Identification of Low Molecular Weight Pyroglutamate Aβ Oligomers in Alzheimer Disease

Abstract

N-terminally truncated A␤ peptides starting with pyroglutamate (A␤pE3) represent a major fraction of all A␤ peptides in the brain of Alzheimer disease (AD) patients. A␤pE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length A␤. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of A␤pE3 and studied the potential involvement of oligomeric A␤pE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall A␤ plaque load and A␤pE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight A␤pE3 oligomers. Alzheimer disease (AD)3 represents the most frequent form of dementia and is characterized by the presence of extracellular amyloid plaques composed of amyloid-␤ (A␤) surrounded by dystrophic neurites and neurofibrillary tangles. The discovery that certain early-onset familial forms of AD may be caused by enhanced levels of A␤ peptides have led to the hypothesis that amyloidogenic A␤ is intimately involved in the AD pathogenic process (1). In the past extracellular A␤ has been regarded as the major culprit, whereas more recent evidence now points to toxic effects of A␤ in intracellular compartments (2-3). In addition, other concepts propose that the soluble oligomers and the ␤-sheet containing amyloid fibrils are the toxic forms of A␤ (4 -6). Supporting this notion, it has been demonstrated that soluble oligomeric A␤42, but not plaque-associated A␤, correlates best with cognitive dysfunction in AD (7-8). Oligomers are formed preferentially intracellulary within neuronal processes and synapses rather than extracellularly (9 -10). Besides full-length A␤ peptides starting with an aspartate at position 1, a variety of different N-truncated A␤ peptides have been identified in AD brains. Ragged peptides including phenylalanine at position 4 of A␤ have been reported as early as 1985 by Masters et al. (11). In contrast, no N-terminal sequence could be obtained from cores purified in a sodium dodecyl sulfate-containing buffer, which led to the assumption that the N terminus could be blocked (12-13). The presence of A␤pE3 (N-terminally truncated A␤ starting with pyroglutamate) in AD brain was subsequently shown using mass spectrometry of purified A␤ peptides, explaining at least partially initial difficulties in sequencing A␤ peptides purified from human brain tissue (14). The author...

Characterization of AD-like phenotype in aged APPSwe/PS1dE9 mice

“…Previous studies reported that 3-month-old APP/PS1 mice exhibit anxiety-like phenotype, although without spatial cognitive dysfunction, when compared with age-matched WT controls (Trinchese et al 2004;Webster et al 2014). The open field and elevated plus maze test confirmed that anxietylike behaviors continue to exist in the late stages of this mouse AD model, similar to the Tg2576 (Gil-Bea et al 2007;Lassalle et al 2008), J20 (Harris et al 2010;Cissé et al 2011), and 5xFAD mice (Wirths et al 2010;Shukla et al 2013), all of which also exhibit anxietyrelated behavior deficits in the elevated plus maze task. However, there is no anxiety-related behavior abnormality in the APP/PS1 double knock-in mice from 7 to 24 months old (Webster et al 2013); while 3xFAD mice show decreased anxiety-related behavior in the open field and elevated plus maze test (Nelson et al 2007;Filali et al 2012).…”

Pyroglutamate Amyloid-β (Aβ): A Hatchet Man in Alzheimer Disease

“…In addition, the titer of IgM autoantibodies against A␤ pE3 correlated with the cognitive status of individuals at risk to develop AD (96). In good agreement, the level of A␤ pE3 oligomers was significantly decreased in the plasma of AD patients (51). However, it is noteworthy that these studies are pilot studies with small group sizes and need to be further replicated and confirmed using larger cohorts of patients and controls.…”

“…A recently generated novel monoclonal antibody (9D5) was used to demonstrate that it is possible to detect low molecular weight pyroglutamate-modified A␤ oligomers (51). The selectivity of the antibody for low molecular weight (4 -10-mers) pyroglutamate-modified A␤ was confirmed by size exclusion chromatography and immunoblot assays.…”

“…Passive immunization of 4.5-month-old 5XFAD mice with the 9D5 antibody for 6 weeks was capable of reducing overall A␤ plaque load and A␤ pE3-x levels, leading to a normalization of the behavioral phenotype. Based on that, 9D5 represents a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight A␤ pE3 oligomers (51).…”

Pyroglutamate Amyloid β (Aβ) Aggravates Behavioral Deficits in Transgenic Amyloid Mouse Model for Alzheimer Disease

“…However, it cannot be excluded that the unmodified A␤ 3-42 also participates in the observed deficits. It should be noted that passive immunization against low molecular weight pyrogutamate-modified A␤ oligomers produces beneficial therapeutic effects in 5XFAD mice (39). These data imply that a change in the amount of A␤ pE3 is sufficient to affect pathology despite the presence of other A␤ isotypes.…”