Monomeric A?42 peptides are more efficiently cleared from the brain than oligomeric A?42. (A) A schematic diagram of experimental setup. Fluorescently labeled A?42 prepared at different time points were injected into the brain followed by in vivo imaging at 5 hpi and 24 hpi. (B,C?) Distribution of injected, fluorescently labeled A?42 (mA?42 or oA?42) in the brains of 3 dpf larvae at 5 hpi (B,C) and 24 hpi (B?,C?) (left, overlays with brightfield; middle, HiLyte Fluor 555; right, thresholded images of A?42 fluorescence). (D,E) Quantification of the area fraction (%) occupied by A?42 fluorescence within square unit (512 × 512 pixels) at different time points. (F) Clearance rate of mA?42 and oA?42 between 5 hpi and 24 hpi by fluorescence quantification. Two-tailed unpaired t-test, p = 0.023. Data are presented as mean ± SEM. N = 7 per group. Data are representative of at least three independent experiments. dpf, days post fertilization; hpi, hours post injection; HB, hindbrain; OT, optic tectum, Scale bars = 100 ?m. * p < 0.05.

Cleared mA?42 accumulates in the pronephros via blood flow. (A,B) mA?42-injected larvae after propranolol (100 ?M) treatment. The mA?42 intensity in the pronephros of propranolol-treated larvae (white dotted lines) decreased (B) compared to control (A). (C,D) mA?42-injected larvae after control morphants (C) and 200 ?M tnnt2a morphants (D). (E) Quantification of heartbeats upon propranolol treatment. n = 9 for control group, n = 10 for propranolol. (F) Quantification of the mA?42 intensity in the pronephros after propranolol treatment. n = 8 per group. (G) Quantification of the mA?42 intensity in the pronephros of control and tnnt2a morphants. Statistical significance was determined by two tailed unpaired t-test. Data are presented as mean ± SEM. n = 9 per group. Data are representative of at least three independent experiments. (H) A schematic diagram of experimental setting of A? and tracer injection. mA?42 or oA?42 (HiLyte) was injected into ventricle and 10 kDa Dextran was injected into caudal vein. (I) Quantification of the A?42 fluorescence intensity in the pronephros normalized by background fluorescence. n = 9 for mA?42, n = 8 for oA?42. Data are representative of at least three independent experiments. (J?K?) Confocal fluorescence images showing the brain and pronephros of zebrafish with lateral view after mA?42 (J) or oA?42 (K) injection at 3 dpf. Red fluorescence indicates A?42-HiLyte Fluor and green fluorescence show 10 kDa dextran tracer injected into caudal vein. (J?) Ventricle-injected mA?42 was seen in the pronephros region (white dotted lines and depicted as p). (K?) Ventricle-injected oA?42 was seen only in the brain region (b), but not detected in the pronephros. Caudal vein-injected dextran accumulated in the pronephros of both mA?42 and oA?42-injected larvae (J?,K?). (J?,K?) show merged images. b, brain; MO, morpholino; p, pronephros; Scale bars = 100 ?m. ** p < 0.001; *** p < 0.0005; **** p < 0.0001.

Cleared mA?42 accumulates in the pronephros via blood flow. (A,B) mA?42-injected larvae after propranolol (100 ?M) treatment. The mA?42 intensity in the pronephros of propranolol-treated larvae (white dotted lines) decreased (B) compared to control (A). (C,D) mA?42-injected larvae after control morphants (C) and 200 ?M tnnt2a morphants (D). (E) Quantification of heartbeats upon propranolol treatment. n = 9 for control group, n = 10 for propranolol. (F) Quantification of the mA?42 intensity in the pronephros after propranolol treatment. n = 8 per group. (G) Quantification of the mA?42 intensity in the pronephros of control and tnnt2a morphants. Statistical significance was determined by two tailed unpaired t-test. Data are presented as mean ± SEM. n = 9 per group. Data are representative of at least three independent experiments. (H) A schematic diagram of experimental setting of A? and tracer injection. mA?42 or oA?42 (HiLyte) was injected into ventricle and 10 kDa Dextran was injected into caudal vein. (I) Quantification of the A?42 fluorescence intensity in the pronephros normalized by background fluorescence. n = 9 for mA?42, n = 8 for oA?42. Data are representative of at least three independent experiments. (J?K?) Confocal fluorescence images showing the brain and pronephros of zebrafish with lateral view after mA?42 (J) or oA?42 (K) injection at 3 dpf. Red fluorescence indicates A?42-HiLyte Fluor and green fluorescence show 10 kDa dextran tracer injected into caudal vein. (J?) Ventricle-injected mA?42 was seen in the pronephros region (white dotted lines and depicted as p). (K?) Ventricle-injected oA?42 was seen only in the brain region (b), but not detected in the pronephros. Caudal vein-injected dextran accumulated in the pronephros of both mA?42 and oA?42-injected larvae (J?,K?). (J?,K?) show merged images. b, brain; MO, morpholino; p, pronephros; Scale bars = 100 ?m. ** p < 0.001; *** p < 0.0005; **** p < 0.0001.

