Single cell analysis reveals the involvement of the long non-coding RNA Pvt1 in the modulation of muscle atrophy and mitochondrial network.

Long non-coding RNAs (lncRNAs) are emerging as important players in the regulation of several aspects of cellular biology. For a better comprehension of their function, it is fundamental to determine their tissue or cell specificity and to identify their subcellular localization. In fact, the activity of lncRNAs may vary according to cell and tissue specificity and subcellular compartmentalization. Myofibers are the smallest complete contractile system of skeletal muscle influencing its contraction velocity and metabolism. How lncRNAs are expressed in different myofibers, participate in metabolism regulation and muscle atrophy or how they are compartmentalized within a single myofiber is still unknown. We compiled a comprehensive catalog of lncRNAs expressed in skeletal muscle, associating the fiber-type specificity and subcellular location to each of them, and demonstrating that many lncRNAs can be involved in the biological processes de-regulated during muscle atrophy. We demonstrated that the lncRNA Pvt1, activated early during muscle atrophy, impacts mitochondrial respiration and morphology and affects mito/autophagy, apoptosis and myofiber size in vivo. This work corroborates the importance of lncRNAs in the regulation of metabolism and neuromuscular pathologies and offers a valuable resource to study the metabolism in single cells characterized by pronounced plasticity.

Metal Dyshomeostasis and Their Pathological Role in Prion and Prion-Like Diseases: The Basis for a Nutritional Approach.

Metal ions are key elements in organisms' life acting like cofactors of many enzymes but they can also be potentially dangerous for the cell participating in redox reactions that lead to the formation of reactive oxygen species (ROS). Any factor inducing or limiting a metal dyshomeostasis, ROS production and cell injury may contribute to the onset of neurodegenerative diseases or play a neuroprotective action. Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are a group of fatal neurodegenerative disorders affecting the central nervous system (CNS) of human and other mammalian species. The causative agent of TSEs is believed to be the scrapie prion protein PrPSc, the ß sheet-rich pathogenic isoform produced by the conformational conversion of the α-helix-rich physiological isoform PrPC. The peculiarity of PrPSc is its ability to self-propagate in exponential fashion in cells and its tendency to precipitate in insoluble and protease-resistance amyloid aggregates leading to neuronal cell death. The expression "prion-like diseases" refers to a group of neurodegenerative diseases that share some neuropathological features with prion diseases such as the involvement of proteins (α-synuclein, amyloid ß, and tau) able to precipitate producing amyloid deposits following conformational change. High social impact diseases such as Alzheimer's and Parkinson's belong to prion-like diseases. Accumulating evidence suggests that the exposure to environmental metals is a risk factor for the development of prion and prion-like diseases and that metal ions can directly bind to prion and prion-like proteins affecting the amount of amyloid aggregates. The diet, source of metal ions but also of natural antioxidant and chelating agents such as polyphenols, is an aspect to take into account in addressing the issue of neurodegeneration. Epidemiological data suggest that the Mediterranean diet, based on the abundant consumption of fresh vegetables and on low intake of meat, could play a preventive or delaying role in prion and prion-like neurodegenerative diseases. In this review, metal role in the onset of prion and prion-like diseases is dealt with from a nutritional, cellular, and molecular point of view.

Primary cultures from fetal bovine brain.

This study describes a method of obtaining primary cultures from the cerebral cortex and the hypothalamus of bovine fetuses. We describe here the influence of tissue origin, developmental stage and culture medium conditions on cell differentiation and prevalence of neurons vs glial cells. To identify optimal conditions for obtaining and growing viable neurons and astrocytes in culture, we tested early, middle and late stages of development. Explants from cortex, early stages (week 10 of pregnancy out of 36) and low fetal calf serum concentration (1%) yielded maximum amounts of neurons. Fresh and thawed tissues gave comparable results.

Production in Escherichia coli, folding, purification and characterization of notexin with wild type sequence and with N-terminal and catalytic site mutations.

Notexin (Ntx) is a group I phospholipase A2 (PLA2) protein, main component of the Australian snake Notechis scutatus scutatus venom. It is both a presynaptic neurotoxin and a myotoxin. In this work, for the first time, a method for the production and folding of recombinant Ntx was developed. Ntx was produced with wild type sequence (rNtx), with an extra peptide (T7-Ntx) or a methionine (M-Ntx) before Asn-1, and with Asn-1 substituted by alanine (Ntx-A1) or by serine (Ntx-S1). The proteins were analyzed for their catalytic and toxic activities. rNtx activity resulted to be comparable to that of the venom extracted protein. The Ntx N-terminus was found to have a major influence on both the catalytic and toxic activities of the protein. The first amino acid of snake venom PLA2s is highly conserved: it is an asparagine in about all group I PLA2s, while in most (>70%) of group II PLA2s it is a serine or an asparagine. Interestingly, Ntx-S1 resulted to be, for both enzymatic and toxic activities, the mutant most similar to the wild type protein. The role of the catalytic activity of Ntx in its toxicity was investigated by replacing the aspartic acid 49, involved in the coordination of the cofactor calcium ion, by a lysine. The obtained mutant (Ntx-K49) is deprived of catalytic activity but possesses a residual toxicity.

Copper(II) binding to the human Doppel protein may mark its functional diversity from the prion protein.

Doppel (Dpl) is the first described homologue of the prion protein, the main constituent of the agent responsible for prion diseases. The cellular prion protein (PrP(C)) is predominantly present in the central nervous system. Although its role is not yet completely clarified, PrP(C) seems to be involved in Cu(2+) recycling from synaptic clefts and in preventing neuronal oxidative damage. Conversely, Dpl is expressed in heart and testis and has been shown to regulate male fertility by intervening in gametogenesis and sperm-egg interactions. Therefore, despite a high sequence homology and a similar three-dimensional fold, the functions of PrP(C) and Dpl appear unrelated. Here we show by electron paramagnetic resonance and fluorescence spectroscopy that the in vitro binding of copper(II) to human recombinant Dpl occurs with a different pattern from that observed for recombinant PrP. At physiological pH values, two copper(II)-binding sites with different affinities were found in Dpl. At lower pH values, two additional copper(II)-binding sites can be identified as follows: one complex is present only at pH 4, and the other is observed in the pH range 5-6. As derived from the electron paramagnetic resonance characteristics, all Dpl-copper(II) complexes have a different coordination sphere from those present in PrP. Furthermore, in contrast to the effect shown previously for PrP(C), addition of Cu(2+) to Dpl-expressing cells does not cause Dpl internalization. These results suggest that binding of the ion to PrP(C) and Dpl may contribute to the different functional roles ascribed to these highly homologous proteins.