Structural basis of the 24B3 antibody against the toxic conformer of amyloid ß with a turn at positions 22 and 23.

Amyloid ß-protein (Aß) oligomers are involved in the early stages of Alzheimer's disease (AD) and antibodies against these toxic oligomers could be useful for accurate diagnosis of AD. We identified the toxic conformer of Aß42 with a turn at positions 22/23, which has a propensity to form toxic oligomers. The antibody 24B3, developed by immunization of a toxic conformer surrogate E22P-Aß9-35 in mice, was found to be useful for AD diagnosis using human cerebrospinal fluid (CSF). However, it is not known how 24B3 recognizes the toxic conformation of wild-type Aß in CSF. Here, we report the crystal structure of 24B3 Fab complexed with E22P-Aß11-34, whose residues 16-26 were observed in electron densities, suggesting that the residues comprising the toxic turn at positions 22/23 were recognized by 24B3. Since 24B3 bound only to Aß42 aggregates, several conformationally restricted analogs of Aß42 with an intramolecular disulfide bond to mimic the conformation of toxic Aß42 aggregates were screened by enzyme immunoassay. As a result, only F19C,A30homoC-SS-Aß42 (1) bound significantly to 24B3. These data provide a structural basis for its low affinity to the Aß42 monomer and selectivity for its aggregate form.

Structure Optimization of the Toxic Conformation Model of Amyloid ß42 by Intramolecular Disulfide Bond Formation.

Amyloid ß (Aß) oligomers play a critical role in the pathology of Alzheimer's disease. Recently, we reported that a conformation-restricted Aß42 with an intramolecular disulfide bond through cysteine residues at positions 17/28 formed stable oligomers with potent cytotoxicity. To further optimize this compound as a toxic conformer model, we synthesized three analogues with a combination of cysteine and homocysteine at positions 17/28. The analogues with Cys-Cys, Cys-homoCys, or homoCys-Cys, but not the homoCys-homoCys analogue, exhibited potent cytotoxicity against SH-SY5Y and THP-1 cells even at 10 nM. In contrast, the cytotoxicity of conformation-restricted analogues at positions 16/29 or 18/27 was significantly weaker than that of wild-type Aß42. Furthermore, thioflavin-T assay, non-denaturing gel electrophoresis, and morphological studies suggested that the majority of these conformation-restricted analogues exists in an oligomeric state in cell culture medium, indicating that the toxic conformation of Aß42, rather than the oligomeric state, is essential to induce cytotoxicity.

Synthesis and Characterization of Propeller- and Parallel-Type Full-Length Amyloid ß40 Trimer Models.

Oligomers of the amyloid ß (Aß) protein play a critical role in the pathogenesis of Alzheimer's disease. However, their heterogeneity and lability deter the identification of their tertiary structures and mechanisms of action. Aß trimers and Aß dimers may represent the smallest aggregation unit with cytotoxicity. Although propeller-type trimer models of E22P-Aß40 tethered by an aromatic linker have recently been synthesized, they unexpectedly exhibited little cytotoxicity. To increase the flexibility of trimeric propeller-type models, we designed and synthesized trimer models with an alkyl linker, tert-butyltris-l-alanine (tButA), at position 36 or 38. In addition, we synthesized two parallel-type trimer models tethered at position 38 using alkyl linkers of different lengths, α,α-di-l-norvalyl-l-glycine (di-nV-Gly) and α,α-di-l-homonorleucyl-l-glycine (di-hnL-Gly), based on the previously reported toxic dimer model. The propeller-type E22P,V36tButA-Aß40 trimer (4), which was designed to mimic the C-terminal anti-parallel ß-sheet structures proposed by the structural analysis of 150 kDa oligomers of Aß42, and the parallel-type E22P,G38di-nV-Gly-Aß40 trimer (6) showed significant cytotoxicity against SH-SY5Y cells and aggregative ability to form protofibrillar species. In contrast, the E22P,G38tButA-Aß40 trimer (5) and E22P,G38di-hnL-Gly-Aß40 trimer (7) exhibited weak cytotoxicity, though they formed quasi-stable oligomers observed by ion mobility-mass spectrometry and native polyacrylamide gel electrophoresis. These results suggest that 4 and 6 could have some phase of the structure of toxic Aß oligomers with a C-terminal hydrophobic core and that the conformation and/or aggregation process rather than the formation of stable oligomers contribute to the induction of cytotoxicity.

