Journal of Clinical Medicine Article Small Neuron-Derived Extracellular Vesicles from Individuals with Down Syndrome Propagate Tau Pathology in the Wildtype Mouse Brain Aurélie Ledreux 1, *, Sarah Thomas 1 , Eric D. Hamlett 2 , Camille Trautman 1 , Anah Gilmore 1 , Emily Rickman Hager 3 , Daniel A. Paredes 1 , Martin Margittai 3 , Juan Fortea 4 and Ann-Charlotte Granholm 1 1 Knoebel Institute for Healthy Aging, University of Denver, Denver, CO 80208, USA; sarahjthomas123@gmail.com (S.T.); cwtrautman@gmail.com (C.T.); anah.gilmore@du.edu (A.G.); daniel.paredes@du.edu (D.A.P.); lotta.granholm-bentley@du.edu (A.-C.G.) 2 Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA; hamlette@musc.edu 3 Department of Chemistry and Biochemistry, University of Denver, Denver, CO 80208, USA; emilyh108@yahoo.com (E.R.H.); martin.margittai@du.edu (M.M.) 4 Hospital de la Santa Creu i Sant Pau and Catalan Down Syndrome Foundation, 08041 Barcelona, Spain; JFortea@santpau.cat * Correspondence: Aurelie.Ledreux@du.edu  Abstract: Individuals with Down syndrome (DS) exhibit Alzheimer’s disease (AD) pathology at a  young age, including amyloid plaques and neurofibrillary tangles (NFTs). Tau pathology can spread Citation: Ledreux, A.; Thomas, S.; via extracellular vesicles, such as exosomes. The cargo of neuron-derived small extracellular vesicles Hamlett, E.D.; Trautman, C.; Gilmore, (NDEVs) from individuals with DS contains p-Tau at an early age. The goal of the study was to A.; Rickman Hager, E.; Paredes, D.A.; investigate whether NDEVs isolated from the blood of individuals with DS can spread Tau pathology Margittai, M.; Fortea, J.; Granholm, in the brain of wildtype mice. We purified NDEVs from the plasma of patients with DS-AD and A.-C. Small Neuron-Derived controls and injected small quantities using stereotaxic surgery into the dorsal hippocampus of adult Extracellular Vesicles from wildtype mice. Seeding competent Tau conformers were amplified in vitro from DS-AD NDEVs but Individuals with Down Syndrome not NDEVs from controls. One month or 4 months post-injection, we examined Tau pathology in Propagate Tau Pathology in the Wildtype Mouse Brain. J. Clin. Med. mouse brains. We found abundant p-Tau immunostaining in the hippocampus of the mice injected 2021, 10, 3931. https://doi.org/ with DS-AD NDEVs compared to injections of age-matched control NDEVs. Double labeling with 10.3390/jcm10173931 neuronal and glial markers showed that p-Tau staining was largely found in neurons and, to a lesser extent, in glial cells and that p-Tau immunostaining was spreading along the corpus callosum and Academic Editor: Giuseppe Lanza the medio-lateral axis of the hippocampus. These studies demonstrate that NDEVs from DS-AD patients exhibit Tau seeding capacity and give rise to tangle-like intracellular inclusions. Received: 1 August 2021 Accepted: 28 August 2021 Keywords: Down syndrome; aging; biomarkers; neuropathology; Alzheimer’s disease Published: 31 August 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- iations. Down syndrome (DS) is the most common aneuploidy and cause of intellectual dis- ability of genetic origin, with an incidence of 1 in 600–700 babies [1]. The presence of a third copy of a segment of or the entire chromosome 21 (Hsa21) results in the overexpression of several genes, including the amyloid precursor protein (APP) gene. Due to the overexpres- sion of several different genes in Hsa21, individuals with DS develop Alzheimer’s disease Copyright: © 2021 by the authors. (AD) pathology, including neuroinflammation, neuronal cell loss, amyloid plaques and Licensee MDPI, Basel, Switzerland. neurofibrillary tangles (NFTs) [2–7]. The prevalence of AD in DS (DS-AD) is significantly This article is an open access article higher than that seen in the general population and occurs at a much earlier age [8]. The distributed under the terms and presence of the amyloid precursor protein (APP) gene on Hsa21 plays a role in this early conditions of the Creative Commons Attribution (CC BY) license (https:// onset AD [4,9], but other genes on this chromosome (e.g., RCAN1 and Dyrk1A) might creativecommons.org/licenses/by/ also play a role in the development of AD pathology and clinical manifestations of de- 4.0/). mentia [10–12]. Pathological alterations of the microtubule-associated protein Tau occurs J. Clin. Med. 2021, 10, 3931. https://doi.org/10.3390/jcm10173931 https://www.mdpi.com/journal/jcm