Nogo-A is secreted in extracellular vesicles, occurs in blood and can influence vascular permeability

Abstract

Nogo-A is a transmembrane protein with multiple functions in the central nervous system (CNS), including restriction of neurite growth and synaptic plasticity. Thus far, Nogo-A has been predominantly considered a cell contact-dependent ligand signaling via cell surface receptors. Here, we show that Nogo-A can be secreted by cultured cells of neuronal and glial origin in association with extracellular vesicles (EVs). Neuron- and oligodendrocyte-derived Nogo-A containing EVs inhibited fibroblast spreading, and this effect was partially reversed by Nogo-A receptor S1PR2 blockage. EVs purified from HEK cells only inhibited fibroblast spreading upon Nogo-A over-expression. Nogo-A-containing EVs were found in vivo in the blood of healthy mice and rats, as well as in human plasma. Blood Nogo-A concentrations were elevated after acute stroke lesions in mice and rats. Nogo-A active peptides decreased barrier integrity in an in vitro blood-brain barrier model. Stroked mice showed increased dye permeability in peripheral organs when tested 2 weeks after injury. In the Miles assay, an in vivo test to assess leakage of the skin vasculature, a Nogo-A active peptide increased dye permeability. These findings suggest that blood borne, possibly EV-associated Nogo-A could exert long-range regulatory actions on vascular permeability.

Keywords: Nogo-A; S1PR2; blood-brain barrier; exosomes; leakage; stroke.

Figure 1.

Validation of EV isolation. EVs were isolated from pre-cleared cell culture supernatants through two rounds of ultracentrifugation at 100 000 g for 70 min and re-suspended in PBS for analysis. (a) Representative Western blots for EV marker proteins (Alix, CD63, Flotillin-2), and proteins used as negative controls (Calnexin, GM130, Ago2). Similar results were observed in >3 independent experiments. b and b’ Representative TEM images showing the morphology of the isolated EVs. (b’) is cropped and magnified from the region in (b) indicated by the dotted line. (c) and (d) Representative TEM images of EVs with immuno-gold labelling for CD63. Red Arrows indicate positive labeling. Scale bars = 100nm in all TEM images. (e) Size distribution of immune-gold (CD63⁺) labelled EVs from TEM analysis from n = 3 independent EV isolations. (f) Representative size distribution of the isolated EVs from NTA. Red dotted line represents the standard deviation from 3 independent measurements. – CL= ultracentrifugation supernatant; P100= pellet of 100 000 g centrifugation.

Figure 2.

Nogo-A is present in extracellular vesicles (EVs). (a) Representative western blots of EVs from N2a and Oli-Neu cells, pelleted through ultracentrifugation. Detection of Nogo-A/-B was performed with the Rb172A serum. (b) Density gradient fractionation of the supernatant of N2a cells. F1= top fraction, F10 = bottom fraction (not shown). Note the overlap in fractions positive for Nogo-A and Flotillin-1 (F4-F7). (c) Quantification of the total yield of EVs per 10⁶ secreting cells, measured as µg of EV protein. Statistical test performed was an unpaired student’s t-test, n = 3, p = 0.013.

Figure 3.

The C-terminus of Nogo-A is on the luminal side of extracellular vesicles (EVs), and the proteolytically sensitive N-terminus faces the extracellular space. (a) Map of the C-terminally flash-flag tagged construct of Nogo-A with the tag insertion site indicated in green arrow heads. (b) Map of the N-terminally flash-flag tagged Nogo-A construct. (c) Transmission electron microscopy (TEM) micrographs of EVs collected from HEK cells transfected with N-terminally (top) or C-terminally (bottom) tagged Nogo-A constructs. Scale bars = 100 nm. Red Arrows indicate positive labeling. (d) Quantification of the percentage of labelled EVs for N- and C-terminally tagged Nogo-A constructs. The statistical test performed was an unpaired student’s t-test, p = 0.0072. n = 3 independent EV isolations and (e) Western blots of from cells and EVs over-expressing either the N- or the C-terminally flag-tagged construct. Red labels F1–F4 indicate cleaved fragments of Nogo-A present in cell lysates and EVs. Green label F4 indicates a C-terminal cleaved fragment only found in EVs. For the limited proteolysis of EV samples, the EVs were treated with Proteinase K with or without permeabilization in 0.05% Triton X-100.