Brain lymphatic endothelial cells take up monomeric A?42, but not oligomeric A?42. (A) A schematic diagram of the larval brain with the dorsal view. Red lines in the dotted box depict the loop structure of the brain lymphatic endothelial cells (BLECs) in the optic tectum. (B) Quantification of the co-localization of A?42 with BLECs. Data are presented as mean ± SEM. n = 9 for mA?42 and n = 7 for oA?42. Statistical significance was determined by two-tailed unpaired t-test. (C) Quantification of the numbers of mrc1a+ BLECs in the optic tectum after mA?42 or oA?42 injection in the Tg(mrc1a:mCherry). Data are presented as mean ± SEM. n = 9 per group. (D) Quantification of the number of A?42 positive BLECs nearby (within 10 ?m) mesencephalic vein (MsV) region showing mrc1a+ positivity. Statistical significance was determined by ordinary one-way ANOVA with Tukey?s test. n = 10 for mA?42 and n = 8 for oA?42. (E,F) Confocal projections of prox1a:RFP+ BLECs and A?42-HiLyte Fluor 488. (E?,F?) High magnification of dotted boxes in (E,F) showing endocytic vesicles of BLECs. mA?42-injected BLECs showed robust uptake of mA?42 into endocytic vesicles ((E?), yellow arrows) whereas oA?42-injected did weak uptake ((F?), white arrows). Scale bars in (E?F?) = 10 ?m. (G,H) Confocal fluorescence images of the brain optic tectum region with double transgenic Tg(kdrl:EGFP); Tg(mrc1a:mCherry) larvae after A?42 injection (HiLyte Fluor 647-A?42) at 3 dpf. mA?42 fluorescence detected in the neighboring (within 10 ?m) kdrl:EGFP+ cerebrovasculature was mostly co-localized with mrc1a:mCherry+ BLECs (yellow arrowheads) (G), whereas oA?42 (H) fluorescence neighboring kdrl:EGFP+ cerebro-vasculatures was not. HB, hindbrain; OT, optic tectum; MsV, mesencephalic vein; Scale bars in (G,H) = 50 ?m. ns, not significant; **** p < 0.0001.

Brain lymphatic endothelial cells take up monomeric A?42, but not oligomeric A?42. (A) A schematic diagram of the larval brain with the dorsal view. Red lines in the dotted box depict the loop structure of the brain lymphatic endothelial cells (BLECs) in the optic tectum. (B) Quantification of the co-localization of A?42 with BLECs. Data are presented as mean ± SEM. n = 9 for mA?42 and n = 7 for oA?42. Statistical significance was determined by two-tailed unpaired t-test. (C) Quantification of the numbers of mrc1a+ BLECs in the optic tectum after mA?42 or oA?42 injection in the Tg(mrc1a:mCherry). Data are presented as mean ± SEM. n = 9 per group. (D) Quantification of the number of A?42 positive BLECs nearby (within 10 ?m) mesencephalic vein (MsV) region showing mrc1a+ positivity. Statistical significance was determined by ordinary one-way ANOVA with Tukey?s test. n = 10 for mA?42 and n = 8 for oA?42. (E,F) Confocal projections of prox1a:RFP+ BLECs and A?42-HiLyte Fluor 488. (E?,F?) High magnification of dotted boxes in (E,F) showing endocytic vesicles of BLECs. mA?42-injected BLECs showed robust uptake of mA?42 into endocytic vesicles ((E?), yellow arrows) whereas oA?42-injected did weak uptake ((F?), white arrows). Scale bars in (E?F?) = 10 ?m. (G,H) Confocal fluorescence images of the brain optic tectum region with double transgenic Tg(kdrl:EGFP); Tg(mrc1a:mCherry) larvae after A?42 injection (HiLyte Fluor 647-A?42) at 3 dpf. mA?42 fluorescence detected in the neighboring (within 10 ?m) kdrl:EGFP+ cerebrovasculature was mostly co-localized with mrc1a:mCherry+ BLECs (yellow arrowheads) (G), whereas oA?42 (H) fluorescence neighboring kdrl:EGFP+ cerebro-vasculatures was not. HB, hindbrain; OT, optic tectum; MsV, mesencephalic vein; Scale bars in (G,H) = 50 ?m. ns, not significant; **** p < 0.0001.

