Secretion of VGF relies on the interplay between LRRK2 and post-Golgi v-SNAREs

Filippini, Francesca; Nola, Sébastien; Zahraoui, Ahmed; Roger, Kévin; Esmaili, Mansoore; Sun, Ji; Wojnacki, José; Vlieghe, Anaïs; Bun, Philippe; Blanchon, Stéphanie; Rain, Jean-Christophe; Taymans, Jean-Marc; Chartier-Harlin, Marie-Christine; Guerrera, Chiara; Galli, Thierry

doi:10.1016/j.celrep.2023.112221

Cited by 5 publications

(7 citation statements)

References 86 publications

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“…The majority of the selected genes code for SNARE proteins, but several small GTPases, the phospholipases C and D, the tight junction proteins claudins, phospholipid scramblases, sorting nexins, and several PDZ domain containing proteins, including syntenin-1, were also included. Some of these proteins have been shown to affect sEV release in other cell lines (Rab27, syntenin, VAMP7, PLD2, YKT6, SNAP23 and STX4) [ 26 , 27 , 30 , 33 , 54 – 57 ]. The criteria that we used to select proteins affecting sEV release was that at least three of the four siRNAs against the same protein showed an effect in the same direction.…”

Section: Resultsmentioning

confidence: 99%

“…2 a). VAMP7 depletion also caused a reduction of sEV release, but this protein has previously been reported to reduce sEV secretion in other cell lines and was not further investigated here [ 26 , 30 ]. For validation, sEVs released from control and knockdown (kd) cells were isolated by sequential centrifugation and pelleted at 100,000× g [ 41 ].…”

Section: Resultsmentioning

confidence: 99%

“…VAMP7 has previously been described to be involved in exosome secretion in the human leukemic cell line K562 [ 26 , 30 ]. This SNARE was also identified in our screening, further indicating that the screening assay worked as intended.…”

Section: Discussionmentioning

confidence: 99%

“…Generally, fusion involves one R-SNARE (usually v-SNARE), and three Q-SNAREs (usually t-SNAREs) [ 21 ]. Previous studies have shown that the R-SNAREs VAMP7 (vesicle-associated membrane protein 7) and YKT6 are associated with exosome release [ 26 – 30 ]. Moreover, some studies have investigated the role of Q-SNAREs and showed the involvement of syntaxin (STX) 3, STX6, STX4, STX1A and SNAP23 in exosome release or in sorting of molecules to exosomes [ 31 – 37 ].…”

Section: Introductionmentioning

confidence: 99%

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siRNA screening reveals that SNAP29 contributes to exosome release

Hessvik

Sagini

Romero

et al. 2023

Cell. Mol. Life Sci.

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Cells release extracellular vesicles (EVs) of different sizes. Small EVs (< 200 nm) can originate from the fusion of multivesicular bodies with the plasma membrane, i.e. exosomes, and from budding of the plasma membrane, i.e. small ectosomes. To investigate the molecular machinery required for the release of small EVs, we developed a sensitive assay based on incorporation of radioactive cholesterol in EV membranes and used it in a siRNA screening. The screening showed that depletion of several SNARE proteins affected the release of small EVs. We focused on SNAP29, VAMP8, syntaxin 2, syntaxin 3 and syntaxin 18, the depletion of which reduced the release of small EVs. Importantly, this result was verified using gold standard techniques. SNAP29 depletion resulted in the largest effect and was further investigated. Immunoblotting analysis of small EVs showed that the release of several proteins considered to be associated with exosomes like syntenin, CD63 and Tsg101 was reduced, while the level of several proteins that have been shown to be released in ectosomes (annexins) or by secretory autophagy (LC3B and p62) was not affected by SNAP29 depletion. Moreover, these proteins appeared in different fractions when the EV samples were further separated by a density gradient. These results suggest that SNAP29 depletion mainly affects the secretion of exosomes. To investigate how SNAP29 affects exosome release, we used microscopy to study the distribution of MBVs using CD63 labelling and CD63-pHluorin to detect fusion events of MVBs with the plasma membrane. SNAP29 depletion caused a redistribution of CD63-labelled compartments but did not change the number of fusion events. Further experiments are therefore needed to fully understand the function of SNAP29. To conclude, we have developed a novel screening assay that has allowed us to identify several SNAREs involved in the release of small EVs.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

siRNA screening reveals that SNAP29 contributes to exosome release

Hessvik

Sagini

Romero

et al. 2023

Cell. Mol. Life Sci.

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“…In terms of gender, CD99-derived cell adhesion peptides were enriched in males, a nding reliably observed across human brain and other tissue types 37,38 . Proteoforms derived from vesicle-associated membrane protein 2 (VAMP2) and VGF were also enriched in males vs females, two proteins which have recently shown secretory interplay 39 . Of the 348 peptides associated with education, the majority were cytoskeletal, with neuro lament light chain (NEFL) proteoforms making up 26 of the peptides signi cantly positively associated with increasing years in education.…”

Section: Differential Enrichment Of Proteoforms Between Discrete Diag...mentioning

confidence: 99%

Interrogation of the human cortical peptidome uncovers cell-type specific signatures of cognitive resilience against Alzheimer’s disease

Morgan,

Carlyle

2024

Preprint

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Alzheimer’s disease (AD) is characterised by age-related cognitive decline. Brain accumulation of amyloid-β plaques and tau tangles is required for a neuropathological AD diagnosis, yet up to one-third of AD-pathology positive community-dwelling elderly adults experience no symptoms of cognitive decline during life. Conversely, some exhibit chronic cognitive impairment in absence of measurable neuropathology, prompting interest into cognitive resilience – retained cognition despite significant neuropathology – and cognitive frailty – impaired cognition despite low neuropathology. Synapse loss is widespread within the AD-dementia, but not AD-resilient, brain. Recent evidence points towards critical roles for synaptic proteins, such as neurosecretory VGF, in cognitive resilience. However, VGF and related proteins often signal as peptide derivatives. Here, nontryptic peptidomic mass spectrometry was performed on 102 post-mortem cortical samples from individuals across cognitive and neuropathological spectra. Neuropeptide signalling proteoforms derived from VGF, somatostatin (SST) and protachykinin-1 (TAC1) showed higher abundance in AD-resilient than AD-dementia brain, whereas signalling proteoforms of cholecystokinin (CCK) and chromogranin (CHG) A/B and multiple cytoskeletal molecules were enriched in frail vs control brain. Integrating our data with publicly available single nuclear RNA sequencing (snRNA-seq) showed enrichment of cognition-related genes in defined cell-types with established links to cognitive resilience, including SST interneurons and excitatory intratelencephalic cells.

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Unveiling defects of secretion mechanisms in Parkinson’s disease

Filippini,

Galli

2024

Journal of Biological Chemistry

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Secretion of VGF relies on the interplay between LRRK2 and post-Golgi v-SNAREs

Cited by 5 publications

References 86 publications

siRNA screening reveals that SNAP29 contributes to exosome release

siRNA screening reveals that SNAP29 contributes to exosome release

Interrogation of the human cortical peptidome uncovers cell-type specific signatures of cognitive resilience against Alzheimer’s disease

Unveiling defects of secretion mechanisms in Parkinson’s disease

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