Fig 1: Blood-derived SAP is transported through the BBB into the hippocampus of SAMP8 mice. (A) Volcano plot showing upregulated and downregulated peptides in the sera of SAMP8 mice. Cyan-colored dots indicate shared DEPs between the sera and hippocampus of SAMP8 mice. (B) Venn diagram indicating the shared DEPs between the sera and hippocampus of 6-month-old SAMP8 mice. The peptides that are not detected in the transcriptomic data of hippocampus of SAMR1 and SAMP8 mice are highlighted with red. (C) Violin graphs showing ELISA for SAP volume using the lysates of the hippocampi of SAMR1 and SAMP8 mice. The SAP volume is normalized with respect to the total volume of protein in lysates. The dashed line indicates the median, dot lines indicate quartiles, and dots indicate the results of each mouse. Statistic uses unpaired and two-tailed t test. (D) Confocal and IMARIS-3D images of blood vessels stained with SAP (green), CD31 (red), and GFAP (blue) in the hippocampi of 6-month-old SAMR1 and SAMP8 mice, showing the extravasation of SAP from the blood to the astrocytes (arrows) and brain parenchyma. (E) Quantification of SAP staining intensity in the astrocytes of the hippocampus of SAMP8 mice. The dashed line indicates the median, dot lines indicate quartiles, and dots indicate the results of each mouse. Statistic uses unpaired and two-tailed t test. (F) Confocal images of blood vessels stained with occludin (green) and CD31 (magenta) in the hippocampi of 6-month-old SAMR1 and SAMP8 mice, showing the gap of vulnerable tight junction (arrowheads) in SAMP8 mice. (G) Quantification of occludin-positive tight junction length in CD31+ endothelial cell area. The dashed line indicates the median, dot lines indicate quartiles, and dots indicate the results of each mouse. Statistic uses unpaired and two-tailed t test. (H) Schematic diagram of the study design to quantify the changes in BBB resistance after SAP treatment into Boyden chambers using an in vitro 3D BBB model, which is composed by primary endothelial cells, pericytes, and astrocytes. (I) Line graph showing the changes of electrical resistance values in the in vitro BBB model treated with 30, 60, and 120 nM SAP at 0, 1, 4, 12, and 24 h after treatment (left). Data represent mean ± SEM. Violin graph showing the area under the curve (AUC) of the electrical resistance value changes for each SAP concentration (right). The dashed line indicates the median, dot lines indicate quartiles, and dots indicate the results from each chamber prepared with four independent cultures. Statistics use two-way ANOVA. (J) Violin graphs showing ELISA for SAP volume using the media in the outer compartment of in vitro BBB model treated with 60 nM SAP into the inner chamber. The SAP volume is normalized with respect to the total volume of protein in media. The dashed line indicates the median, dot lines indicate quartiles, and dots indicate the results of each culture. Statistic uses unpaired and two-tailed t test. (K) Confocal images of blood vessels stained with occludin (green) and CD31 (magenta) in primary brain endothelial cells treated with 120 nM SAP for 4 h showing the disorder of occludin-positive tight junction. (L) Quantification of occludin-positive tight junction area in CD31+ primary brain endothelial cell area. The dashed line indicates the median, dot lines indicate quartiles, and dots indicate the results from each independent culture. Statistic uses paired and two-tailed t test
Fig 2: Proposed model of the blood-derived neurotoxic SAP property in SAMP8 mice at early aging stages. Based on transcriptomic analysis of the hippocampus of SAMP8 mice, changes in the expression of BBB maintenance-associated genes were observed, suggesting that SAMP8 mice exhibit dysregulation of BBB function. Consistent with this, the hippocampus of SAMP8 mice showed the vulnerability of tight junctions between endothelial cells. The integration of proteomic and transcriptomic analyses of the hippocampi and sera from SAMP8 mice suggests transport of SAP, carboxylesterase 1, and orosomucoid 1 from the blood into the brain parenchyma at 6 months of age. Particularly, SAP is abundantly detected around the hippocampal BBB components, including astrocytes. In addition, SAP has multiple functions, including attenuating the tight junction strength and inducing neuronal apoptosis and synaptic decline. Taken together, we propose a model: at early stages of aging in SAMP8 mice, upregulated serum SAP is transported to the brain parenchyma through vulnerable tight junctions or accelerated endocytosis in endothelial cells, leading to neurodegeneration
