Cerebral vascular amyloid seeds drive amyloid β-protein fibril assembly with a distinct anti-parallel structure

Cerebrovascular accumulation of amyloid β-protein (Aβ), a condition known as cerebral amyloid angiopathy (CAA), is a common pathological feature of patients with Alzheimer's disease. Familial Aβ mutations, such as Dutch-E22Q and Iowa-D23N, can cause severe cerebrovascular accumulation of amyloid that serves as a potent driver of vascular cognitive impairment and dementia. The distinctive features of vascular amyloid that underlie its unique pathological properties remain unknown. Here, we use transgenic mouse models producing CAA mutants (Tg-SwDI) or overproducing human wild-type Aβ (Tg2576) to demonstrate that CAA-mutant vascular amyloid influences wild-type Aβ deposition in brain. We also show isolated microvascular amyloid seeds from Tg-SwDI mice drive assembly of human wild-type Aβ into distinct anti-parallel β-sheet fibrils. These findings indicate that cerebrovascular amyloid can serve as an effective scaffold to promote rapid assembly and strong deposition of Aβ into a unique structure that likely contributes to its distinctive pathology.

region of Ab16-22 (red) containing 1-13 C labeled Leu2 (corresponding to Leu17 in the Ab40 sequence) and unlabeled Ab16-22 (black). Fibrils were harvested after incubating at 37 °C for 10 days. The 1626 cm -1 resonance in the spectrum of the unlabeled peptide is characteristic of b-sheet secondary structure. The splitting into two intense bands at 1602 and 1630 cm -1 is associated with anti-parallel b-sheet 2,3 . a.u. = arbitrary units.
(b) The seven-residue GNNQQNY peptide, a fragment of the yeast prion protein Sup35p, forms fibrils with parallel, in-register b-strands. The parallel, in-register structure in this peptide, which was also established by solid-state NMR measurements 5 is likely driven by hydrogen bonding interactions between the side chain amide groups of Asn and Gln. In this case, isotope labeling does not shift the major symmetric 1629 cm -1 band but results in a weak isotope shifted resonance at 1596 cm -1 . The broad intensity at ~1650 cm -1 is attributed to the side chain Asn and Gln amide vibrations.
Molecular structures of the parallel and anti-parallel b-strands are shown below the FTIR spectra to illustrate that the hydrogen bonding arrangement is different in the two geometries. This difference in hydrogen-bonding gives rise to the differences in the amide I vibration, which corresponds largely to the C=O stretching vibration.
The differences in the vibrational spectra for anti-parallel and parallel b-strands within b-sheet secondary structure observed for these two model peptides are the same those found by vibrational calculations on anti-parallel and parallel b-sheets 2 . Namely, for anti-parallel structure the major component of the amide I normal mode splits into two equally intense bands (one with higher frequency and one with lower frequency). In contrast, for parallel structure the major b-sheet peak does not change frequency or intensity, but a very weak isotope shifted band is observed at a lower frequency than the intense low-frequency component of the amide I band observed in the spectra of the anti-parallel structure. The differences arise in how the internal coordinates couple within the amide I normal mode. This coupling is dependent on the geometry. FTIR spectroscopy of Ab40-DI containing 1-13 C-Gly33 provides a probe for anti-parallel b-sheet secondary structure. Here we describe the protocol for obtaining both antiparallel and parallel fibrils of Ab40-DI, and compare FTIR spectra and TEM images of the resulting fibrils.