J. Clin. Med. 2021, 10, 3931 2 of 23 early in DS [5,13], leading to the intracellular accumulation of phosphorylated forms of Tau (p-Tau) into neurofibrillary tangles (NFTs) [6,14]. The Tau protein is a neuronal protein important for microtubule polymerization and stabilization [15,16]. The adult human brain has six Tau isoforms that all derive from one gene (MAPT) by alternative splicing of exons 2, 3 and 10 [17–20]. Under normal physiolog- ical conditions, there is a balance between Tau phosphorylation and dephosphorylation. The abnormal hyperphosphorylation of Tau contributes greatly to the conversion of normal Tau into paired helical filaments (PHFs) [17,21]. When Tau is hyperphosphorylated, it loses biological activity and becomes resistant to degradation, leading to conformational changes that render Tau insoluble and prone to aggregation [20,22–26]. Tau pathology plays a central role in several different neurodegenerative diseases including AD [20,27], DS- AD [5,13,14], frontotemporal dementia (FTD) [20,28] and chronic traumatic encephalopathy (CTE) [17,29–33]. The complicated seeding capacity of toxic Tau species has not yet been ex- plored in depth but could provide a viable theory as to why AD and other tauopathies start in one part of the brain and spread in a network-like manner to other brain areas [34]. P-Tau pathology has been suggested to spread by a prion-like mechanism in the brain [35,36], and toxic forms of Tau have been shown to spread trans-synaptically [28,37]. Investigators have reported the seeding and spreading of human p-Tau into the brain of wildtype (WT) mice from brain homogenates prepared from individuals with various forms of tauopathies and have shown transfer of p-Tau pathology into neurons and, to some extent, also glial cells [16,18,27,35,36,38,39]. Ferrer and collaborators reported that injected p-Tau could spread to the ipsilateral hippocampus, corpus callosum and as far as the septal nuclei and the contralateral corpus callosum in adult WT mice [35]. Albert and colleagues have shown that the prion-like seeding and spreading of toxic p-Tau in the brain of WT mice could be blocked by a Tau neutralizing antibody, Tau antibody D, which recognizes an epitope in the central region of Tau [40]. They found that the Tau antibody D could not only neutralize the pathological species of Tau, but also prevent its spread to distant brain regions following injection [40]. However, biological mechanisms for the seeding of p-Tau from extracellular vesicles (EVs) have not been explored in depth. Recently, EVs—including exosomes—have been recognized as carriers of AD-related pathological proteins as well as non-coding RNAs, lipids and surface markers [41–44]. There are at least the following three major types of EVs, based on their size, composi- tion and biogenesis mechanisms: exosomes, microvesicles and apoptotic bodies [45,46]. Exosomes are a type of EV with a diameter of 40–150 nm and whose major functions include cell-cell communication and cell maintenance [46]. The biogenesis of exosomes occurs via the intracellular formation of multivesicular bodies (MVBs) and is regulated by two pathways—namely, the endosomal sorting complexes required for transport (ESCRT)- dependent or ESCRT-independent pathways [47]. The endosomal sorting machinery is important for the biogenesis and function of exosomes [41]. The vast differences in the contents of exosomal cargo and release mechanisms have been shown to be influenced by the cell of origin but also by disease conditions [48,49]. After being released from cells, small EVs and their cargo are taken up by other cells via interaction with various exosomal surface proteins and cellular receptors on the recipient cell membrane. Once attached, exosomes can be endocytosed, fused with the recipient cell and can promote intracellular signaling pathways, affecting gene expression, cell metabolism and other im- portant functions of the recipient cell [50–52]. It is known that essentially all the cells of the CNS release exosomes, including neurons [30,53], astrocytes [54–58], microglia [54,59] and oligodendrocytes [60]. While some exosomes exert effects in their proximal milieu, others travel long distances in the brain to affect other cell types or networks [61]. Exosomes are involved in normal functions of the CNS such as cell-cell communication, transcription regulation, neurogenesis