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Machado-Joseph disease (MJD; also called spinocerebellar ataxia type 3) is a dominantly inherited late-onset neurodegenerative disorder caused by expansion of polyglutamine (polyQ)-encoding CAG repeats in the MJD1 gene (also known [...]

Trans-soluble AT-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complexes formed between the SNARE motifs of synaptobrevin II, SNAP-25, and syntaxin play an essential role in Ca2+-regulated exocytosis. Apart from the well studied interactions of the SNARE domains, little is known about the functional relevance of other evolutionarily conserved structures in the SNARE proteins. Here, we show that substitution of two highly conserved tryptophan residues within the juxtamembrane domain (JMD) of the vesicular SNARE Synaptobrevin II (Sybil) profoundly impairs priming of granules in mouse chromaffin cells without altering catecholamine release from single vesicles. Using molecular dynamic simulations of membrane-embedded Sybil, we show that Trp residues of the JMD influence the electrostatic surface potential by controlling the position of neighboring lysine and arginine residues at the membrane-water interface. Our observations indicate a decisive role of the tryptophan moiety of Sybil in keeping the vesicles in the release-ready state and support a model wherein tryptophan-mediated proteinlipid interactions assist in bridging the apposing membranes before fusion. [ABSTRACT FROM AUTHOR]

Summary: Assembly of SNARE proteins between opposing membranes mediates fusion of synthetic liposomes, but it is unknown whether SNAREs act during exocytosis at the moment of Ca2+ increase, providing the molecular force for fusion of secretory vesicles. Here, we show that execution of pre- and postfusional steps during chromaffin granule exocytosis depends crucially on a short molecular distance between the complex-forming SNARE motif and the transmembrane anchor of the vesicular SNARE protein synaptobrevin II. Extending the juxtamembrane region of synaptobrevin by insertion of flexible “linkers” reduces priming of granules, delays initiation of exocytosis upon stepwise elevation of intracellular calcium, attenuates fluctuations of early fusion pores, and slows rapid expansion of the pore in a linker-length dependent fashion. These observations provide evidence that v-SNARE proteins drive Ca2+-triggered membrane fusion at millisecond time scale and support a model wherein continuous molecular pulling by SNAREs guides the vesicle throughout the consecutive stages of exocytosis. [Copyright &y& Elsevier]

Angelman syndrome (AS) is a severe neurodevelopmental disorder featuring ataxia, cognitive impairment, and drug-resistant epilepsy. AS is caused by mutations or deletion of the maternal copy of the paternally imprinted UBE3A gene, with current precision therapy approaches focusing on re-expression of UBE3A . Certain phenotypes, however, are difficult to rescue beyond early development. Notably, a cluster of microRNA binding sites was reported in the untranslated Ube3a1 transcript, including for miR-134, suggesting that AS may be associated with microRNA dysregulation. Here, we report levels of miR-134 and key targets are normal in the hippocampus of mice carrying a maternal deletion of Ube3a ( Ube3a m-/p+ ). Nevertheless, intracerebroventricular injection of an antimiR oligonucleotide inhibitor of miR-134 (Ant-134) reduced audiogenic seizure severity over multiple trials in 21- and 42-day-old AS mice. Interestingly, Ant-134 also improved distance traveled and center crossings of AS mice in the open-field test. Finally, we show that silencing miR-134 can upregulate targets of miR-134 in neurons differentiated from Angelman patient-derived induced pluripotent stem cells. These findings indicate that silencing miR-134 and possibly other microRNAs could be useful to treat clinically relevant phenotypes with a later developmental window in AS., Competing Interests: The Royal College of Surgeons in Ireland (D.C.H.) holds a patent for the inhibition of microRNA-134 for the treatment of seizure-related disorders and other neurologic injuries (US 9,803,200 B2). J.W., M.A.J., and M.T.M. are employees of F Hoffman-La Roche Ltd. All other authors have no competing interests., (© 2022 The Author(s).)