Characterization of APPPS1 and APPPS1–Adamts13^−/− mice

To examine the levels of human APP in brains of transgenic mice, we subjected brain homogenates to immunoblot analyses using the human-Aβ-specific antibody 6E10 [22,23]. The presence of full-length 100/105 kDa human APP can only be seen in homogenates from APPPS1 and APPPS1–Adamts13^−/− mice (S1A Fig). Immunoblot analyses with the antibody that recognizes both human and mouse APP indicated a marked increase in APP protein expression in the brains of transgenic mice compared with nontransgenic mice (S1B Fig). Immunoblot probed with antibody to the C-terminal loop domain revealed the full-length human PS1 (S1C Fig, filled arrow) and the human C-terminal fragments (S1C Fig, open arrow) in the brains of transgenic mice [24]. Disruption of ADAMTS13 was confirmed by PCR (S1D Fig), and ADAMTS13 mRNA was detected by reverse transcription-Polymerase Chain Reaction (RT-PCR) in the liver of (wild-type) WT and APPPS1 mice but not in APPPS1–Adamts13^−/− mice (S1E Fig).

ADAMTS13 activities and VWF levels in plasma

We analyzed mice for plasma ADAMTS13 activity. We found a significant reduction in plasma ADAMTS13 activity in 12-month-old APPPS1 mice compared with age-matched WT mice and 2- and 6-month-old APPPS1 mice (Fig 1A). A slight but not statistically significant decrease in ADAMTS13 activity was observed in 6-month-old APPPS1 mice compared with 2-month-old APPPS1 mice, suggesting a reduction of ADAMTS13 activity with age in APPPS1 mice. Because the only known substrate for ADAMTS13 is VWF [9], we analyzed plasma VWF levels. This analysis indicated that Adamts13^−/− and APPPS1–Adamts13^−/− mice at 2, 6, and 12 months of age exhibited greater increases in plasma VWF levels compared with age-matched WT and APPPS1 mice (Fig 1B). Deficiency of ADAMTS13 in APPPS1 mice resulted in an age-dependent increase in plasma VWF levels. Plasma VWF levels also were significantly increased in 12-month-old APPPS1 mice compared with age-matched WT mice and 2-month-old APPPS1 mice. Moreover, we observed a marked increase in the ratio of plasma UL-VWF to low–molecular-weight VWF multimers in 12-month-old APPPS1–Adamts13^−/− mice compared with age-matched APPPS1 littermates (Fig 1C and 1D).

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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.

https://doi.org/10.1371/journal.pbio.3000313.g001

ADAMTS13 deficiency leads to early and progressive BBB breakdown in APPPS1 mice

We quantified BBB permeability by in vivo multiphoton imaging assays of a vascular tracer, fluorescein isothiocyanate (FITC)-dextran (molecular weight (MW) = 40,000 Da). In contrast to the intact BBB observed in 2-month-old WT, Adamts13^−/−, or APPPS1 mice, the BBB was leaky in age-matched APPPS1–Adamts13^−/− littermate mice (Fig 2A and 2C). Similarly, immunostaining of fibrin with platelet endothelial cell adhesion molecule-1 (CD31) showed significant perivascular fibrin deposits in the hippocampus and cortex of 2-month-old APPPS1–Adamts13^−/− mice (Fig 2B and 2D and S2A Fig). Additionally, immunoblot analysis of fibrin in capillary-depleted brain tissue revealed that fibrin accumulated significantly in the brain parenchyma of 2-month-old APPPS1–Adamts13^−/− mice but not in WT, Adamts13^−/−, or APPPS1 littermate mice (S2B and S2C Fig). Multiphoton microscopy analysis of FITC-dextran leakage and quantification of perivascular fibrin deposits indicated that BBB permeability increased with increasing age in APPPS1–Adamts13^−/− mice (Fig 2A, 2B, 2C and 2D and S2A, S2B and S2C Fig). APPPS1 mice showed increased BBB permeability at 12 months of age, consistent with earlier reports that BBB breakdown was present in this transgenic mice at 9 to 12 months of age [19,25–27]. However, in APPPS1–Adamts13^−/− mice, the brains were substantially more permeable at this age. Together, these data indicate that ADAMTS13 deletion is sufficient to induce early and progressive BBB damage in APPPS1 mice.

