Glial activation in response to ADan deposition. (A–F) GFAP immunohistochemistry reveals an activation of astrocytes (arrowheads) at sites of Congo-red stained ADan deposition (red) in 4-month-old (D), 10-month-old (E), and 18-month-old (F) ADanPP7 tg mice. No such hypertrophic and darkly stained astrocytes are seen in non-tg littermates (A, B, and C, respectively). (G–L) Similarly, activation of Iba1-positive microglia in ADanPP7 mice closely follows the temporal-spatial pattern of ADan deposition (arrowheads in J, K, and L). Corresponding non-tg littermates in (G, H, and I, respectively). (M and N) Higher magnification of GFAP⁺ astroglia (M) and Iba1⁺ microglia (N) with congophillic amyloid (red). (O) Confocal imaging of Iba1 (red) and amyloid (green; methoxy staining) gives the impression that microglia are capable of engulfing ADan (shown is a z-stack of 28 slices). [Scale bars: 300 μm (A–L), 50 μm (M and N), 5 μm (O).]

ADan lesions and their impact on the neuropil and vasculature. (A–D) Various types of ADan lesions in 18-month-old ADanPP7 tg mice (Ab 5282) at light microscopic level. (E–G) Ultrastructural appearance of the ADan lesions. Amyloid fibrils (asteriks), microglia cell nucleus (N); dystrophic neurites (DN); microglia cell with the typical features of phagosomal/lysosomal material (arrowhead in G). (H and I) Confocal microscopy of double-immunolabeled vessels (red, smooth muscle-cell actin; green, ADan) in a non-tg control (H) and tg (I) mouse. Note the focal disappearance of smooth muscle cells at sites of ADan deposition (arrowheads, I). (J) Hemosiderin-positive microglia (blue) reveal the occurrence of cerebral microhemorrhages. (K) Hemorrhage frequency per hemisphere in tg and non-tg 18-month-old mice [F(1,14) = 14.440, P < 0.002; n = 8, four females and four males per group). (L) Dystrophic synaptophysin-positive structures in the vicinity of both parenchymal and vascular ADan deposits (arrowhead), but also throughout the parenchyma (arrow). (M and N) Cresyl violet staining of the hippocampus of an 18-month-old tg mouse (N) compared with a non-tg littermate (M). (O) Number of CA3 neurons in 18-month-old ADanPP7 mice did not reveal significant changes [F(1,10) = 0.326, P = 0.5806; n = 6 non-tg, 6 tg; five males, seven females). [Scale bars: 20 μm (A–D, J, L), 2 μm (E, G), 3 μm (F), 10 μm (H, I), 200 μm (M, N).]

Aβ deposition is reduced and not coincident with ADan deposition in double-tg ADanPP7/APPPS1 mice. (A and B) Aβ immunostaining of 4-month-old ADanPP7⁺/APPPS1⁺ double-tg mice compared with single-tg ADanPP7⁻/APPPS1⁺ littermates reveals a decrease in Aβ deposition. Higher magnifications additionally show a change in plaque morphology (i.e., less but occasionally larger plaques in the double-tg mice). (C) Quantification of neocortical Aβ load reveals a drastic decrease in Aβ deposition in the double-tg mice [F(1,10) = 17.852, P < 0.0019; all females; n = 5 single tg, n = 7 double tg]. (D and E) Western blotting of APP and Aβ40/42 (Ab 6E10) in ADanPP7⁺/APPPS1⁺ mice in comparison with ADanPP7⁻/APPPS1⁺ siblings reveals no change in APP but a decrease in the double-tg mice in both Aβ40 and Aβ42. Shown are three mice from each genotype. (F) Densitometry of Aβ levels of all of the mice confirms the significant decrease in both Aβ40 [71.4%, F(1,10) = 7.444, P < 0.0213] and Aβ42 [47.9%, F(1,10) = 8.106, P < 0.0173] species (all females; n = 5 single tg, 7 double tg). (G and H) Western blotting of ADanPP (Ab Itm2b) and ADan peptide (Ab 5282) reveals no change in ADanPP and ADan levels between genotypes. Shown are three mice each. (I) Densitometry of ADan levels of all of the mice indicates no significant difference (all females; n = 4 single tg, 7 double tg). (J–M) Confocal microscopy reveals deposition of both peptides (green, ADan; red, Aβ) in some amyloid plaques in the neocortex of double-tg mice; however, plaques consistently contained separate amyloid foci in which the two proteins do not colocalize (J). Other amyloid deposits in the hippocampus demonstrated deposition of only ADan or only Aβ in close vicinity to each other (K). Plaques consisting of only Aβ were often decorated with ADanPP/ADan⁺ dystrophic boutons (arrowheads in L). Vascular amyloid consistently contained either ADan alone, or fascinatingly, both ADan and Aβ as separate foci along the same vessel, without colocalization. [Scale bars: 500 and100 μm (A and B), and 20 μm (J–M).]

