Proximity labeling reveals unique and shared proximal proteomes associated with pE3-Aβ and 6E10 amyloid pathology in human Alzheimer’s disease hippocampus

Abstract Amyloid plaques are a hallmark neuropathological feature of Alzheimer's disease (AD), composed of insoluble amyloid beta (Aβ) peptides. Aβ undergoes post-translational modifications that alter its biophysical properties, aggregation kinetics, and neurotoxicity, creating a heterogeneous pool of species that differentially affect AD pathogenesis. Pyroglutamate-modified Aβ at glutamate position 3 (pE3-Aβ) is a particularly aggregation-prone and proteolytically resistant variant that preferentially accumulates within plaque cores, is implicated in early plaque seeding, and is a major target of emerging anti-amyloid immunotherapies. However, the molecular environment surrounding pE3-Aβ vs. 6E10-positive amyloid pathology in the human hippocampus remains incompletely defined. Here, we used Biotinylation by Antibody Recognition (BAR), an in-situ proximity labeling approach, to map and compare the proximal proteomes associated with pE3-Aβ and 6E10-positive amyloid pathology in formalin-fixed postmortem human hippocampal tissue from pathologically confirmed AD cases and cognitively normal (CN) controls. Differential proteomic analysis identified 48 significantly enriched proteins in AD pE3-Aβ captures, 28 in AD 6E10 captures, and 15 in CN 6E10 captures. Whereas no significant enrichment was detected in CN pE3-Aβ captures, supporting pE3-Aβ as a pathology-associated species. pE3-Aβ in AD demonstrated the largest variant-specific signature with 31 unique proteins; 6E10 had 11 unique proteins in AD and 14 unique proteins in CN; 16 proteins were shared between AD pE3-Aβ and AD 6E10, with PCSK1N shared across AD pE3-Aβ and AD/CN 6E10. Pathway enrichment analysis of proximal proteomes revealed broader biological disruptions linked to pE3-Aβ, including synaptogenesis signaling, clathrin-mediated endocytosis, mitochondrial division signaling, and neurotransmitter release. Shared pathways included SNARE signaling, glutamatergic receptor signaling, and netrin signaling. These findings demonstrate that pE3-Aβ is associated with an expanded, variant-specific proximal proteome in human AD hippocampus and may implicate intracellular trafficking, synaptic signaling, and mitochondrial pathways as network-level vulnerabilities relevant to pE3-Aβ pathology in AD. Comparison of CN and AD 6E10-positive amyloid further distinguished proximal protein networks associated with 6E10-positive Aβ in CN tissue vs. pathological deposition.