Abstract
Aggregation and deposition of amyloid-β (Aβ) peptides in extracellular plaques and in the cerebral vasculature are prominent neuropathological features of Alzheimer’s disease (AD) and closely associated with the pathogenesis of AD. Amyloid plaques in the brains of most AD patients and transgenic mouse models exhibit heterogeneity in the composition of Aβ deposits, due to the occurrence of elongated, truncated, and post-translationally modified Aβ peptides. Importantly, changes in the deposition of these different Aβ variants are associated with the clinical disease progression and considered to mark sequential phases of plaque and cerebral amyloid angiopathy (CAA) maturation at distinct stages of AD. We recently showed that Aβ phosphorylated at serine residue 26 (pSer26Aβ) has peculiar characteristics in aggregation, deposition, and neurotoxicity. In the current study, we developed and thoroughly validated novel monoclonal and polyclonal antibodies that recognize Aβ depending on the phosphorylation-state of Ser26. Our results demonstrate that selected phosphorylation state-specific antibodies were able to recognize Ser26 phosphorylated and non-phosphorylated Aβ with high specificity in enzyme-linked immunosorbent assay (ELISA) and Western Blotting (WB) assays. Furthermore, immunofluorescence analyses with these antibodies demonstrated the occurrence of pSer26Aβ in transgenic mouse brains that show differential deposition as compared to non-phosphorylated Aβ (npAβ) or other modified Aβ species. Notably, pSer26Aβ species were faintly detected in extracellular Aβ plaques but most prominently found intraneuronally and in cerebral blood vessels. In conclusion, we developed new antibodies to specifically differentiate Aβ peptides depending on the phosphorylation state of Ser26, which are applicable in ELISA, WB, and immunofluorescence staining of mouse brain tissues. These site- and phosphorylation state-specific Aβ antibodies represent novel tools to examine phosphorylated Aβ species to further understand and dissect the complexity in the age-related and spatio-temporal deposition of different Aβ variants in transgenic mouse models and human AD brains.
Highlights
Alzheimer’s disease (AD) is the most common form of dementia worldwide (Alzheimer’s Association, 2020)
The critical role of Aβ in the pathogenesis of AD is strongly supported by the identification of early-onset familial AD (FAD)causing mutations within the genes encoding either the amyloid precursor protein (APP) itself or presenilin 1 and 2 (PS1 and PS2) that commonly alter the production of Aβ peptides in quantitative and qualitative ways (Bateman et al, 2011; Benilova et al, 2012; Katsnelson et al, 2016; De Strooper and Karran, 2016)
Our results show that selected phosphorylation state-specific antibodies recognize Ser26-phosphorylated and non-phosphorylated Aβ with high specificity in enzyme-linked immunosorbent assay (ELISA), Western Blotting (WB), and immunofluorescence staining
Summary
Alzheimer’s disease (AD) is the most common form of dementia worldwide (Alzheimer’s Association, 2020). There is strong evidence that the genetic risk for AD that has been associated with polymorphisms in both Apoliprotein E (ApoE) and Clusterin (CLU) is at least partly attributable to effects of these proteins on Aβ deposition (Ray et al, 1998; Tanzi, 2012; Bettens et al, 2013; Karch et al, 2014; Tcw and Goate, 2017; Belloy et al, 2019). These genetic studies indicate that the accumulation and aggregation of Aβ can be a trigger in the pathogenesis of AD-related dementia
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