Fig 2: KIBRA Knockout decreases Aß from monomers, oligomers to extracellular deposits in 5XFAD mice. (a) Schematic representation of Aß-specific antibody recognition sites. (b) Representative images and quantification analysis of ThS-stained insoluble Aß plaques in whole brain of 3–5-month-old mice. Scale bars = 500 µm. 5XFAD (n = 6M/6F), KIBRA+/-5XFAD (n = 4M/5F), KIBRA-/-5XFAD (n = 6M/5F) mice. (c) ELISA analysis of Aß40 and Aß42 in formic-acid (FA)-soluble fractions isolated from the whole brain of 3–5-month-old mice. Brain lysates from six mice per group were analyzed. (d) Images and quantification analysis of the whole brains from 3–5-month-old mice of corresponding genotypes, labeled with the anti-APP/Aß antibody (clone 4G8). Scale bar = 500 µm. Eight mice per group were analyzed. (e) ELISA analysis of Aß40 and Aß42 in TBS-soluble fractions isolated from the whole brain of 3–5-month-old mice. Brain lysates from six mice per group were analyzed. (f and g) Analysis of Aß oligomer in the whole brain of the indicated groups. Dot blot analysis of anti-amyloidogenic protein oligomer A11 and quantification analysis of oligomer A11 (f), and ELISA analysis of 82E1-specific Aß oligomers (g) from the whole brain lysates of 3–5-month-old mice (n = 6) in the whole brain lysates of the indicated groups. (h) WB analysis of brain lysates from indicated 5XFAD mice for full length APP (FL-APP), oligomer Aß, CTF-ß, and Aß monomer (anti-Aß: clone 6E10). (i) WB analysis of brain lysates from the indicated 5XFAD mice for APP/APP-CTFß (anti-C1/6.1) and BACE1.Data are represented as the mean ± SE. n.s.=0.05, ???P < 0.001, ??P < 0.01, and *P < 0.05 as determined by one-way ANOVA followed by Tukey's post hoc comparisons tests.

Fig 3: KIBRA Knockout reduces secretion of EVs harboring APP-CTFß/Aß both in vivo and in vitro. (a) WB analysis of small EVs from cell culture supernatants from equal number of KIBRA-KD and CTRL HT22 cells overexpressing APPswe. Whole cell lysates (WCLs) and small EVs were blotted for the EVs markers Alix, CD63, CD9, and APP-CTFß and for the endoplasmic reticulum marker Calnexin. (b) Quantification of EVs protein levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APPswe in three independent experiments. (c) Representative NTA traces of EVs isolated from cell culture supernatants from equal numbers of KIBRA-KD and CTRL cells overexpressing APPswe. Quantification of NTA of four independent experiments. (d) Simoa analysis was used to measure human Aß40 and Aß42 levels in small EVs purified from sucrose step gradient centrifugation from equal numbers of KIBRA-KD and CTRL cells with APPswe overexpression. (n = 3, from three independent experiments). (e) Quantification of exosomal APP-CTFß levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APPswe in three independent experiments. (f) Sucrose step gradient fraction d isolated from the extracellular space of the brain from 3–5-month-old of the indicated groups mice were blotted for the exosomal markers CD63, CD9, Calnexin, and APP-CTFß. (g) Quantification of exosomal protein levels in the small EVs obtained from the indicated groups mice in three independent experiments. (h) Human Aß40 and Aß42 levels were measured by MSD analysis from sucrose step gradient fraction d isolated from the extracellular space of the brain from 3–5-month-old indicated groups mice (n = 6, from three biological replicates). (i) Quantification of exosomal APP-CTFß levels in the small EVs obtained from the indicated groups mice in three independent experiments. Data are represented as the mean ± SE. In a-e, ??P < 0.01, ???P < 0.001, and ????P < 0.0001 in two-tailed Student's t test. In f-h, *P < 0.05, ??P <0.01, ???P < 0.001, and ????P < 0.0001 as determined by one-way ANOVA followed by Tukey's post hoc comparisons tests.