Fig 3: Blood-derived SAP promotes neuronal apoptosis and synaptic density decline in a primary cortical neuron culture. (A) Schematic diagram showing the study design to quantify apoptosis and synaptic density in E15.5 mouse-derived primary cortical neurons treated with the sera of SAMR1 and SAMP8 mice for 48 h, recombinant SAP for 6 h, and the neutralizing anti-SAP antibody for 2 h before sera treatment. (B) Confocal images of cleaved-caspase 3 (red) and MAP2 (green) stained cortical neurons treated with vehicle, 1000 µg/mL total serum protein of SAMR1 and SAMP8 mice, 60 nM recombinant SAP, or 500 µg/mL total serum protein of SAMP8 mice and the neutralizing anti-SAP antibody, showing that apoptotic cells with cleaved-caspase 3+ granules (arrowheads). Cleaved-caspase 3; MAP2 double positive neurons (arrows) are counted as cleaved-caspase 3+ neurons. (C) Ratio of cleaved-caspase 3+ apoptotic neurons in primary cortical neurons treated with 100, 250, 500, or 1000 µg/mL serum protein of SAMR1 and SAMP8 mice. Data represent mean ± SEM from at least three independent cultures. Statistic uses two-way ANOVA. (D) ELISA for SAP concentration in the sera of 6-month-old SAMR1 and SAMP8 mice. The SAP volume is normalized with fluid volume of sera. The dashed line indicates the median, dot lines indicate quartiles, and dots indicate the results of each mouse. Statistic uses unpaired and two-tailed t test. (E) Ratio of cleaved-caspase 3+ apoptotic neurons in primary cortical neuron cultures treated with vehicle and 60 nM recombinant SAP. Data represent mean ± SEM from four independent cultures, and dots indicate the results from each experimental culture. Statistic uses unpaired and two-tailed t test. (F) Ratio of cleaved-caspase 3+ apoptotic neurons in primary cortical neurons treated with control rabbit IgG and neutralizing anti-SAP antibody 2 h before the treatment with 500 µg/mL serum protein of SAMR1 and SAMP8 mice. Data represent mean ± SEM from four independent cultures, and dots indicate the results from each experimental culture. Statistic uses two-way ANOVA. (G) Confocal images of synaptophysin (magenta) and MAP2 (green)-, and PSD95 (magenta) and MAP2 (green)-stained cortical neurons treated with vehicle, 500 µg/mL serum protein of SAMR1 and SAMP8 mice, 60 nM recombinant SAP, or 500 µg/mL serum protein of SAMP8 mice and the neutralizing anti-SAP antibody. (H, I) Colocalized area of Synaptophysin+ presynapses or PSD95+ postsynapses with MAP2+ cytoplasmic and neurite area in primary cortical neurons treated with vehicle and 500 µg/mL serum protein of SAMR1 and SAMP8 mice. Data represent mean ± SEM from four independent cultures. Statistics use two-way ANOVA. (J, K) Colocalized area of Synaptophysin + presynapses or PSD95+ postsynapses with MAP2+ neuronal area in primary cortical neurons treated with vehicle and 60 nM recombinant SAP. Data represent mean ± SEM from five independent cultures, and dots indicate the results from each experimental culture. Statistic uses unpaired and two-tailed t test. (L, M) Colocalized area of Synaptophysin+ presynapses or PSD95+ postsynapses with MAP2+ neuronal area in primary cortical neurons treated with control rabbit IgG and neutralizing anti-SAP antibody 2 h before the treatment with 500 µg/mL sera of SAMR1 and SAMP8 mice. Data represent mean ± SEM from four independent cultures, and dots indicate the results from each experimental culture. Statistics uses two-way ANOVA. ns, not significant
Supplier Page from Abcam for Mouse SAP ELISA Kit