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Abstract Isolation of neuron‐derived extracellular vesicles (NDEVs) with L1 Cell Adhesion Molecule (L1CAM)‐specific antibodies has been widely used to identify blood biomarkers of CNS disorders. However, full methodological validation requires demonstration of L1CAM in individual NDEVs and lower levels or absence of L1CAM in individual EVs from other cells. Here, we used multiple single‐EV techniques to establish the neuronal origin and determine the abundance of L1CAM‐positive EVs in human blood. L1CAM epitopes of the ectodomain are shown to be co‐expressed on single‐EVs with the neuronal proteins β‐III‐tubulin, GAP43, and VAMP2, the levels of which increase in parallel with the enrichment of L1CAM‐positive EVs. Levels of L1CAM‐positive EVs carrying the neuronal proteins VAMP2 and β‐III‐tubulin range from 30% to 63%, in contrast to 0.8%–3.9% of L1CAM‐negative EVs. Plasma fluid‐phase L1CAM does not bind to single‐EVs. Our findings support the use of L1CAM as a target for isolating plasma NDEVs and leveraging their cargo to identify biomarkers reflecting neuronal function.

Molecular diagnostic tools with non-invasive properties that allow detection of pathological events in Alzheimer’s disease (AD) and other neurodegenerative tauopathies are essential for the development of therapeutics. Several diagnostic strategies based on the identification of biomarkers have been proposed. However, its specificity among neurodegenerative disorders is disputable as the association with pathological events remains elusive. Recently, we showed that Amphiphysin-1 (AMPH1) protein’s abundance is reduced in the central nervous system (CNS) of the tauopathy mouse model JNPL3 and AD brains. AMPH1 is a synaptic protein that plays an important role in clathrin-mediated endocytosis and associates with BIN1, one of the most important risk loci for AD. Also, it has been associated with a rare neurological disease known as Stiff-Person Syndrome (SPS). Auto-antibodies against AMPH1 are used as diagnostic biomarkers for a paraneoplastic variant of SPS. Therefore, we set up to evaluate the presence and abundance of auto-AMPH1 antibodies in tau-mediated neurodegeneration. Immunoblots and enzyme-linked immunosorbent assays (ELISA) were conducted to detect the presence of auto-AMPH1 antibodies in sera from euthanized mice that developed neurodegeneration (JNPL3) and healthy control mice (NTg). Results showed increased levels of auto-AMPH1 antibodies in JNPL3 sera compared to NTg controls. The abundance of auto-AMPH1 antibodies correlated with motor impairment and AMPH1 protein level decrease in the CNS. The results suggest that auto-AMPH1 antibodies could serve as a biomarker for the progression of tau-mediated neurodegeneration in JNPL3 mice.

Abstract Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (

Circulating neuronal extracellular vesicles (NEVs) of Alzheimer’s disease (AD) patients show high Tau and β-amyloid (Aβ) levels, whereas their astrocytic EVs (AEVs) contain high complement levels. To validate EV proteins as AD biomarkers, we immunocaptured NEVs and AEVs from plasma collected from fifteen wild type (WT), four 2xTg-AD, nine 5xFAD, and fifteen 3xTg-AD mice and assessed biomarker relationships with brain tissue levels. NEVs from 3xTg-AD mice had higher total Tau (p = 0.03) and p181-Tau (p = 0.0004) compared to WT mice. There were moderately strong correlations between biomarkers in NEVs and cerebral cortex and hippocampus (total Tau: cortex, r = 0.4, p = 0.009; p181-Tau: cortex, r = 0.7, p < 0.0001; hippocampus, r = 0.6, p < 0.0001). NEVs from 5xFAD compared to other mice had higher Aβ42 (p < 0.005). NEV Aβ42 had moderately strong correlations with Aβ42 in cortex (r = 0.6, p = 0.001) and hippocampus (r = 0.7, p < 0.0001). AEV C1q was elevated in 3xTg-AD compared to WT mice (p = 0.005); AEV C1q had moderate-strong correlations with C1q in cortex (r = 0.9, p < 0.0001) and hippocampus (r = 0.7, p < 0.0001). Biomarkers in circulating NEVs and AEVs reflect their brain levels across multiple AD mouse models supporting their potential use as a “liquid biopsy” for neurological disorders.

We have previously shown that blood astrocytic-origin extracellular vesicles (AEVs) from Alzheimer’s disease (AD) patients contain high complement levels. To test the hypothesis that circulating EVs from AD patients can induce complement-mediated neurotoxicity involving Membrane Attack Complex (MAC) formation, we assessed the effects of immunocaptured AEVs (using anti-GLAST antibody), in comparison with neuronal-origin (N)EVs (using anti-L1CAM antibody), and nonspecific CD81+ EVs (using anti-CD81 antibody), from the plasma of AD, frontotemporal lobar degeneration (FTLD), and control participants. AEVs (and, less effectively, NEVs) of AD participants induced Membrane Attack Complex (MAC) expression on recipient neurons (by immunohistochemistry), membrane disruption (by EthD-1 assay), reduced neurite density (by Tuj-1 immunohistochemistry), and decreased cell viability (by MTT assay) in rat cortical neurons and human iPSC-derived neurons. Demonstration of decreased cell viability was replicated in a separate cohort of autopsy-confirmed AD patients. These effects were not produced by CD81+ EVs from AD participants or AEVs/NEVs from FTLD or control participants, and were suppressed by the MAC inhibitor CD59 and other complement inhibitors. Our results support the stated hypothesis and should motivate future studies on the roles of neuronal MAC deposition and AEV/NEV uptake, as effectors of neurodegeneration in AD.