Optimization of the Linker Length in the Dimer Model of E22P-Aß40 Tethered at Position 38.

Since amyloid ß (Aß) oligomers are more cytotoxic than fibrils, various dimer models have been synthesized. We focused on the C-terminal region that could form a hydrophobic core in the aggregation process and identified a toxic conformer-restricted dimer model (E22P,G38DAP-Aß40 dimer) with an l,l-2,6-diaminopimelic acid linker (n = 3) at position 38, which exhibited moderate cytotoxicity. We synthesized four additional linkers (n = 2, 4, 5, 7) to determine the most appropriate distance between the two Aß40 monomers for a toxic dimer model. Each di-Fmoc-protected two-valent amino acid was synthesized from a corresponding dialdehyde or cycloalkene followed by ozonolysis, using a Horner-Wadsworth-Emmons reaction and asymmetric hydrogenation. Then, the corresponding Aß40 dimer models with these linkers at position 38 were synthesized using the solid-phase Fmoc strategy. Their cytotoxicity toward SH-SY5Y cells suggested that the shorter the linker length, the stronger the cytotoxicity. Particularly, the E22P,G38DAA-Aß40 dimer (n = 2) formed protofibrillar aggregates and exhibited the highest cytotoxicity, equivalent to E22P-Aß42, the most cytotoxic analogue of Aß42. Ion mobility-mass spectrometry (IM-MS) measurement indicated that all dimer models except the E22P,G38DAA-Aß40 dimer existed as stable oligomers (12-24-mer). NativePAGE analysis supported the IM-MS data, but larger oligomers (30-150-mer) were also detected after a 24 h incubation. Moreover, E22P,G38DAA-Aß40, E22P,G38DAP-Aß40, and E22P,G38DAZ-Aß40 (n = 5) dimers suppressed long-term potentiation (LTP). Overall, the ability to form fibrils with cross ß-sheet structures was key to achieving cytotoxicity, and forming stable oligomers less than 150-mer did not correlate with cytotoxicity and LTP suppression.

Characterization of a Conformation-Restricted Amyloid ß Peptide and Immunoreactivity of Its Antibody in Human AD brain.

Characterization of amyloid ß (Aß) oligomers, the transition species present prior to the formation of Aß fibrils and that have cytotoxicity, has become one of the major topics in the investigations of Alzheimer's disease (AD) pathogenesis. However, studying pathophysiological properties of Aß oligomers is challenging due to the instability of these protein complexes in vitro. Here, we report that conformation-restricted Aß42 with an intramolecular disulfide bond at positions 17 and 28 (SS-Aß42) formed stable Aß oligomers in vitro. Thioflavin T binding assays, nondenaturing gel electrophoresis, and morphological analyses revealed that SS-Aß42 maintained oligomeric structure, whereas wild-type Aß42 and the highly aggregative Aß42 mutant with E22P substitution (E22P-Aß42) formed Aß fibrils. In agreement with these observations, SS-Aß42 was more cytotoxic compared to the wild-type and E22P-Aß42 in cell cultures. Furthermore, we developed a monoclonal antibody, designated TxCo-1, using the toxic conformation of SS-Aß42 as immunogen. X-ray crystallography of the TxCo-1/SS-Aß42 complex, enzyme immunoassay, and immunohistochemical studies confirmed the recognition site and specificity of TxCo-1 to SS-Aß42. Immunohistochemistry with TxCo-1 antibody identified structures resembling senile plaques and vascular Aß in brain samples of AD subjects. However, TxCo-1 immunoreactivity did not colocalize extensively with Aß plaques identified with conventional Aß antibodies. Together, these findings indicate that Aß with a turn at positions 22 and 23, which is prone to form Aß oligomers, could show strong cytotoxicity and accumulated in brains of AD subjects. The SS-Aß42 and TxCo-1 antibody should facilitate understanding of the pathological role of Aß with toxic conformation in AD.

Amyloid ß toxic conformer has dynamic localization in the human inferior parietal cortex in absence of amyloid plaques.

