Can human pluripotent stem cell-derived cardiomyocytes advance understanding of muscular dystrophies?

Muscular dystrophies (MDs) are clinically and molecularly a highly heterogeneous group of single-gene disorders that primarily affect striated muscles. Cardiac disease is present in several MDs where it is an important contributor to morbidity and mortality. Careful monitoring of cardiac issues is necessary but current management of cardiac involvement does not effectively protect from disease progression and cardiac failure. There is a critical need to gain new knowledge on the diverse molecular underpinnings of cardiac disease in MDs in order to guide cardiac treatment development and assist in reaching a clearer consensus on cardiac disease management in the clinic. Animal models are available for the majority of MDs and have been invaluable tools in probing disease mechanisms and in pre-clinical screens. However, there are recognized genetic, physiological, and structural differences between human and animal hearts that impact disease progression, manifestation, and response to pharmacological interventions. Therefore, there is a need to develop parallel human systems to model cardiac disease in MDs. This review discusses the current status of cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSC) to model cardiac disease, with a focus on Duchenne muscular dystrophy (DMD) and myotonic dystrophy (DM1). We seek to provide a balanced view of opportunities and limitations offered by this system in elucidating disease mechanisms pertinent to human cardiac physiology and as a platform for treatment development or refinement

The Remains of the Day: The International Economic Order in the Era of Disintegration

The last two decades of the XX century have been marked by a vigorous acceleration of international economic integration both at a global and regional level. States accepted pervasive constraints on their national decision-making in the hope that stability and predictability would favor economic growth. This model of international economic integration, however, has recently shown worrying signs of ‘disintegration’. Disintegration manifests itself both as disintegration of the international legal regimes which compose the international economic order; and disintegration through law, namely the social, economic and environmental disintegration phenomena,triggered or at leastfacilitated by these regimes. Relying on the paradox integration/disintegration as an analytical framework, this article draws a blueprint of the various disintegration phenomena, which are further analyzed in the individual contributions to this Special Issue. It seeks to identify a relationship betweenthetwo dimensions of disintegration and detect possible correlation patterns. Last, after engaging with the different normative alternatives put forward by the contributors, it concludes by calling for a rethinking of the traditional approach to international economic integration. This reconceptualization should be premised on the full realization that the current model entails a great deal of environmental and social ‘hidden costs’

The remains of the day: The international economic order in the era of disintegration

The last two decades of the XX century have been marked by a vigorous acceleration of international economic integration both at a global and regional level. States accepted pervasive constraints on their national decision-making in the hope that stability and predictability would favor economic growth. This model of international economic integration, however, has recently shown worrying signs of 'disintegration'. Disintegration manifests itself both as disintegration of the international legal regimes which compose the international economic order; and disintegration through law, namely the social, economic and environmental disintegration phenomena, triggered or at least facilitated by these regimes. Relying on the paradox integration/disintegration as an analytical framework, this article draws a blueprint of the various disintegration phenomena, which are further analyzed in the individual contributions to this Special Issue. It seeks to identify a relationship between the two dimensions of disintegration and detect possible correlation patterns. Last, after engaging with the different normative alternatives put forward by the contributors, it concludes by calling for a rethinking of the traditional approach to international economic integration. This reconceptualization should be premised on the full realization that the current model entails a great deal of environmental and social 'hidden costs'

Investigating the Impact of Delivery Routes for Exon Skipping Therapies in the CNS of DMD Mouse Models

Nucleic acid-based therapies have demonstrated great potential for the treatment of monogenetic diseases, including neurologic disorders. To date, regulatory approval has been received for a dozen antisense oligonucleotides (ASOs); however, these chemistries cannot readily cross the blood–brain barrier when administered systemically. Therefore, an investigation of their potential effects within the central nervous system (CNS) requires local delivery. Here, we studied the brain distribution and exon-skipping efficacy of two ASO chemistries, PMO and tcDNA, when delivered to the cerebrospinal fluid (CSF) of mice carrying a deletion in exon 52 of the dystrophin gene, a model of Duchenne muscular dystrophy (DMD). Following intracerebroventricular (ICV) delivery (unilateral, bilateral, bolus vs. slow rate, repeated via cannula or very slow via osmotic pumps), ASO levels were quantified across brain regions and exon 51 skipping was evaluated, revealing that tcDNA treatment invariably generates comparable or more skipping relative to that with PMO, even when the PMO was administered at higher doses. We also performed intra-cisterna magna (ICM) delivery as an alternative route for CSF delivery and found a biased distribution of the ASOs towards posterior brain regions, including the cerebellum, hindbrain, and the cervical part of the spinal cord. Finally, we combined both ICV and ICM injection methods to assess the potential of an additive effect of this methodology in inducing efficient exon skipping across different brain regions. Our results provide useful insights into the local delivery and associated efficacy of ASOs in the CNS in mouse models of DMD. These findings pave the way for further ASO-based therapy application to the CNS for neurological disease

