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Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs.

Membrane depolarization is the signal that triggers release of neurotransmitter from nerve terminals. As a result of depolarization, voltage-dependent Ca 2؉ channels open, level of intracellular Ca 2؉ increases. and release of neurotransmitter commences. Previous study had shown that in rat brain synaptosomes, muscarinic acetylcholine (ACh) receptors (mAChRs) interact with soluble NSF attachment protein receptor proteins of the exocytic machinery in a voltage-dependent manner. It was suggested that this interaction might control the rapid, synchronous release of acetylcholine. The present study investigates the mechanism for such a voltagedependent interaction. Here we show that depolarization shifts mAChRs, specifically the m2 receptor subtype, to a low affinity state toward its agonists. At resting potential, mAChRs are in a high affinity state (K d of ϳ20 nM) and they shift to a low affinity state (K d of tens of M) upon membrane depolarization. In addition, interaction between m2 receptor subtype and the exocytic machinery increases with receptor occupancy. Both phenomena are independent of Ca 2؉ influx. We propose that these results may explain control of ACh release from nerve terminals. At resting potential the exocytic machinery is clamped due to its interaction with the occupied mAChR and depolarization relieves this interaction. This, together with Ca 2؉ influx, enables release of ACh to commence.Neurotransmitter release from nerve terminals is a unique instance of a secretory process where external stimuli must be handled with extreme speed and accuracy. Many proteins have been implicated to be involved in neurotransmitter release, but only a few have been found to be essential (1-4). The minimal essential core common to all secretory processes consists of the SNARE 1 (SNAP receptors) proteins (5): VAMP, syntaxin, and SNAP-25. In nerve terminals the essential core contains also voltage-dependent Ca 2ϩ channels and synaptotagmin (reviewed in Refs. 1, 2, 6, and 7). This set of proteins comprises the exocytic apparatus, henceforth called ExA. The ExA proteins have a combinatorial large repertoire of interactions that dynamically change throughout the process of neurotransmitter release (1, 6 -8). Such interactions are modulated by a variety of ions (9, 10), second messengers and signaling cascades (11-16), and membrane potential (17).Despite the overwhelming amount of accumulated data, a major challenge still remains to explain how the depolarization signal is transduced into molecular interactions that eventually lead to a rapid synchronous release of neurotransmitter.We have recently addressed this question and identified a group of membrane receptors not previously considered part of the control machinery of neurotransmitter release. We showed that, in rat brain synaptosomes, at Ca 2ϩ -depleted solution, presynaptic muscarinic ACh receptors (mAChRs) directly interact with the ExA, specifically with syntaxin and SNAP-25, and that this interaction is voltage-dependent (18). A related, large body of work...

1. Release of neurotransmitter into the synaptic cleft is the last step in the chain of molecular events following the arrival of an action potential at the nerve terminal. The neurotransmitter exerts negative feedback on its own release. This inhibition would be most effective if exerted on the first step in this chain of events, i.e. a step that is mediated by membrane depolarization. Indeed, in numerous studies feedback inhibition was found to be voltage dependent. 2. The purpose of this study is to investigate whether the mechanism underlying feedback inhibition of transmitter release resides in interaction between the presynaptic autoreceptors and the exocytic apparatus, specifically the soluble NSF-attachment protein receptor (SNARE) complex. 3. Using rat synaptosomes we show that the muscarinic ACh autoreceptor (mAChR) is an integral component of the exocytic machinery. It interacts with syntaxin, synaptosomalassociated protein of 25 kDa (SNAP-25), vesicle-associated membrane protein (VAMP) and synaptotagmin as shown using both cross-linking and immunoprecipitation. 4. The interaction between mAChRs and both syntaxin and SNAP-25 is modulated by depolarization levels; binding is maximal at resting potential and disassembly occurs at higher depolarization. 5. This voltage-dependent interaction of mAChRs with the secretory core complex appears suitable for controlling the rapid, synchronous neurotransmitter release at nerve terminals.

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The cytoarchitecture, synaptic connectivity, and physiological properties of neurons are determined during their development by the interactions between the intrinsic properties of the neurons and signals provided by the microenvironment through which they grow. Many of these interactions are mediated and translated to specific growth patterns and connectivity by specialized compartments at the tips of the extending neurites: the growth cones (GCs). The mechanisms underlying GC formation at a specific time and location during development, regeneration, and some forms of learning processes, are therefore the subject of intense investigation. Using cultured Aplysia neurons we studied the cellular mechanisms that lead to the transformation of a differentiated axonal segment into a motile GC. We found that localized and transient elevation of the free intracellular calcium concentration ([Ca2+]i) to 200-300 microM induces GC formation in the form of a large lamellipodium that branches up into growing neurites. By using simultaneous on-line imaging of [Ca2+]i and of intraaxonal proteolytic activity, we found that the elevated [Ca2+]i activate proteases in the region in which a GC is formed. Inhibition of the calcium-activated proteases prior to the local elevation of the [Ca2+]i blocks the formation of GCs. Using retrospective immunofluorescent methods we imaged the proteolysis of the submembrane spectrin network, and the restructuring of the cytoskeleton at the site of GC formation. The restructuring of the actin and microtubule network leads to local accumulation of transported vesicles, which then fuse with the plasma membrane in support of the GC expansion.

GNE myopathy is an adult onset neuromuscular disorder characterized by slowly progressive distal and proximal muscle weakness, caused by missense recessive mutations in the GNE gene. Although the encoded bifunctional enzyme is well known as the limiting factor in the biosynthesis of sialic acid, no clear mechanisms have been recognized to account for the muscle atrophic pathology, and novel functions for GNE have been hypothesized. Two major issues impair studies on this protein. First, the expression of the GNE protein is minimal in human and mice muscles and there is no reliable antibody to follow up endogenous expression. Second, no reliable animal model is available for the disease and cellular models from GNE myopathy patients’ muscle cells (expressing the mutated protein) are less informative than expected. In order to broaden our knowledge on GNE functions in muscle, we have taken advantage of the CRISPR/Cas9 method for genome editing to first, add a tag to the endogenous Gne gene in mouse, allowing the determination of the spatiotemporal expression of the protein in the organism, using well established and reliable antibodies against the specific tag. In addition we have generated a Gne knock out murine muscle cell lineage to identify the events resulting from the total lack of the protein. A thorough multi-omics analysis of both cellular systems including transcriptomics, proteomics, phosphoproteomics and ubiquitination, unraveled novel pathways for Gne, in particular its involvement in cell cycle control and in the DNA damage/repair pathways. The elucidation of fundamental mechanisms of Gne in normal muscle may contribute to the identification of the disrupted functions in GNE myopathy, thus, to the definition of novel biomarkers and possible therapeutic targets for this disease.

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