Amyloid ß (Aß) plays a critical role in the pathogenesis of Alzheimer's disease. Nevertheless, its distribution and clearance before Aß plaque formation needs to be elucidated. Using an optimized immunofluorescent staining method, we examined the distribution of Aß in the post-mortem parietal cortex of 35 subjects, 30 to 65 years of age, APOE ε3/ε3, without AD lesions. We used 11A1, an antibody against an Aß conformer which forms neurotoxic oligomers. 11A1 immunoreactivity (IR) was present in cortical neurons, pericapillary spaces, astrocytes and the extracellular compartment at 30 years of age. The percentage of neurons with 11A1 IR did not change with age, but the number and percentage of astrocytes with 11A1 IR gradually increased. Notably, the percentage of pericapillary spaces labeled with 11A1 IR declined significantly in the 5th decade of the life, at the same time that 11A1 IR increased in the extracellular space. Our findings indicate that the Aß toxic conformer is normally present in various cell types and brain parenchyma, and appears to be constitutively produced, degraded, and cleared from the inferior parietal cortex. The decrease in pericapillary Aß and the concomitant increase of extracellular Aß may reflect an age-associated impairment in Aß clearance from the brain.

Synthesis and biochemical characterization of quasi-stable trimer models of full-length amyloid ß40 with a toxic conformation.

Here, we report the first synthesis of quasi-stable trimer models of full-length Aß40 with a toxic conformation using a 1,3,5-phenyltris-l-alanyl linker at position 34, 36, or 38. The only trimer to exhibit weak neurotoxicity against SH-SY5Y cells was the one which was linked at position 38. This suggests that such a propeller-type trimer model is not prone to forming oligomers with potent neurotoxicity, which is in contrast with its corresponding dimer model.

Synthetic Models of Quasi-Stable Amyloid ß40 Oligomers with Significant Neurotoxicity.

The formation of soluble oligomers of amyloid ß42 and 40 (Aß42, Aß40) is the initial event in the pathogenesis of Alzheimer's disease (AD). Based on previous systematic proline replacement and solid-state NMR, we proposed a toxic dimer structure of Aß42, a highly aggregative alloform, with a turn at positions 22 and 23, and a hydrophobic core in the C-terminal region. However, in addition to Aß42, Aß40 dimers can also contribute to AD progression because of the more abundance of Aß40 monomer in biological fluids. Here, we describe the synthesis and characterization of three dimer models of the toxic-conformation constrained E22P-Aß40 using l,l-2,6-diaminopimeric acid (DAP) or l,l-2,8-diaminoazelaic acid (DAZ) linker at position 30, which is incorporated into the intermolecular parallel ß-sheet region, and DAP at position 38 in the C-terminal hydrophobic core. E22P-A30DAP-Aß40 dimer (1) and E22P-A30DAZ-Aß40 dimer (2) existed mainly in oligomeric states even after 2 weeks incubation without forming fibrils, unlike the corresponding monomer. Their neurotoxicity toward SH-SY5Y neuroblastoma cells was very weak. In contrast, E22P-G38DAP-Aß40 dimer (3) formed ß-sheet-rich oligomeric aggregates, and exhibited more potent neurotoxicity than the corresponding monomer. Ion mobility-mass spectrometry suggested that high molecular-weight oligomers (12-24-mer) of 3 form, but not for 1 and 2 after 4 h incubation. These findings indicate that formation of the hydrophobic core at the C-terminus, rather than intermolecular parallel ß-sheet, triggers the formation of toxic Aß oligomers. Compound 3 may be a suitable model for studying the etiology of Alzheimer's disease.

Monoclonal antibody with conformational specificity for a toxic conformer of amyloid ß42 and its application toward the Alzheimer's disease diagnosis.

Amyloid ß-protein (Aß42) oligomerization is an early event in Alzheimer's disease (AD). Current diagnostic methods using sequence-specific antibodies against less toxic fibrillar and monomeric Aß42 run the risk of overdiagnosis. Hence, conformation-specific antibodies against neurotoxic Aß42 oligomers have garnered much attention for developing more accurate diagnostics. Antibody 24B3, highly specific for the toxic Aß42 conformer that has a turn at Glu22 and Asp23, recognizes a putative Aß42 dimer, which forms stable and neurotoxic oligomers more potently than the monomer. 24B3 significantly rescues Aß42-induced neurotoxicity, whereas sequence-specific antibodies such as 4G8 and 82E1, which recognizes the N-terminus, do not. The ratio of toxic to total Aß42 in the cerebrospinal fluid of AD patients is significantly higher than in control subjects as measured by sandwich ELISA using antibodies 24B3 and 82E1. Thus, 24B3 may be useful for AD diagnosis and therapy.