Journal: bioRxiv

Article Title: SSAO-mediated decrease of endothelial BDNF release affects neuronal GluA1 and PSD95 expression: a peripheral mechanism inducing NVU and CNS disturbances

doi: 10.1101/2025.03.04.641410

Figure Lengend Snippet: Neurovascular unit (NVU) in vitro model A, Extended experimental timeline. Upper arrow represents neuron-glia mixed culture timeline, indicated as days in vitro (DIV). Medium changes needed at each time point are indicated. Middle arrow represents hCMEC/D3 endothelial cell line culture timeline. Seeding, confluence, and deprivation steps are indicated and were synchronized to mixed culture development. Day 0 is considered the day when cells achieve confluence and after this, time of this culture is indicated as days after confluence (DAC). Lower arrow represents the timeline once co-culture system is set up, and time is indicated as days in vitro since co-culture (DIV - CC). Treatments are indicated the day they were performed after setting up the co-culture. B, Co-culture experimental scheme with representative phase contrast and immunocytochemistry images from WT endothelial cells (Occ: Occludin, ZO-1: Zonula occludens, H: Hoechst) and neuron-glia mixed culture (GluA1, MAP2) before setting up the co-culture. According to the time line in A , WT endothelial cells seeded in the transwell device (TW) were placed inside the neuron-glia mixed culture well. Both parts can communicate through the semipermeable membrane at the bottom of the transwell.

Article Snippet: Mixed cultures were then incubated in a humidified chamber overnight at 4 °C with primary antibodies anti-GluA1 (1:1000; AB1504; RRID: AB_2113602; Merck-Millipore); anti-PSD95 (1:1000; clone 6G6-1C9; ab2723; RRID: AB_303248; Abcam); anti-Synaptophysin1 (1:1000; 101 002; RRID: AB_887905; Synaptic Systems); and anti-MAP2 (1:500, M1406; RRID: AB_477171 Sigma; and 1:1000; 188 002; RRID: AB_2138183; Synaptic Systems) diluted in PBS supplemented with 2 or 5 % normal Goat Serum (Sigma).

Techniques: In Vitro, Co-Culture Assay, Immunocytochemistry, Membrane

Journal: bioRxiv

Article Title: SSAO-mediated decrease of endothelial BDNF release affects neuronal GluA1 and PSD95 expression: a peripheral mechanism inducing NVU and CNS disturbances

doi: 10.1101/2025.03.04.641410

Figure Lengend Snippet: Classification of subpopulation types according to GluA1-labeling mean intensity values. A, Table showing theoretical GluA1-positive cells (C1, C2, Cn) and the mean intensity of each one (X1, X2, Xn), with representative image showing the examples of the three subtypes. Mixed primary cells co-cultured with non-treated WT endothelial cells were used as control condition. Scale bar = 10μm. B , Mean intensity values from control condition were used to establish the thresholds (25 th and 75 th percentile intensity values) and to generate the subpopulation types (Type I, Type II, Type III) for each independent culture. These threshold values were applied to the rest of conditions of the same experiment to classify cells into the proper subpopulation types.

Article Snippet: Mixed cultures were then incubated in a humidified chamber overnight at 4 °C with primary antibodies anti-GluA1 (1:1000; AB1504; RRID: AB_2113602; Merck-Millipore); anti-PSD95 (1:1000; clone 6G6-1C9; ab2723; RRID: AB_303248; Abcam); anti-Synaptophysin1 (1:1000; 101 002; RRID: AB_887905; Synaptic Systems); and anti-MAP2 (1:500, M1406; RRID: AB_477171 Sigma; and 1:1000; 188 002; RRID: AB_2138183; Synaptic Systems) diluted in PBS supplemented with 2 or 5 % normal Goat Serum (Sigma).