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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.

https://doi.org/10.1371/journal.pbio.3000313.g002

To investigate whether the BBB breakdown could be related to altered endothelial junctions, we analyzed the expression of junctional proteins in APPPS1–Adamts13^−/− mice. Immunoblotting showed progressive age-dependent loss of the tight junction protein Zonula Occludens-1 (ZO-1) and the adherens junction protein vascular endothelial-cadherin (VE-cadherin) in isolated brain capillaries of APPPS1–Adamts13^−/− mice compared with age-matched WT, Adamts13^−/−, or APPPS1 littermate mice (Fig 2E and 2F and S3A Fig), beginning at 2 months and peaking at 12 months. Double immunostaining of CD31⁺ endothelium and ZO-1 (S3B, S3C and S3D Fig.) or VE-cadherin (S3E and S3F Fig) confirmed significant age-dependent reductions in both junctional proteins in the cortex and hippocampus of APPPS1–Adamts13^−/− mice. No significant changes in ZO-1 and VE-cadherin were observed in 2- and 6-month-old APPPS1 mice. At 12 months, APPPS1 mice also showed reductions in ZO-1 and VE-cadherin, but they were less marked than the reductions observed in APPPS1–Adamts13^−/− mice. In addition, quantification of the coverage of pericyte on brain microvessels in the cortex (Fig 2G) and hippocampus (Fig 2H and S4 Fig) revealed no significant differences in 2-, 6-, and 12-month-old Adamts13^−/− and APPPS1 mice compared with age-matched WT littermates. Microvascular pericytes in the cortex and hippocampus were significantly reduced in 6- and 12-month-old APPPS1–Adamts13^−/− mice compared with those in age-matched WT, Adamts13^−/−, and APPPS1 littermate mice.

ADAMTS13 deficiency leads to microvascular and cerebral blood flow reductions in APPPS1 mice

To address whether the early BBB breakdown and loss of the BBB properties in APPPS1–Adamts13^−/− mice were associated with microvascular degeneration, brain capillary density was analyzed by CD31 staining. We found greater than 36% to 39% and 29% to 38% reductions in capillary density in 6-month-old APPPS1–Adamts13^−/− mice in the cortex and hippocampus, respectively, when compared with other genotypes (Fig 3A and 3C and S5A and S5B Fig). At 12 months, APPPS1 mice displayed 31% and 36% reductions in cortical and hippocampal capillary density, respectively, when compared with WT littermates, as reported [28,29]. ADAMTS13 deficiency in APPPS1 mice resulted in even greater than 25% and 41% reductions in cortical and hippocampal capillary density compared with APPPS1 mice. There was no difference in capillary density among 2-month-old WT, Adamts13^−/−, APPPS1, and APPPS1–Adamts13^−/− mice.

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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.

https://doi.org/10.1371/journal.pbio.3000313.g003

To test whether the reduced capillary density reflects a decrease in brain capillary perfusion, we assessed the cortical vasculature using in vivo multiphoton microscopy imaging of intravenously injected FITC-dextran (MW = 2,000,000 Da). This analysis revealed a significant capillary perfusion deficit with a 34% to 40% reduction in the length of perfused cortical capillaries in 6-month-old APPPS1–Adamts13^−/− mice compared with WT, Adamts13^−/−, and APPPS1 littermate mice (Fig 3B and 3D). Compared with WT littermate mice, 12-month-old APPPS1 mice showed a 37% reduction in the length of perfused cortical capillaries, but this was less marked than the 59% reduction observed in age-matched APPPS1–Adamts13^−/− mice. Dynamic susceptibility contrast perfusion MRI of intravenously injected contrast agent gadopentetate dimeglumine revealed a significantly greater 43% reduction in hippocampal blood flow in 12-month-old APPPS1–Adamts13^−/− mice compared with WT littermates that was more potent than the 24% reduction found in APPPS1 mice (Fig 3E and 3F). These data are consistent with previous reports showing that regional brain capillary density correlates with blood flow [30,31].

ADAMTS13 deficiency accelerates AD pathology and impairs cognitive function in APPPS1 mice

Next, we examined whether ADAMTS13 deficiency causing brain vascular damage and regional cerebral blood flow reduction can influence Aβ pathology in APPPS1 mice. At 12 months of age, soluble and insoluble Aβ40 and Aβ42 were increased by 62%, 52%, 62%, and 36% in brains of APPPS1–Adamts13^−/− mice, respectively, compared with those in APPPS1 littermates (Fig 4A and 4B). Aβ immunostaining and thioflavin S (ThS) staining confirmed this observation, showing that the cortical and hippocampal plaque load were markedly elevated in APPPS1–Adamts13^−/− mice compared with APPPS1 mice (Fig 4C, 4D and 4E). Furthermore, APPPS1–Adamts13^−/− mice developed 2.3-fold and 1.7-fold increases in cerebral amyloid angiopathy (CAA) and parenchymal amyloid deposition, respectively, as measured by in vivo multiphoton imaging of methoxy-X04⁺ amyloid plaques and Tetramethylrhodamine (TMR)-dextran angiopathy (Fig 4F and 4G). Notably, the CAA load was positively correlated with the BBB permeability determined by the permeability surface (PS) product for FITC-dextran in APPPS1 and APPPS1–Adamts13^−/− mice (Fig 4H).

