Single-extracellular vesicle (EV) analyses validate the use of L1 Cell Adhesion Molecule (L1CAM) as a reliable biomarker of neuron-derived EVs.

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 ß-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 ß-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.

Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum.

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 (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age.

Flow Cytometry of Synaptoneurosomes (FCS) Reveals Increased Ribosomal S6 and Calcineurin Proteins in Activated Medial Prefrontal Cortex to Nucleus Accumbens Synapses.

Learning and behavior activate cue-specific patterns of sparsely distributed cells and synapses called ensembles that undergo memory-encoding engram alterations. While Fos is often used to label selectively activated cell bodies and identify neuronal ensembles, there is no comparable endogenous marker to label activated synapses and identify synaptic ensembles. For the purpose of identifying candidate synaptic activity markers, we optimized a flow cytometry of synaptoneurosome (FCS) procedure for assessing protein alterations in activated synapses from male and female rats. After injecting yellow fluorescent protein (YFP)-expressing adeno-associated virus into medial prefrontal cortex (mPFC) to label terminals in nucleus accumbens (NAc) of rats, we injected 20 mg/kg cocaine in a novel context (cocaine+novelty) to activate synapses, and prepared NAc synaptoneurosomes 0-60 min following injections. For FCS, we used commercially available antibodies to label presynaptic and postsynaptic markers synaptophysin and PSD-95 as well as candidate markers of synaptic activity [activity-regulated cytoskeleton protein (Arc), CaMKII and phospho-CaMKII, ribosomal protein S6 (S6) and phospho-S6, and calcineurin and phospho-calcineurin] in YFP-labeled synaptoneurosomes. Cocaine+novelty increased the percentage of S6-positive synaptoneurosomes at 5-60 min and calcineurin-positive synaptoneurosomes at 5-10 min. Electron microscopy verified that S6 and calcineurin levels in synaptoneurosomes were increased 10 min after cocaine+novelty. Pretreatment with the anesthetic chloral hydrate blocked cocaine+novelty-induced S6 and calcineurin increases in synaptoneurosomes, and novel context exposure alone (without cocaine) increased S6, both of which indicate that these increases were due to neural activity per se. Overall, FCS can be used to study protein alterations in activated synapses coming from specifically labeled mPFC projections to NAc.SIGNIFICANCE STATEMENT Memories are formed during learning and are stored in the brain by long-lasting molecular and cellular alterations called engrams formed within specific patterns of cue-activated neurons called neuronal ensembles. While Fos has been used to identify activated ensemble neurons and the engrams within them, we have not had a similar marker for activated synapses that can be used to identify synaptic engrams. Here we developed a procedure for high-throughput in-line analysis of flow cytometry of synaptoneurosome (FCS) and found that ribosomal S6 protein and calcineurin were increased in activated mPFC-NAc synapses. FCS can be used to study protein alterations in activated synapses within specifically labeled circuits.

Neuronal and Astrocytic Extracellular Vesicle Biomarkers in Blood Reflect Brain Pathology in Mouse Models of Alzheimer's Disease.

Circulating neuronal extracellular vesicles (NEVs) of Alzheimer's disease (AD) patients show high Tau and ß-amyloid (Aß) 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 Aß42 (p < 0.005). NEV Aß42 had moderately strong correlations with Aß42 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.

Astrocyte- and Neuron-Derived Extracellular Vesicles from Alzheimer's Disease Patients Effect Complement-Mediated Neurotoxicity.

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.

Novel autoimmune response in a tauopathy mouse model.

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.

Amphiphysin-1 protein level changes associated with tau-mediated neurodegeneration.

Tauopathies are a family of neurodegenerative diseases that have the pathological hallmark of intraneuronal accumulation of filaments composed of hyperphosphorylated tau proteins that tend to aggregate in an ultrastructure known as neurofibrillary tangles. The identification of mutations on the tau gene in familial cases of tauopathies underscores the pathological role of the tau protein. However, the molecular process that underlines tau-mediated neurodegeneration is not understood. Here, a proteomics approach was used to identify proteins that may be affected during the course of tau-mediated neurodegeneration in the tauopathy mouse model JNPL3. The JNPL3 mice express human tau proteins bearing a P301L mutation, which mimics the neurodegenerative process observed in humans with tauopathy. The results showed that the protein amphiphysin-1 (AMPH1) is significantly reduced in terminally ill JNPL3 mice. Specifically, the AMPH1 protein level is reduced in brain regions known to accumulate aggregates of hyperphosphorylated tau proteins. The AMPH1 protein reduction was validated in Alzheimer's disease cases. Taken together, the results suggest that the reduction of the AMPH1 protein level is a molecular event associated with the progression of tau-mediated neurodegeneration.