ADan accelerates the occurrence of Tau lesions in double-tg ADanPP7/TauP301S mice. (A) Gallyas silver staining of 12-month-old ADanPP7⁺/TauP301S⁺ mice reveals increased Tau lesions in both neocortex and brainstem in comparison with single-tg ADanPP7⁻/TauP301S⁺ littermates, with no staining in ADanPP7⁺/TauP301S⁻ mice. Inserts are higher magnifications. (B) Quantification of neocortical Gallyas-positive cells shows a dramatic increase in ADanPP7⁺/TauP301S⁺ mice [all female; n = 6 double tg, n = 7 single tg; F(1,11) = 5.377, P < 0.041]. (C) Western blotting of the sarcosyl insoluble fraction of forebrain “F” and hindbrain “H” homogenates using Ab HT7 reveals an increase in the forebrain of ADanPP7⁺/TauP301S⁺ mice (Lane 3) in comparison with ADanPP7⁻/TauP301S⁺ littermates (Lane 1), while the increase in the hindbrain appears smaller (Lanes 4 and 2, respectively). (D and E) No difference in neocortical ADan pathology (Ab 5282) between ADanPP7⁺/TauP301S⁺ mice and ADanPP7⁺/TauP301S⁻ littermates is observed (D) consistent with the stereological quantification [E; F(1,10) = 1.014, P = 0.338; all females; n = 6 double tg, 6 single tg]. (F) Western blotting also does not reveal a difference in ADan peptide levels between ADanPP7⁺/TauP301S⁺ mice (Lanes 3 and 4) in comparison with ADanPP7⁺/TauP301S⁻ littermates (Lanes 5 and 6). (G) Phenotyping of the Tau-lesions in the neocortex of ADanPP7⁺/TauP301S⁺ mice reveals a Tau conformation folded at the N terminus (Ab Alz-50) with mostly an intact C terminus (Ab Tau-46.1). The Tau lesions are Thiazin red (TR)-negative, while ADan deposits in the wall of blood vessels are TR-positive (see merger of Alz-50, Tau46.1, and TR staining). The lesions consist of hyperphosphorylated Tau at Ser396-404 (Ab PHF1) and Ser199-202 (Ab pSer199-202). For comparison, phenotyping of the Tau lesions in the neocortex of 12-month-old APPPS1⁺/TauP301S⁺ mice reveals the same conformation and phosphorylation pattern. For a list of antibodies and stains, see Table S1. [Scale bars: 100 and 20 μm (A–F), 10 μm (G).]

Age-related deposition of cerebral Dan-amyloid in ADanPP-tg mice. (A) Mutant BRI2 contains a 10-nucleotide duplication just before the stop codon generating a longer ORF (277 instead of 266 amino acids) (1). Normal cleavage at position 243 by a furin-like protease releases the amyloidogenic 34 amino acid long ADan peptide in FDD (2, 3). In addition, BRI2 undergoes processing by ADAM10 to release the extracellular Brichos domain from the membrane-bound N-terminal part that, in turn, undergoes regulated intramembrane proteolysis by SPPL2a/b (42). The recognition sites of various antibodies (Ab) are indicated in green. (B) Western blotting at 2 months of age reveals higher levels of ADanPP in line 7 compared to line 6 (Upper, Ab 1139). Transgene expression in line 7 is several-fold higher than endogenous murine Itm2b (Lower, Ab 1141 recognizes both murine and human BRI2). (C) Western blot of ADanPP7 mouse brain at 2, 4, 10, and 18 months demonstrates that levels of precursor protein remain relatively the same (Upper, Ab 1139) with increasing accumulation of ADan peptide, which runs at the same height as the ADan monomer (M) of FDD patients (Lower, Ab 5282). (D) ADanPP expression in a 2-month-old ADanPP7 mouse (Ab Itm2b). (E) Methoxy staining of ADan lesions in an 18-month-old ADanPP7 mouse. (F–I) Quantification of the amyloid lesions in ADanPP7 mice in hippocampus was done using ADan-specific Ab 5282 in combination with Congo red. At 4 months of age amyloid deposits are seen in the stratum lacunosum moleculare of CA3 (F); at 10 months of age the amyloid appears largely associated with vessels and occurs throughout the dentate gyrus (G); at 18 months of age the entire hippocampus is covered with amyloid lesions (H). Stereological analysis of the Congo-red ADan material (I) revealed a significant increase from 2 to 18 months of age [F(3,24) = 56.404, P < 0.0001] with no gender difference [F(1,24) = 0.017, P = 0.8979; n = 4 females and 4 males, per age group]. (Scale bar, 200 μm.)