Background

Alzheimer's disease (AD) has previously been linked to dysregulated pancreatic hormones, including insulin, which can exacerbate AD pathology by causing brain insulin resistance [1–2]. Amylin, a centrally acting neuroendocrine hormone co-synthesized and co-secreted with insulin from pancreatic β-cells [3], readily crosses from the blood to the brain parenchyma [4] and is reportedly involved in the central regulation of satiation [5]. In nearly all (>95% prevalence) patients with type-2 diabetes mellitus (T2DM), amylin forms cytotoxic amyloid plaques in the pancreas, which contributes to the pathogenesis of T2DM by inducing β-cell apoptosis and subsequent β-cell mass depletion [6–8]. In both sporadic and early-onset familial AD, amylin synergistically forms co-aggregates with vascular and brain parenchymal Aβ in the brain [9–14]. Consistent with findings in human AD brains, APPswe/PS1dE9 (APP/PS1) rats expressing human amylin in the pancreas (since murine amylin is non-amyloidogenic) revealed that chronic exposure to circulating amylin promoted cerebrovascular and parenchymal amylin-Aβ deposition [9–13]. These results suggest an association between Aβ pathology and amyloidogenic human amylin but the exact mechanism responsible for amylin-Aβ co-aggregation remains to be determined.

Previous research demonstrated that heterologous seeding between amylin and common Aβ fragments (e.g., Aβ42 and Aβ40) promotes amyloid formation in a manner that is comparable to that of homogenous amylin amyloid [15–17]. In vivo amylin-Aβ hetero-amyloid formation and cytotoxicity are facilitated by the co-expression of the two peptides in cells [21–22]. The hypothesis that amylin and Aβ peptides aggregate in the human AD brain to form amylin-Aβ hetero-oligomers has previously been verified by co-immunoprecipitation experiments using human brain samples from AD patients [9]. These converging data [9,15–22] support the presence of amylin-Aβ hetero-oligomers in the human brain and establish a need for their quantification. Proper detection and quantification of amylin-Aβ hetero-oligomers may further establish the connection between amylin and Aβ and their collective role in AD pathology.

While the need for a means to reliably quantify the molecular amylin-Aβ interaction in AD has been acknowledged [9–13], currently employed molecular techniques (namely immunoprecipitation, western blot, circular dichroism, and electron microscopy) limit studies to reduced sample sizes due to their inherent complexity, non-scalability, and in some cases, cost. Additionally, the denaturing conditions commonly used in methods such as western blot prove to be additional obstacles, as the size and structural stability of hetero-oligomers can be altered. Therefore, there is still an unmet need for an assay method that can detect and quantify hetero-oligomers of amylin-Aβ without interrupting molecular interactions. Previously, we reported the creation of a novel enzyme-linked immunosorbent assay (ELISA) to detect amylin-Aβ hetero-oligomers in blood and brain tissue [23] in AD model animals and human donor tissue. Here, we provide a detailed, stepwise protocol for our assay. Relative to the currently employed techniques, this assay is scalable, high-throughput, cost-effective, and non-complex, all while critically maintaining hetero-oligomer stability. The assay detailed here may prove to be a useful tool to detect, analyze, and eventually disentangle the role of the amyloidogenic amylin and Aβ proteins in AD and other pathological conditions.