Fig 4: KIBRA Knockout stabilizes intracellular Aß/AßPP in 5XFAD mice. (a and b) Representative images and quantification analysis of cortical and hippocampal regions of the indicated 3–5-month-old mice. Intraneuronal Aß/AßPP in the brain sections were stained with the anti-4G8 antibody (green color), and neuronal nuclear with the DAPI (blue color). Scale bar = 200 µm in upper panel, 50 µm in lower panel. (b-c) Quantification analysis of the same cortical (b) and hippocampal regions (c) of indicated 3–5-month-old mice, respectively. More than seven mice per group were analyzed. (d) WB and quantification analysis of cell lysates from the KIBRA-KD and CTRL HT22 cells with APPswe overexpression for APP/APP-CTFß (anti-C1/6.1). (e) Quantification analysis of Aß40 and Aß42 levels in cell lysates from the KIBRA-KD and CTRL HT22 cells with APPswe overexpression by Simoa technique. (n = 3, from three independent experiments). Data are represented as the mean ± SE. n.s.=0.05 was determined by one-way ANOVA followed by Tukey's post hoc comparisons tests (b-c) or two-tailed Student's t test (d-e).

Fig 5: Inhibition of lysosome function in KIBRA knockout cells rescues secretion of EVs harboring APP-CTFß/Aß. (a) WB analysis of WCLs in KIBRA-KD and CTRL cells overexpressing APPswe, and treated with 20 nM bafilomycinA1 (BafA1) for 12 h, when indicated, and WCLs were blotted for Alix, CD63, and CD9. (b) Representative NTA traces of EVs derived from KIBRA-KD and CTRL cells overexpressing APPswe, and treated with BafA1. Right panel represents quantification of NTA of three independent experiments. (c) Human Aß40 and Aß42 levels measured by Simoa analysis from small EVs purified from sucrose step gradient centrifugation from equal number of KIBRA-KD and CTRL cells overexpressing APPswe, and treated with BafA1. (n = 3, from three independent experiments). (d) WB analysis of EVs secretion in KIBRA-KD and CTRL cells overexpressing APPswe, and treated with 20 nM bafilomycinA1 (BafA1) for 12 h, when indicated. EVs were blotted for Alix, CD63, CD9, and APP-CTFß. (e) Quantification of EVs protein levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APPswe, and treated with lysosome inhibitor BafA1 in three independent experiments. (f) Quantification of APP-CTFß levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APPswe, and treated with lysosome inhibitor BafA1 in three independent experiments. (g and h) WB analysis of EVs secretion in KIBRA-KD and CTRL cells overexpressing APPswe transfected with control small interfering RNAs (siRNAs) or siRNAs targeting Rab7. WCLs (g) and EVs (h) were blotted for Alix, CD63, CD9, Rab7, and APP-CTFß. (i) Quantification of EVs protein levels (Alix, CD63, and CD9) in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APPswe, and transfected with siRNAs in four independent experiments. (j) Quantification of APP-CTFß levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APPswe, and transfected with siRNAs in three independent experiments. (k) Representative NTA traces of EVs derived from KIBRA-KD and CTRL cells overexpressing APPswe, and treated with siRNAs targeting Rab7. Right panel represents quantification of NTA of three independent experiments. (l) Human Aß40 and Aß42 levels measured by Simoa analysis from small EVs purified from sucrose step gradient centrifugation from equal numbers of KIBRA-KD and CTRL cells overexpressing APPswe, and treated with siRNAs in three independent experiments. The quantification results were plotted as dot plots, showing the mean ± SE. *P < 0.05, ??P < 0.01, ???P < 0.001, ????P < 0.0001, n.s., not significant (P > 0.05) as determined by one-way ANOVA followed by Tukey's post hoc comparisons tests.