†These authors contributed equally.
Alzheimer’s disease is an age-dependent neurodegenerative disease. Recently, different non-coding RNAs (ncRNAs), including microRNAs, long non-coding RNAs, and circular RNAs, have been found to contribute to Alzheimer’s disease’s pathogenesis. Extracellular vehicles could be enriched in ncRNAs and in their role in mediating intercellular communication. Signatures of extracellular vesicular ncRNAs have shown them to be a potential biomarker in Alzheimer’s disease. This perspective discusses the potential role of extracellular vehicle ncRNAs in Alzheimer’s disease, providing a theoretical basis for extracellular vesicular ncRNAs in Alzheimer’s disease, from pathogenesis to diagnosis and treatment.
Alzheimer’s disease (AD) is an age-dependent neurodegenerative disease with a
prevalence rate of 32% in people aged 85 or older, accounting for 60-80% of all
dementia cases. AD is characterized by the occurrence of senile plaques (SPs) and
neurofibrillary tangles (NFTs), synaptic dysfunction, neuronal death, chronic
inflammation and brain atrophy . SPs are composed of amyloid-beta (A
With the advances in next-generation sequencing (NGS) techniques, several novel
classes of non-coding RNAs (ncRNAs) have emerged, including microRNAs (miRNAs),
circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) . miRNAs are
Extracellular vehicles (EVs) are heterogeneous membranous structures of endosomal origin circulating in the extracellular space, considered a novel mode of intercellular communication. EVs comprise a diversity of subpopulations distinguished by their size, morphology, composition, biological origin and function. EVs can be broadly divided into microvesicles (MVs) and exosomes. MVs are 50-500 nm in diameter and are secreted directly from the plasma membrane by outward budding. Exosomes are 50-150 nm in diameter and are secreted from the plasma membrane through fusion with multivesicular bodies (MVBs) or late endosomes. EVs circulate in various biological fluids and deliver their contents to recipient cells to elicit functional responses. EVs carry specific proteins, lipids or RNA species, which determine their fate and functions in turn. In the brain, several cell types are capable of releasing EVs. EVs derived from microglia, which account for approximately 10% of the brain’s cells, are considered part of the inflammatory response. Moreover, oligodendrocytes, neurons, astrocytes, and embryonic neural stem cells have been described to release EVs [23, 24].
The biogenesis of exosomes starts within the endosomal system. Several cellular steps are needed to release exosomes, including the generation of intraluminal vesicles (ILVs) within MVBs, MVB trafficking along microtubules, and docking and fusion between the plasma membrane and MVBs (Fig. 1). Lipid raft microdomains play a critical role in MVB formation. Neutral sphingomyelinase 2 (nSMase2) mediated generation of ceramide from sphingomyelin hydrolysis induces negative membrane curvature and leads to ILV budding into MVBs .
Schematic representation of EVs biogenesis and release. Exosomes are generated within MVBs and transported to the plasma membrane. MVB biogenesis is regulated by nSMas2, ESCRTs, Vps4, ALIX, syndecan-syntenin, ARF6, and PLD2. MVB trafficking, docking and fusion are controlled by kinesins, Arl8, RABs, and SNARE proteins. MVs bud directly from the plasma membrane .
The ESCRT machinery is essential for ubiquitination dependent MVB biogenesis from endosome-derived vesicles. The ESCRT system consists of ESCRT-0 (tumor susceptibility 101, TSG101), ESCRT-I (Signal transducing adapter molecule 1, STAM1), ESCRT-II (Vacuolar protein sorting 25, Vps25), ESCRT-III (Vps20, Vps24, Vps2, and Vacuolar sorting protein, Snf7) and ATPase Vps4 complex. ESCRT-0 and ESCRT-I recognize and retain ubiquitylated transmembrane cargoes on the limiting membrane into MVBs and recruit ESCRT-II/III subcomplexes form a spiral-shaped structure. The ESCRT-III associated ALIX (ALG-2 interacting protein X) affects specific cargo selection [26, 27].