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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.

https://doi.org/10.1371/journal.pbio.3000313.g004

To determine whether ADAMTS13 deficiency in APPPS1 mice affected Aβ production and APP processing, we examined APP metabolism. Consistent with previous reports [32,33], significant increases in the levels of full-length APP and β-secretase (BACE1) and APP C-terminal fragments (β-CTFs and α-CTFs) were observed in brain homogenates from 12-month-old APPPS1 mice compared with age-matched WT controls (Fig 4I and S6 Fig). However, the levels of these proteins were similar in APPPS1 and APPPS1–Adamts13^−/− mice, suggesting that accelerated Aβ brain accumulation and CAA observed in APPPS1–Adamts13^−/− mice did not result from increased Aβ production and processing. Another key mechanism underlying increased Aβ brain accumulation is reduced clearance of Aβ from the brain [2,19,34]. Therefore, we tested whether the expression of the Aβ BBB-transporting proteins is altered in APPPS1–Adamts13^−/− mice. Western blot analysis of Aβ efflux transporters in isolated brain capillaries revealed significant down-regulation of low-density lipoprotein receptor-related protein 1 (LRP1) and P-glycoprotein (P-gp) in APPPS1 mice compared with WT mice (Fig 4I and 4J), consistent with earlier reports [35–37]. We then found a marked decrease in LRP1 and P-gp in APPPS1–Adamts13^−/− mice compared with APPPS1 mice. The level of the Aβ influx transporter receptor for advanced glycation end products (RAGE) did not change (S6 Fig). Using established protocols [38, 39], we next examined the brain Aβ elimination into the blood of mice using an Aβ-stabilizing anti-Aβ antibody HJ5.1 (HJ5.1) that binds brain-derived Aβ within the blood to prevent its rapid degradation. This analysis showed a significant reduction in the plasma levels of Aβ40 and Aβ42 at 40 minutes after injection of HJ5.1 in APPPS1–Adamts13^−/− mice compared with APPPS1 mice (Fig 4K and 4L), indicating that ADAMTS13 deficiency impeded Aβ clearance from the brain into blood. Finally, we tested whether overexpression of LRP1 in brain microvessels in APPPS1–Adamts13^−/− mice affects Aβ pathology. Consistent with findings that LRP1 is a key Aβ clearance protein [31], adenoviral-mediated overexpression of a human LRP1 minigene in the hippocampus of APPPS1–Adamts13^−/− mice (S7A Fig) resulted in a significant reduction in Aβ load (Fig 4M and S7B Fig).

To assess the functional outcome of ADAMTS13 deficiency in APPPS1 mice, we first examined spatial learning and memory using the Morris water maze (MWM) test. At 12 months, APPPS1 mice had significant impairments of learning and memory (Fig 5A, 5B and 5C), consistent with earlier reports [40,41]. Compared with APPPS1 mice, APPPS1–Adamts13^−/− mice showed significantly worse performance, as indicated by a significant increase in the escape latency time during the hidden platform test (Fig 5A), fewer platform crossings (Fig 5B), and shorter time spent in the platform quadrants (Fig 5C) during the probe trials. We next used the Y-maze test. APPPS1–Adamts13^−/− mice exhibited a significantly lower rate of spontaneous alternations than other genotypes (Fig 5D), indicating a more severe working memory deficit. We also performed the passive avoidance test to assess the cognitive functions of animals [42]. This analysis showed that all mice exhibited comparable initial latencies to enter the dark compartment (Fig 5E). Compared with other groups, APPPS1–Adamts13^−/− mice took a shorter time to reenter the dark compartment during the retention session, showing an impairment of memory. In addition, animals were tested in the open-field test. We found a significantly lower number of rearings (Fig 5F) and a shorter traveling distance (S8 Fig) in APPPS1–Adamts13^−/− mice compared with other genotypes. These data indicate that APPPS1–Adamts13^−/− mice had worse locomotor activity than the control APPPS1 mice. To examine whether early cerebrovascular changes in APPPS1–Adamts13^−/− mice are associated with cognitive decline, the Y-maze test was performed in 5-month-old mice. Consistent with previous reports [43–45], APPPS1 mice exhibited a significant impairment in working memory (Fig 5G). ADAMTS13 deficiency caused further worsening of performance in APPPS1 mice.