Techniques: Labeling, Cell Culture, Control

Journal: bioRxiv

Article Title: SSAO-mediated decrease of endothelial BDNF release affects neuronal GluA1 and PSD95 expression: a peripheral mechanism inducing NVU and CNS disturbances

doi: 10.1101/2025.03.04.641410

Figure Lengend Snippet: Characterization of the neuronal part of the NVU that constitutes the in vitro co-culture model. A, Representative immunofluorescence of mixed glial-neuron primary culture staining astrocytes (GFAP, green), neurons (MAP2, red) and nuclei (Hoechst, blue). B, Proportion of the of astrocyte-neuron quantification present in mixed-cortical primary cultures at 15DIV. n=45 C, Representative immunoblot images showing neuron maturity at different DIV using both pre- and post-synaptic markers GluA1 (∼110-kDa band, top panel), PSD95 (∼95-kDa band, middle-top panel), and Synaptophysin (∼38-kDa band, middle-bottom panel). β-actin was used as a loading control (∼42-kDa band, bottom panel). D, Levels of synaptic markers relative to those observed at 9DIV, corresponding to western blot quantification. Bars represent mean ± SEM; n=4-8 experiments. E, Representative confocal images of double-staining of GluA1, PSD95, and Synaptophysin with MAP2, used as a cytoskeleton marker at different DIV showing neuron maturity on mixed-cortical culture (scale bar = 10 μm). F, Quantification of each synaptic marker intensity relative to those observed at 9DIV. Bars represent mean ± SEM; n=3-7 experiments.

Article Snippet: Mixed cultures were then incubated in a humidified chamber overnight at 4 °C with primary antibodies anti-GluA1 (1:1000; AB1504; RRID: AB_2113602; Merck-Millipore); anti-PSD95 (1:1000; clone 6G6-1C9; ab2723; RRID: AB_303248; Abcam); anti-Synaptophysin1 (1:1000; 101 002; RRID: AB_887905; Synaptic Systems); and anti-MAP2 (1:500, M1406; RRID: AB_477171 Sigma; and 1:1000; 188 002; RRID: AB_2138183; Synaptic Systems) diluted in PBS supplemented with 2 or 5 % normal Goat Serum (Sigma).

Techniques: In Vitro, Co-Culture Assay, Immunofluorescence, Staining, Western Blot, Control, Double Staining, Marker

Journal: bioRxiv

Article Title: SSAO-mediated decrease of endothelial BDNF release affects neuronal GluA1 and PSD95 expression: a peripheral mechanism inducing NVU and CNS disturbances

doi: 10.1101/2025.03.04.641410

Figure Lengend Snippet: AβD treatment on endothelial cells reduces GluA1 and PSD95 protein levels in the co-cultured neurons. A, Scheme showing co-culture set up at 9DIV of neuron-glia mixed culture and 5μM AβD addition to the endothelial cells the following day in the apical compartment, with system maintenance until 15DIV, when cell lysates were collected. B, Representative immunoblot images showing levels of GluA1 (∼110-kDa band, top panel), PSD95 (∼95-kDa band, middle-top panel) and Synaptophysin (∼38-kDa band, middle-bottom panel) in total homogenates and PSD fractions from non-treated (-) and treated (AβD) endothelial cells (WT or hSSAO/VAP-1) co-cultured with neurons. Lack of synaptophysin was used as a control of PSD fraction purity. β-tubulin was used as a loading control (∼54-kDa band, bottom panel). C, Quantification of PSD95 and GluA1 protein levels relative to co-culture with non-treated WT and hSSAO/VAP-1 endothelial cells. Bars represent mean ± SEM of n=4-9 independent experiments, analyzed by two-way ANOVA (#, source of variation) followed by Tukey’s multiple comparisons post-hoc test (*).

Article Snippet: Mixed cultures were then incubated in a humidified chamber overnight at 4 °C with primary antibodies anti-GluA1 (1:1000; AB1504; RRID: AB_2113602; Merck-Millipore); anti-PSD95 (1:1000; clone 6G6-1C9; ab2723; RRID: AB_303248; Abcam); anti-Synaptophysin1 (1:1000; 101 002; RRID: AB_887905; Synaptic Systems); and anti-MAP2 (1:500, M1406; RRID: AB_477171 Sigma; and 1:1000; 188 002; RRID: AB_2138183; Synaptic Systems) diluted in PBS supplemented with 2 or 5 % normal Goat Serum (Sigma).