(a) FTIR spectrum of Ab40-DI with anti-parallel structure. Inset shows the amide I region (see also Supplementary Fig. 3). The anti-parallel fibrils of Ab40-DI were obtained using the following procedure modified from that of Tycko and colleagues 1 . Pure Ab40-DI peptide was dissolved in DMSO at a concentration of 2 mM (~10 mgs in 0.5 mL DMSO). A 100 µL aliquot was taken and diluted into 5 mL 10 mM phosphate buffer (10 mM NaCl) such that the final concentration of DMSO was 2%, and the concentration of Ab40-DI was 100 µM. The solution was filtered with a 0.22 µM filter and allowed to fibrillize overnight. The solution was then transferred into a glass vial and bath sonicated (15 min). Another 5 mL of cold phosphate buffer was added to the sonicated solution along with another 100 microliters of the DMSO stock solution of Ab40-DI. This material was allowed to fibrillize for 3 h in the cold room and then filtered twice with 0.2 µM filters. Another 100 microliters of DMSO stock in 5 mL of cold phosphate buffer was added to the filtered solution. Comparison of the amide 1 region (1600-1700 cm -1 ) of the spectrum obtained at 22 °C after incubating for 1 week in panel (a) with the spectrum obtained of fibrils immediately following the anti-parallel fibril preparation at 4-6 °C (see Supplementary Fig. 3a) shows there is considerably more random coil structure in the sample prepared and kept at low temperature. The high frequency region of the FTIR spectrum (~3000 cm -1 ) contains water vibrations and indicates that the samples contain comparable amounts of residual hydration. (b) FTIR spectrum of Ab40-DI with parallel b-sheet structure obtained by incubation at 37 °C. (c) TEM images of antiparallel fibrils at 22 °C. Scale bar = 100 nm. (d) TEM images of parallel fibrils obtained by incubating Ab40-DI at 37 °C. The anti-parallel fibrils have a more curved appearance than the parallel fibrils as described by Tycko and coworkers 6 . Scale bar = 100 nm. Anti-parallel fibrils of Ab40-I were previously found to be meta-stable 6 .
Here we show that for anti-parallel fibrils of Ab40-DI we observe a conversion to parallel structure upon increasing the temperature from 4 °C to 37 °C. (a) FTIR spectra of the anti-parallel seeds prepared at 4-6 °C using the protocol described in Supplementary  Fig. 2. (b) Increasing the temperature to 22 °C leads to the formation of fibrils as observed by TEM (Fig. 1b). The FTIR spectrum does not change over a week of incubation at this temperature. (c-e) Increasing the temperature to 37 °C leads to a conversion of the anti-parallel fibrils to parallel fibrils. The change is detected by the shift (and loss of intensity) of the 1610 cm -1 band to 1600 cm -1 . This conversion indicates that the anti-parallel fibrils are meta-stable.
AFM images of the Ab40-D and Ab40-I peptides (unpublished) indicate that the antiparallel fibrils are composed of laterally associated oligomers as observed for Ab42 2 . We suggest that the lower intensity of the ~1604-1610 cm -1 band in the in vitro experiments compared to the seeded fibrils is due to a mixture of both parallel and antiparallel ß-strands within the "protofibrils" formed in vitro. In contrast to the fibrils, the fibrils seeded from vascular amyloid are stable at 37 °C. The anti-parallel peak observed in Fig. 7e (main text) is more intense than observed above and does not change upon incubation at 37 °C.

Supplementary Figure 4
Supplementary Figure 4. Transient anti-parallel structure occurs in the formation of parallel Ab40-DI fibrils. Intramolecular antiparallel b-strands can be found within the Ab peptide due to the formation of b-hairpin secondary structure in pre-fibrillar intermediates 7 and intermolecular antiparallel b-strands can occur within the cross bsheets of fibrils 6 . We have recently shown that b-hairpin secondary structure develops within the high MW oligomers and protofibrils of Ab42 during the conversion to mature fibrils 1 . In this supporting figure, we show for Ab40-DI that transient anti-parallel structure also occurs prior to the formation of stable b-sheet secondary structure (i.e. before the formation of mature fibrils). This transient appearance of anti-parallel structure in non-fibrillar intermediates is distinct from the anti-parallel structure observed in the fibrils generated from vascular amyloid seeds.