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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.

https://doi.org/10.1371/journal.pbio.3000313.g005

Reduced dendritic spines have been implicated in cognitive impairment of AD [46,47]. We next examined whether impaired vascular functions by ADAMTS13 deficiency could induce synaptic dysfunction. Golgi staining revealed a 24% reduction in dendritic length (Fig 5H, 5I and 5J) and a 30% loss of spine density (Fig 5K) in the field CA1 of hippocampus (CA1) of APPPS1 mice at 12 months compared with age-matched WT littermates as well as a more severe 46% reduction in dendritic length and a 43% loss of spine density in APPPS1–Adamts13^−/− mice compared with age-matched WT littermates. In contrast, 2-month-old APPPS1–Adamts13^−/− mice did not show evidence of Aβ deposition and synaptic loss (S9A, S9D and S9E Fig). The levels of soluble Aβ40 and Aβ42 in brains of 2-month-old APPPS1–Adamts13^−/− were comparable to that of age-matched APPPS1 mice (S9B and S9C Fig). To further determine whether ADAMTS13 deficiency may affect the synaptic function, we performed whole-cell patch clamp recordings on hippocampal CA1 neurons in brain slices from 2-month-old mice. There were no significant changes in miniature excitatory postsynaptic current (mEPSC) frequency and amplitude among WT, Adamts13^−/−, APPPS1, and APPPS1–Adamts13^−/− mice (S9F, S9G and S9H Fig). However, BBB breakdown and loss of endothelial junctions in APPPS1–Adamts13^−/− mice were already detectable at this age (Fig 2A, 2B, 2C, 2D, 2E and 2F and S2A, S2B, S2C, S3A, S3B, S3C, S3D, S3E and S3F Figs). These data indicate that vascular damage precedes the development of plaque load and synaptic dysfunction in the brain of APPPS1–Adamts13^−/− mice.

Virus-mediated expression of ADAMTS13 reverses BBB breakdown and microvascular and cerebral blood flow reductions in APPPS1 mice

Next, we studied the role of ADAMTS13 in APPPS1 mice by injecting recombinant adeno-associated virus serotype 8 (AAV8)-mediated expression of a murine ADAMTS13 variant (AAV8-ADAMTS13) into the hippocampi of 9-month-old APPPS1 mice, which already exhibited vascular damage, plaque deposition, and cognitive deficits [25,48,49]. After 3 months of virus injection, the mice were tested at 12 months of age. Confocal microscopy analysis for red fluorescent protein (RFP) indicated that the cortex and hippocampus were extensively transduced at 3 months after injection (Fig 6A). Expression of the truncated ADAMTS13 variant in the cortex and hippocampus was demonstrated by immunoblot analyses, using the antibody that recognizes the metalloproteinase domain of ADAMTS13 (Fig 6B). Multiphoton microscopy analysis of FITC-dextran indicated a substantial reduction in BBB permeability in APPPS1 mice treated with AAV8-ADAMTS13 compared with control mice treated with AAV8-RFP (Fig 6C and 6D). No significant differences were observed in microvascular pericytes between control and AAV8-ADAMTS13 adenovirus groups (Fig 6E). Administration of AAV8-ADAMTS13 into APPPS1 mice also caused a significant increase in the capillary density in the cortex and hippocampus and the length of perfused cortical microvessels (Fig 6F, 6G, 6H and 6I). Contrast perfusion MRI revealed that AAV8-ADAMTS13 treatment restored hippocampal blood flow in APPPS1 mice (Fig 6J and 6K).

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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.

https://doi.org/10.1371/journal.pbio.3000313.g006

Virus-mediated expression of ADAMTS13 reduces Aβ deposition to improve cognitive deficits of APPPS1 mice

We asked whether the improved vascular function and regional cerebral blood flow by AAV8-ADAMTS13 could improve AD pathology. At 12 months, APPPS1 mice that received AAV8-ADAMTS13 exhibited reduced levels of brain-soluble and -insoluble Aβ40 and Aβ42 relative to AAV8-RFP–treated controls (Fig 7A and 7B). The cerebral Aβ plaque load was markedly reduced by 41% and 46% in the cortex and hippocampus, respectively, in APPPS1 mice subjected to AAV8-ADAMTS13 injection (Fig 7C and S10A Fig). Plaque staining with ThS confirmed the reduced amyloid plaques in mice injected with AAV8-ADAMTS13 (Fig 7D and S10B Fig). Furthermore, the profiles of CAA and parenchymal amyloid deposition in mice injected with AAV8-ADAMTS13 were significantly reduced compared with those of the control mice (Fig 7E and 7F). Western blot analysis of isolated brain capillaries revealed that AAV8-ADAMTS13 injection led to significantly higher levels of Aβ BBB-transporting proteins LRP1 and P-gp than did AAV8-RFP injection (Fig 7G and 7H). In line with these data, we found significantly increased Aβ elimination from brain to blood in mice injected with AAV8-ADAMTS13 (Fig 7I and 7J). Collectively, these results suggest that the ADAMTS13 expression by virus administration likely decreased Aβ accumulation in the brains of APPPS1 mice via increased BBB-mediated Aβ clearance.