Ubiquitination independent MVB biogenesis has also been extensively described. Syndecan clustering was triggered by heparanase mediated trimming of heparan sulfate chains. Syntenin further binds syndecan to ALIX and participates in exosome formation mediated by ESCRT-III. Selective cargo sorting of CD63 incorporation into exosomes is regulated by the small GTPase ARF6 (ADP ribosylation factor 6) and the effector protein PLD2 (phospholipase D2) .
Upon maturation, MVBs can be transported to the plasma membrane along microtubules by multiple kinesin isoforms to secrete exosomes. MVBs transportation, docking, and fusion are regulated by Arl8 (ADP ribosylation factor-like 8), Rabs (RAB7, RAB27, RAB35), and SNARE complexes (YKT6, Syntaxin-1a, Syntaxin-4, Syntaxin-5, synaptotagmin-7, SNAP23, and VAMP7) .
The diameter of MVs are incredibly heterogeneous, ranging from 50 nm to 1,000 nm
(up to 10
We searched studies on the PubMed database using the following keywords: extracellular Vesicle, EV, exosome, microvesicle, MVB, circular RNA, circRNA, Alzheimer’s disease, and AD. EVs mediate horizontal transfer of RNA between donor and recipient cells, as first identified by Valadi et al.  and Skog et al. . ncRNAs are highly enriched in EVs. Pegtel et al.  reported the exosome-mediated miRNA transfer from Epstein-Barr virus-infected cells to uninfected recipient dendritic cells. These transferred miRNAs can regulate the gene expression of recipient cells . RNA sequencing of EVs has revealed abundant lncRNA and circRNA in human blood . Dysregulation of extracellular vesicular ncRNA has been identified in several neurodegenerative disorders, including AD. Two lncRNAs, PCA3 and RP11-462G22.1, were increased in Parkinson’s disease (PD) leukocytes . Similarly, Gui et al.  found that these two lncRNAs were also elevated in CSF exosomes in AD and PD. Known dysregulated miRNAs and lncRNAs verified by RT-PCR from serum EVs, serum exosomes, and CSF exosomes are summarized in Table 1.
|plasma EVs||miR-424-5p, miR-3065-5p, miR-93-5p||up|||
|miR-1306-5p, miR-342-3p, miR-15b-3p||down|
|plasma EVs||miR-23a-3p, miR-126-3p, let-7i-5p, miR-151a-3p||down|||
|plasma exosome||miR-135a, miR-384||up|||
|plasma neural exosome||miR-132||up||[67, 71]|
|CSF exosome||miR-27a-3p, miR-30a-5p, miR-34c||up||[68, 72]|
|CSF exosome||lncRNA RP11-462G22.1, lncRNA PCA3||up|||
Extracellular vesicular ncRNAs are shuttled between donor and recipient cells and function actively in recipient cells, suggesting a novel mechanism of intercellular communication . Since the first discovery of EVs in AD physiopathology, their multifaceted roles in this setting have been explored , including their role in mediating neuroinflammation . Exosomal miRNAs occurring in the blood have been investigated, and those from the central nervous system (CNS), including neurons, astrocytes, and CSF. They are considered promising diagnostic biomarkers in AD, as detected by RT-PCR or deep sequencing [20, 39, 40, 41]. Moreover, exosomes derived from the CNS have also been isolated in the blood (termed plasma-derived neural exosomes), furthering their appeal as target biomarkers in AD .
Role of EV miRNAs in the differential diagnosis of AD. Blue color represents serum EVs, and orange color represents CSF EVs. EV miRNAs in red color were upregulated in AD, while EV miRNAs in green were downregulated in AD.
EV miRNAs may be potential biomarkers for the differential diagnosis of AD (Fig. 2). Lugli et al.  applied NGS to investigate the differently expressed serum exosomal miRNAs in AD relative to controls, identifying 20 miRNAs. miR-342-3p was highlighted particularly, given that its downregulation has also been reported in previous studies. Cheng et al.  explored serum exosomal miRNA expression in AD from the AIBL cohort and identified 17 dysregulated serum exosomal miRNAs. Both  and  support the potential biomarker capability of miR-342-3p. Another two miRNAs, miR-21-5p and miR-451a, were found to be decreased in plasma EVs in AD relative to those in dementia with Lewy bodies (DLB), with area under curve (AUC) values of 0.93 and 0.95, respectively, suggesting these could be potential biomarkers to discriminate these diseases . Li et al.  examined the expression of 18 miRNAs in plasma EVs in vascular dementia (VD), AD, and mild cognitive impairment (MCI). They found that among the three miRNAs found to be decreased in AD compared to healthy control, only miR-1306-5p was differentially expressed between AD, MCI, and VD.
