fxr1 and NACA transcripts. Reconstitution of normal levels of Rbm24 in cells overexpressing microRNA-222 rescues muscle-specific splicing. In conclusion, we have identified a new function of microRNA-222 leading to alteration of myogenic differentiation at the level of alternative splicing, and we provide evidence that this effect is mediated by Rbm24 protein.

In Balb 3T3 murine fibroblasts infected with retroviruses carrying the v-src oncogene, treatment with the glucocorticoid hormone dexamethasone induces a 10-fold increase in the number of transformed foci and of anchorage-independent colonies. In contrast, in NIH-3T3-infected cells the number of foci and of colonies growing in soft agar is considerably reduced by the addition of the hormone. The effect of dexamethasone on both Balb 3T3 and NIH 3T3 cells is dose-dependent and mediated by specific receptors. The expression of glucocorticoid receptors as well as transactivation of a mouse mammary tumor virus promoter in the presence of dexamethasone is comparable in the two cell lines. Dexamethasone does not change the expression and kinase activity of v-Src proteins either in freshly infected Balb 3T3 and NIH 3T3 cells or in morphologically normal clones or in transformed foci derived from infected Balb 3T3 cells stably expressing v-Src. However, in cocultivation assays of phenotypically normal clones of v-Src expressing Balb 3T3 cells mixed with a large excess of parental Balb 3T3 cells, the hormone is able to rescue the ability to form transformed foci of these otherwise normal cells. The present data point out a new role of glucocorticoid hormones in controlling transformation in a cell-specific manner through epigenetic mechanisms.

Eps8 is a recently identified substrate of receptor and nonreceptor tyrosine kinases implicated in the control of cell proliferation. To investigate potential functions of Eps8, its intracellular localization has been examined in several cell types. In cycling fibroblasts immunolabeling with antibodies to Eps8 reveals a punctate pattern within the perinuclear region and staining of motile peripheral cell extensions and cell-cell contact regions. Stimulation of quiescent Swiss 3T3 fibroblasts with serum induces a striking reorganization of the actin cytoskeleton which is accompanied by the enrichment of Eps8 and cortactin in membrane ruffles and lamellipodia. A similar accumulation of Eps8 to membrane ruffles is observed in cells treated with phorbol esters, which also induce marked changes of the F-actin cytoskeleton. The localization of Eps8 at the cell cortex is largely independent from the binding of Eps8 to an EGFR/ErbB-2 chimeric receptor. Moreover, fractionation studies reveal that a portion of the Eps8 molecules present in the cell periphery, unlike cortactin and the receptor, is resistant to mild extraction with detergent. Upon cellular transformation by the tyrosine kinase v-Src, a pool of Eps8 is recruited to newly formed specialized regions of the cytoskeleton, such as actin bodies in terminally differentiated myotubes and podosomes in fibroblasts, where cortactin and a variety of cytoskeletal proteins are also found. Extraction with Triton X-100 preserves the association of Eps8 to podosomes and leaves the majority of the v-Src tyrosine-phosphorylated Eps8 in the detergent-resistant fraction. The observed recruitment of Eps8 to highly dynamic cytoskeletal structures of normal and transformed cells suggests that Eps8 may play a role in the reorganization of the cytoskeleton, perhaps acting as a docking site for other signaling molecules.

The conversion of skeletal myoblasts to terminally differentiated myocytes is negatively controlled by several growth factors and oncoproteins. In this study, we have investigated the molecular mechanisms by which v-Src, a prototypic tyrosine kinase, perturbs myogenesis in primary avian myoblasts and in established murine C2C12 satellite cells. We determined the expression levels of the cell cycle regulators pRb, cyclin D1 and D3 and cyclin-dependent kinase inhibitors p21 and p27 in v-Src-transformed myoblasts and found that, in contrast to myogenin, they are normally modulated by differentiative cues, implying that v-Src affects myogenesis independent of cell proliferation. We then examined the levels of expression, DNA-binding ability and transcription-activation potentials of myogenic regulatory factors in transformed myoblasts and in myotubes after reactivation of a temperature-sensitive allele of v-Src. Our results reveal two distinct potential modes of repression targeted to myogenic factors. On the one hand, we show that v-Src reversibly inhibits the expression of MyoD and myogenin in C2C12 cells and of myogenin in quail myoblasts. Remarkably, these loci become resistant to activation of the kinase in the postmitotic compartment. On the other hand, we demonstrate that v-Src efficiently inhibits muscle gene expression by repressing the transcriptional activity of myogenic factors without affecting MyoD DNA-binding activity. Indeed, forced expression of MyoD and myogenin allows terminal differentiation of transformed myoblasts. Finally, we found that ectopic expression of the coactivator p300 restores transcription from extrachromosomal muscle-specific promoters.

SH3-containing proteins are involved in signal transduction by a number of growth factor receptors and in the organization of the cytoskeleton. The recently identified Eps8 protein, which contains an SH3 domain, is coupled functionally and physically to the EGFR and is tyrosine phosphorylated by this receptor and other receptors as well. Here, we examined the regulation of eps8 expression in response to mitogenic or differentiative signals. We show that Eps8 is expressed at low levels in resting fibroblasts, but its expression is strongly induced during activation by serum, phorbol esters and the v-src oncogene. Conversely, expression of Eps8, but not of other EGFR substrates such as Shc or Eps15, is virtually extinguished in non-proliferating, terminally differentiated murine myogenic cells. The putative role of Eps8 protein as a v-Src substrate was analysed in murine fibroblasts and in quail myogenic cells expressing a temperature-sensitive variant of the tyrosine kinase. Tyrosine phosphorylation of Eps8 was detected only at the permissive temperature. A non-myristylated, transformation-defective mutant of v-Src did not phosphorylate Eps8, whereas it phosphorylated Shc. Together, these findings indicate that Eps8 may be a critical substrate of v-Src. They further establish Eps8 as an example of a signal transducer whose expression senses the balance between growth and differentiation and might, therefore, be involved in the determination of the phenotype.