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; PDGFRa, 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 = a1, LAMB1 = ß1, LAMC1 = ?1). 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 Aß transport across the blood-CSF barrier in vivo and in vitro. a Quantification of Aß1-40 and Aß1-42 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 Aß transcytosis analysis in an CP epithelial transport system using fluorescently labeled Aß peptides. f movement of scrambled Aß1-425-FAM (left) and Aß1-42TAMRA (middle) from the basolateral to the apical chamber, and normalized Aß transcytosis quotient of Aß1-42 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