and plasticity, as well as the response to inflammation [62], but also neuron-glia interface [54], synaptic plasticity [63] and the regeneration/protection of neurons [64]. Exosomes have also been found to play a significant role in the transmis- J. Clin. Med. 2021, 10, 3931 3 of 23 sion and propagation of protein aggregates associated with several neurodegenerative diseases [65–67]. Recently, multiple investigators have reported that exosomes isolated from AD plasma samples, transgenic Tau mouse brains or AD brain tissues can spread AD pathology, includ- ing Aβ and p-Tau, leading to cell damage and contribute to neuronal loss in AD [68–72]. Aβ peptides are present in exosome cargo, and these exosomes are accumulating around and within neuritic plaques in the brain of those with AD [65]. These findings suggest that exosomes, and potentially other EVs, can act as vehicles for the neuron-to-neuron or neuron-to-glia transfer of toxic species of amyloid and Tau in neurodegenerative disorders. On the other hand, exosomes can also exert a neuroprotective role in neurodegenerative disorders, for example, by reducing Aβ levels via the uptake of Aβ-containing exosomes secreted from neurons into microglia for amyloid metabolism [73], to spread growth factors to remote areas of the brain [74,75] or as a vehicles for the delivery of pharmacological therapies providing cell-free neurotherapy [76]. Based on the reported Tau alterations observed in individuals with DS [5,13,77–79], the current study was focused on the following two central questions: (1) Do neuron-derived EVs (NDEVs) isolated from the plasma of persons with DS-AD contain Tau species that have seeding capacity in the WT mouse brain, and (2) If so, to which cell types in the CNS do the toxic species of Tau spread? To answer these questions, we utilized several methods to characterize the NDEVs from controls and persons with DS-AD combined with stereotaxic injections into the WT mouse brain. 2. Materials and Methods 2.1. Isolation of NDEVs from Human Plasma Plasma samples were obtained from persons with DS-AD or non-DS age-matched control participants via the Down Syndrome Biobank Consortium (DSBC) (study protocol approved by University of Denver Institutional Review Board). Plasma aliquots (0.25 mL) were treated with thrombin (System Biosciences, Palo Alto, CA, USA) for 30 min at room temperature, then mixed with 0.22 µm of filtered Dulbecco’s Phosphate Buffered Saline (DPBS) containing a protease and phosphatase inhibitor cocktail (Thermo Scientific, Inc., Waltham, MA, USA) [13,30,80]. After centrifugation, the supernatant was mixed with ExoQuick polymer solution (System Biosciences, CA, USA) and the resulting pellet was resuspended in ultra-pure H2 O containing a protease and phosphatase inhibitor cocktail as previously described [13]. To selectively isolate neuronal EVs, samples were incubated for 1 h with mouse biotinylated anti-human CD171 monoclonal antibody (clone 5G3, L1CAM, eBiosciences, CA, USA). Then, Streptavidin-Plus Ultra-link beads (Thermo Scientific, Inc.) were added to each suspension and incubated for 4 h with gentle mixing. Immuno- absorbed samples were centrifuged at 400× g for 10 min, washed once with DPBS, then NDEVs were eluted with 0.1 M glycine-HCl (pH 3.0), incubated at 4 ◦ C for 5 min and centrifuged for 5 min at 4500× g [13]. The supernatant was transferred to a fresh tube containing 1 M Tris-HCl and 3% BSA in DPBS. The volume was brought to 0.25 mL by adding DPBS, and NDEV preparations to be used for stereotaxic injections were stored at −80 ◦ C until use for the intracranial surgeries. To determine the CD81, Tau and p-Tau levels in each exosome preparation, another aliquot of plasma was treated as described but resuspended in M-PER lysis buffer (ThermoFisher) instead of DPBS and underwent one freezing/thawing cycle. NDEVs were isolated from an initial volume of 0.25 mL of plasma and were resuspended in a final volume of 0.25 mL. Therefore, all the NDEV biomarker values refer to the original plasma volume. Levels of CD81 were measured using CD81 Elisa kit (Cusabio, Houston, TX, USA), while Tau (N-terminal to mid-domain (R1) Tau) and p-Tau T231 levels were measured using the Simoa SR-X instrument (Quanterix, MA, USA) according to the manufacturer’s instructions. J. Clin. Med. 2021, 10, x FOR PEER REVIEW 4 of 23 J. Clin. Med. 2021, 10, 3931 4 of 23 2.2. Validation of NDEVs 2.2. Validation