Laminin and α-Dystroglycan Mediate Acetylcholine Receptor Aggregation via a MuSK-Independent Pathway

Specific isoforms of laminin (LN) are concentrated at neuromuscular junctions (NMJs) where they may participate in synaptic organization or function. In myotubes from C2 cells, LN is concentrated within the majority of spontaneous acetylcholine receptor (AChR) aggregates. Neural agrin substantially increases this colocalization, suggesting that agrin can recruit LN into AChR aggregates. Addition of LN to C2 myotubes induces a more than twofold increase in the number of AChR aggregates. These aggregates have a larger size and are more dense than are those induced by agrin, suggesting that LN is involved in the growth and/or stabilization of AChR aggregates. Consistent with this hypothesis, an antiserum to LN reduces the size of individual AChR aggregates but increases their number. In C2 myotubes, extracellular matrix receptors containing the integrin beta1 subunit are poorly colocalized with AChR aggregates, suggesting that integrins may not be involved in LN-induced aggregation. In contrast, almost all AChR aggregates are associated with dystroglycan immunoreactivity, and monoclonal antibody (mAb) IIH6 against alpha-dystroglycan (alpha-DG), a LN and agrin receptor, causes a concentration-dependent inhibition of LN-induced aggregation. Moreover, S27 cells, which lack a functional alpha-DG, and two C2-derived cell lines expressing antisense DG mRNA fail to aggregate AChRs in response to LN. Finally, LN-induced AChR aggregation does not involve the phosphorylation of the muscle-specific tyrosine kinase receptor (MuSK) or the AChR beta subunit. We hypothesize that the interaction of LN with alpha-DG contributes to the growth and/or stabilization of AChR microaggregates into macroaggregates at the developing NMJ via a MuSK-independent mechanism

Evaluation of the dystrophin carboxy-terminal domain for micro-dystrophin gene therapy in cardiac and skeletal muscles in the DMDmdx rat model

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the gene encoding dystrophin. Gene therapy using micro-dystrophin (MD) transgenes and recombinant adeno-associated virus (rAAV) vectors hold great promise. To overcome the limited packaging capacity of rAAV vectors, most MD do not include dystrophin carboxy-terminal (CT) domain. Yet, the CT domain is known to recruit α1- and β1-syntrophins and α-dystrobrevin, a part of the dystrophin-associated protein complex (DAPC), which is a signaling and structural mediator of muscle cells. In this study, we explored the impact of inclusion of the dystrophin CT domain on ΔR4-23/ΔCT MD (MD1), in DMDmdx rats, which allows for relevant evaluations at muscular and cardiac levels. We showed by LC-MS/MS that MD1 expression is sufficient to restore the interactions at a physiological level of most DAPC partners in skeletal and cardiac muscles, and that inclusion of the CT domain increases the recruitment of some DAPC partners at supra-physiological levels. In parallel, we demonstrated that inclusion of the CT domain does not improve MD1 therapeutic efficacy on DMD muscle and cardiac pathologies. Our work highlights new evidences of the therapeutic potential of MD1 and strengthens the relevance of this candidate for gene therapy of DMD

The role of dystroglycan in muscular dystrophy and synaptogenesis /

alpha- and beta-dystroglycan (DG) were first identified as members of an oligomeric, transmembrane complex expressed in muscle and linking laminin (LN) in the extracellular matrix (ECM) to dystrophin in the submembraneous cytoskeleton. This dystrophin-associated glycoprotein complex (DGC) has been proposed to perform a structural role in skeletal muscle, its loss leading to loss of membrane integrity, muscle fiber degeneration and muscular dystrophy. alpha- and beta-DG appear to form the core of the DGC since alpha-DG is a high affinity LN receptor while beta-DG is a transmembrane protein that anchors alpha-DG to the membrane and interacts with dystrophin intracellularly. In order to determine the involvement of DG in skeletal muscle homeostasis and in LN assembly, we generated mouse muscle cell lines deficient in DG expression. Extensive characterization of these cells revealed that DG is essential for LN assembly on the surface of mature myotubes but that it is not involved in the maintenance of membrane integrity in culture. However, DG-deficient cells show increased apoptotic cell death during and after the period of myoblast differentiation into myotubes, indicating that DG is important for muscle cell survival.The ECM molecule agrin has been implicated in the induction of acetylcholine receptor (AChR) aggregation at the neuromuscular junction (NMJ). The C-terminus of agrin shares significant homology with the region of LN that interacts with alpha-DG; we therefore reasoned that alpha-DG could be an agrin receptor. Ligand overlay assays revealed that alpha-DG binds agrin with high affinity and antibody perturbation experiments indicated that alpha-DG is involved in agrin-induced aggregation of AChRs. The role of alpha-DG in AChR aggregation was further studied using the DG deficient muscle cell lines. We found that alpha-DG is involved in the later stages of agrin-induced AChR aggregation.We further localized DG and two of its intracellular ligands, dystrophin and its autosomal homologue utrophin, to a synaptic layer in the retina suggesting a role for DG in central nervous system synapses. DG, utrophin and LN are also co-expressed around blood vessels indicating a possible function in blood-brain barrier homeostasis

Prospettive e criticità della tutela penale dei dati personali.