BLECs depletion decreases peripheral transport of mA?42 to the pronephros. (A) A schematic diagram of zebrafish 3 dpf larvae with dorsal view. Dotted box depicts the loop structure of BLECs in the optic tectum. Blue lines depict the pronephros. (B,C) Confocal fluorescence images of the brain optic tectum region with Tg(prox1a:TagRFP); Tg(fli1a:EGFP) that labels BLECs and brain vasculatures simultaneously. Control morphant (B) and ccbe1 morphant (C) at 3 dpf with BLECs depleted in the brain with intact vasculatures. Scale bar in C = 50 ?m. (D?G) Dorsal view of the larval brains of control (D,E) and ccbe1 morphants (F,G) 4 h after A?42 injection at 3 dpf. Red fluorescence represents A?42-HiLyte Fluor 647. mA?42 is seen in BLECs (arrowheads) in the control morphants (D) but not in the ccbe1 morphants (F). (D?,E?,F?,G?) Confocal images of zebrafish pronephros (dotted lines) after A?42 (the same fish with brain images). The robust pronephric accumulation of A?42 was detectable in mA?42-injected control (D?), but the reduced pronephric delivery of mA?42 was observed in ccbe1 morphants (F?) compared to control. oA?42 injection into both control and ccbe1 morphants show almost no pronephric accumulation of A?42 (E?,G?). Scale bars = 100 ?m. (H) Quantification of A?42 intensity in the pronephros, normalized by the intensity of non-fluorescent background. (I) Quantification of the relative intensity ratio between the pronephros and hindbrain. Statistical significance was determined by ordinary one-way ANOVA with Tukey?s test. HB, hindbrain; MO, morpholino; OT, optic tectum; n, independent biological samples or animals. Numbers within bar bottom graphs represent n. **, p < 0.01; ****, p < 0.0001; ns, not significant.

Selective ablation of BLECs decreased internalization of mA?42 and pronephric accumulation. (A) A schematic diagram of the experimental setting. BLEC-specific ablation using the confocal laser. (B,C) Confocal images of BLECs in the double transgenic Tg(mrc1a:mCherry); Tg(kdrl:EGFP) at 3 dpf before laser irradiation (B) and after ablation (C). Yellow arrows indicate the ablated BLECs. Scale bars = 50 ?m. (D) Confocal images of mrc1a+ BLECs in the loop of the optic before ablation (D) and after ablation and mA?42 injection (D??D?). (D?) shows red channel, (D?) shows mA?42 (HiLyte647, white) and (D?) is a merged image. Dotted lines denote the loop of BLECs. (E) Confocal images of mrc1a+ BLECs with mA?42 injection (non-ablated control). Scale bars = 50 ?m. (E?) shows mA?42 (HiLyte647, white) and (E??) shows merged images of BLECs and mA?42. (F) Quantification of the relative intensity ratio between the pronephros and hindbrain. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed unpaired t-test. n = 15 for non-ablated control, n = 8 for ablated. p = 0.0092. (G) Quantification of area fraction (%) occupied by mrc1a+ BLECs. Data are presented as mean ± SEM. Statistical significance was determined by ordinary one-way ANOVA with Tukey?s test. **, p < 0.01; ****, p < 0.0001.

Mannan administration reduces uptake of mA?42 by BLECs and peripheral transport. (A) A schematic diagram of dorsal view of the 3 dpf larval brain and the experimental setup. Dotted gray box denotes the region of interest. (B,C) Confocal images of mrc1a:mCherry+ BLECs co-injected with pHrodoGreen and mA?42 (HiLyte Fluor 647). Arrows indicate colocalization of pHrodoGreen and mA?42 (B). Empty arrowheads show that the mannan administration interferes with colocalization of pHrodoGreen and mA?42. Data are representative of at least three independent experiments. Scale bars = 10 ?m. (D,E) Confocal images of the zebrafish pronephros after mA?42 injection. Dotted lines depict the pronephros structure. Arrowheads indicate the accumulation of mA?42. Mannan treatment prior to mA?42 injection (E) resulted in a reduced pronephric accumulation compared to PBS control (D). Scale bars = 100 ?m. (F) Quantification of the relative ratio of the intensity between the pronephros and brain. Statistical significance was determined by two-tailed unpaired t-test. p = 0.0092. Data are presented as mean ± SEM. n = 9 per group. Data are representative of at least two independent experiments. **, p < 0.01.

EPPS treatment enhances the BLEC localization and pronephric transport of oA?42. (A) A schematic diagram of EPPS treatment after oA?42 (HiLyte-Fluor 647) injection. Gray dotted box depicts the region of interest and blue lines indicate the pronephros. (B?E) Confocal images of prox1a:RFP+ BLECs in the loop of the optic tectum of oA?42-injected larval brain with control (B,D) and EPPS treatment (C,E). (B,C) Co-localization of BLECs with oA?42 increased upon EPPS treatment (arrows) in c compared to control (B). Scale bars in (B,C) = 50 ?m. (D,E) Confocal images of prox1a:RFP+ BLECs with EPPS treatment (250 mM, e) after oA?42 injection with high magnification revealed the internalized oA?42 in BLECs compared to control (D). Scale bars in (D,E) = 5 ?m. (F) Quantification of oA?42 co-localization in BLECs (%) upon EPPS treatment. Statistical significance was determined by ordinary one-way ANOVA with Tukey?s test. (G,H) Confocal images of the pronephros (white dotted lines) injected with oA?42 in control (G) and with EPPS treatment for 24 h (H). (I) Quantification of the relative ratio of the intensity between the pronephros and brain. Two-tailed unpaired t-test, p = 0.0002. Data are presented as mean ± SEM. Data are representative of at least two independent experiments. Numbers within bar bottom graphs represent n. ***, p < 0.001; ****, p < 0.0001.