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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.

https://doi.org/10.1371/journal.pbio.3000313.g007

We next determine the roles of ADAMTS13 in Aβ uptake and degradation in human cerebral microvascular endothelial (hCMEC/D3) cells. Aβ uptake was assessed by the addition of Aβ1–42 labeled with 5(6)-carboxyfluorescein (FAM-Aβ42) to the media of hCMEC/D3 cells in the presence and absence of human recombinant ADAMTS13 (rADAMTS13). This analysis showed that the levels of intracellular Aβ42 was enhanced in a time-dependent manner (Fig 7K). Treatment with rADAMTS13 significantly increased Aβ42 uptake following 30, 45, and 60 minutes of FAM-Aβ42 incubation compared with vehicle-treated hCMEC/D3 cells. To quantify Aβ42 degradation, hCMEC/D3 cells were incubated with FAM-Aβ42 for 1 hour, followed by additional incubation for 2 hours without FAM-Aβ42. The levels of Aβ42 remaining were measured by ELISA and defined as degraded Aβ. We found that more Aβ42 was degraded by rADAMTS13-treated hCMEC/D3 cells (Fig 7L). Because astrocytes can take up and degrade Aβ [50], we also examined whether ADAMTS13 affects Aβ uptake and degradation in astrocytes. We observed a time-dependent increase in Aβ42 uptake in astrocytes (S11A Fig). However, Aβ42 uptake was not significantly change by rADAMTS13. ELISA analysis showed that rADAMTS13 also did not increase Aβ42 degradation in astrocytes (S11B Fig). Taken together, our results indicate that ADAMTS13 increased both the uptake and clearance of Aβ42 in cerebral microvascular endothelial cells but not in astrocytes. However, we cannot rule out the possibility that ADAMTS13 may also play a role in mediating Aβ clearance in other cell types.

The dendritic spine density in the hippocampal CA1 region was significantly increased in APPPS1 mice injected with AAV8-ADAMTS13 (Fig 7M and 7N). We then tested whether the reversal of dendritic spine deficits in APPPS1 mice could induce functional changes. In MWM tests, APPPS1 mice injected with AAV8-ADAMTS13 took a shorter time to find the hidden platform in training sessions (Fig 7O), crossed the platform region more times (S12A Fig), and spent a longer time in the platform quadrants (S12B Fig) in the probe trials compared with AAV8-RFP–treated controls. In the Y-maze test, ADAMTS13 expression by virus administration reduced the percentage of spontaneous alternations (Fig 7P). In the open-field test, mice injected with AAV8-ADAMTS13 showed a significantly longer traveling distance (S12C Fig) and a higher number of rearings (S12D Fig) than AAV8-RFP–injected mice. Therefore, virus-mediated expression of ADAMTS13 in APPPS1 mice effectively ameliorated behavioral deficits.

Ethics statement

All animal experiments were approved by the Animal Care and Use Committee of Institutes of Brain Science, Fudan University (approval number 20150119–011).

Animals

Adamts13^−/− mice were purchased from the Jackson Laboratory. APPPS1 transgenic mice, which express a chimeric mouse/human amyloid precursor protein (Mo/Hu APP 695swe) with the mutant human presenilin 1 (PS1-dE9), were purchased from the Model Animal Research Center of Nanjing University. All mice were maintained on a C57BL/6 background. Male hemizygous APPPS1 mice were crossed to female hemizygous Adamts13^−/− mice to generate APPPS1, Adamts13^−/−, and APPPS1–Adamts13^−/− mice and littermate nontransgenic controls. Mice were kept on a 12-hour light/dark cycle and had ad libitum access to food and water.

In vivo multiphoton imaging

In vivo time-lapse multiphoton images were taken as we previously described [17]. Briefly, in the parietal bone, a 2-mm–diameter window was opened, and a sterile cover glass was placed above the brain. Image experiments were performed using a multiphoton laser-scanning microscope (FV1200MPE, Olympus, Japan). Cerebrovascular permeability was evaluated by time-lapse imaging taken every 180 seconds for 20 minutes after FITC-dextran (40,000 Da, Sigma-Aldrich; 0.1 ml of 10 mg/ml) injection. To analyze perfusion of cortical microvessels, 0.1 ml bolus of FITC-dextran (2,000,000 Da, Sigma-Aldrich; 10 mg/ml) was injected intravenously. Multiphoton z-stack images were obtained from 200 μm below the surface of the cortex to a depth of 500 μm.

To visualize amyloid plaques, methoxy-XO4 (Tocris Bioscience, Bristol, UK; 10 mg/kg) was injected intraperitoneally into mice. Twelve hours later, the mice were anesthetized and implanted with a cranial window, and amyloid imaging was performed [56]. TMR-conjugated dextran (70,000 Da; Invitrogen, MA; 0.1 ml of 10 mg/ml) was intravenously injected before imaging to provide a fluorescent angiogram. In vivo images (500 × 500 μm) were acquired using a 25× objective. Each image was traced using ImageJ software. The thresholded TMR-dextran and methoxy-XO4 images were superimposed in 2 different layers. The TMR-dextran⁺ area was selected, and the mean value of the pixels of the methoxy-XO4 image was calculated within the TMR-dextran selection. CAA load was presented as a percentage of methoxy-XO4⁺ area covering TMR-dextran⁺ area. See details in S1 Text.

