Fig 1.
ADAMTS13 activities and VWF levels in murine plasma.
(A) ADAMTS13 activities in plasma from 2-, 6-, and 12-month-old WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice (n = 6). (B) VWF levels in plasma from 2-, 6-, and 12-month-old WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice (n = 6). (C) Representative immunoblotting analysis of plasma VWF multimers from 12-month-old APPPS1 and APPPS1–Adamts13−/− mice. (D) Quantitative analysis of the ratio of UL to LMW) VWF multimers in each group (n = 5). Relevant data values are included in S1 Data. Values are mean ± SD. *P < 0.05. ADAMTS13, a disintegrin and metalloprotease with thrombospondin type I motif, member 13; APPPS1, amyloid precursor protein–presenilin 1; LMW, low molecular weight; UL, ultralarge; VWF, von Willebrand factor; WT, wild type.
Fig 2.
ADAMTS13 deficiency leads to early and progressive BBB breakdown in APPPS1 mice.
(A) In vivo multiphoton microscopy images of intravenously injected FITC-dextran (MW = 40,000 Da; green) leakage in cortical vessels in 2-, 6-, and 12-month-old WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice. Scale bar, 100 μm. (B) Representative images of fibrin extravascular deposits (green) and CD31+ microvessels (white) in the hippocampus of 2-, 6-, and 12-month-old WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice. Scale bar, 10 μm. (C) Quantification of the PS product of FITC-dextran in WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice (n = 6). Relevant data values are included in S1 Data. (D) Quantification of fibrin extravascular deposits in the hippocampus of WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice (n = 6). (E) Representative immunoblots of the tight junction protein ZO-1 and the adherens junction protein VE-cadherin in isolated brain microvessels of WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice. (F) Immunoblot analysis of the tight junction protein ZO-1 in isolated brain microvessels of WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice (n = 6). Relevant data values are included in S1 Data. (G–H) Quantification of PDGFR-β+ pericyte coverage on CD31+ microvessels in the cortex (G) and hippocampus (H) in WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice (n = 6). Relevant data values are included in S1 Data. Values are mean ± SD. *P < 0.05. ADAMTS13, a disintegrin and metalloprotease with thrombospondin type I motif, member 13; APPPS1, amyloid precursor protein–presenilin 1; BBB, Blood-brain barrier; CD31, platelet endothelial cell adhesion molecule-1; FITC, fluorescein isothiocyanate; MW, molecular weight; NS, not significant; PDGFR-β, plate-derived growth factor receptor-β; PS, permeability surface; VE, vascular endothelial-cadherin; WT, wild type; ZO-1, Zonula Occludens-1. The 12m APPPS1 panel of Figure 2A is excluded from this article's CC-BY license. See the accompanying retraction notice for more information.
Fig 3.
ADAMTS13 deficiency leads to microvascular and cerebral blood flow reductions in APPPS1 mice.
(A) Confocal images of CD31+ microvessels in the cortex of 2-, 6-, and 12-month-old WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice. Scale bar, 50 μm. (B) In vivo multiphoton microscopy images of perfused cortical capillaries with intravenously injected FITC-dextran (MW = 2,000,000 Da). (C–D) Quantification of microvascular density (C) and perfused capillary length (D) in 6- and 12-month-old WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice (n = 6). Relevant data values are included in S1 Data. (E–F) Representative rCBF maps generated from dynamic susceptibility contrast perfusion MRI (E) and quantification of rCBF at the hippocampal levels (F) in 12-month-old WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice (n = 5). Scale bar, 1 mm. Relevant data values are included in S1 Data. Values are mean ± SD. *P < 0.05. ADAMTS13, a disintegrin and metalloprotease with thrombospondin type I motif, member 13; APPPS1, amyloid precursor protein–presenilin 1; CD31, platelet endothelial cell adhesion molecule-1; FITC, fluorescein isothiocyanate; MW, molecular weight; rCBF, relative cerebral blood flow; WT, wild type. The 6m WT panel of Figure 3B is excluded from this article's CC-BY license. See the accompanying retraction notice for more information.
Fig 4.
Accelerated AD pathology and impaired brain Aβ clearance in ADAMTS13-deficient APPPS1 mice.
