N-terminally truncated Aβ4-x proteoforms and their relevance for Alzheimer’s pathophysiology

BACKGROUND: The molecular heterogeneity of Alzheimer’s amyloid-β (Aβ) deposits extends well beyond the classic Aβ1-40/Aβ1-42 dichotomy, substantially expanded by multiple post-translational modifications that increase the proteome diversity. Numerous truncated fragments consistently populate the brain Aβ peptidome, and their homeostatic regulation and potential contribution to disease pathogenesis are largely unknown. Aβ4-x peptides have been reported as major components of plaque cores and the limited studies available indicate their relative abundance in Alzheimer’s disease (AD). METHODS: Immunohistochemistry was used to assess the topographic distribution of Aβ4-x species in well-characterized AD cases using custom-generated monoclonal antibody 18H6—specific for Aβ4-x species and blind for full-length Aβ1-40/Aβ1-42—in conjunction with thioflavin-S and antibodies recognizing Aβx-40 and Aβx-42 proteoforms. Circular dichroism, thioflavin-T binding, and electron microscopy evaluated the biophysical and aggregation/oligomerization properties of full-length and truncated synthetic homologues, whereas stereotaxic intracerebral injections of monomeric and oligomeric radiolabeled homologues in wild-type mice were used to evaluate their brain clearance characteristics. RESULTS: All types of amyloid deposits contained the probed Aβ epitopes, albeit expressed in different proportions. Aβ4-x species showed preferential localization within thioflavin-S-positive cerebral amyloid angiopathy and cored plaques, strongly suggesting poor clearance characteristics and consistent with the reduced solubility and enhanced oligomerization of their synthetic homologues. In vivo clearance studies demonstrated a fast brain efflux of N-terminally truncated and full-length monomeric forms whereas their oligomeric counterparts—particularly of Aβ4-40 and Aβ4-42—consistently exhibited enhanced brain retention. CONCLUSIONS: The persistence of aggregation-prone Aβ4-x proteoforms likely contributes to the process of amyloid formation, self-perpetuating the amyloidogenic loop and exacerbating amyloid-mediated pathogenic pathways.