Fig 1: Analysis of extracellular vesicle (EV) secretion after intracerebroventricular (icv) injection of Aß oligomers (AßO). (a) ELISA analysis of soluble Aß1–42 levels in the cerebrospinal fluid (CSF) of 7 (n = 10) and 40 (n = 15) weeks old APP/PS1 mice. (b) Nanoparticle Tracking Analysis (NTA; NanoSight) quantification of the amount of particles in CSF 2 and 6 h after icv injection of scrambled peptide (black) or AßO (grey) in C57BL/6J mice (n = 4), analyzed using 2-way ANOVA. (c) ExoView analysis of the amount of CD81 captured—CD81 positive and CD9 captured—CD81 positive EVs in CSF 6 h after icv injection of scrambled peptide (black) or AßO (grey) in C57BL/6J mice (n = 6). For each biological replicate, the presented result is the average from three different technical replicates on the chip. (d) NTA (NanoSight) quantification of the particles in the medium of primary CPE cells after 2 h of stimulation with scrambled peptide (black) or AßO (grey) and 24 h of incubation (n = 6). (e) Representative confocal images of ALIX, AnnexinA2 (ANXA2), CD63, Flotillin1 (FLOT1) and RAB5 (red) in the CP 6 h after the icv injection of scrambled peptide or AßO in C57BL/6J mice (n = 3). Cell nuclei are counterstained with Hoechst (blue). The ependymal cells that line the ventricle are indicated by a white line. Scale bar represents 100 µm in the overview images and 10 µm in the zoomed-in images. (f, g) Quantification (f) and representative transmission electron microscopy (TEM) images (g) of CP tissue, isolated from mice 4 h after the icv injection with scrambled peptide or AßO in C57BL/6J mice (n = 2). Amount of multivesicular bodies (MVBs) per cell section and amount of intraluminal vesicles (ILVs) per cell section are displayed. Two regions of 20 CPE cells were manually counted for each biological replicate, resulting in the average amount of MVBs and ILVs in 40 CPE cells per cell section. White arrow heads point to ILVs present in MVBs. Scale bar represents 10 µm in the overview images and 1 µm in the zoomed-in images
Fig 2: Validation of cell yield and profile in liquid biopsy samples(A) Graph showing the average number of cells collected per “positive” liquid biopsy of CSF at different time points after the release cocktail injection as well as after 3 days of infusion via minipump (∗p < 0.001, one-way ANOVA, n = 5–7 animals per experimental group).(B and C) Graphs showing the cell-type profile of cells collected via liquid biopsy of CSF at different time points after the release cocktail injection, as well as after 3 days of infusion via minipump (n = 5–7 independent samples per group).(D–F) High-magnification images of cells collected via liquid biopsy of CSF after the injection of the release cocktail (0.5 mU Nam + Ab) and immunostained for different markers.(G) Image of a cell colony 10 days after the initial plating of the liquid biopsy sample. Cells show a typical NSPC morphology.(H) Image of a neurosphere 15 days after the initial plating of the liquid biopsy sample. The optical plane is at the surface of the coverslip and shows adherent cells with typical NSPC morphology, some being connected with the overlaying neurosphere via cellular processes, as well as a few erythrocytes.(I) Graph showing the maximum number of passages obtained per liquid biopsy sample from saline-injected animals (gray bars, total of 12 samples), or after milking (black bars, total of 29 samples, release cocktail of 0.5 U Nam + Ab). White arrows, erythrocytes; DCX, neuroblasts; GFAP, astrocytes; PCNA, proliferating cells; PDGFRα, oligodendroblasts; SOX2, neural progenitors. Error bars: SEM. Scale bars, 20 μm. See also Figure S1
Fig 3: CSTB is secreted and induces recruitment of migrating interneurons A–CWestern blot analysis for Cstb in the CSF from 2 different E14 mouse embryos (A), in the CM from E14 cortical cells in culture for 4 days (B), in the CM from hCOs after 4 days in culture (C). The CM was collected from hCOs at different time points in culture (d19, d38, d60, d80). Protein extract (PE) from 60 days hCOs was used as a positive control. GAPDH immunostaining was used as a negative control on hCOs CM. Ponceau Red staining is shown to indicate the presence of proteins at comparable levels in the CM samples.DMicrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14, co-electroporated with mCherry-expressing vector and HA-empty vector or HA-Cstb, and analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles.EDistribution of GFP+ interneurons in the 5 equal bins of the mouse developing cortex—Bin1 corresponded to the apical side and Bin5 to the pial side. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles.FMicrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14, co-electroporated with mCherry- and HA-Cstb-expressing vectors, and analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD67-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles.GDistribution of GFP+ interneurons in the 5 equal bins of the mouse developing cortex comparing electroporated sides with the contralateral ones in the same section to avoid different distribution of interneurons rostro-caudally.Data information: Scale bars: 100 µm in (D and F) and 20 µm in (d' and f'). Data are represented as mean ± SEM. Statistical significance was based on Mann–Whitney test (*P < 0.05). Every dot in the plots refers to independent analyzed mouse brains. Exact P-values in Appendix Table S1.Source data are available online for this figure.
Fig 4: Clustering and positions of laminin-111 nitration sites based on their extent of modification. Panel A): Overview of 6-nitroTrp (left panels) and 3-nitroTyr (right panels) formation, given as site occupancies, in response to increasing molar excesses of ONOOH (as indicated on the horizontal axis) within their cluster of site occupancy level. Panel B): Position of laminin-111 nitration sites in the sequences of the 3 laminin chains (horizontal axis: LAMA1 = α1, LAMB1 = β1, LAMC1 = γ1). Bars indicate the sites of nitration with the color indicating the assigned cluster and thus extent of modification (red, cluster A; yellow, cluster B; black, cluster C; grey, cluster D, in order of decreasing extent of modification, see text for further details). Left hand columns, modifications at Trp residues (W), right hand columns, modifications at Tyr residues (Y). Annotations to the right of the chain sequences in panel B indicate sites with known biological function. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig 5: Low-grade peripheral inflammation affects Aβ transport across the blood-CSF barrier in vivo and in vitro. a Quantification of Aβ1-40 and Aβ1-42 in CSF and plasma (n = 5–8). b Expression of the genes P-gp and Lrp2 in CP (n = 5). c Representative images of LRP2 immunostaining of CP. Scale bar: 20 μm. d Quantification of the relative red staining of LRP2 in CP (n = 5 per group). e Schematic diagram of Aβ transcytosis analysis in an CP epithelial transport system using fluorescently labeled Aβ peptides. f movement of scrambled Aβ1-425-FAM (left) and Aβ1-42TAMRA (middle) from the basolateral to the apical chamber, and normalized Aβ transcytosis quotient of Aβ1-42 from the basolateral to the apical chamber (right) (n = 3). g Representative images of LRP2 staining in primary CP epithelial cells. h Quantification of the relative red staining of LRP2 in primary CP epithelial cells (n = 3). Mean ± SEM, two-way ANOVA Bonferroni’s post hoc test for multiple comparisons (a, b, d), one-way ANOVA Bonferroni’s post hoc test for multiple comparisons f Nonparametric Mann–Whitney U test h. *p < 0.05, **p < 0.01, ***p < 0.001
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