Mammalian brains are highly enriched with sialoglycans, which have been implicated in brain development and disease progression. However, in vivo labeling and visualization of sialoglycans in the mouse brain remain a challenge because of the blood−brain barrier. Here we introduce a liposome-assisted bioorthogonal reporter (LABOR) strategy for shuttling 9-azido sialic acid (9AzSia), a sialic acid reporter, into the brain to metabolically label sialoglycoconjugates, including sialylated glycoproteins and glycolipids. Subsequent bioorthogonal conjugation of the incorporated 9AzSia with fluorescent probes via click chemistry enabled fluorescence imaging of brain sialoglycans in living animals and in brain sections. Newly synthesized sialoglycans were found to widely distribute on neuronal cell surfaces, in particular at synaptic sites. Furthermore, large-scale proteomic profiling identified 140 brain sialylated glycoproteins, including a wealth of synapse-associated proteins. Finally, by performing a pulse−chase experiment, we showed that dynamic sialylation is spatially regulated, and that turnover of sialoglycans in the hippocampus is significantly slower than that in other brain regions. The LABOR strategy provides a means to directly visualize and monitor the sialoglycan biosynthesis in the mouse brain and will facilitate elucidating the functional role of brain sialylation.brain | sialic acid | live imaging | glycoproteomics | histochemistry S ialic acids are a family of negatively charged monosaccharides that are commonly expressed as outer terminal residues of cell surface glycans and widely distributed throughout mammalian tissues (1). Intriguingly, the brain is the organ with the highest level of sialylated glycans and the only organ, in mammals, with more sialic acids carried by glycolipids than glycoproteins (2). Accumulating evidence indicates that sialic acids are an essential nutrient for brain development and cognition (3). Gangliosides (i.e., glycosphingolipids containing α2,3-linked sialic acids) undergo dramatic changes in both structural complexity and expression density as the brain develops and matures (4). Polysialic acid (PSA), a linear α2,8-linked polymer of sialic acid, is predominantly attached to the N-glycans of neural cell adhesion molecule, which regulates neuronal differentiation and migration (5). In addition, α2,3-linked sialic acids and, less commonly, α2,6-linked sialic acids terminate N-glycans and O-glycans on synaptic proteins, mediating neural transmission and synaptic plasticity (6, 7). Aberrant sialylation has been implicated in cancer cell metastasis to the brain (8), lysosomal storage disorders (9), and neurodegenerative diseases (10).Sialic acid metabolism can be probed in vivo using the recently emerged bioorthogonal chemical reporter strategy, in which analogs of sialic acid or its biosynthetic precursor N-acetylmannosamine (ManNAc) containing a chemical reporter (e.g., the azide) are used as metabolic tracers for labeling sialoglycans in live cells and in living animals...
Lentiviral technology has proven to be a powerful tool to express exogenous genes in dividing and non-dividing cells. Currently, most protocols for generating high-titer lentivirus require ultracentrifugation, which can be an instrumental barrier for routine operations in a laboratory. In this study, the effect of relative centrifugal force (RCF) on the concentration efficiency of the lentivirus was systematically explored, and it was found that sucrose gradient centrifugation with a relatively low speed (≤10,000 g) robustly produces a high-titer virus (up to 2 × 108 TU/ml). The optimal sucrose concentration is 10%, and the recovery rate of the functional virus is greater than 80%. The infection efficiency of both concentrated and un-concentrated lentivirus decreases rapidly when the viruses are stored at 4 °C (τ ≈ 1.3 days) or subjected to multiple freeze-thaw cycles (τ = 1.1 rounds). In summary, we describe an efficient and easy-to-handle protocol for high-titer lentivirus purification.
The fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane requires two classes of molecules—SNAP receptor (SNARE) and Sec1/Munc18 (SM) protein. Reconstitution studies suggest that the SM protein Munc18-1 promotes the zippering of trans-SNARE complexes and accelerates the kinetics of SNARE-dependent membrane fusion. However, the physiological role of this trans-SNARE-regulating function in synaptic exocytosis remains to be established. Here we first demonstrate that two mutations in the vesicle-anchored v-SNARE selectively impair the ability of Munc18-1 to promote trans-SNARE zippering, whereas other known Munc18-1/SNARE-binding modes are unaffected. In cultured neurons, these v-SNARE mutations strongly inhibit spontaneous as well as evoked neurotransmitter release, providing genetic evidence for the trans-SNARE-regulating function of Munc18-1 in synaptic exocytosis. Finally, we show that the trans-SNARE-regulating function of Munc18-1 is compromised by a mutation associated with Ohtahara Syndrome, a severe form of epilepsy.
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