NDEV size of NDEVs and concentration were assessed using nanoparticle tracking analysis withNDEV size and concentration the Nanosight were assessed apparatus (Malvern using nanoparticle Instruments). NDEVs were tracking analysis diluted with in filtered the DPBSNanosight apparatus and labeled with(Malvern ExoGlow™ Instruments). membrane NDEVs werekit labelling diluted (Systemin filtered DPBS and Biosciences, CA, labeled with ExoGlow™ membrane labelling kit (System Biosciences, USA) following manufacturer’s protocol. Particle enumeration and sizing were carriedCA, USA) following manufacturer’s protocol. Particle out on a LM10 Nanosight instrumentenumeration and sizing on fluorescence mode.were carriedofout Analysis on a counts particle LM10 Nanosight instrument on fluorescence mode. Analysis of particle counts was conducted with Nanosight NTA 2.3 software, using a detection threshold of five. The was conducted with Nanosight presence NTA 2.3 of exosomal software, markers, using athe including detection threshold tetraspanins CD63of andfive. CD81Thewas presence of also con- exosomal firmed usingmarkers, Westernincluding blottingthe tetraspanins [81] using MiniCD63 andTGX Protean CD81 was gels also confirmed (Bio-Rad, Hercules, using CA, Western blotting [81] using Mini Protean TGX gels (Bio-Rad, Hercules, USA). These were loaded with the maximum volume of NDEV per well and separated at CA, USA). These were 150 Vloaded for 40 with the maximum min. Next, volume proteins were of NDEVtoper transferred PVDFwellmembrane. and separated at 150 Vwere Membranes for 40 min. Next, probed overnight proteins were transferred to PVDF membrane. Membranes at 4 °C with antibodies against CD63 (EXOAB-CD63A-1, System Biosci- were probed overnight ences) and 4 ◦ C with atCD81 antibodies Sigma (SAB3500454, againstAldrich), CD63 (EXOAB-CD63A-1, System with washed and incubated Biosciences) and appropriate CD81 (SAB3500454, Sigma Aldrich), washed and incubated with secondary antibodies for 1 h at room temperature. Membranes were then washed andappropriate secondary antibodies for 1 h at room temperature. Membranes were then washed and developed developed with SuperSignal™ West Femto chemiluminescent substrate (ThermoFisher, with SuperSignal™ West Femto chemiluminescent substrate (ThermoFisher, Hillsboro, OR, Hillsboro, OR, USA) and visualized on a ChemiDoc imaging system (Bio-Rad). USA) and visualized on a ChemiDoc imaging system (Bio-Rad). To further demonstrate NDEV enrichment in our samples, we used four plasma sam- To further demonstrate NDEV enrichment in our samples, we used four plasma ples from healthy volunteers and isolated NDEV as described above and, in addition to samples from healthy volunteers and isolated NDEV as described above and, in addition the final NDEV, collected supernatant after each step (EXQSN, WASH and BDSN) of the to the final NDEV, collected supernatant after each step (EXQSN, WASH and BDSN) of isolation protocol as depicted in Figure 1. EV-specific markers were measured using com- the isolation protocol as depicted in Figure 1. EV-specific markers were measured using mercial ELISA kits: levels of membrane-bound CD81 and luminal Alix were assessed us- commercial ELISA kits: levels of membrane-bound CD81 and luminal Alix were assessed ing ELISA kits (Cusabio, Houston, TX, USA). To demonstrate enrichment for neuron-spe- using ELISA kits (Cusabio, Houston, TX, USA). To demonstrate enrichment for neuron- cific proteins, we measured using single molecule array (Simoa) technology on the SR-X specific proteins, we measured using single molecule array (Simoa) technology on the SR-X instrument (Quanterix, MA, USA) the levels of Tau, UCH-L1 and NF-light in plasma, the instrument (Quanterix, MA, USA) the levels of Tau, UCH-L1 and NF-light in plasma, the threedifferent three differentsupernatants supernatantscollected collectedatateach eachisolation isolationstep stepand andthe thefinal finalNDEV NDEVprepspreps(see (see experimental outline in Figure experimental outline in Figure 1). 