Journal: Journal of Extracellular Vesicles

Article Title: Neuron‐derived extracellular vesicles contain synaptic proteins, promote spine formation, activate TrkB‐mediated signalling and preserve neuronal complexity

doi: 10.1002/jev2.12355

Figure Lengend Snippet: Neuron‐derived EVs activate Akt and RPS6 via TrkB‐mediated signalling. (A) Schematic overview of the experimental procedure. Neurobasal medium from DIV13 cortical neurons was collected and the EVs‐containing medium was treated with 100 µM ANA‐12 or DMSO as vehicle (Veh) for 1 h. EVs were isolated following sequential UC. (B) Representation of Veh‐EVs, with active TrkB as a cargo, and ANA‐12‐EVs, with diminished TrkB phosphorylation. (C) Neuronal lysates, P10K and EVs samples (Veh or ANA‐12) were subjected to WB and EVs markers (Alix, Flotillin‐1, TSG‐101), phosho‐Tyr706‐TrkB and total TrkB were analysed. (D) Cultured cortical neurons were treated with Veh‐EVs or ANA‐12‐EVs for 24 h. (D1) Neurons UT or EVs‐treated were lysed and subjected to WB analysis. (D2) Densitometric analysis of phospho‐Tyr706‐TrkB, phospho‐Ser473‐Akt, phospho‐Ser235/236‐RPS6, and total levels of TrkB, Akt and RPS6. Actin was used as a loading control. Values represent culture replicates of three independent neuronal cultures (mean ± SEM). Full length WB can be found in Figure . Data was analysed by One‐way ANOVA (** P < 0.01 vs. Veh‐EVs and *** P < 0.001 vs. Veh‐EVs).

Article Snippet: The following primary antibodies were used (1:1000 it not stated otherwise): mouse monoclonal anti‐Alix (1:500, Cell Signalling Technology, #2171), mouse monoclonal anti‐TrkB (BD Biosciences, #610102), rabbit polyclonal anti‐calnexin (Abcam, #ab22595), mouse monoclonal anti‐PSD95 (Thermo Fisher Scientific, #MA1‐045), rabbit polyclonal anti‐p75 (Promega, #G323A), rabbit polyclonal anti‐VGLUT‐1 (Synaptic Systems, #135303), mouse monoclonal anti‐Flotillin‐1 (BD Biosciences, #610821), mouse monoclonal anti‐TSG101 (Abcam, #ab83), mouse polyclonal anti‐BDNF (Icosagen, #327‐100), rabbit monoclonal anti‐phospho RPS6 (Ser235/236) (Cell Signalling Technology, #4858), mouse monoclonal anti‐RPS6 (Cell Signalling Technology, #2317), rabbit monoclonal anti‐phospho Akt (Ser473) (Cell Signalling Technology, #4060), rabbit monoclonal anti‐Akt (Cell Signalling Technology, #4691), rabbit monoclonal anti‐phospho TrkB (Tyr706) (1:500, Signalway Antibody #11328), rabbit monoclonal anti‐phospho GluA1 (Ser831) (Merck, #08‐823), rabbit polyclonal anti‐GluA1 (Merck, #ABN241), mouse monoclonal anti‐phospho‐ERK1/2 (Thr202/Tyr204) (Cell Signalling Technology, #9106), rabbit polyclonal anti‐ERK (Santa Cruz Biotechnology, #sc‐93), rabbit polyclonal anti‐phospho‐PKC α/β II (Thr638/641) (Cell Signalling Technology, 9375), rabbit polyclonal anti‐PKCα (Cell Signalling Technology, #2056), rabbit polyclonal anti‐phospho‐p70 S6 kinase (Ser371) (Cell Signalling Technology, #9208), rabbit polyclonal anti‐p70 S6 kinase (Cell Signalling Technology, #9202), rabbit monoclonal anti‐phospho‐4E‐BP1 (Thr37/46) (Cell Signalling Technology, #2855) and rabbit monoclonal anti‐4E‐BP1 (Cell Signalling Technology, #9644).

Techniques: Derivative Assay, Isolation, Cell Culture