Spatial Single-Cell Proteomics Reveals Molecular Trajectories Of Tangle-Bearing Neurons In Alzheimer's Disease

Neurofibrillary tangles composed of hyperphosphorylated tau are a defining pathological hallmark of Alzheimer's disease (AD); however, the pathways and mechanisms associated with the transition from physiological tau to tangle pathology remain unclear. Here, we integrate laser microdissection of post-mortem, fixed human AD brain tissue labelled with an antibody recognizing tangle-associated phospho-tau (AT8) with mass spectrometry-based proteomics, applied to individual neurons and to small neuronal pools. This approach identified ~2,000 and ~5,000 proteins, respectively, and enabled direct detection of disease-associated tau phosphorylation sites without prior enrichment. A layered analysis of the proteome of tangle-positive and tangle-negative neurons revealed heterogeneous disease-associated states. Pseudotime analysis, combined with an AI-driven analytical framework, indicates that neurons do not segregate into discrete classes but instead organize along a continuum of proteomic changes that correlate with tau abundance. This organization enabled the construction of a trajectory of pathological neuronal responses that can be resolved within an individual brain. Early stages of this trajectory are characterized by coordinated remodeling of proteostasis networks, including reduced proteasome component abundance and increased lysosomal acidification machinery, followed by disruption of synaptic pathways. Notably, despite extensive proteomic remodeling, neurons bearing tangles show little evidence of activated cell-death programs, suggesting prolonged molecular adaptation rather than acute degeneration. Together, these findings establish a framework for single-cell-resolved proteome analysis of human brain disease in situ and define a continuum of neuronal states underlying tau pathogenesis, revealing early vulnerabilities and adaptive responses during AD progression.