Fig 2: (A,B) Schematic overview of the microfluidic chip. (A) The open top curved channel (blue) is separated from the straight bottom channel (yellow) by a polyester membrane (5 µm pores). (B) Cross-section connected channels. (C,D,E) neurovascular unit (NVU)-on-a-chip microfluidic device. (C) Open-top microfluidic chip in a 6-well plate, in the bottom channel (yellow) endothelial cells can be cultured, while the open top channel (blue) is used for neuronal differentiation. (D) Staining performed on endothelial cell monoculture on bottom channel of the microfluidic chip on a polyester membrane. Based on the staining pattern, a monolayer was present. Blue: Nuclei; Red: F-actin. (E) Staining performed on neurons differentiated in the top channel of the microfluidic chip on a polyester membrane. Blue: Nuclei; Green: Microtubule Associated Protein 2 (MAP2); Red: Synapsin-1/2 (SYN1/2). (F): Staining performed on neurons co-cultured with astrocytes differentiated on a 24-well plate. Blue: Nuclei; Green: MAP2; Red: Glial fibrillary acidic protein (GFAP), a 1:1 ratio of neurons and astrocytes is visible. (G): Staining performed on neurons differentiated on a 24-well plate. Blue: Nuclei; Green: MAP2; Red: SYN1/2. Scale bars, 50 µm.

Fig 3: Aß-derived fragments differently affect phosphorylation of Syn1 at serine 551 and calcineurin activity. a Representative images of mature cortical neurons (17–22 DIV) treated with vehicle (Ctrl), rat Aß1-40, rat Aß1-42, human Aß1-16, human/rat Aß17-40, human/rat Aß17-42 for 1 h at 37 °C and stained with antibodies recognizing Syn1 phosphorylation at serine 551 (pSyn1S551, green). Syn1,2 (magenta) was used as a synaptic marker. Scale bar 5 µm. b Quantification of the density and c IF intensity of synaptic puncta positive for pSyn1S551. (D) Quantification of calcineurin activity in cortical neurons (21DIV) treated with Aß peptides, BgTx and PNU-120596. Data were normalized to the Ctrl. In plots, the boxes show the interquartile range and median, whiskers minimum and maximum values, and + indicates the mean. Values in brackets are the in b and c number of analysed cells obtained from three independent experiments and in d numbers of independent experiments. One-way ANOVA followed by Dunnett´s post hoc test was applied to assess statistical significance of comparison to Ctrl; *p < 0.05, **p < 0.01, ****p < 0.0001

Fig 4: N-terminal portion of Aß requires choline and nicotine to increase TRP and Ca2+ influx via a7nAChRs. a Images show TRP labelled by Syt1 Ab loading (magenta) in rat cortical neurons treated either with vehicle or 200 pMAß1-16 (1 h, 37 °C) in the neuronal growth media, in Tyrode´s buffer or in Tyrode´s buffer supplemented with 28 µM choline. Synapses were identified by co-staining for Syn1,2 (green). Scale bar 5 µm. b Quantification of the experiment described in a. c Ca2+ imaging was done using Cal-520 AM indicator in HEK293T expressing a7nAChRs. Traces show the time course of the experiment. Arrows depict the sequential applications of human Aß1-16, human/rat Aß17-42, human Aß1-42 (200 pM, 115 µl/s) or vehicle solution and nicotine (100 µM, 115 µl/s). d Quantification of nicotine-induced Cal-520 fluorescence in experiment shown in graph (C). Values in brackets refer to the number of analysed cells (b) or recorded wells (d) obtained from two (b) or four (D) independent preparations. Boxes indicate the interquartile range with median, whiskers minimum and maximum values, and + shows the mean. Significance was assessed by one-way ANOVA followed by Dunnett´s post hoc test; marks (***p < 0.001, **p < 0.01) above boxes indicate comparisons to the Ctrl

Fig 5: Apocynin treatment rescues synaptic phenotype. (A) Representative images of 8-oxo-dG stainings and 8-oxo-dG quantification for untreated and APO treated iNeurons relative to respective untreated control cells at DIV21. n = 39 for C1; n = 46 for C1 + APO; n = 42 for KdVS1; n = 51 for KdVS1 + APO; n = 26 for CRISPR1; n = 25 for CRISPR1 + APO; n = 26 for C2; n = 25 for C2+ APO; n = 28 for KdVS2; n = 27 for KdVS2+ APO; n = 31 for KdVS3; n = 23 for KdVS3+ APO. Scale bar: 20 µm. (B) Representative images of SQSTM1 stainings of iNeurons at DIV21 and fluorescence quantification in untreated and APO treated iNeurons relative to untreated control cells at DIV21. n = 43 for C1; n = 34 for C1+ APO; n = 34 for KdVS1; n = 38 for KdVS1+ APO; n = 40 for CRISPR1; n = 39 for CRISPR1+ APO; n = 16 for C2; n = 19 for C2+ APO; n = 25 for KdVS2; n = 27 for KdVS2+ APO; n = 34 for KdVS3; n = 24 for KdVS3+ APO. Scale bar: 20 µm. (C) Representative images of dendrites stained for MAP2 and SYN1-SYN2 for C1, KdVS1 and CRISPR1 at DIV21 either untreated or APO treated and SYN1-SYN2 quantification. n = 11 for C1; n = 9 for C1+ APO; n = 12 for KdVS1; KdVS1+ APO; n = 10 for CRISPR1; CRISPR1+ APO; n = 7 for C2; C2+ APO; KdVS2; KdVS2+ APO; KdVS3, KdVS3+ APO. Scale bar: 20 µm. Two-way ANOVA was used to determine statistically significant changes. (D) Representative voltage clamp recordings at Vh = -60 mV showing sEPSCs at DIV21 with and without APO treatment during differentiation. (E) sEPSC frequency and (F) amplitude quantifications. n = 8 for C1; C1+ APO; n = 9 for KdVS1; KdVS1+ APO; n = 11 for CRISPR1; n = 8 for CRISPR1+ APO; n = 15 for C2; n = 14 for C2+ APO; n = 8 for KdVS2; n = 9 for KdVS2+ APO; n = 8 for KdVS3; KdVS3+ APO. (G) Representative raster plots for untreated C1 network and untreated and APO treated CRISPR1 network at DIV30 (3 min. of recording). Quantification of (H) mean firing rate, (I) network burst frequency, (J) percentage of random spikes, and (K) CV of inter-network burst interval. n = 15 for C1; n = 17 for C1 + APO; n = 16 for CRISPR1; n = 15 for CRISPR1 + APO. All data presented in this figure were generated in at least 2 independent experiments and statistically significant differences were tested through Kruskal-Wallis and Dunn’s multiple comparison test, if not mentioned differently. All samples were always compared to the respective untreated control; only significant differences were indicated. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001.