1). Figure1.1. Experimental Figure Experimental workflow workflowforforexosome exosomevalidation validationstudies. studies.Thrombin-treated Thrombin-treatedplasma plasmawas was firstprocessed first processedwith withExoQuick ExoQuickpolymer polymerreagent, reagent,whereafter whereafterthetheEV-depleted EV-depletedplasma plasma(EXQSN) (EXQSN)waswas collected. The EV pellets were then mixed with L1CAM biotinylated antibody, then streptavidin- collected. The EV pellets were then mixed with L1CAM biotinylated antibody, then streptavidin- coated beads. The bead supernatant (BDSN) was collected for biomarker analyses and the bead coated beads. The bead supernatant (BDSN) was collected for biomarker analyses and the bead complex was next washed with Dulbecco’s Phosphate Buffered Saline (DPBS) (WASH was col- complex was next washedsmall lected). Neuron-derived with Dulbecco’s Phosphate extracellular vesicles Buffered (NDEVs)Saline were (DPBS) (WASH was then recovered collected). from the mix. Neuron-derived Exosome-related proteins and neuron-specific proteins were measured at each step of theExosome- small extracellular vesicles (NDEVs) were then recovered from the mix. isolation related proteins procedure. The and neuron-specific results proteins were from these validation measured studies at eachinstep are presented of the2isolation Figures and 3. procedure. The results from these validation studies are presented in Figures 2 and 3. J. Clin. Med. 2021, 10, 3931 5 of 23 2.3. In Vitro Seeding and Conformational Tau Analysis Seeding competent Tau conformers were amplified from NDEVs using a modified version of our previously published amplification protocol [24]. NDEVs from control and DS subjects were mixed with 0.1% Triton X-100, sonicated for 2 min on ice (tip sonicator) and then combined with recombinant Tau (hTau40) and heparin (Celsus, average molecular weight 4400 Da). Final volume was 700 µL, all mixtures contained 10 µM Tau, 40 µM heparin and NDEVs at a protein concentration of 150 µg/mL. The samples were subjected to 30 cycles of fracture and growth at 37 ◦ C, utilizing a bath sonicator with a microplate horn (QSonica). Each cycle included a 5-second sonication step at 5% amplitude, followed by a 30-minute step of quiescent incubation. The amplified material was then sedimented by ultracentrifugation (30 min at 130,000× g). Pellets were analyzed using SDS-PAGE and Coomassie staining. 2.4. Stereotaxic Surgery At eight months of age, male C57BL/6 mice were anesthetized deeply with ke- tamine/xylazine (120 mg/kg/6 mg/kg) and immobilized in a mouse stereotaxic frame (Kopf Instruments) connected to a syringe pump system (Harvard Apparatus, Holliston, MA, USA). Mice were randomly assigned to be injected unilaterally with either 2 µL of NDEVs isolated from the blood of a DS-AD individual (n = 8) or 2 µL of NDEV from an age- matched control donor (n = 7). Burr holes were made using a Drexel drill and a 10-microliter Hamilton syringe was lowered to the dorsal layer of the hippocampus (AP −1.3 mm; ML 1.5 mm; DV −2.0 mm relative to Bregma) according to the Paxinos mouse atlas [82]. After the injection, the syringe was kept in place for 10 min to limit diffusion back into the needle track. The mice were maintained for either 1 month (n = 7) or 4 months (n = 8) after injection. All animal procedures followed the guidelines of the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the University of Denver Institutional Animal Care and Use Committee (IACUC). 2.5. Immunohistochemistry and Image Analysis Mice were euthanized following NIH guidelines using carbon dioxide inhalation. Brains were rapidly removed and fixed for 48 h in 4% paraformaldehyde at 4 ◦ C, then transferred to 30% sucrose solution in phosphate buffered saline (PBS, pH 7.4) for 1 week. Coronal sections (35-micron thickness) were obtained using a cryostat (Microm). Tau im- munostaining was analyzed using two different commercial phospho-Tau antibodies. NFTs represent cytoplasmic inclusions of abnormal insoluble aggregates in the brain of patients with AD. These aggregates are paired helical filaments (PHFs), mostly consisting of Tau protein. Conformational changes of Tau involve hyperphosphorylation and truncation resulting in PHF assembly. In the current study, we examined different stages of Tau aggregation using two immunological markers of specific N-terminus phosphorylation Tau sites, including a marker for early stages of abnormal Tau processing, T231, and a marker for a later stage, S396 [83]. Coronal sections were blocked for 1 h with 10% normal serum, then permeabilized in 0.1% Triton-X in TBS. Sections were incubated overnight at 4 ◦ C with primary antibodies against p-Tau S396 (Invitrogen, Cat# 44-752G) and P-Tau T231 (Invitrogen, Cat# 44-746G), washed and then