Measurements of cerebral blood flow using MRI

Mice were anesthetized with 1% to 1.5% isoflurane in air/O₂ (2:1). MRI experiments were performed on a 9.4 T/400 mm scanner (Agilent Technologies, Santa Clara, CA), using a quadrature conformal surface coil (7.5mm–12.5mm) [57]. A series of T2-weighted MR images were collected before and after intravenous injection of contrast agent gadopentetate dimeglumine (Shanghai Xudong Haipu Pharmaceutical Co., Shanghai, China, 0.42 mmol/kg) via tail vein. See details in S1 Text.

Injection of adeno-associated virus vectors

Recombinant AAV8 encoding a C-terminal truncated murine ADAMTS13 variant (aa 1–2400) (pAAV8-CMV-ADAMTS13-P2A-mCherry-3FLAG) under a cytomegalovirus (CMV) promoter and control adenovirus only expressing the RFP AAV (pAAV8-CMV-P2A-mCherry-3FLAG) were prepared by Obio Technology (Shanghai, China). Previous studies have documented the safety and long-term expression of this AAV8-mediated expression of ADAMTS13 variant in a murine model of thrombotic thrombocytopenic purpura [58]. ADAMTS13 and RFP AAV8 were stereotactically bilaterally injected into the hippocampus (5 × 10¹² viral genomes/ml, 2 μl per hippocampus) of the mice at 9 months of age. The following coordinates were used: anteroposterior +2.0 mm, mediolateral ±1.5 mm, and dorsoventral −1.5 mm [59]. The adenovirus carrying a human LRP1 minigene (Adv-mLRP1) and empty adenovirus (Adv-Con) were prepared (Obio Technology, Shanghai, China) and injected into the hippocampus of APPPS1–Adamts13^−/− mice at 12 months of age, as described by Winkler and colleagues [31]. Injection of Adv-mLRP1 effectively induced the expression of LRP1 minigene in the hippocampus of APPPS1–Adamts13^−/− mice (S7A Fig).

MWM

Experiments were conducted by investigators blinded to genotypes or treatments of mice for all behavioral measurements. The MWM was used to test spatial learning and reference memory functions [59]. See details in S1 Text.

Y-maze test

Y-maze tests are used to assess short-term spatial working memory [60]. The Y-maze consists of 3 black horizontal arms (36 cm long, 5 cm wide, and 10 cm high) at 120° angles to each other. See details in S1 Text.

Passive avoidance test

The passive avoidance test was assessed in a step-through box apparatus (Med Associates, St Albans, VT) consisting of a light compartment and a dark compartment [42]. The floor of each compartment contained a grid, with only the dark one being electrified by a generator. The mice were placed into the light compartment, and after 60 seconds, the guillotine door was opened. The latency to enter the dark compartment was recorded. Once entering the dark compartment, the mice received an immediate electrical shock (0.5 mA for 1 second). Mice were then returned to the home cages. After 24 hours, each mouse was again placed into the light compartment, and the latency to enter into the dark compartment was recorded for up to 300 seconds.

Open-field test

Mice were tested for 5 minutes in a clear 27.3 × 27.3 × 40 cm activity chamber (Med Associates, VT) with three 16-beam infrared arrays. See details in S1 Text.

Tissue preparation and ThS staining

Mice were deeply anesthetized; brains were removed and immersed with 4% paraformaldehyde in PBS at 4°C. Frozen coronal sections of the brain were cut at 15-μm thickness with a cryostat (Leica Microsystems, IL) and stored at −80°C until use. For ThS staining, brain sections were incubated in 1% ThS (T1892, Sigma-Aldrich, MO) for 10 minutes and rinsed in PBS. See details in S1 Text.

Immunohistochemistry

Frozen coronal brain sections were immunostained as described previously [17,61]. See details in S1 Text.

Analysis of extravascular fibrin deposition

Cerebral tissue cryosections were blocked with 5% normal donkey serum in PBS and incubated with a combination of antibodies of rat anti-CD31 (PECAM-1; 553370, 1:400; BD Pharmingen, NJ) and rabbit anti-fibrin (ogen) (AP00766PU-N, 1:2,000; Acris Antibodies, Germany) overnight at 4°C. The sections were washed and incubated with Alexa Fluor 594 donkey anti-rat IgG and Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen, CA). To quantify extravascular deposits of fibrin, the images were contrast enhanced to clearly differentiate positivity from background and quantified using the NIH ImageJ integrated density analysis tool. For each animal, 6 fields from the cortex and 4 fields from the hippocampus in 6 sections (100 μm apart) were analyzed.