Stante che la società odierna è andata incontro a profonde trasformazioni in seguito all’evoluzione vertiginosa dell’utilizzo delle nuove tecnologie, del ruolo sempre più di primo piano che rivestono e della stessa natura di Internet, anche in virtù sui meccanismi da economia di scala che ormai ne sono parte integrante, emerge la necessità di un adattamento del diritto ad esso, affinché si abbia una regolamentazione efficace. Siffatta operazione risulta imprescindibile per preservare i valori e i principi fondamentali invalsi nell’orizzonte di senso dello Stato di diritto, con il corollario della protezione, da parte di questi, dei diritti dell’individuo: viene abbandonata l’idea della Rete come spazio libero dal diritto. Stabilito ciò, il criterio di massima da seguire nel tentativo di trasporre la sovranità statale nel mondo della Rete è stato sintetizzato nella massima per cui che ciò che è illecito off line deve essere illecito anche on line. Inoltre, la rete si è rivelata essere un ambiente particolarmente favorevole a comportamenti illeciti, lesivi dei diritti dei singoli: in virtù di questo e parallelamente all’evoluzione tecnologica, è cambiato negli anni l’approccio alla criminalità informatica, diventata essa stessa “criminalità nel cyberspace”; categoria che non può essere circoscritta a un numero chiuso o limitato di reati e quindi di vittime potenziali, ma che invece include una molteplicità indefinita di illeciti e di modalità di offesa di diritti e interessi altrui. L’intervento sanzionatorio presenta diverse sfide, sia sotto il profilo dell’individuazione del locus commissi delicti (e quindi dell’autorità competente) sia sotto quello della conformità ai principi che informano il diritto penale, con particolare riferimento al principio di determinatezza e a quello di offensività. Per questo, si è proceduto a una ricognizione del settore del diritto penale dell’informatica. Nel campo specifico dei dati personali, la prima operazione da compiere consiste nella ricostruzione della normativa a riguardo e soprattutto nell’individuazione e trattazione del bene giuridico potenzialmente leso da condotte abusive aventi ad oggetto i dati degli utenti. In questo senso, al tradizionale diritto alla riservatezza, ormai consolidato a livello costituzionale, si affianca il diritto alla protezione dei dati personali, da intendersi come corretta gestione degli stessi, che gode di riconoscimento espresso ad opera delle massime fonti europee. Si tratta di una elaborazione in cui numerosi contributi sono stati offerti dalla dottrina. Di fronte all’importanza del bene giuridico, si analizza come l’ordinamento italiano abbia elaborato un sistema di tutele e quanta influenza vi abbia avuto l’opera del legislatore europeo. La disciplina europea riveste infatti un’importanza fondamentale nel settore della protezione dei dati personali, in ispecie dopo l’emanazione del Regolamento 679/2016, che ha ridefinito l’intera materia e fornito nuove direttrici di intervento. L’attuale panorama italiano, conseguente alla novella che nel 2018 ha interessato il Codice in materia di protezione dei dati personali, si è orientato verso un approccio complessivamente più attento alla prevenzione del rischio di reato. Nondimeno, ampio spazio è riservato all’analisi degli illeciti previsti nell’ordinamento italiano per sanzionare condotte di illecito trattamento dei dati personali degli utenti, anche considerando il contributo offerto dalla dottrina e dalla giurisprudenza. Proprio questa impostazione generale, unitamente all’analisi della struttura della rete e delle caratteristiche dei soggetti che vi operano, porta naturalmente ad approfondire il discorso riguardante la figura degli intermediari. Questi soggetti si differenziano infatti dai singoli sia per il potenziale impatto delle loro azioni sia per l’essere a tutti gli effetti delle imprese, ergo delle persone giuridiche. Partendo da questo, si sono esaminati vari possibili paradigmi di tutela, partendo dalla possibilità, ampiamente esaminata dalla dottrina, della configurazione in capo agli stessi di una responsabilità omissiva impropria; opzione poi rigettata in virtù sia dell’inconsistenza sul piano delle categorie penalistiche, sia della conseguenza indiretta di investire tali soggetti del ruolo di censori della rete. La risposta preferibile appare invece essere, in consonanza con la linea tracciata dal legislatore europeo, quella basata sulla prevenzione e gestione del rischio. Si è quindi analizzata l’opportunità di inserire i reati in materia di trattamento dei dati personali nel novero dei reati presupposto ex D. lgs. 231/2001, ripercorrendo sia i punti di contatto tra le due discipline e quindi le plausibili ricadute positive, sia le criticità. Partendo da queste ultime, uno sguardo in ottica comparatistica sembrerebbe suggerire la possibilità di ascrivere direttamente all’ente la responsabilità per abusi o illeciti trattamenti dei dati