Neurotoxicity and Physicochemical Properties of Aβ Mutant Peptides from Cerebral Amyloid Angiopathy

Abstract

Cerebral amyloid angiopathy (CAA) due to ␤-amyloid (A␤) is one of the specific pathological features of familial Alzheimer's disease. A␤ mainly consisting of 40-and 42-mer peptides (A␤40 and A␤42) exhibits neurotoxicity and aggregative abilities. All of the variants of A␤40 and A␤42 found in CAA were synthesized in a highly pure form and examined for neurotoxicity in PC12 cells and aggregative ability. All of the A␤40 mutants at positions 22 and 23 showed stronger neurotoxicity than wild-type A␤40. Similar tendency was observed for A␤42 mutants at positions 22 and 23 whose neurotoxicity was 50 -200 times stronger than that of the corresponding A␤40 mutants, suggesting that these A␤42 mutants are mainly involved in the pathogenesis of CAA. Although the aggregation of E22G-A␤42 and D23N-A␤42 was similar to that of wild-type A␤42, E22Q-A␤42 and E22K-A␤42 aggregated extensively, supporting the clinical evidence that Dutch and Italian patients are diagnosed as hereditary cerebral hemorrhage with amyloidosis. In contrast, A21G mutation needs alternative explanation with the exception of physicochemical properties of A␤ mutants. Attenuated total reflection-Fourier transform infrared spectroscopy spectra suggested that ␤-sheet content of the A␤ mutants correlates with their aggregation. However, ␤-turn is also a critical secondary structure because residues at positions 22 and 23 that preferably form two-residue ␤-turn significantly enhanced the aggregative ability. Alzheimer's disease (AD)1 is neuropathologically characterized by the progressive deposition of amyloid in the brain parenchyma and cortical blood vessels (1). This deposition mainly consists of 40-and 42-mer ␤-amyloid peptides (A␤40 and A␤42) generated from amyloid precursor protein by two proteases, ␤-and ␥-secretases (2, 3). Cerebral amyloid angiopathy (CAA) in familial Alzheimer's disease is linked to missense mutations inside the A␤-coding region in the amyloid precursor protein. The mutations of A␤ sequence are concentrated at positions 21-23 and are called Flemish (A21G) (4), Arctic (E22G) (5, 6), Dutch (E22Q) (7), Italian (E22K) (8), and Iowa (D23N) (9) mutations. These A␤ mutant peptides may play a pathological role in the CAAs because wild-type A␤ peptides induce neuronal death in vitro (10). Neurotoxicity and formation of amyloid fibrils of some CAA-related A␤40 mutants have been independently reported by several groups (11-15). However, there are no reports on the neurotoxicity and aggregation of the CAA-related A␤42 mutants with the exception of Dutch mutation (E22Q) (11), the investigation of which is essential to reveal the mechanism of CAA because wild-type A␤42 shows considerably stronger neurotoxicity and aggregative ability than wild-type A␤40 (11). Moreover, it is indispensable to simultaneously compare neurotoxicity and aggregative ability of all of the CAA-related A␤40 and A␤42 mutants in the same conditions such as pH, peptide concentration, reaction buffer, and temperature.It is difficult to synthesize A␤42 with 14 hydrophobic and/or ...

Analysis of the Secondary Structure of β-Amyloid (Aβ42) Fibrils by Systematic Proline Replacement

“…3A, all of the proline-substituted A␤42 mutants at positions 15-32 with the exception of E22P-A␤42 hardly aggregated after 8 h of incubation. Only the data of the sedimentation assay are shown, because the HPLC data are more reliable than the Th-T fluorescence data as reported previously (22). Similar results were obtained after 16 and 24 h of incubation with the exception of V18P-, F19P-, G25P-, and A30P-A␤42 that showed slow but significant aggregation kinetics (data not shown).…”

“…5. The fibrils of E22P-A␤42 have been reported previously (22). The morphologies of these fibrils resembled each other well.…”

“…Cell Culture of PC12 Cells-Rat pheochromocytoma PC12 cells were obtained from Riken Cell Bank and were cultured as reported previously (15,21,22). For experimental purposes, near-confluent cultures of the cells were plated at ϳ10 4 cells/100 l/well fresh culture medium in a 96-well tissue culture plate coated with collagen and incubated at 37°C under 5% CO 2 overnight before the experiments.…”

“…Sedimentation Assay for Fibril Formation-Each A␤ derivative was dissolved in 0.02% NH 4 OH at 250 M. After a 10-fold dilution by 50 mM sodium phosphate containing 100 mM NaCl at pH 7.4, the resultant peptide solution (25 M) was incubated at 37°C for 4,8,16,24, or 48 h. After centrifugation at 15,000 rpm in an Eppendorf microcentrifuge at 4°C for 10 min, 25 l of the supernatant was then analyzed by HPLC as reported previously (15,21,22). The area of the absorption at 220 nm was integrated and expressed as a percentage of the control.…”

“…Prolines are rarely present in ␤-sheet, whereas they are easily accommodated in a variety of turns, for example, as a Pro-X corner (where X is a variable amino acid residue) (13). Quite recently, Williams et al (14) have investigated a systematic proline replacement of A␤40, showing that the residues 15-21, 24 -28, and 31-36 are likely to include the ␤-sheet portions of the fibril and that the residues at positions 22,23,29, and 30 probably occupy turn positions among these ␤-sheet elements. This conclusion does not contradict our preliminary investigation using proline-substituted A␤42 at positions 19 -26 (15).…”