ABC40 Sigma-AldrichAnti-AIP1/Alix Antibody

Detection of α-synuclein (α-syn) in biological fluids such as saliva may serve as potential biomarker of PD. α-syn pertaining to extracellular vesicles (EVs) has been recently studied in plasma, but not in other biological fluids such as saliva.

The filarial nematode Brugia malayi is an etiological agent of Lymphatic Filariasis. The capability of B. malayi and other parasitic nematodes to modulate host biology is recognized but the mechanisms by which such manipulation occurs are obscure. An emerging paradigm is the release of parasite-derived extracellular vesicles (EV) containing bioactive proteins and small RNA species that allow secretion of parasite effector molecules and their potential trafficking to host tissues. We have previously described EV release from the infectious L3 stage B. malayi and here we profile vesicle release across all intra-mammalian life cycle stages (microfilariae, L3, L4, adult male and female worms). Nanoparticle Tracking Analysis was used to quantify and size EVs revealing discrete vesicle populations and indicating a secretory process that is conserved across the life cycle. Brugia EVs are internalized by murine macrophages with no preference for life stage suggesting a uniform mechanism for effector molecule trafficking. Further, the use of chemical uptake inhibitors suggests all life stage EVs are internalized by phagocytosis. Proteomic profiling of adult male and female EVs using nano-scale LC-MS/MS described quantitative and qualitative differences in the adult EV proteome, helping define the biogenesis of Brugia EVs and revealing sexual dimorphic characteristics in immunomodulatory cargo. Finally, ivermectin was found to rapidly inhibit EV release by all Brugia life stages. Further this drug effect was also observed in the related filarial nematode, the canine heartworm Dirofilaria immitis but not in an ivermectin-unresponsive field isolate of that parasite, highlighting a potential mechanism of action for this drug and suggesting new screening platforms for anti-filarial drug development.

A dysfunctional endosomal pathway and abnormally enlarged early endosomes in neurons are an early characteristic of Down syndrome (DS) and Alzheimer's disease (AD). We have hypothesized that endosomal material can be released by endosomal multivesicular bodies (MVBs) into the extracellular space via exosomes to relieve neurons of accumulated endosomal contents when endosomal pathway function is compromised. Supporting this, we found that exosome secretion is enhanced in the brains of DS patients and a mouse model of the disease, and by DS fibroblasts. Furthermore, increased levels of the tetraspanin CD63, a regulator of exosome biogenesis, were observed in DS brains. Importantly, CD63 knockdown diminished exosome release and worsened endosomal pathology in DS fibroblasts. Taken together, these data suggest that increased CD63 expression enhances exosome release as an endogenous mechanism mitigating endosomal abnormalities in DS. Thus, the upregulation of exosome release represents a potential therapeutic goal for neurodegenerative disorders with endosomal pathology.

The microtubule-associated protein tau has a critical role in Alzheimer disease and related tauopathies. There is accumulating evidence that tau aggregates spread and replicate in a prion-like manner, with the uptake of pathological tau seeds causing misfolding and aggregation of monomeric tau in recipient cells. Here we focused on small extracellular vesicles enriched for exosomes that were isolated from the brains of tau transgenic rTg4510 and control mice. We found that these extracellular vesicles contained tau, although the levels were significantly higher in transgenic mice that have a pronounced tau pathology. Tau in the vesicles was differentially phosphorylated, although to a lower degree than in the brain cells from which they were derived. Several phospho-epitopes (AT8, AT100, and AT180) thought to be critical for tau pathology were undetected in extracellular vesicles. Despite this, when assayed with FRET tau biosensor cells, extracellular vesicles derived from transgenic mice were capable of seeding tau aggregation in a threshold-dependent manner. We also observed that the dye used to label extracellular vesicle membranes was still present during nucleation and formation of tau inclusions, suggesting either a role for membranes in the seeding or in the process of degradation. Together, we clearly demonstrate that extracellular vesicles can transmit tau pathology. This indicates a role for extracellular vesicles in the transmission and spreading of tau pathology. The characteristics of tau in extracellular vesicles and the seeding threshold we identified may explain why tau pathology develops very slowly in neurodegenerative diseases such as Alzheimer disease.

To assess the value of exosomal miRNAs as biomarkers for Alzheimer disease (AD), the expression of microRNAs was measured in a plasma fraction enriched in exosomes by differential centrifugation, using Illumina deep sequencing. Samples from 35 persons with a clinical diagnosis of AD dementia were compared to 35 age and sex matched controls. Although these samples contained less than 0.1 microgram of total RNA, deep sequencing gave reliable and informative results. Twenty miRNAs showed significant differences in the AD group in initial screening (miR-23b-3p, miR-24-3p, miR-29b-3p, miR-125b-5p, miR-138-5p, miR-139-5p, miR-141-3p, miR-150-5p, miR-152-3p, miR-185-5p, miR-338-3p, miR-342-3p, miR-342-5p, miR-548at-5p, miR-659-5p, miR-3065-5p, miR-3613-3p, miR-3916, miR-4772-3p, miR-5001-3p), many of which satisfied additional biological and statistical criteria, and among which a panel of seven miRNAs were highly informative in a machine learning model for predicting AD status of individual samples with 83-89% accuracy. This performance is not due to over-fitting, because a) we used separate samples for training and testing, and b) similar performance was achieved when tested on technical replicate data. Perhaps the most interesting single miRNA was miR-342-3p, which was a) expressed in the AD group at about 60% of control levels, b) highly correlated with several of the other miRNAs that were significantly down-regulated in AD, and c) was also reported to be down-regulated in AD in two previous studies. The findings warrant replication and follow-up with a larger cohort of patients and controls who have been carefully characterized in terms of cognitive and imaging data, other biomarkers (e.g., CSF amyloid and tau levels) and risk factors (e.g., apoE4 status), and who are sampled repeatedly over time. Integrating miRNA expression data with other data is likely to provide informative and robust biomarkers in Alzheimer disease.