incubated for 1 h with appropriate secondary antibodies (Alexa Fluor 594, Life Technologies, 1:250) at room temperature. The stained sections were washed and mounted using ProLong Gold antifade mounting media (Molecular Probes). Images were obtained with a laser scanning confocal micro- scope (Olympus FV3000 Laser Scanning Confocal microscope) using FluoView software (Olympus). The intensity of p-Tau immunofluorescence was estimated using ImageJ [84] by creating a maximum projection of all the slices in the stack, subtracting the background (rolling ball radius of 25 pixels) and measuring mean integrated density. Staining intensity was measured separately in the hilar region (polymorphic layer) and the granule cell layer of the dentate gyrus, on at least two different brain sections per animal, and averaged to obtain a single value for each antibody and animal. J. Clin. Med. 2021, 10, 3931 6 of 23 Double immunofluorescence staining was performed with either p-Tau antibodies (S396 or T231, as described previously) and with GFAP (Glial fibrillary acidic protein, Dako, 1:100), Iba1 (ionized calcium-binding adapter molecule 1, Wako, 1:500) or NeuN (Chemicon, 1:200) to determine whether p-Tau co-localized with astrocytic, microglial or neuronal markers, respectively. Double immunofluorescent labeling was carried out according to our previously published protocols [85]. Sections were incubated overnight at 4 ◦ C with p-Tau 396 or p-Tau T231 and antibodies against astrocytes, microglia or neural cell bodies as described above. p-Tau S396 and p-Tau T31 were visualized with Alexa Fluor 594-labelled secondary antibody (Life Technologies, Carlsbad, CA, USA, 1:250) and GFAP, Iba1 and NeuN were visualized using Alexa Fluor 488 (Life Technologies, 1:250). Mounted sections were imaged in stacks in both channels via laser scanning confocal microscope and processed as described above. The staining intensity for Iba1 and GFAP was determined as described above. The ImageJ Coloc2 plugin was used to determine the pixel intensity correlation in the dentate gyrus area between the two channels (using the same ROI for both channels). Scatter dot plots were generated by plotting the average Pearson’s pixel intensity correlation coefficient between p-Tau S396 or T231 and GFAP, NeuN or Iba1 for each mouse. 2.6. Statistical Analyses All data are presented as mean ± standard error of the mean (SEM). For each time point (1 month and 4 months post-injection), two-way ANOVA (treatment × injection side) were run to determine differences in staining intensities between the DS-AD- and control-injected groups. Statistical analyses were performed using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA). A statistically significant difference was assumed at p < 0.05. 3. Results 3.1. NDEV Validation and Characterization The analysis of NDEVs using fluorescent NTA revealed an enrichment in the expected size range (Figure 2A), with a concentration of ≈109 –1010 particles/mL and an average size of 107 ± 4.2 nm. The size distribution and particle concentration are within the range reported by other investigators [70,86,87]. By Western blotting, we found that the NDEV samples contained both CD63 and CD81, which are members of the tetraspanin family and are located on the exosome surface (Figure 2B,C). Using commercially available ELISA kits, we measured EV-specific markers CD81 and Alix levels in the plasma, NDEV preps as well as the three supernatants collected throughout the isolation procedure (see Figure 1 for experimental design). We found that the CD81 and Alix levels were significantly elevated in the NDEV preps compared to the plasma and to the three supernatants collected through the isolation procedure (Figure 3A,B), demonstrating a specific enrichment for EVs in our preparations. Next, to confirm the enrichment for neuronal EVs in our preparations, we measured the levels of relatively specific neuronal biomarkers (Tau, NF-light and UCH-L1) in the NDEV preps as well as in the matching plasma samples and in the three different supernatants recovered during the isolation process. All three proteins were measurable to some extent in the plasma samples using the Simoa technology (Quanterix, MA) (Figure 3C–E). However, when comparing the NDEV with the plasma levels, we observed a clear enrichment of these three neuronal proteins, with the NDEV levels being between nine and 50 times higher than the plasma levels, strongly suggesting that the NDEV enrichment protocol used herein successfully isolated neuron-derived EVs. 21, 10, x FOR PEER REVIEW 7 of 23 J. Clin. Med. 2021, 10, 3931 7 of 23 Figure Figure 2. NDEV2.characterization. NDEV characterization. (A) nanoparticle (A) Fluorescent Fluorescenttracking nanoparticle analysistracking analysis illustrating illustrating size distribution size and concentra- tion of distribution and concentration extracellular vesicles of extracellular in neuron-derived vesicles exosome samples. Thein neuron-derived single peak at ≈100 nm exosome suggests samples. The small extracellular single peak at ≈100 nm suggests small extracellular vesicle (EV)-specific enrichment. (B) vesicle (EV)-specific enrichment. (B) Representative Western blot image of CD63 tetraspanin protein indicative of EV Representa- tive Western enrichment. blot image CD63 (System of CD63 Biosciences, tetraspanin protein EXOAB-CD63A-1) indicative was detected of EV at 53 kD enrichment. in NDEVs isolatedCD63 (System from human plasma Biosciences, EXOAB-CD63A-1) was detected at 53 kD in NDEVs isolated from human via Western blot. (C) Representative Western blot showing tetraspanin CD81 (SAB3500454, Sigma Aldrich) at 25 kD plasma via in total EVs (EV)Western blot. (C) from human Representative plasma Western (System Biosciences, blot showing EXOP-500A-1) andtetraspanin CD81EVs neuron-derived (SAB3500454, (NDEV) isolated Sigma fromAl- human plasma.drich) at 25 kD in total (D) Amplification of TauEVs (EV) fibrils from from human NDEVs plasma obtained (System from blood. M Biosciences, = marker, CEXOP-500A-1) = control NDEVs, and DSneu- = NDEVs ron-derived EVs (NDEV) isolated from plasma from a subject with Down syndrome. from human plasma. (D) Amplification of Tau fibrils from NDEVs obtained from blood. M = marker, C = control NDEVs, DS = NDEVs from plasma from a subject In the NDEV preps to be used for stereotactic injections, the levels of CD81, Aβ42, Tau with Down syndrome. and p-Tau T231 were measured using a standard ELISA kit (CD81, Cusabio) or Simoa tech- nology on the SR-X instrument (Quanterix, MA). Importantly, the levels of AD-associated biomarkers were higher in the DS-AD NDEVs than in the control NDEVs (Table 1), sup- porting the morphological results presented below. d. 2021, 10,J.xClin. FORMed. 2021, PEER 10, 3931 REVIEW 8 of 23 8 of 23 Figure 3. Quantification Figure 3. Quantification of biomarkersof showing biomarkers showing enrichment forenrichment for neuron-derived neuron-derived EVs. measured EVs. Biomarkers were Biomarkers in plasma, were EV-depleted measured plasma in plasma, (exqsn), EV-depleted bead supernatant plasma (bdsn), (exqsn), wash bead supernatant supernatant (bdsn), (wash) and in NDEVs wash supernatant (described in Figure 1). (wash) Levels of small and in NDEVs EV-associated (described tetraspanin in Figure CD81 (A) and1). Levels Alix of small (B) were EV-associated measured tetraspanin using commercial ELISACD81kits(A) (Cusabio). and Alix (B) were Levels of neuron-specific measured proteins using NF-light (C),commercial UCH-L1 (D)ELISA and Taukits (E)(Cusabio). in plasma Levels of neuron-specific were measured using Simoa pro- assay kits teins NF-light (C), UCH-L1 (D) and Tau (E) in plasma were measured using Simoa assay kits on the on the SR-X instrument (Quanterix). Note that NDEVs contained significantly higher levels of all components including the SR-X markers, neuron-specific instrument (Quanterix). strongly Note suggesting thatthat theNDEVs contained significantly immunoprecipitation higher procedure for levels NDEVs hadof all compo- succeeded. The same nents including the neuron-specific markers, strongly suggesting that the immunoprecipitation pro- process was used to obtain the NDEVs for injection into mouse brain. cedure for NDEVs had succeeded. The same process was used to obtain the NDEVs for injection into mouse brain. Table 1. Levels of biomarkers measured in NDEV enriched from plasma samples from one participant with DS-AD and one control. In the NDEV preps to be used for stereotactic injections, the levels of CD81, Aβ42, Tau and p-Tau T231 were measured using a standard ELISA Control kit (CD81, Cusabio)DS-AD NDEV or Simoa NDEV technology on the SR-XCD81 instrument (ng/mL) (Quanterix, MA). Importantly, 15.21 the levels of AD-asso- 23.51 ciated biomarkers wereAβ42 higher in the DS-AD NDEVs than (pg/mL)