Preparation of brain microvessels and capillary-depleted brain homogenates

Mouse brain capillaries were isolated as previously described [17,30]. Briefly, brains were removed, and the meninges and large surface vessels were discarded. Brain tissue was homogenized in ice-cold 16% dextran (Sigma-Aldrich, MO) in PBS containing 2% fetal bovine serum (FBS) with a glass dounce tissue grinder. Homogenates were centrifuged at 6,000g for 15 minutes at 4°C. The supernatant was collected and centrifuged again to obtain capillary-depleted brain homogenates. Pellets were resuspended in PBS containing 1% BSA and passed through a 100-μm and 40-μm cell strainer (BD Falcon, San Jose, CA). Microvessels were collected from the 40-μm strainer membrane and used for experiments.

Western blotting

Brain tissues, brain capillaries, and capillary-depleted brain homogenates were lysed in RIPA lysis buffer (Millipore, MO) containing protease inhibitor cocktails (Roche Diagnostics, Basel, Switzerland). Equal amounts of protein were separated by SDS-PAGE electrophoresis, blotted onto a nitrocellulose membrane, and incubated with primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies (1:2,000; Cell Signaling Technology, MA). Signals were detected by an enhanced chemiluminescence detection reagent (PerkinElmer) and imaged using a ChemiDoc Touch imaging system with Quantity One software (Bio-Rad, Hercules, CA). See details in S1 Text.

ADAMTS13 activity assay

ADAMTS13 activity in plasma was determined using a commercial FRETS-VWF73 peptide (Peptides International, Louisville, KY) according to the manufacturer’s instructions. Briefly, FRETS-VWF73 (100 μl of 4 mM, final concentration 2 μM) was incubated with 4 μl plasma in reaction buffer (5 mM Bis-Tris, 25 mM CaCl2, 0.005% Tween-20 [pH 6.0]). Fluorescence intensities were detected with a fluorescence spectrophotometer (Bio-Tech, Winooski, VT) every 5 minutes for 1 hour using excitation at 340 nm and emission at 450 nm.

Assay of VWF levels

VWF levels in plasma was measured as described by Zhu and colleagues [62]. Briefly, microtiter plates were coated with rabbit anti-VWF antibody (A0082, DAKO, CA) overnight at 4°C. Plasma samples (1:40 diluted with 3% BSA in PBS) were incubated in the wells for 2 hours at room temperature. Then, the plates were incubated with 100 μl HRP-conjugated VWF (P0226, 1:2,000; DAKO, CA) for 1 hour at room temperature. After washing, 100 μl tetramethylbenzidine substrate (Sigma-Aldrich) was added to the wells. After 15 minutes, the reaction was stopped with 50 μl 2N HCL. Absorbance was read at 450 nm in an ELISA reader (Bio-Rad, CA). We could not achieve satisfactory analyses for ADAMTS13 activity and VWF levels in mice brains with commercially available antibodies, and thus these analyses were precluded from the study.

Assay of VWF multimers

The VWF multimers analysis was performed as described by Xu and colleagues [17]. Citrated plasma was diluted in sample buffer (70 mM Tris-HCl [pH 6.5], containing 2.4% sodium dodecyl sulfate, 4% urea, and 4 mM EDTA) and was incubated at 60°C for 20 minutes. The sample was fractionated on a 1.2% SeaKem HGT agarose (50041, Rockland, ME) gel by electrophoresis and transferred onto nitrocellulose membrane (A10006746, Life Science, NJ). VWF multimers were visualized using a rabbit anti-VWF antibody (A0082, DAKO, CA). The signal was obtained using a ChemiDoc Touch imaging system (Bio-Rad, CA).

Aβ ELISA

Cortex and hippocampus were homogenized in ice-cold Tris buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 50 mM EDTA, and 1% Triton X-100) with protease inhibitors (Roche Diagnostics, Basel, Switzerland) and centrifuged at 21,000g for 30 minutes at 4°C. The supernatant was used for measurements of soluble Aβ fraction. For insoluble Aβ, the pellet was resuspended in 70% formic acid (10% v/w), homogenized, and ultracentrifuged at 100,000g for 60 minutes at 4°C; formic acid-containing supernatants were neutralized with 1 M Tris buffer (pH 11). The levels of Aβ 40 and Aβ 42 in both fractions were quantified by Ultrasensitive Human ELISA Kits (KHB3482 and KHB3544, Invitrogen, MA) according to the instructions of the manufacturer.

Analysis of the elimination of brain Aβ into plasma

The efflux efficacy of brain-derived Aβ in blood was determined as described [38,39]. Briefly, an Aβ-stabilizing anti-Aβ antibody (HJ5.1, 150 μg/mouse; provided by Dr. David M. Holtzman, Washington University School of Medicine, St. Louis, MO) was injected intravenously into 12-month-old APPPS1 transgenic mice. The anti-Aβ antibody HJ5.1 can rapidly bind brain-derived Aβ in the blood to prevent its degradation and allow direct measurement of the rate Aβ enters blood from the brain [38]. Blood was collected before, 10 minutes after, and 40 minutes after HJ5.1 antibody injection by retro-orbital bleeding. Plasma levels of Aβ 40 and Aβ 42 were quantified by using Ultrasensitive Human ELISA Kits (KHB3482 and KHB3544, Invitrogen, MA).