(A–B) Amount of soluble and insoluble Aβ40 (A) and Aβ42 (B) in 12-month-old APPPS1 and APPPS1–Adamts13−/− mice (n = 6). Relevant data values are included in S1 Data. (C) Representative micrographs of amyloid plaques labeled with antibody against Aβ from the cortex and hippocampus of 12-month-old APPPS1 and APPPS1–Adamts13−/− mice. Scale bar, 100 μm. (D–E) Quantification of Aβ load (D) and ThS+ plaques (E) (n = 6). Relevant data values are included in S1 Data. (F–G) In vivo multiphoton microscopy images of methoxy-X04 labeled Aβ plaques (white) and cerebral angiopathy with TMR-dextran (red) (F) and quantification of CAA load and parenchymal X04+ amyloid load (G) in the cortex of APPPS1 and APPPS1–Adamts13−/− mice (n = 6). Scale bar, 100 μm. Relevant data values are included in S1 Data. (H) Linear relationship between the CAA load and the BBB permeability determined by the PS product for FITC-dextran in APPPS1 and APPPS1–Adamts13−/− mice. Relevant data values are included in S1 Data. (I) Immunoblot analysis of full-length APP, CTFs, and BACE1 in brain tissue and Aβ BBB-transporting proteins RAGE, LRP1, and P-gp in isolated brain microvessels of WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice. (J) Quantification of LRP1 and P-gp (n = 5). Relevant data values are included in S1 Data. (K–L) Plasma accumulation of brain-derived Aβ40 (panel K) and Aβ42 (panel L) before, 10 minutes after, and 40 minutes after the Aβ stabilizing anti-Aβ antibody HJ5.1 injection (n = 6). Relevant data values are included in S1 Data. (M) Quantification of Aβ load in the hippocampus of 12-month-old APPPS1–Adamts13−/− mice treated with Adv-Con and Adv-mLRP1 (n = 5). Relevant data values are included in S1 Data. Values are mean ± SD. *P < 0.05. AD, Alzheimer disease; ADAMTS13, a disintegrin and metalloprotease with thrombospondin type I motif, member 13; Adv-Con, control adenovirus; Adv-mLRP1, adenovirus carrying an LRP1 minigene; APP, amyloid precursor protein; APPPS1, amyloid precursor protein–presenilin 1; Aβ, amyloid-β; BACE1, β-secretase; BBB, Blood-brain barrier; CAA, cerebral amyloid angiopathy; CTF, C-terminal fragment; FITC, fluorescein isothiocyanate; FL-APP, Full length APP; HJ5.1, Aβ-stabilizing anti-Aβ antibody HJ5.1; Hpc, hippocampus; LRP1, lipoprotein receptor-related protein 1; P-gp, P-glycoprotein; PS, permeability surface; r, Pearson’s coefficient; RAGE, receptor for advanced glycation end products; ThS, thioflavin S; TMR, Tetramethylrhodamine; WT, wild type.
Fig 5.
ADAMTS13 deficiency accelerates cognitive impairment and synaptic loss in APPPS1 mice.
(A–F) WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice (n = 12–15 mice at 12 months) were subjected to the MWM, Y-maze, passive avoidance, and open-field test. Escape latency during platform trials (A), number of platform crossings (B), and time spent in the target quadrant in probe trial (C) of the MWM test. (D) Alternation of arms in the spontaneous alternation test of the Y-maze test. (E) Latencies to enter the dark compartment in the passive avoidance test. (F) Number of rearing in open-field test. (G) Y-maze test in 5-month-old WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice (n = 12). Relevant data values are included in S1 Data. (H–I) Representative images of dendritic length (H) and dendritic spines (I) in the hippocampal CA1 region of WT, Adamts13−/−, APPPS1, and APPPS1–Adamts13−/− mice at 12 months. Scale bar, 100 μm (H) and 10 μm (I). (J–K) Quantification of total dendritic length (J) and spine number per 10 μm of dendrite (K) for each group (n = 3). Relevant data values are included in S1 Data. Values are mean ± SD. *P < 0.05. ADAMTS13, a disintegrin and metalloprotease with thrombospondin type I motif, member 13; APPPS1, amyloid precursor protein–presenilin 1; CA1, field CA1 of hippocampus; MWM, Morris Water Maze; NS, not significant; WT, wild type.
Fig 6.
ADAMTS13 adeno-associated virus treatment reduces vascular dysfunction in APPPS1 mice.
