Small-scale isolation of synaptic vesicles from mammalian brain

Ahmed, Saheeb; Holt, Matthew; Riedel, Dietmar; Jahn, Reinhard

doi:10.1038/nprot.2013.053

Cited by 81 publications

(88 citation statements)

References 27 publications

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“…Many studies have investigated the proteome of endosomes, synaptic or secretory vesicles, leading to an exhaustive list of proteins present in these various organelles91415161718192021. However, the molecular composition of vesicles that are transported in neurons has not been defined.…”

Section: Resultsmentioning

confidence: 99%

Self-propelling vesicles define glycolysis as the minimal energy machinery for neuronal transport

et al. 2016

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The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) facilitates fast axonal transport in neurons. However, given that GAPDH does not produce ATP, it is unclear whether glycolysis per se is sufficient to propel vesicles. Although many proteins regulating transport have been identified, the molecular composition of transported vesicles in neurons has yet to be fully elucidated. Here we selectively enrich motile vesicles and perform quantitative proteomic analysis. In addition to the expected molecular motors and vesicular proteins, we find an enrichment of all the glycolytic enzymes. Using biochemical approaches and super-resolution microscopy, we observe that most glycolytic enzymes are selectively associated with vesicles and facilitate transport of vesicles in neurons. Finally, we provide evidence that mouse brain vesicles produce ATP from ADP and glucose, and display movement in a reconstituted in vitro transport assay of native vesicles. We conclude that transport of vesicles along microtubules can be autonomous.

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Section: Resultsmentioning

confidence: 99%

Self-propelling vesicles define glycolysis as the minimal energy machinery for neuronal transport

et al. 2016

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“…Synaptic vesicles from mice brain were purified as described in detail elsewhere46. Briefly, mice brains were homogenized in homogenization buffer supplemented with protease inhibitors, using a glass-Teflon homogenizer.…”

Section: Methodsmentioning

confidence: 99%

MicroRNA exocytosis by large dense-core vesicle fusion

Gumurdu

Yıldız

Eren

et al. 2017

Sci Rep

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Neurotransmitters and peptide hormones are secreted into outside the cell by a vesicle fusion process. Although non-coding RNA (ncRNA) that include microRNA (miRNA) regulates gene expression inside the cell where they are transcribed, extracellular miRNA has been recently discovered outside the cells, proposing that miRNA might be released to participate in cell-to-cell communication. Despite its importance of extracellular miRNA, the molecular mechanisms by which miRNA can be stored in vesicles and released by vesicle fusion remain enigmatic. Using next-generation sequencing, vesicle purification techniques, and synthetic neurotransmission, we observe that large dense-core vesicles (LDCVs) contain a variety of miRNAs including miR-375. Furthermore, miRNA exocytosis is mediated by the SNARE complex and accelerated by Ca2+. Our results suggest that miRNA can be a novel neuromodulator that can be stored in vesicles and released by vesicle fusion together with classical neurotransmitters.

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“…Finally, we used a recent protocol to isolate SVs from mouse brains (Ahmed et al . ). The ~35 nm vesicles isolated with this protocol were recognized by the anti‐synaptophysin antibody, a transmembrane glycoprotein present in almost all neurons that participates in synaptic transmission.…”

Section: Resultsmentioning

confidence: 97%

“…SVs were purified according to the protocol described by Ahmed et al . () as described in detail in the Supporting information. Briefly, the brains of two young (6‐8 weeks) male wild‐type C57BL/6J mice (stock 000664, The Jackson Laboratory, RRID: IMSR_JAX:000664) were obtained and homogenized in 4 mM HEPES buffer pH 7.4, 320 mM sucrose at 4°C supplemented with protease inhibitor.…”

Section: Methodsmentioning

confidence: 99%

Amyloid oligomerization of the Parkinson's disease related protein α‐synuclein impacts on its curvature‐membrane sensitivity

Gallea

Ambroggio

Vilcaes

et al. 2018

Journal of Neurochemistry

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The amyloid aggregation of the presynaptic protein α-synuclein (AS) is pathognomonic of Parkinson's disease and other neurodegenerative disorders. Physiologically, AS contributes to synaptic homeostasis by participating in vesicle maintenance, trafficking, and release. Its avidity for highly curved acidic membranes has been related to the distinct chemistry of the N-terminal amphipathic helix adopted upon binding to appropriated lipid interfaces. Pathologically, AS populate a myriad of toxic aggregates ranging from soluble oligomers to insoluble amyloid fibrils. Different gain-of-toxic function mechanisms are linked to prefibrillar oligomers which are considered as the most neurotoxic species. Here, we investigated if amyloid oligomerization could hamper AS function as a membrane curvature sensor. We used fluorescence correlation spectroscopy to quantitatively evaluate the interaction of oligomeric species, produced using a popular method based on lyophilization and rehydration, to lipid vesicles of different curvatures and compositions. We found that AS oligomerization has a profound impact on protein-lipid interaction, altering binding affinity and/or curvature sensitivity depending on membrane composition. Our work provides novel insights into how the formation of prefibrillar intermediate species could contribute to neurodegeneration due to a loss-of-function mechanism. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

show abstract

Small-scale isolation of synaptic vesicles from mammalian brain

Cited by 81 publications

References 27 publications

Self-propelling vesicles define glycolysis as the minimal energy machinery for neuronal transport

Self-propelling vesicles define glycolysis as the minimal energy machinery for neuronal transport

MicroRNA exocytosis by large dense-core vesicle fusion

Amyloid oligomerization of the Parkinson's disease related protein α‐synuclein impacts on its curvature‐membrane sensitivity

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