SOBA: Development and testing of a soluble oligomer binding assay for detection of amyloidogenic toxic oligomers

Abstract

The formation of toxic Amyloid β-peptide (Aβ) oligomers is one of the earliest events in the molecular pathology of Alzheimer's Disease (AD). These oligomers lead to a variety of downstream effects, including impaired neuronal signaling, neuroinflammation, tau phosphorylation, and neurodegeneration, and it is estimated that these events begin 10 to 20 y before the presentation of symptoms. Toxic Aβ oligomers contain a nonstandard protein structure, termed α-sheet, and designed α-sheet peptides target this main-chain structure in toxic oligomers independent of sequence. Here we show that a designed α-sheet peptide inhibits the deleterious effects on neuronal signaling and also serves as a capture agent in our soluble oligomer binding assay (SOBA). Pre-incubated synthetic α-sheet-containing Aβ oligomers produce strong SOBA signals, while monomeric and β-sheet protofibrillar Aβ do not. α-sheet containing oligomers were also present in cerebrospinal fluid (CSF) from an AD patient versus a noncognitively impaired control. For the detection of toxic oligomers in plasma, we developed a plate coating to increase the density of the capture peptide. The proof of concept was achieved by testing 379 banked human plasma samples. SOBA detected Aβ oligomers in patients on the AD continuum, including controls who later progressed to mild cognitive impairment. In addition, SOBA discriminated AD from other forms of dementia, yielding sensitivity and specificity of 99% relative to clinical and neuropathological diagnoses. To explore the broader potential of SOBA, we adapted the assay for a-synuclein oligomers and confirmed their presence in CSF from patients with Parkinson's disease and Lewy body dementia.

Keywords: Alzheimer’s disease; SOBA; alpha-sheet; detection; toxic oligomer.

Fig. 1.

Milestones in the molecular pathology of AD and targeting the toxic Aβ oligomers with a de novo designed α–sheet peptide. (A) Timing of different pathological milestones along the AD continuum. There is consensus regarding this general order of events (–19), except that some studies and models put tau phosphorylation before Aβ plaque deposition. The Aβ oligomers are depicted in red as a hexamer, noting that hexamers and dodecamers (stacked hexamers) are the dominant α–sheet toxic oligomers (13) and they are approximately spherical by atomic force microscopy (32). (B) Structural changes in 75 µM Aβ42 in PBS during aggregation from unstructured monomer, to low-molecular-weight α–sheet soluble oligomers, to high-molecular weight β-sheet protofibrillar aggregates. Samples were diluted to 25 µM immediately before CD. (C) Preincubated Aβ42 samples in PBS (75 µM) spiked into plasma corresponding to the time points in panel B show that the α–sheet oligomers produced high SOBA signals while the monomer and β-sheet protofibrils did not (100 pM Aβ42 applied to SOBA in each case). (D) Effects of pre-incubated Aβ42 oligomers (75 µM in PBS, inclubated for 24 h at 25 °C) on electrical activity of neuronal cells and maintenance of signal with co-administration of α–sheet design. Aβ42 oligomers (30 µM diluted in differentiation medium, see Methods) caused a 20% drop in the average number of spikes (P < 0.002 vs. other conditions). AP193 alone and Aβ42+AP193 (2:1, excess Aβ) were within the variability of what was observed in baseline measurements prior to adding compounds. (E) Size exclusion chromatograms (SEC) of neat human CSF on Superdex 200 column monitored at 254 nm. AD and CO control samples with assignments for peaks that are SOBA positive with three Aβ toxic oligomer peaks indicated, based on molecular weight standards. (F) Oligomer peaks for samples before and after SOBA, showing the depletion of 3-, 6-, and 12-mer peaks in the AD sample from binding of α–sheet oligomers and no change in these same peaks in the CO control sample.

Fig. 2.

SOBA discriminates between controls, non-AD CI, and MCI/AD cases. (A) Aβ toxic oligomers detected with SOBA plotted for plasma samples (n = 379) and colored by clinical diagnosis (see legend at right). SOBA-positive CO samples correspond to false positives or preclinical AD “converters,” PC-AD, which are colored purple if validated through clinical follow-up or autopsy. Averages of raw luminescent readings are provided. Samples with signals above the threshold (28,207 signal, denoted by the dashed horizontal line) are SOBA positive and those below are SOBA negative. (B–F) Plots for the CSF biomarkers associated with the plasma samples in panel A: Aβ42, tau, p-tau181, tau/Aβ42, and p-tau181/Aβ42, respectively, were determined using commercial Luminex and/or InnoGenetics multiplex assays. Note overlap between the CO, MCI, and AD groups as well as the lack of discrimination between PC-AD and CO cases. (G) SOBA values (n = 379) binned by final diagnosis but colored by original clinical diagnosis. Neuropathological diagnoses were used when available, clinical otherwise. The toxic oligomer-positive, SOBA-positive CO cases correspond to an early preclinical stage along the AD continuum. The non-AD CI cases are also included. Note the 3 cases contrary to the final diagnoses determined with neuropathological and biomarker follow up. Two are SOBA-negative with one clinically diagnosed as AD (red) and the other clinically diagnosed as non-AD CI (blue) but determined to be AD from the neuropathological assessment. The other is a SOBA-positive case that was determined to be non-AD CI by autopsy. (H) Case studies of CO versus PC-AD (CO ➙ MCI converters) and non-AD CI versus AD. CO v. PC-AD: Comparison of biomarker levels between two individuals (ID20 and ID93) in the control group and the values when ID93 later converted to MCI. AD v. non-AD CI and CO: Case study of two cognitively impaired individuals diagnosed with dementia, one due to AD (ID37) and the other FTLD (ID57), representing a non-AD case of cognitive impairment. A control CO subject is provided for reference (ID32).

Fig. 3.

SOBA Aβ toxic oligomer signals over time, overall performance in distinguishing controls (CO) and non-AD dementia participants from those on the Alzheimer’s Disease continuum (PC-AD, MCI, and AD), and extension of the assay to detect α–synuclein toxic oligomers associated with PD and Lewy body disorders. (A) Profile plot of SOBA-AD signals for longitudinal samples separated by clinical diagnosis at study entry and change in diagnosis over follow up for 59 participants with at least one follow-up visit. Note the difference in the scales of the y-axes. (B) SOBA results for CSF from patients with PD and/or LBD patients and controls with a modified version of the assay that replaces the 6E10 Aβ detection antibody for the 4B12 a-synuclein antibody (SOBA-PD). CSF was diluted 1:10 in PBS. The purple values correspond to four CO individuals (two of which had two samples each, see panel C) from the AD plasma sample set who were later diagnosed with PD and/or LBD. (C) Using SOBA-AD and SOBA-PD for discrimination of AD and PD/LBD, potential comorbidity, and preclinical detection of PD/LBD for two CO controls from the AD plasma set who later converted to PD. SOBA-PD detected a-synuclein toxic oligomers in the CSF 6 and 7 y, respectively, before clinical diagnosis. In addition, one of the subjects also tested positive for Aβ toxic oligomers by SOBA-AD (plasma), suggesting potential comorbidity. Furthermore, two CO cases were later found to have LBD upon autopsy 13 and 16 y later. Their CO CSF samples were SOBA-PD positive. (D) Receiver operating characteristic analysis and associated results for SOBA-screened plasma in this study, n = 379 samples. Comparison was made against the neuropathology diagnoses where available and confirmed clinical diagnoses when not (confirmed = follow-up including neuropsychological and biomarker assessments). Further comparisons are provided in the SI Appendix, Data set.