(a) Ab40-DI forms fibrils rapidly in solution. Thioflavin T measurements show there is a rapid rise fluorescence associated with fibril formation at 37 °C starting from solutions of monomeric Ab40-DI (100 µM). The halfway point in the transition occurs at ~0.5 h and the transition is largely complete by 3-4 hours. (b) Formation of b-sheet secondary structure accompanies fibril formation. Circular dichroism spectra were obtained of the Ab40-DI peptide as a function of incubation time. The Ab40-DI peptide is largely random coil before 1 h and converts to b-sheet secondary structure after ~1 h. At 37 °C, Ab40-DI forms b-sheet structure in which the b-strands have a parallel orientation ( Supplementary Fig. 2). (c) FTIR spectra of Ab40-DI containing 1-13 C Ile31 and 1-13 C Val39 obtained as a function of incubation time reveal several features. The spectra obtained at 0 h and 0.5 h exhibit an isotope-shifted resonance at 1613 cm -1 characteristic of antiparallel structure. This band losses intensity and shifts to 1597 cm -1 as fibrils form. (d) Expansion of the FTIR spectra in the region of 1580-1630 cm -1 showing the amide I vibrational bands associated with the conversion of ant-parallel bhairpin structure (observed prior to fibril formation) to b-sheet with parallel b-strands (after fibril formation).

Supplementary Figure 5
Supplementary Figure 5. Dutch/Iowa CAA mutant cerebral microvascular amyloid in Tg-SwDI mice does not accumulate administered biotin and aged wild-type mice do not accumulate biotin-labeled wild-type Ab peptides. In this supporting figure, we show that unlike biotin-labeled wild-type Ab peptides, intrahippocampal administered biotin alone does not accumulate on pre-exisiting cerebral capillary amyloid deposits in Tg-SwDI mice. Further, we show that intrahippocampal administered biotin-labeled wild-type Ab peptides do not accumulate on cerebral capillaries of aged wild-type mice that lack vascular amyloid deposits.
Biotin was injected into the hippocampal region of twelve months old Tg-SwDI mice (a-c). Alternatively, biotin-labeled wild-type Ab40 (d-f) or biotin-labeled wild-type Ab42 (g-i) was injected into the hippocampus of similarly aged wild-type mice lacking capillary amyloid deposits. Brain sections were prepared and fibrillar amyloid was detected using Amylo-Glo (blue) and immunolabeled with an antibody to collagen IV for detection of cerebral blood vessels using Alexa Fluor 594-conjugated donkey anti-rabbit (red). Biotin or biotin-labeled wild-type Ab40 or Ab42 peptides were detected using streptavidin-Alexa Fluor 488 (green). Scale bars = 50 µm. The studies described in the main text set the stage for parallel studies on vascular amyloid derived from human brain associated with sporadic CAA or associated with CAA resulting from the Ab40-D or Ab40-I mutations. (a) Microvessels isolated from human brain associated with sporadic CAA stained for fibrillar amyloid using thioflavin S (green) and immunolabeled for cerebral blood vessels using an antibody to collagen IV (red). Scale bar = 50 µm. (b) Microvascular amyloid deposits after digestion and removal of the microvessels stained for fibrillar amyloid using thioflavin S (green). Scale bar = 50 µm. (c) Thioflavin T fluorescence showing rapid fibril grow of Ab42 in the presence and absence of human microvascular amyloid seeds derived from the human brain tissue. (d) FTIR spectra of Ab fibrils formed using soluble wild-type Ab42 labeled with 1-13 C Gly33 added to seeds from human microvascular amyloid deposits show anti-parallel signature. form 1 of Ab42 can nucleate rapid fibril growth of Ab40 monomers, but not seeds derived from fibril form 2. The ability of seeds from fibril form 1 to efficiently nucleate Ab40 growth demonstrates that these two isoforms are not inherently incapable of cross-seeding. Nevertheless, the inability of the seeds of form 2 to efficiently nucleate Ab40 growth provides a potential explanation for observation that parenchymal plaques are comprised of predominantly Ab42 11 , i.e. the Ab42 fibril structure in parenchymal plaques may be substantially different from the structure in vascular amyloid. For Ab42, the seeds from both fibril forms result in rapid fibril growth using Ab42 monomers. These experiments were undertaken with 40 µM monomer Ab solutions and 5% seeds.