N-truncated Abeta starting with position four: early intraneuronal accumulation and rescue of toxicity using NT4X-167, a novel monoclonal antibody

Background: The amyloid hypothesis in Alzheimer disease (AD) considers amyloid beta peptide (A beta) deposition causative in triggering down-stream events like neurofibrillary tangles, cell loss, vascular damage and memory decline. In the past years N-truncated A beta peptides especially N-truncated pyroglutamate A beta(pE3-42) have been extensively studied. Together with full-length A beta(1-42) and A beta(1-40), N-truncated A beta(pE3-42) and A beta(4-42) are major variants in AD brain. Although A beta(4-42) has been known for a much longer time, there is a lack of studies addressing the question whether A beta(pE3-42) or A beta(4-42) may precede the other in Alzheimer's disease pathology. Results: Using different A beta antibodies specific for the different N-termini of N-truncated A beta, we discovered that A beta(4-x) preceded A beta(pE3-x) intraneuronal accumulation in a transgenic mouse model for AD prior to plaque formation. The novel A beta(4-x) immunoreactive antibody NT4X-167 detected high molecular weight aggregates derived from Ntruncated A beta species. While NT4X-167 significantly rescued A beta(4-42) toxicity in vitro no beneficial effect was observed against A beta(1-42) or A beta(pE3-42) toxicity. Phenylalanine at position four of A beta 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 PS1.9 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. Conclusions: A beta(4-x) precedes A beta(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 A beta(4-x) and represents a novel tool in Alzheimer research.