Characterization of a Mouse Model lacking Acetylcholine Receptor Activity during Embryonic Development

During embryonic development, acetylcholine (ACh) and the acetylcholine receptor (AChR) play a pivotal role in the establishment, maturation and maintenance of the neuromuscular junction (NMJ). In humans, genetic mutations affecting AChR-mediated signal transduction give rise to a variety of phenotypes mainly defined by muscle weakness, and known as congenital myasthenic syndromes (CMS), demonstrating the importance of AChR for the correct development of nerve/muscle contacts. However, thus far it has been impossible to determine the specific role played by AChR-mediated postsynaptic activity in NMJs during embryonic development. In this work, the effects caused by lack of postsynaptic activity in the NMJ were studied on a reporter mouse line generated by homologous recombination and expressing a structurally intact but functionally silent GFP-tagged AChR. In these animals, the subunit of the AChR, normally expressed during embryonic development, carries a point mutation (P121L) which causes fast-channel CMS in human patients. Homozygous -fc animals die at birth, and a wide variety of severe physiological abnormalities appears during embryonic development, caused by the silencing of AChR-mediated postsynaptic potentials. The size, shape and density of the NMJ were profoundly altered by lack of postsynaptic activity, although the overall number of receptors did not seem to change. A vastly increased outgrowth of motor axons could also be detected, and this alteration is associated with the absence of motoneuron death at late embryonic stages. Further alterations could be found in the disorganization of muscle fiber architecture, and the presence of multiple innervation sites on single muscle fibers. These results clarify the role of AChR-mediated postsynaptic activity in the proper development and maturation of nerve/muscle contacts, and support the possibility that presynaptic development is influenced by putative reciprocal signaling between nerve and muscle. The reporter mice provide a new tool to distinguish in an as-yet unknown resolution between activity-dependent and putative structurally-dependent pathways during NMJ maturation, leading to important implications in the study of synapse formation and maintenance, as well as in the field of receptor studies

Novel Mouse Model Reveals Distinct Activity-Dependent and –Independent Contributions to Synapse Development

The balanced action of both pre- and postsynaptic organizers regulates the formation of neuromuscular junctions (NMJ). The precise mechanisms that control the regional specialization of acetylcholine receptor (AChR) aggregation, guide ingrowing axons and contribute to correct synaptic patterning are unknown. Synaptic activity is of central importance and to understand synaptogenesis, it is necessary to distinguish between activity-dependent and activity-independent processes. By engineering a mutated fetal AChR subunit, we used homologous recombination to develop a mouse line that expresses AChR with massively reduced open probability during embryonic development. Through histological and immunochemical methods as well as electrophysiological techniques, we observed that endplate anatomy and distribution are severely aberrant and innervation patterns are completely disrupted. Nonetheless, in the absence of activity AChRs form postsynaptic specializations attracting motor axons and permitting generation of multiple nerve/muscle contacts on individual fibers. This process is not restricted to a specialized central zone of the diaphragm and proceeds throughout embryonic development. Phenotypes can be attributed to separate activity-dependent and -independent pathways. The correct patterning of synaptic connections, prevention of multiple contacts and control of nerve growth require AChR-mediated activity. In contrast, myotube survival and acetylcholine-mediated dispersal of AChRs are maintained even in the absence of AChR-mediated activity. Because mouse models in which acetylcholine is entirely absent do not display similar effects, we conclude that acetylcholine binding to the AChR initiates activity-dependent and activity-independent pathways whereby the AChR modulates formation of the NMJ

Covid-19 and the role of smoking: the protocol of the multicentric prospective study COSMO-IT (COvid19 and SMOking in ITaly).

The emergency caused by Covid-19 pandemic raised interest in studying lifestyles and comorbidities as important determinants of poor Covid-19 prognosis. Data on tobacco smoking, alcohol consumption and obesity are still limited, while no data are available on the role of e-cigarettes and heated tobacco products (HTP). To clarify the role of tobacco smoking and other lifestyle habits on COVID-19 severity and progression, we designed a longitudinal observational study titled COvid19 and SMOking in ITaly (COSMO-IT). About 30 Italian hospitals in North, Centre and South of Italy joined the study. Its main aims are: 1) to quantify the role of tobacco smoking and smoking cessation on the severity and progression of COVID-19 in hospitalized patients; 2) to compare smoking prevalence and severity of the disease in relation to smoking in hospitalized COVID-19 patients versus patients treated at home; 3) to quantify the association between other lifestyle factors, such as e-cigarette and HTP use, alcohol and obesity and the risk of unfavourable COVID-19 outcomes. Socio-demographic, lifestyle and medical history information will be gathered for around 3000 hospitalized and 700-1000 home-isolated, laboratory-confirmed, COVID-19 patients. Given the current absence of a vaccine against SARS-COV-2 and the lack of a specific treatment for -COVID-19, prevention strategies are of extreme importance. This project, designed to highly contribute to the international scientific debate on the role of avoidable lifestyle habits on COVID-19 severity, will provide valuable epidemiological data in order to support important recommendations to prevent COVID-19 incidence, progression and mortality

Time Lapse in Vivo Visualization of Developmental Stabilization of Synaptic Receptors at Neuromuscular Junctions

The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing stabilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synaptically during postnatal development and thus could mediate the stabilization of end plate AChR