Figure 4.

EV-associated Nogo-A is active as an inhibitor of fibroblast spreading. Fibroblasts were plated in the presence of N2a EVs or proteins from the supernatant of ultracentrifugation (SN100) for 1 h at 37 °C, and cell spreading was quantified thereafter as cytoplasmic area. PBS or spinal cord extract (‘myelin’) were used as negative and positive controls respectively. (a) Images of fibroblasts treated PBS, myelin or N2a cell-derived EVs. Green=phalloidin (cytoskeleton), Blue=DAPI (nuclei). (b) Quantification of cell spreading in the presence of myelin or N2a EVs from n = 3 independent experiments. (c) Quantification of cell spreading in the presence of SN100, either coated or in solution as indicated). (d) Dose-response curve of fibroblast spreading in the presence of N2a-derived EVs. Each curve represents one independent EV isolation, 3 replicate wells at each concentration and (e) the values determined from one myelin preparation, 3 replicate wells at each concentration. Scale bar = 20 µm.

Figure 5.

Nogo-A+ EV-induced spreading inhibition is partially recovered by S1PR2 blockade. Fibroblasts were pre-treated with 10 µM JTE-013 or vehicle (DMSO) and plated at 37 °C for 1 h in the presence of EVs or control (PBS). (a) Images of fibroblast spreading with vehicle or JTE-013 treatment. Green=phalloidin (cytoskeleton), blue = DAPI (nuclei). (b) Quantification of the inhibition achieved with EVs, and the recovery with JTE-013 treatment, normalized to the control (PBS) condition and (c) Analysis of the percent change in cytoplasmic area upon JTE treatment relative to the DMSO control. The statistical test performed was a one-way ANOVA with a Bonferroni multiple comparisons test. Scale bar = 20.µm.

Figure 6.

Nogo-A in serum of adult mouse, rat, human and in mice after focal cerebral stroke. (a) Assay development: Binding of Nogo-A antibody to Nogo-A Δ20, limit of detection and quantification. (b) Capture ELISA for Nogo-A; results for blood-derived samples of mice, rats, and humans. (c) Detection of CD63-associated with Nogo-A⁺ EVs by capture ELISA, results for blood serum in mice and plasma in humans. (d) Scheme of experimental design of stroke study in mice. (e) Representative micrograph of stroked brain visualized with Nissl staining, scale bar: 500 µm. Quantitative measure of stroke volume in mm³. (f) Representative immunofluorescence images of contralesional, intact (upper row) and ipsilesional, stroked (lower row) cortex with astrocytes (GFAP), microglia (Iba1) and vasculature (CD31) at 15 days post injury (dpi). Scale bar 100 µm and (g) Quantitative assessment of Nogo-A in mouse serum at baseline (intact), at 7 dpi and 15 dpi. The statistical test performed was a one-way ANOVA with a Bonferroni multiple comparisons.

Figure 7.

Nogo-A regulates vascular permeability in CNS and non-CNS vasculature. (a) Schematic representation of the in vitro BBB model. (b) Real-time impendence measurement at 10’000 Hz after luminal administration of cell culture medium (Ctrl), VEGF (100 ng/ml) or Nogo-A Δ20 (1 µM, 10 µM) measuring ion flow between cells. (c) Transendothelial electrical resistance measurement (TEER) at 12.5 Hz after luminal (endothelial side) and abluminal (astrocyte side) administration of cell culture medium (Ctrl), VEGF (100 ng/ml) or Nogo-A Δ20 (1 µM, 10 µM) measuring paracellular ion permeability after 6 h. (d) Permeability measurement for fluorescent marker molecules sodium fluorescein (SF) and Evans blue labeled albumin (EBA) after luminal and abluminal VEGF or Nogo-A Δ20 treatment. (e) Vascular permeability for systemic Evans Blue (EB) in intact vs. stroke mice. Quantitative assessment of EB signal in peripheral organs incl. heart, kidney, liver, lung, muscle, and skin and (f) Intradermal injection of VEGF or Nogo-A Δ20 induces local permeability changes (Miles assay). The statistical test performed were t-tests to compare two groups (in e), and a one-way ANOVA with Bonferroni multiple comparisons test to compare multiple groups (in c,d,f).