Proximity labeling reveals unique and shared interactomes of unmodified and pyroglutamate amyloid beta in human hippocampus in Alzheimer's disease.

Amyloid plaques are a hallmark neuropathological feature of Alzheimer's disease (AD), composed of insoluble amyloid beta (Aβ) peptide. Aβ undergoes post-translational modifications that alter their biophysical properties, aggregation kinetics, and neurotoxicity, creating a heterogeneous pool of species that differentially affect AD pathogenesis. Pyroglutamate-modified Aβ (pEAβ) 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 pEAβ versus unmodified Aβ (pan-Aβ) 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 protein-protein interactions (proteomes) of pEAβ and pan-Aβ 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 pEAβ captures, 28 in AD pan-Aβ captures, and 15 in CN pan-Aβ captures. Whereas no significant enrichment was detected in CN pEAβ captures, supporting pEAβ as a pathology-associated species. pEAβ in AD demonstrated the largest variant-specific signature with 31 unique proteins, pan-Aβ showed 11 unique proteins in AD, and 14 unique proteins in CN, 16 proteins were shared between AD pEAβ and AD pan-Aβ, with PCSK1N shared across AD pEAβ, and AD/CN pan-Aβ. Pathway enrichment analysis revealed broader biological disruptions linked to pEAβ, 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 pEAβ engages an expanded, variant-specific interactome in human AD hippocampus and designate intracellular trafficking, synaptic signaling, and mitochondrial pathways as network-level vulnerabilities relevant to pEAβ pathology in AD. Notably, comparison of CN versus AD pan-Aβ further distinguished protein networks associated with physiological Aβ engagement versus pathological pan-Aβ deposition.