Fig. 4

Complete endothelial knockout of PICALM eliminates all beneficial effects of artesunate in 5XFAD mice. A PICALM immunostaining is barely detectable in lectin-positive endothelium in isolated brain capillaries from Picalm^lox/lox; Cdh5-Cre mice compared to Picalm^lox/lox littermate controls. Immunostaining is representative of n = 3 for each mouse line. B PICALM immunoblotting of endothelial cells isolated from brain capillaries from Picalm^lox/lox; Cdh5-Cre and Picalm^lox/lox mice. The blot is representative of n = 2 cultures for each mouse line. C, D PICALM immunoblotting of brain capillaries (C) and capillary depleted (CD) brain (D) isolated from Picalm^lox/lox; Cdh5-Cre and Picalm^lox/lox mice. n = 4 for each mouse line. E Experimental treatment paradigm for Picalm^lox/lox; Cdh5-Cre; 5XFAD mice with artesunate (Art) or vehicle. Mice were injected i.p. with 32 mg/kg/day Art or vehicle for 2 months, starting at 3 months of age, followed by behavior tests, evaluation of cerebral blood flow (CBF) responses, and tissue collection and assays at 5 mo of age. F, G Amyloid-β 42 (Aβ 42) (F) and Aβ 40 (G) levels in the hippocampus (Hpp) and cortex (Ctx) in Picalm^lox/lox; Cdh5-Cre; 5XFAD mice treated with vehicle or artesunate (Art). H, I Representative images of Aβ immunostaining (H), and quantification of Aβ load in the Hpp and Ctx (I) in Picalm^lox/lox; Cdh5-Cre; 5XFAD mice treated with vehicle or Art. Scale bar in (H) is 500 μm. J, K Representative images of Thioflavin S-positive amyloid deposits (J) and quantification of Thioflavin S amyloid load in the Hpp and Ctx (K) in Picalm^lox/lox; Cdh5-Cre; 5XFAD mice treated with vehicle or Art. Scale bar in (I) is 500 μm. L, M Representative images (L) and quantification (M) of Aβ vascular load in small pial arteries and penetrating cortical arterioles in Picalm^lox/lox; Cdh5-Cre; 5XFAD mice treated with vehicle or Art. Aβ vascular load is expressed as fraction of Aβ-positive area relative to α-smooth muscle actin (αSMA)-positive area of the vessel wall, normalized to the vehicle-treated group. Scale bar in (L) is 25 μm. (N) Cerebral blood flow (CBF) response to whisker stimulation in Picalm^lox/lox; Cdh5-Cre; 5XFAD mice treated with vehicle or Art. O-R Novel object location (O) and recognition (P), burrowing (Q), and nesting score (R) in Picalm^lox/lox; Cdh5-Cre; 5XFAD mice treated with vehicle or Art. In F, G, I, K, M, N, n = 7 mice per condition (Vehicle: 4 male, 3 female; Art: 5 male, 2 female). In O-R, n = 8 mice per condition (Vehicle: 4 male, 4 female; Art: 6 male, 2 female). Single points per mouse indicated by circles in C, D, F, G, I, K, M-R, with boxes representing mean ± SD. Significance determined by two-tail t-test. Full blots for B-D shown in supp. Fig. 5