The amyloid hypothesis in Alzheimer disease (AD) considers amyloid β peptide (Aβ) deposition causative in triggering down-stream events like neurofibrillary tangles, cell loss, vascular damage and memory decline. In the past years N-truncated Aβ peptides especially N-truncated pyroglutamate Aβ_pE3-42 have been extensively studied. Together with full-length Aβ_1–42 and Aβ_1–40, N-truncated Aβ_pE3-42 and Aβ_4–42 are major variants in AD brain. Although Aβ_4–42 has been known for a much longer time, there is a lack of studies addressing the question whether Aβ_pE3-42 or Aβ_4–42 may precede the other in Alzheimer’s disease pathology.

Using different Aβ antibodies specific for the different N-termini of N-truncated Aβ, we discovered that Aβ_4-x preceded Aβ_pE3-x intraneuronal accumulation in a transgenic mouse model for AD prior to plaque formation. The novel Aβ_4-x immunoreactive antibody NT4X-167 detected high molecular weight aggregates derived from N-truncated Aβ species. While NT4X-167 significantly rescued Aβ_4–42 toxicity in vitro no beneficial effect was observed against Aβ_1–42 or Aβ_pE3-42 toxicity. Phenylalanine at position four of Aβ was imperative for antibody binding, because its replacement with alanine or proline completely prevented binding. Although amyloid plaques were observed using NT4X-167 in 5XFAD transgenic mice, it barely reacted with plaques in the brain of sporadic AD patients and familial cases with the Arctic, Swedish and the presenilin-1 PS1Δ9 mutation. A consistent staining was observed in blood vessels in all AD cases with cerebral amyloid angiopathy. There was no cross-reactivity with other aggregates typical for other common neurodegenerative diseases showing that NT4X-167 staining is specific for AD.

Aβ_4-x precedes Aβ_pE3-x in the well accepted 5XFAD AD mouse model underlining the significance of N-truncated species in AD pathology. NT4X-167 therefore is the first antibody reacting with Aβ_4-x and represents a novel tool in Alzheimer research.