Zhang, Ziteng

Abstract Background Brain cells secrete extracellular vesicles (EVs) containing signaling and pathological proteins related to Alzheimer's disease (AD) and related disorders. Recent studies demonstrate brain‐derived EVs (BDEVs) contain misfolded tau and are transmissible of tau pathology in the brain. However, the underlying mechanism regarding EV‐mediated tau pathology is still largely uncharacterized. Method We conducted the immuno‐affinity purification of tau in human brain‐derived EVs (BDEVs) isolated from age and sex‐matched 14 AD and 14 CTRL cases and performed tandem mass‐tag mass spectrometry to profile unbiased tau interactome in BDEVs. We used Nanotemper Monolith to validate the interaction of EV‐tau interactors with tau, and super‐resolution microscopy (Nanoimager) to detect their colocalization in a single‐EV level. We next designed siRNAs to silence EV‐tau interactors in SH‐SY5Y cells overexpressing human P301L tau (SH‐SY5Y‐P301Ltau) and assessed tau loading to EVs by Flow Nanoanalyzer. These EVs were tested for their uptake and tau seeding by human iPSC‐derived neurons (iNeurons). We finally tested the inhibitors of identified molecules on BDEV‐mediated tau propagation in vivo . Result A total of 764 proteins were identified in BDEV‐associated tau interactome from CTRL and AD patients. Sixty‐five proteins were significantly downregulated in AD BDEVs; whereas 5 proteins were significantly upregulated in AD BDEVs compared to CTRL BDEVs. The most enriched tau‐interacting proteins were significantly positively correlated with Braak stage. We confirmed the direct binding of candidate proteins to tau with micromolar binding affinities by Monolith, and their colocalization with tau in human BDEVs using Nanoimager. Silencing of candidate molecules in human SH‐SY5Y‐P301Ltau cells showed reduced loading of tau in EVs, their reduced uptake by iNeurons, and tau seeding activities. Finally, intracerebroventricular injection of neutralizing antibody against the most promising target results in reduction of tau pathology in PS19 mice expressing P301S human tau mutant. Conclusion Our study identified tau‐interacting molecules highly enriched in AD BDEVs compared to CTRL BDVs. Silencing of candidate molecule reduced tau loading to the EVs and their uptake by iNeurons and neutralizing antibody suppressed tau dissemination, highlighting them as promising therapeutic targets to halt tau pathology in AD and related tauopathy.

Abstract Background Extracellular vesicles (EVs) are key mediators in transferring pathological proteins associated with Alzheimer's disease (AD). The apolipoprotein E (APOE) gene, particularly the ε4 allele, is a major genetic risk factor for late‐onset AD. However, the influence of APOE genotype on the biological characteristics and cargo composition of brain‐derived extracellular vesicles (BDEVs) in AD remains poorly understood. Method In this study, BDEVs were isolated from human AD temporal cortex with APOE3/3 and APOE4/4 genotypes ( N = 20 / group). Nanoflow cytometry and super‐resolution microscopy were used to evaluate the tau load within these BDEVs. EV‐mediated tau transmission was assessed in aged human Tau KI and APP NL‐G‐F : Tau KI mice in vivo . BDEV uptake and tau seeding were evaluated in iPSC‐derived neurons through live imaging. Additionally, shotgun lipidomics and data independent acquisition proteomics were analyzed lipidome and proteome of BDEVs, respectively. Key molecules linked to APOE genotype were identified through integrated weighted gene co‐expression network and trait correlation analysis. Result APOE4/4 BDEVs exhibited a higher proportion of phosphorylated tau (pS396) at single EV levels. Upon injection of 300pg tau of BDEVs into aged Tau KI and APP NL‐G‐F : Tau KI mice, APOE4/4 BDEVs significantly increased AT8 + phosphorylated tau levels, and induced neuroinflammation in vivo . Moreover, APOE4/4 BDEVs demonstrated stronger neuronal uptake and tau transfer compared to APOE3/3 BDEVs, impairing the neuronal activity of iPSC‐derived neurons. Lipidomic revealed that APOE4/4 BDEVs were significantly more enriched in pro‐inflammatory long‐chain polyunsaturated fatty acids (PUFA) compared to APOE3/3 with Braak stage association. Furthermore, gene ontology pathway analysis using differentially expressed proteins in APOE4/4 BDEVs showed increased activity in oxidative phosphorylation and sterol‐binding. Multi‐omics integrated data revealed that upregulation of PUFA was significantly associated with elevated cell adhesion molecules and ATPase transporter activity with neural cell adhesion molecule as one of key molecule. Notably, neutralizing antibody against the molecule effectively reduced APOE4/4 BDEV internalization and mitigated EV‐mediated tau pathology in recipient cells. Conclusion Our comprehensive analysis suggested that AD APOE4/4 BDEV may enhance transmissibility of tau pathology and neuroinflammation via enriched pro‐inflammatory PUFA and cell adhesion molecules. Targeting these pathways presents a promising therapeutic strategy to mitigate disease progression, particularly in APOE4 individuals.