Liang, Zhewei

Abstract P2x purinoreceptor 7 (P2RX7), an ATP-gated ion channel, is known to play pivotal roles in the progression of Alzheimer’s disease (AD), although its cell type-specific pathological mechanisms have yet to be elucidated. Here, we show that genetic deletion of P2rx7 mitigates brain atrophy, tau accumulation and cognitive impairment in PS19 tauopathy mice. Specific deletion of P2rx7 in microglia, but not astrocytes, significantly suppresses tau propagation from the entorhinal cortex to CA1 in the hippocampus, an early event in AD pathology. Single-cell (sc)-RNA sequencing of mouse brains revealed specific P2rx7 expression in microglia, inducing inflammatory changes accompanied by elevated extracellular vesicles (EVs) secretion in PS19 mice. Brain-derived EVs (BDEVs) proteome demonstrated that P2RX7 increases EV cargo loading of tau and mitochondrial molecules in BDEVs from PS19 mice, which was further validated by single-molecule super-resolution. Notably, following the injection of BDEVs isolated from PS19 mice with or without P2rx7 deficiency, the microglial transcriptome of recipient mice revealed enriched DNA-sensing and type II interferon signaling in response to BDEVs from PS19 mice, which was diminished in the group injected with P2rx7-deficient BDEVs. Thus, our results indicate that P2RX7 regulates EV-mediated tau and mitochondrial transfer and inflammatory activation in microglia with increased EV secretion, thereby contributing to tauopathy and neurodegeneration, highlighting the therapeutic potential of targeting the P2RX7-EV axis in AD.

Abstract Background Alpha fetoprotein (AFP) is a circulating tumor marker utilized clinically to detect liver cirrhosis, liver cancer and testicular seminomas. AFP molecule contains one single N-glycosylation site, with highly variable glycan structures, resulting in diverse AFP glycoforms in circulating blood. The core fucosylation of AFP (AFP-L3) is a clinical biomarker to monitor and predict risks of liver cancer progression and recurrence. Clinical testing for AFP-L3 utilizes lectin-binding and gel-shift electrophoresis. We developed a novel method to measure AFP glycoforms, based on immune-enrichment and liquid chromatography with high resolution mass spectrometry (LC-HRMS). Methods Immune enrichment of AFP was achieved by anti-AFP antibody, immobilized to magnetic beads. After binding and enrichment, the magnetic beads were washed, AFP was eluted, and the samples were analyzed by LC-HRMS (Exploris 480 MS and Vanquish UHPLC, Thermo Fisher Scientific), using full scan, positive polarity acquisition mode. Chromatographic separation was performed on PLRP column (Agilent); flow rate 200 µL/min. The HRMS data were processed and deconvoluted with intact mass analysis workflow in Biopharma Finder 5.1 (Thermo Fisher Scientific) to derive the intact AFP masses. The method’s sensitivity and imprecision were assessed by replicate analysis of serial diluted patient serum samples. AFP and its associated glycoforms were matched and identified with an in-house developed bioinformatics workflow. The deconvoluted intact masses and the sum intensity of detected charged species for each AFP glycoform were used to quantify relative abundance of AFP glycoforms. Results The method specificity for AFP was confirmed by bottom-up analysis after immune-enrichment with observed peptide sequence coverage over 35%. The recovery of the immune enrichment was shown to be independent of the AFP glycoform compositions. The identified AFP glycoforms and their relative abundance were consistent among replicates between AFP concentration 100 ng/mL and 5000 ng/mL. Analysis of authentic serum specimens from patients with different chronic liver diseases and liver cancer (n=102), revealed multiple complex biantennary branched AFP glycan modifications, with different numbers of terminal sialic acid and core fucose. Four AFP glycoforms were identified in over 60% patient samples with elevated AFP concentrations. Although occurring consistently, relative abundance of these AFP glycoforms varied significantly among patients. Assessment of the AFP fucosylation and sialylation features suggested that the two are orthogonal processes. Clinical correlations suggested that fewer terminal sialic acids were associated with impaired liver functions, while the fucosylation was linked to inflammatory responses and presence of liver malignancy. Strong linear correlation (Spearman’s rs=0.952, N=35) was observed between AFP-L3% quantitated by the LC-HRMS method, and AFP-L3% determined by the lectin-binding based isotachophoresis assay (Wako Diagnostics). Discrepancies between the two methods were observed in specimens containing < 400 ng/mL of AFP, in which the LC-HRMS method detected only a single predominant glycoform. Conclusions We developed and evaluated the LC-HRMS method for profiling circulating AFP glycoforms in patients with chronic liver diseases and liver cancer. The detected AFP glycoforms and their relative abundance were used to establish glycan features, which can be further linked to cancer development, impaired liver function, and inflammation.