Proteomic identification and validation of novel neuronal EV-based markers for Alzheimer’s disease biomarker discovery

Abstract Extracellular vesicles (EVs) circulate in biofluids and carry tissue-specific molecular cargo, offering significant potential for the discovery of minimally invasive biomarkers. However, translation in neurodegenerative diseases has been hindered by the lack of validated neuronal EV surface markers that enable selective isolation from plasma. We hypothesized that proteomic profiling of EVs released from human induced pluripotent stem cell (hiPSC)-derived neurons would identify 1. robust Alzheimer’s disease (AD)-associated signatures that reflect disease pathogenesis, and 2. surface-accessible neuronal markers capable of enriching disease-relevant cargo. Neurons differentiated from AD patients and age-matched cognitively normal (CN) individuals were used to isolate EVs, which were characterized and analyzed by LC-MS proteomics in both total and membrane-enriched fractions. Proteomic profiling identified numerous dysregulated proteins, with a subset validated across independent AD datasets. We identified CNTNAP2 and STX1B as neuronal, brain-enriched EV surface proteins accessible for selective capture and confirmed their presence in EVs from post-mortem human brain, supporting them as bona fide brain-derived EV markers. Immuno-isolation of plasma EVs showed that CNTNAP2-positive EVs had a robust AD-associated increase in phosphorylated tau, identifying CNTNAP2 as a highly discriminative brain-derived EV marker and supporting its potential for blood-based AD diagnostics. Graphical Abstract Previous studies indicate that extracellular vesicles (EVs) released from neurons carry disease-relevant cargo, yet the search for the optimal neuronal surface markers for the selective isolation of EVs pertinent to Alzheimer’s disease (AD) from blood is ongoing. To address this need, we performed proteomic profiling of EVs derived from hiPSC-neurons (iNEVs) of AD patients and cognitively normal individuals (CN). LC-MS analysis of whole and membrane-enriched EV fractions revealed robust protein dysregulation and identified CNTNAP2 and STX1B as surface-exposed neuronal EV proteins, confirmed in human brain-derived EVs (BDEVs). Immuno-isolation of plasma EVs demonstrated that CNTNAP2-positive EVs are enriched for phosphorylated tau in AD, whereas the previously used markers; NrCAM and ATP1A3-positive EVs showed limited discrimination. These findings establish a robust framework for proteomic discovery and nominate CNTNAP2 as a promising EV selection marker for blood-based AD diagnostics.