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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV110065	1/1	Homo sapiens	NAY	(d)(U)C	Sullivan CP	2011	11%
Study summary Full title Retromer disruption promotes amyloidogenic APP processing All authors Sullivan CP, Jay AG, Stack EC, Pakaluk M, Wadlinger E, Fine RE, Wells JM, Morin PJ Journal Neurobiol Dis Abstract Retromer deficiency has been implicated in sporadic AD and animals deficient in retromer components (show more...)Retromer deficiency has been implicated in sporadic AD and animals deficient in retromer components exhibit pronounced neurodegeneration. Because retromer performs retrograde transport from the endosome to the Golgi apparatus and neuronal A? is found in late endosomal compartments, we speculated that retromer malfunction might enhance amyloidogenic APP processing by promoting interactions between APP and secretase enzymes in late endosomes. We have evaluated changes in amyloid precursor protein (APP) processing and trafficking as a result of disrupted retromer activity by knockdown of Vps35, a vacuolar sorting protein that is an essential component of the retromer complex. Knocking down retromer activity produced no change in the quantity or cellular distribution of total cellular APP and had no affect on internalization of cell-surface APP. Retromer deficiency did, however, increase the ratio of secreted A?42:A?40 in HEK-293 cells over-expressing APP695, due primarily to a decrease in A?40 secretion. Recent studies suggest that the retromer-trafficked protein, Wntless, is secreted at the synapse in exosome vesicles and that these same vesicles contain A?. We therefore hypothesized that retromer deficiency may be associated with altered exosomal secretion of APP and/or secretase fragments. Holo-APP, Presenilin and APP C-terminal fragments were detected in exosomal vesicles secreted from HEK-293 cells. Levels of total APP C-terminal fragments were significantly increased in exosomes secreted by retromer deficient cells. These data suggest that reduced retromer activity can mimic the effects of familial AD Presenilin mutations on APP processing and promote export of amyloidogenic APP derivatives. (hide) EV-METRIC 11% (30th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ TSG101 non-EV: Proteomics no Show all info Study aim Other/Role of retromer disruption in APP processing Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed No Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101

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EV-TRACK ID	EV110065
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	11