(A) Confocal images of the fluorescent signals in the brain of a 12-month-old mouse receiving AAV8-CMV-ADAMTS13 injection. Scale bar, 200 μm. (B) Immunoblotting analysis of transduced ADAMTS13 variant in the Hpc and cortex of mice treated with AAV8-CMV-ADAMTS13 or AAV8 control. (C–D) In vivo multiphoton microscopy images of FITC-dextran (MW = 40,000 Da; green) leakage in cortical vessels (C) and quantification of the PS product of FITC-dextran (D) in 12-month-old APPPS1 mice treated with AAV8-ADAMTS13 or control (n = 6). Scale bar, 100 μm. Relevant data values are included in S1 Data. (E) Quantification of PDGFR-β+ pericyte coverage on CD31+ microvessels in the cortex and Hpc in 12-month-old APPPS1 mice treated with AAV8-ADAMTS13 or control (n = 6). Relevant data values are included in S1 Data. (F–G) Confocal images of CD31+ microvessels in the cortex (F) and quantification of microvascular density in the cortex and Hpc (G) of 12-month-old APPPS1 mice treated with AAV8-ADAMTS13 or control (n = 6). Relevant data values are included in S1 Data. (H–I) In vivo multiphoton microscopy images of perfused cortical capillaries with intravenously injected FITC-dextran (MW = 2,000,000 Da) (H) and quantification of perfused capillary length (I) in 12-month-old APPPS1 mice treated with AAV8-ADAMTS13 or control (n = 6). Scale bar, 100 μm. Relevant data values are included in S1 Data. (J–K) Representative rCBF maps generated from dynamic susceptibility contrast perfusion MRI (J) and quantification of rCBF at the hippocampal levels (K) in 12-month-old APPPS1 mice treated with AAV8-ADAMTS13 or control (n = 5). Scale bar, 1 mm. Relevant data values are included in S1 Data. Values are mean ± SD. *P < 0.05. AAV8, adeno-associated virus serotype 8; ADAMTS13, a disintegrin and metalloprotease with thrombospondin type I motif, member 13; CD31, platelet endothelial cell adhesion molecule-1; CMV, cytomegalovirus; FITC, fluorescein isothiocyanate; Hpc, hippocampus; MW, molecular weight; NS, not significant; PDGFR-β, plate-derived growth factor receptor-β; PS, permeability surface; rCBF, relative cerebral blood flow. The AAV-Control panel of Figure 6C is excluded from this article's CC-BY license. See the accompanying retraction notice for more information.
Fig 7.
ADAMTS13 adeno-associated virus treatment reduces AD pathology and improves cognitive function in APPPS1 mice.
(A–C) Quantification of soluble and insoluble Aβ40 (panel A) and Aβ42 (panel B) and Aβ load (panel C) in 12-month-old APPPS1 mice treated with AAV8-ADAMTS13 or control (n = 6). (D) Quantification of ThS+ plaques in the cortex and Hpc (n = 6). (E–F) In vivo multiphoton microscopy images of methoxy-X04 labeled Aβ plaques (white) and cerebral angiopathy with TMR-dextran (red) (panel E) and quantification of CAA load and parenchymal X04+ amyloid load (panel F) in the cortex of APPPS1 mice treated with AAV8-ADAMTS13 or control (n = 6). Scale bar, 100 μm. (G–H) Immunoblots analysis (panel G) and quantification (panel H) of LRP1 and P-gp in isolated brain microvessels of APPPS1 mice treated with AAV8-ADAMTS13 or control (n = 5). (I–J) Plasma accumulation of brain-derived Aβ40 (panel I) and Aβ42 (panel J) before, 10 minutes after, and 40 minutes after the Aβ-stabilizing anti-Aβ antibody HJ5.1 injection in APPPS1 mice treated with AAV8-ADAMTS13 or control (n = 6). (K) Internalization levels of Aβ42 were analyzed by flow cytometry in vehicle and rADAMTS13-treated hCMEC/D3 cells after incubation with FAM-Aβ42 for 15, 30, 45, and 60 minutes (n = 5). (L) hCMEC/D3 cells were incubated with FAM-Aβ42 for 1 hour, washed, and then incubated with FAM-Aβ42–free medium for 2 hours in the presence or absence of rADAMTS13. The residual Aβ42 was measured by ELISA (n = 5). (M–N) Representative images of Golgi-stained dendritic spines (panel M) and quantification of spine number per 10 μm of dendrite (panel N) in the hippocampal CA1 region of APPPS1 mice treated with AAV8-ADAMTS13 or control. Scale bar, 10 μm (n = 3). (O) Escape latency during platform trials of the MWM test (n = 13–15). (P) Alternation of arms in the Y-maze test in 12-month-old APPPS1 treated with AAV8-ADAMTS13 or control viruses (n = 13–15). Relevant data values are included in S1 Data. Values are mean ± SD. *P < 0.05. AAV8, adeno-associated virus serotype 8; AD, Alzheimer disease; ADAMTS13, a disintegrin and metalloprotease with thrombospondin type I motif, member 13; APPPS1, amyloid precursor protein–presenilin 1; Aβ, amyloid-β; CA1, field CA1 of hippocampus; CAA, cerebral amyloid angiopathy; ELISA, enzyme-linked immunosorbent assay; FAM-Aβ42, Aβ1–42 labeled with 5(6)-carboxyfluorescein; hCMEC/D3, human cerebral microvascular endothelial; HJ5.1, Aβ-stabilizing anti-Aβ antibody HJ5.1; Hpc, hippocampus; LRP1, lipoprotein receptor-related protein 1; MWM, Morris Water Maze; P-gp, P-glycoprotein; rADAMTS13, recombinant ADAMTS13; ThS, thioflavin S; TMR, Tetramethylrhodamine.