Cell cultures

hCMEC/D3 was purchased from DSMZ (CVCL-U985, Braunschweig, Germany). Cells were grown in EBM-2 medium (Lonza, Walkersville, MD) and supplemented with 5% FBS (Gibco, Rockville, MD), 1% penicillin (Gibco, MA), 1% streptomycin (Gibco, MA), 1.4 μM hydrocortisone (Sigma-Aldrich, MO), 5 μg/ml ascorbic acid (Sigma-Aldrich, MO), and 10 mM HEPES (Gibco, MA). Cultures were maintained at 37°C in a humidified 95% air and 5% CO₂, and the medium was changed every 2 to 3 days. Primary cultures of astrocytes were prepared using cortices of postnatal day 1 C57BL/6 mice as described by Zhao and colleagues [63]. Briefly, cortices were minced and incubated with 2.5% trypsin (Gibco, MA) at 37°C for 15 minutes. Dissociated cells were suspended in astrocyte growth medium containing Dulbecco's Modified Eagle Media (DMEM) and Ham's F12 (1:1 v/v) (Gibco, MA) with 10% FBS. The cells were passed through a 100-μm mesh sieve (BD Biosciences, NJ) and plated on poly-D-lysine-coated 75-flask in DMEM/F12, supplemented with 10% FBS, penicillin (100 U/ml, Gibco, MA), and streptomycin (100 μg/ml, Gibco, MA). The culture medium was changed every 2 to 3 days. When confluent, the cells were shaken at 260 rpm for 18 hours, and the medium was aspirated to remove microglial cells. Astrocyte purity was routinely >95% as assessed by glial fibrillary acidic protein (GFAP) immunostaining.

Preparation of Aβ42 peptides

Aβ1–42 labeled with 5(6)-carboxyfluorescein (FAM-Aβ42, AnaSpec, Fermont, CA) were dissolved in 1 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and incubated at room temperature for 2 hours. After the HFIP solution was removed by vacuum centrifugation (RVC 2–25 CD plus, Christ, Osterode, Germany), the remaining Aβ peptide was dissolved in DMSO at 5 mM and sonicated for 10 minutes. The peptide solution was incubated at 37°C for 24 hours and then centrifuged at 14,000g for 10 minutes at 4°C. The supernatant containing soluble oligomers was stored at 4°C.

Aβ uptake assays

hCMEC/D3 cells or astrocytes were seeded in 6-well plates and grown to confluence. FAM-Aβ42 (100 nM) was added 2 hours after addition of rADAMTS13 (60 ng/ml, R&D Systems, LA). After incubation for 15, 30, 45, and 60 minutes (for hCMEC/D3 cells) or 30, 60, and 120 minutes (for astrocytes), cells were rinsed 3 times with cold PBS and enzymatically detached using trypsin (Gibco, MA). Cells were centrifuged and resuspended in PBS and then incubated with 0.2% trypan blue (Gibco, MA) in PBS and fixed with 4% paraformaldehyde. Uptake was measured for fluorescence on a BD LSR Fortessa (BD Biosciences, NJ), and data were analyzed with FlowJo 10.0.8 software (FlowJo, https://www.flowjo.com).

Aβ efflux assays

hCMEC/D3 cells or astrocytes were initially incubated with FAM-Aβ42 for 1 hour (for hCMEC/D3 cells [54]) or 2 hours (for astrocytes). The medium was then removed, and cells were washed with warmed medium. The cells were then incubated in FAM-Aβ42–free medium for 2 hours at 37°C in the presence or absence of rADAMTS13 (60 ng/ml). After washing, RIPA lysis buffer (Millipore, MO) containing protease inhibitor cocktails (Roche Diagnostics, Basel, Switzerland) was added to each well. After centrifugation, protein concentration was determined with the BCA protein assay (Thermo Scientific, MA). FAM-Aβ42 were quantified by Ultrasensitive Human ELISA Kits (KHB3544, Invitrogen, MA) according to the instructions of the manufacturer.

Statistical analysis

All data are represented as mean ± SD. Statistical tests were performed using GraphPad Prism(GraphPad Software, https://www.graphpad.com). Two-group comparisons were evaluated using the unpaired two-tailed Student t test. Multiple comparisons were analyzed by one-way ANOVA followed by the Bonferroni multiple comparison test. A linear correlation between 2 variables was performed using Pearson’s correlation coefficients. Differences with P < 0.05 were considered significant.