Techniques: Cell Culture, Co-Culture Assay, Western Blot, Control

Journal: bioRxiv

Article Title: SSAO-mediated decrease of endothelial BDNF release affects neuronal GluA1 and PSD95 expression: a peripheral mechanism inducing NVU and CNS disturbances

doi: 10.1101/2025.03.04.641410

Figure Lengend Snippet: Endothelial AβD treatment reduces intensity of GluA1 staining in neurons co-cultured with hSSAO/VAP-1-expressing cells. A, Representative confocal images of neurons showing GluA1 (green) and nuclei (Hoechst, blue) staining at different experimental conditions with classification type associated to each cell body. Scale bar = 10 μm. B, Quantification of the percentage of GluA1-positive neurons classified as type I (lower) to type III (higher) intensity, according to their treatment (AβD) or not (-), and their co-culture with endothelial cells (WT or hSSAO/VAP-1). For Type III neurons: NT WT 24.88±0.05 %; AβD WT 17.61±1.96 %; NT SSAO 19.40±1.91 %; AβD SSAO 14.12±1.79 %; For Type I neurons: NT WT 24.88±0.05 %; AβD WT 39.78±8.18 %; NT SSAO 33.49±5.62 %; AβD SSAO 43.18±6.55 %. C, Quantification of the percentage of intensity contribution to global GluA1 intensity staining from non-treated (-) and treated (AβD) co-cultured neurons with endothelial cells (WT or hSSAO/VAP-1). Bars represent mean ± SEM of n=5-9 independent experiments (16 images per experiment), analyzed by two-way ANOVA (#, source of variation) followed by Tukey’s multiple comparisons post-hoc test (*). For Type III neurons: NT WT 50.72±1.43 %; AβD WT 42.42±3.46 %; NT SSAO 43.00±3.02 %; AβD SSAO 33.32±1.87 %; For Type I neurons: NT WT 9.15±0.58 %; AβD WT 18.15±4.85 %; NT SSAO 15.37±3.83 %; AβD SSAO 21.75±5.64 %.

Article Snippet: Mixed cultures were then incubated in a humidified chamber overnight at 4 °C with primary antibodies anti-GluA1 (1:1000; AB1504; RRID: AB_2113602; Merck-Millipore); anti-PSD95 (1:1000; clone 6G6-1C9; ab2723; RRID: AB_303248; Abcam); anti-Synaptophysin1 (1:1000; 101 002; RRID: AB_887905; Synaptic Systems); and anti-MAP2 (1:500, M1406; RRID: AB_477171 Sigma; and 1:1000; 188 002; RRID: AB_2138183; Synaptic Systems) diluted in PBS supplemented with 2 or 5 % normal Goat Serum (Sigma).

Techniques: Staining, Cell Culture, Expressing, Co-Culture Assay

Journal: bioRxiv

Article Title: SSAO-mediated decrease of endothelial BDNF release affects neuronal GluA1 and PSD95 expression: a peripheral mechanism inducing NVU and CNS disturbances

doi: 10.1101/2025.03.04.641410

Figure Lengend Snippet: Global mean intensity of GluA1 ( A ) and PSD95 ( B ) for each subpopulation type. Bars represent mean ± SEM for n=3-9 independent experiments. Data was analyzed by two-way ANOVA analysis followed by Tukey post-hoc test.

Article Snippet: Mixed cultures were then incubated in a humidified chamber overnight at 4 °C with primary antibodies anti-GluA1 (1:1000; AB1504; RRID: AB_2113602; Merck-Millipore); anti-PSD95 (1:1000; clone 6G6-1C9; ab2723; RRID: AB_303248; Abcam); anti-Synaptophysin1 (1:1000; 101 002; RRID: AB_887905; Synaptic Systems); and anti-MAP2 (1:500, M1406; RRID: AB_477171 Sigma; and 1:1000; 188 002; RRID: AB_2138183; Synaptic Systems) diluted in PBS supplemented with 2 or 5 % normal Goat Serum (Sigma).

Techniques: