Link to original article: Huilin Shao, Hyungsoon Im, Cesar M. Castro, Xandra Breakefield, Ralph Weissleder and Hakho Lee. New Technologies for Analysis of Extracellular Vesicles. Chem Rev. 2018 Feb 28;118(4):1917-1950. doi: 10.1021/acs.chemrev.7b00534.
This review was published in the journal Chemical Reviews with an impact factor as high as
This review, published in Chemical Reviews with an impact factor of 54.301, summarizes the research methods of extracellular vesicles in a very comprehensive way, and basically describes all the research methods currently used in EVs research! The corresponding author is Prof. Hakho Lee, Harvard University.
Extracellular Vesicles (EVs) are diverse nanoscale membrane vesicles actively released by cells. Vesicles of similar size can be further categorized based on their biogenesis, size and biophysical properties (e.g. exosomes, microvesicles). While EVs were initially considered cellular debris and therefore underappreciated, EVs are now increasingly recognized as important carriers of intercellular communication and circulating biomarkers for disease diagnosis and prognosis.
The review covers:
Biofluids contain a large number of EVs that can transfer different molecules from Parental Cells to other cells, including: proteins, mRNA/miRNA, DNA, etc.
The formation of EVs determines their membrane composition.
The membrane composition of microvesicles best reflects the plasma membrane of their Parental Cells.
In contrast, specific endosomal protein molecules have been identified in exosomes that reflect the mechanism of exosome formation. The endosomal sorting complex (ESCRT) has been widely recognized to regulate and direct specific molecules into the luminal vesicles of the MVB.ESCRT and its four major complexes (ESCRT 0, I, II, and III) are responsible for the delivery of ubiquitinated proteins for lysosomal degradation and protein recycling. Recent studies have shown that depletion of specific ESCRT family proteins can alter the protein content of exosomes and the rate at which cells release exosomes. More interestingly, exosomes were found to be enriched in components of the ESCRT system (e.g., TSG101 and Alix) that can be used as markers for exosome recognition.
ESCRT is not the only mechanism mediating exosome formation. Other ESCRT-independent processes appear to participate in its formation and secretion in an intertwined manner. Exosomes are also enriched for ESCRT-independent molecules. For example, the four transmembrane proteins CD9, CD63, and CD81 have been shown to be involved in endosomal vesicle transport. The Rab family of small GTPases is involved in vesicle transport and fusion with the plasma membrane suggesting a role for these proteins in the release of exosomes. In addition, elevated ceramide levels in exosomes and reduced exosome release induced by inhibition of sphingomyelin suggest that sphingomyelinases are involved in vesicle release.
Both exosomes and microvesicles contain nucleic acids, including miRNAs, mRNAs, DNA, and other non-coding RNAs. since the initial discovery that EVs contain RNA, there has been a great deal of interest in the use of EV RNA as a diagnostic biomarker. In seminal work, Skog et al. found that serum exosomes from glioblastoma patients contain characteristic mutant mRNAs (EGFRvIII mRNA) and miRNAs that can be used to provide diagnostic information. The discovery of these nucleic acids led to the hypothesis that EVs can transfer genetic information between cells. Indeed, Varadi et al. and Skog et al. showed that EVs contain mRNAs that remain translatable after transfer into the host cell. retrotransposons and other noncoding RNAs are also expressed in EVs. Retrotransposon sequences and miRNAs, as well as translatable mRNAs, are transferred through EVs, and these results highlight the importance of EVs as carriers and transmitters of genetic information.
Although the diffraction limit of conventional optical microscopy is close to the size of EVs, it does not produce clear images. High-resolution EV images need to be obtained by electron microscopy (EM) or atomic force microscopy (AFM). However, the throughput of these methods is limited because specialized staining protocols and equipment are required.
(a) Scanning electron microscopy (SEM) provides surface topography information in three dimensions.
(b) Transmission electron microscopy (TEM) has excellent image resolution and can be used in conjunction with immunogold labeling to provide molecular characterization.
(c) Cryo-EM can analyze EV morphology without extensive processing.
Dynamic light scattering (DLS), also known as Photon Correlation Spectroscopy or Quasi-Elastic Light Scattering, is a physical characterization tool used to measure particle size distributions in solutions or suspensions, and to measure the behavior of complex fluids, such as polymer concentrated solutions. It can also be used to measure the behavior of complex fluids such as concentrated polymer solutions. When light is directed at small particles that are much smaller than their wavelength, the light is scattered in all directions (Rayleigh scattering). If the light source is a laser, we can observe fluctuations in the intensity of the scattered light in one direction over time because the tiny particles in the solution are in Brownian motion and the distance between each of the scattered particles has been changing over time. Scattered light from different particles interferes constructively or destructively depending on the phase. The resulting intensity fluctuation curve over time carries information about the movement of the particles causing the scattering over time. Dynamic light scattering experiments are susceptible to dust or impurities, so filtration and centrifugation of the sample is important.
Dynamic light scattering is used to characterize the size of proteins, polymers, micelles, sugars, and nanoparticles. If the system is monodisperse, the average effective diameter of the particles can be derived, a measurement that depends on the core of the particles, the surface structure, the concentration of the particles, and the type of ions in the medium.DLS can also be used for stability studies, where measurement of the particle size distribution over time reveals the tendency of the particles to coalesce and sink over time. As particles settle, there are more particles with larger sizes. Similarly, DLS can be used to analyze the effect of temperature on stability.
Dynamic Light Scattering collects the fluctuations of scattered light intensity from particles in Brownian motion in a solution, and converts the fluctuations of light intensity into a correlation curve through a correlator to obtain the speed of the fluctuations of light intensity, and to compute information about the diffusion speed of the particles and the particle size of the particles. Small particle samples have faster Brownian motion, faster light intensity fluctuations, and faster decay of the correlation curve, and vice versa for large particles.
In exosome studies, dynamic light scattering measurements are more sensitive, with a lower limit of measurement of 10 nanometers. Compared to the SEM technique, the sample preparation is simple, only requires simple filtration, and the measurement is faster. However, the dynamic light scattering technique measures the fluctuation data of light intensity, so the fluctuation signals of light intensity of large particles will mask the fluctuation signals of light intensity of smaller particles, so dynamic light scattering is not suitable for the measurement of complex exosome samples of varying sizes, and is only suitable for the measurement of the sizes of homogeneous exosomes prepared by chromatography, and it is not possible to measure the concentration of exosomes in the samples.
Nanoparticle Tracking Analysis (NTA) is an optical particle tracking method used to determine the concentration and size distribution of particles. A beam of light is used to illuminate particles in a sample. As the particles scatter the light and undergo Brownian motion, the camera records the path of each particle to determine the average velocity and diffusivity. Unlike the body scattering measurements of DLS, NTA tracks the scattering of individual particles.
This information is then used to mathematically calculate the concentration (i.e., the number of particles in the field of view) and the size distribution (i.e., the hydrodynamic diameter via the Strokes-Einstein equation, Figure 5b). To accurately quantify the concentration and size of heterogeneous vesicles, the NTA procedure requires precise optimization of the camera and analysis settings. Separate measurements using different settings may be required to obtain accurate readings of the EV subset in heterogeneous mixtures.
EVs are heterogeneous in size, origin, and molecular composition; in addition to this, they are found in diverse and complex biological fluids, including blood, pleural fluid, ascites, breast milk, saliva, cerebrospinal fluid, and urine. These fluids also contain a large number of non-vesicular macromolecular structures that may interfere with EV analysis. That is why the separation and enrichment of EV is particularly important.
Ultracentrifugation (80%) and density gradient centrifugation (20%) are the two most common high-throughput hybrid separation methods. Based on their separation mechanisms, these methods can be categorized into three main groups:density, affinity, and size.
The particles are separated by different centrifugal forces: cellular debris is removed at a lower centrifugal force (300g), while the EV is precipitated and concentrated at a higher centrifugal force (100,000g). Although this method is the most widely used gold standard, it has many drawbacks such as large size, expensive instrumentation, long processing time, cumbersome process, contamination by aggregated protein and nucleoprotein particles, and the need for a large number of samples.
Sucrose gradient centrifugation is a more rigorous ultracentrifugation method that facilitates further separation of vesicles of different densities and is commonly used to isolate exosomes (suspension densities of 1.15 to 1.19 g/mL). In this method, a sample containing vesicles of different sizes and macromolecules is layered on a surface with a gradient of increasing density from top to bottom. During centrifugation, different molecules are deposited through the gradient at different rates. Because of its higher resolution, this method is thought to allow for the separation of higher purity EVs (especially exosomes); however, it faces many of the limitations associated with ultracentrifugation. More recent isotonic gradient (e.g., iodoflavanol gradient) methods are thought to work better.
More recently, commercial kits based on polymer*** precipitation methods (e.g., ExoQuick, Exo-Spin) have been developed for EV enrichment. These kits use reduced EV hydration (and thus reduced solubility) to cause precipitation, and then at low centrifugal forces, the precipitated EV products can be readily and reproducibly isolated, thus avoiding long ultracentrifugation operations. However, these kits are expensive for large-scale use and lack specificity for EV. The method also tends to produce non-homogeneous polymer particles. Since all of these reagents reduce the solubility of EV and proteins, the method also ***precipitates lipoproteins and Ago-2 RNA complexes. Thus, ***precipitation is limited as a method of EV isolation.
Size exclusion chromatography separates vesicles and other molecules by gel filtration based on their molecular size. This gel consists of spherical beads containing specific size-distributed pores. When the sample enters the gel, small molecules diffuse into the pores, while large molecules elute directly. As a result, large molecules leave the column earlier than small molecules, which makes it possible to correlate the residence time of the molecules with the size of the column. In recent years, this separation method has been applied to isolate and purify vesicles from complex biological media. commercial companies such as Sepharose, GE Healthcare; qEV, iZon, and others are also developing commercial products to simplify EV enrichment, and the exclusion columns for all of these products are approximately 75-nanometer pore-size resins. Proteins and other smaller contaminating molecules are retained in the pore size, while larger vesicles (>75 nm) can quickly pass through and be eluted in the void. The size exclusion method separates EVs from soluble proteins; to improve the efficiency and resolution of the separation, a variety of factors need to be considered, including the media type, pore size, interaction between the EV and the media, column size, column packing, and flow rate.
In order to improve the efficiency and specificity of EV separation of complex biofluids, a variety of new EV enrichment methods have been developed. However, most of these new methods have low throughput rates compared to conventional methods and should be addressed to make them practical.
Molecular size-based separation is a promising approach to separate EVs from large cellular debris. Various microfluidic filtration systems have been developed for separating EV from large cellular debris and protein aggregates, and most of these systems are based on molecular size differences. For example, Rho et al. constructed a microfluidic device that separates EVs by screening unprocessed blood samples using a membrane filter. the size of the membrane filter was ?1 μm. a capillary tube was inserted below the membrane to guide the filtered EVs into the collection channel. The membrane filter and capillary guide are sandwiched between two ring magnets; this setup allows for easy replacement of the filter set when a large number of samples are processed.
Lee et al. recently used acoustic waves to subdivide EVs in a contactless manner. This separation utilizes ultrasonic standing waves to apply different acoustic interaction forces to the vesicles depending on their size and density. The device consists of a pair of interleaved transducer (IDT) electrodes to generate standing surface acoustic waves across the flow channel.
EV proteins are mainly derived from cytoplasmic membranes, cytoplasmic alcohols, and not from other intracellular organelles (e.g., Golgi, endoplasmic reticulum, nucleus, etc.) The composition of EV proteins is suggestive of vesicle biogenesis and cargo sorting (which is a bit of an odd translation). Therefore the International Organization for Extracellular Vesicles suggests that EV proteins, especially transmembrane and cytoplasmic proteins, should be carefully characterized.
In mammals, both transmembrane proteins and lipid-bound extracellular proteins (e.g., endoprosthetic proteins) are associated with microvesicles and exosomes. The transmembrane proteins of exosomes are enriched in tetrameric proteins (e.g., CD9, CD63, and CD81) a superfamily of proteins with four transmembrane structural domains. Tetrameric proteins are involved in cell membrane trafficking and biosynthetic maturation and are highly expressed in exosomes, a property that has led to the use of tetrameric proteins for the quantification and characterization of exosomes. However, it is important to note that tetrameric proteins are not uniquely expressed only in exosomes. On the other hand, microvesicles are enriched in integrins, selectins, and CD40 ligands, suggesting that they are derived from the plasma membrane of the cell, and EV is enriched in specific transmembrane protein receptors (e.g., epidermal growth factor receptors/ EGFRs) and adhesion proteins (e.g., epithelial cell adhesion molecules/EpCAM). As many transmembrane proteins are involved in normal physiology and disease pathogenesis, they are used as important pathophysiologic EV biomarkers.
EV-associated intracapsular proteins have multiple functions. They include cytoplasmic proteins involved in vesicular transport with membrane or receptor binding capacity, such as TSG101, ALIX, annexin, and Rabs. EV is also enriched in cytoskeletal proteins (e.g., endostatin, myosin, and tubulin), molecular chaperone proteins (e.g., heat-shock proteins/HSPs), and metabolic enzymes (e.g., enolase, glyceraldehyde 3-phosphate dehydrogenase/GAPDH, and ribosomal proteins). ribosomal proteins). Interestingly, recent studies have revealed that EV proteins can be efficiently transported and received by receptor cells, thereby eliciting strong cellular responses in vivo and in vitro. This presents new opportunities for EV as a therapeutic and drug carrier.
Quantification and characterization of EV proteins is important not only for elucidating EV biogenesis and cargo sorting, but also for identifying physiological and pathological markers. However, conventional protein analyses, including Western blotting and enzyme-linked immunosorbent assay (ELISA), usually require large sample sizes, extensive processing and/or bulky specialized instrumentation, and thus are less suitable for clinical applications.
Western blotting is probably the most commonly used technique in EV protein assessment to signal the presence of target proteins associated with EV. In this process, purified vesicle preparations (usually prepared by the current gold standard of gradient ultracentrifugation) can be treated with a buffered lysate containing denaturants and protease inhibitors. Protein lysates are then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS?PAGE) and transferred to membranes for immunoblotting of specific protein targets. Although this method has a long preparation and processing time (> 10h), Western blotting can provide useful information about the molecular size of proteins.
Unlike Western blotting, where ELISA can only quantify target proteins on a relatively small scale, mass spectrometry enables high-throughput peptide profiling. Purified EV preparations undergo enzymatic digestion and peptide separation, followed by ionization analysis by mass spectrometry. Multiple steps in this complex process heavily influence EV proteomic analysis. In addition to effective EV purification, peptide fractionation prior to mass spectrometry analysis is considered an important prerequisite for the identification of vesicular proteins. This is usually achieved by three main methods: (1) SDS?PAGE, (2) two-dimensional liquid chromatography and (3) isoelectric focusing-based fractionation.
It is worth noting that since mass spectrometry analysis can identify digested peptide fragments, proper protein identification, quantification, and validation are necessary. Two technical approaches have been used for quantification: label-based and label-free. In label-based quantitative analysis, labels (isobaric or isotopic) are used for comparative analysis. In label-free quantitative analysis, spectral counting of chromatographic intensities is applied. Identified candidate proteins can be validated using other traditional protein techniques such as Western blotting. Mass spectrometry is usually less sensitive than antibody-based techniques in terms of detection sensitivity.
Although mass spectrometry analysis requires a significant amount of preparation and processing time (days), it can provide high-throughput, quantitative, and EV-comparative proteomic analysis. To date, thousands of vesicle proteins have been systematically classified and protein-protein interactions analyzed. Detailed discussions of mass spectrometry-based proteomic analyses of mammalian and bacterial EVs have been highlighted in several reviews. The study of these networks and interactions has helped to elucidate the functional activities of EV vectors and their important role in long-distance cell-to-cell communication.
To address the technical challenges associated with EV protein quantification, a new generation of biosensors is under development. These biosensors utilize unique sensing mechanisms to detect EVs of various sizes and molecular contents compared to traditional protein detection methods. many of these technologies require only smaller sample sizes and fewer sample handling procedures, making them ideally suited for medical applications.
Flow cytometry is a powerful technique for sorting individual large particles (e.g., cells or micrometer-sized entities) based on light scattering and fluorescence activation; however, conventional flow cytometry has limited sensitivity and resolution for detecting small particles less than 500 nm in diameter. In addition, it suffers from a high optical background due to the presence of small particles (~200 nm) in the sheath fluid. When quantifying EVs by conventional flow cytometry, a large number of small EVs may be overlooked or undercounted: multiple small vesicles may be illuminated at the same time and counted as a single event, a phenomenon referred to as "population theory".
To address the drawbacks of traditional flow cytometry, micron-sized latex beads were used to bind multiple vesicles. The bound EVs are then stained with fluorescent antibodies and their protein markers identified. However, this method lacks the ability to analyze individual vesicles and does not distinguish between different subpopulations of vesicles, which can lead to loss of characterization.
This technique is primarily based on magnetic nanoparticles (MNPs). Since most biological matter naturally lacks a ferromagnetic background, this sensing is virtually unaffected by interference from other biological samples in the same system. Thus, even optically turbid samples are transparent to the magnetic field; when target molecules are targeted by specific MNPs, they contrast strongly with the natural biological background. In magnetic detection based on nuclear magnetic **** vibration (NMR), MNPs are placed in the NMR magnetic field, which generates a localized magnetic field that alters the transverse relaxation rate of the surrounding water molecules and amplifies the analytical signal. As a result, NMR***vibration reduces sample processing, improves detection sensitivity, and has been developed for several point-of-care applications (e.g., detection of circulating tumor cells and bacteria directly from blood samples).
However, applying this technique to EV detection has been challenging because EVs are significantly smaller than tumor cells by 1 to 2 orders of magnitude.Shao et al. developed a new analytical technique specifically for EV detection and protein analysis. This method employs a two-step bioorthogonal click chemistry approach to label EVs. This small-molecule (<200 Da) labeling strategy does not significantly increase the size of antibodies or MNPs, thereby improving the efficiency of retaining target vesicles from unbound antibodies and MNPs. The abundance of EV biomarkers was determined by direct measurement of EV using micro-NMR*** vibrations (μNMR) on a microfluidic chip.
The μNMR system exhibits better detection sensitivity than conventional protein techniques: 10 3-fold more sensitive than WB and ELASA.Using this integrated technique, Shao et al. could study EV in a glioblastoma multiforme (GBM) cell line grown in a petri dish.Comparative protein analyses confirmed that EV indeed reflects the protein profile of its parental cells, and that a combination GBM of four markers (EGFR, EGFRvIII, PDPN, and IDH1)
R132H) can be used to differentiate cancer-derived EVs from host-cell-derived EVs.
In view of the small size of the EVs, a new rapid and label-free detection scheme for EVs: surface plasma **** vibration (SPR) has been proposed.SPR is a method for the detection of EVs at a metal dielectric interface under incident light irradiation and the collective oscillation of conduction electrons at a metal dielectric interface. Unlike other optical detection methods based on time-sensitive fluorescent and chemiluminescent probes, SPR sensing detects localized refractive index changes associated with biomolecule binding in the vicinity of the metal-dielectric interface, and is applied to label-free and real-time detection.
RNA is the main nucleic acid carried by EVs. RNAs transported by eEVs are typically shorter (usually <200 nucleotides, but also up to 5 kb long) compared to RNAs in cells. They are predominantly non-coding rna and include microRNAs (miRNAs), tRNAs (tRNAs), long stranded non-coding RNAs (lnRNAs), and fragmented mRNAs. coding mRNAs (mRNAs) have been identified in transcriptomes ranging in length from 200 to 1,000 nucleotides. mRNAs can be translated into proteins, and miRNAs regulate the expression of target mRNAs in recipient cells. translation of target mRNAs in somatic cells. the amount and nature of RNAs in EVs can vary depending on the cell type from which they originate.
Because they retain their function in receptor cells, researchers have raised the interesting hypothesis that there may be specialized mechanisms for assigning different RNAs to EVs for transport to specific receptor cells, or that they may be utilized to transport therapeutic RNAs to specific sites. This is an active area of research and has been addressed in a number of reviews.
Studies in recent years have found that EVs contain a significant proportion of mRNAs from the mother cell, many of which are cell-specific. these mRNA molecules are often present in EVs as fragments, protecting them from degradation by RNA enzymes and making them powerful circulating biomarkers.
In addition, it has been demonstrated in multiple studies that some <2 kb mRNA molecules in the EV are capable of encoding polypeptides that support protein synthesis (i.e., the function of protein translation). These studies emphasize the multiple roles of EVs as specific cellular messengers in influencing recipient cells and facilitating intercellular communication.
miRNAs are a class of small non-coding rna (typically 17 - 24 nucleotides) that mediate post-transcriptional gene silencing, usually by targeting the 3' untranslated region of mRNAs. By inhibiting protein translation, EV miRNAs are potent regulators in many biological processes. Unlike circulating mRNAs in the EV, miRNAs can exist in body fluids in a variety of stable forms. In addition to being encapsulated in the EV, circulating miRNAs can be loaded onto high-density lipoproteins or bound to extravesicular AGO2 proteins. Current evidence suggests that although most circulating miRNAs bind to RNA-binding proteins, small amounts of miRNAs can also be found in the EV. however, the distribution of miRNAs in the EV remains unclear. As is the case for mRNAs, miRNA expression in EVs reflects their cellular origin but differs slightly from that of the parental cells. Some miRNAs have been found to be preferentially expressed in EVs and to maintain function in recipient cells to regulate protein translation. Recent studies have also found that fetal bovine serum, commonly used in mammalian cell culture, may lead to miRNA artifacts during in vitro EV preparation.
Through NGS, we have also found that there are other types of RNAs present in EV. These RNAs include tRNAs, rRNAs, small nuclear RNAs (snRNAs), small nuclear kernel RNAs (snoRNAs), and long chain non-coding RNAs (lncRNAs). See the table above.
Recent studies have shown that some EVs may contain DNA fragments. These DNA are double-stranded fragments ranging from 100 base pairs (bp) to 2.5 k bp, with some associated >2.5 k bp DNA fragments outside the EV. These fragments represent the entire genomic DNA and can be used to identify mutations present in parental tumor cells. Although there is credible evidence for the presence of DNA in EV, its function has not been determined.
EV nucleic acid has been extensively studied as a potential circulating biomarker and regulator between receptor cells. Conventional tools for nucleic acid extraction and analysis have been successful in providing an important foundation for our understanding of EV nucleic acids. Due to the low levels of nucleic acids in EVs, it is important to develop efficient extraction methods and sensitive detection strategies, especially for rare target molecules in small samples.
With the growing interest in utilizing EV nucleic acids as minimally invasive diagnostic markers, new biosensor technologies have been developed to make extraction and analysis more efficient and rapid. Many of these new platforms offer sensitive quantification of targeted nucleic acid markers and the ability to identify disease markers in complex biological contexts, even including single nucleotide point mutations. This opens up many new opportunities for personalized clinical medicine.
While conventional PCR is a powerful technique for detecting genetic/transcriptional mutations (e.g., EGFRvIII deletion mutations), its sensitivity is limited and its detection of single-nucleotide mutations leaves much to be desired. This problem is particularly relevant to EVs because of the low proportion of mutant transcripts in the general context of wild-type transcripts.Chen et al. recently used a droplet digital PCR (ddPCR) technique to detect rare mutations in EVs.
Shao et al. recently developed an integrated microfluidic platform for on-site EV nucleic acid analysis that integrates three functional modules: targeted enrichment of EVs, on-chip RNA isolation, and real-time RNA analysis. This platform is called the immunomagnetic exosomal RNA (iMER) analysis platform: cancer-specific EVs are isolated from host-derived vesicles using antibody-functionalized magnetic beads, and then the immunomagnetic bead-adsorbed vesicles are lysed on the chip. As the EV lysate passes through a glass bead filter, EV RNA is selectively adsorbed and eluted from the filter for reverse transcription and qPCR analysis. To simplify the analysis process, all key components are integrated into a single chip cassette.
With the development of this system, the authors investigated two mRNA targets of nuclear proteins, MGMT (6-methylguanine)
Tumors are complex structures consisting of malignant cells and surrounding stromal cells such as endothelial cells, fibroblasts, and immune cells. Recent studies have shown that EVs promote intercellular communication in the tumor microenvironment, thereby regulating disease onset, progression, and playing an important role in therapeutic response.
The rest of this large block can be read in the original literature if you are interested.
In most neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, frontotemporal dementia) there is a similar model of disease progression, in which misfolded proteins self-associate to form ordered aggregates that accumulate in cells. The formation of the Abeta peptide of amyloid in Alzheimer's disease (AD) is probably the best known of these protein aggregates. In Parkinson's disease (PD), another type of aggregate forms within the cell, consisting mainly of alpha-synuclein (synaptic nucleoprotein), called Lewy bodies. Recent studies have shown that misfolded proteins involved in many neurodegenerative diseases appear in EVs. Thus, these vesicles hold new promise for detecting and monitoring neurodegenerative diseases.
AD is a delayed-onset neurological disorder: a progressive loss of memory and cognitive abilities due to neurodegeneration. Although the exact cause of AD remains a controversial topic, it is clear that plaque deposition associated with Aβ peptides and neurofibrillary tangles associated with tau proteins are important for disease progression. These amyloid peptides originate from the protein hydrolysis process of amyloid precursor protein (APP). This