Moreover, upregulation of miR-424-5p, miR-93-5p, and miR-3065-5p might predict AD over other forms of dementia and healthy control . Barbagallo et al.  found that miR-34b in serum exosomes was higher in AD than VD. Yang et al. examined the expression of miR-193b, miR-135a, and miR-384 in plasma exosomes from MCI, AD, PD, and VD patients, finding that miR-384 may be the best miRNA discriminating AD, PD, and VD . Wei et al.  examined three miRNAs in plasma exosomes from dementia and controls, finding that miR-223 in AD was lower than in VD. Moreover, the miR-223 in untreated AD patients was significantly lower than those who had already received medical care. Schneider et al.  examined the expression of 752 miRNAs in CSF exosomes in the GENFI AD cohort and sporadic frontotemporal dementia (FTD). mir-632 was significantly increased in AD compared with sporadic FTD, with an AUC value of 0.88.
Role of EV miRNAs in the pathogenesis of AD. EV miRNAs in red color were promotive, while EV miRNAs in green color were suppressive for the complementary aspects.
Dysregulated EV ncRNAs have been linked to AD pathogenesis (Fig. 3).
miR-15b-3p, miR-342-3p, and miR-1306-5p from plasma EVs are decreased in AD
patients . miR-1306 suppresses the expression of
The blood-brain barrier comprises specialized endothelial cells that interface
with astrocytes and pericytes to keep an optimal environment for neuronal
function by supplying nutrients and other metabolic requirements while
eliminating toxic substances. The blood-brain barrier makes the delivery of
therapeutics to the CNS challenging, however. Efficient delivery of drugs to the
CNS is limited to lipophilic compounds of no more than 400 Da . Rabies virus
glycoprotein (RVG) can target the brain specifically, as demonstrated in previous
studies in which RVG was engineered to localize at the surface of EVs by fused
protein RVG-Lamp2b (lysosome-associated membrane glycoprotein 2b) .
Yang et al.  co-transfected RVG-Lamp2b and circSCMH1 overexpressing
plasmids into HEK293T cells to collect EVs containing circSCMH1. These collected
EVs were labeled with Dil and injected into mice via the tail vein. In the brain,
There has been an exponential increase in studies of the roles of EVs and extracellular vesicular ncRNAs in the pathogenesis of AD and their biomarker potential. Extracellular vesicular ncRNAs appear to be attractive novel biomarkers for diagnosing and discriminating AD, VD, and MCI. Biomarkers based on serum EV ncRNA deserve further investigation. Recent studies investigating EV ncRNAs mainly focused on miRNAs. The roles of EV related lncRNA and circRNA are as yet rarely explored.
Some challenges remain, however. Microglial EVs play a beneficial role in the early stage of AD while having a detrimental action in the later stages [65, 69]. The detailed roles of EVs from different sources and at different stages of AD are still unknown. Moreover, the different sorting mechanisms of MVB biogenesis determine the incorporation of specific cargo, but the detailed mechanisms involved in the selective sorting of ncRNAs remain unclear. Riancho et al.  compared miRNA levels in exosome-enriched CSF fractions with miRNAs in raw CSF samples, finding that miR-598 and miR-9-5p were shifted from raw CSF to exosome-enriched CSF fractions in AD, indicating that the changes of exosomal miRNAs may be caused by altered exosome trafficking.  circRNAs from EVs in AD has not yet been reported, while miRNAs have been widely studied. Exosome-mediated delivery of ncRNAs for the treatment of AD also deserves further investigation. Further studies may improve our understanding of the role of EVs and extracellular vesicular ncRNAs in both the etiology and progression of AD.
X.W. and Y.H. conceived and designed the study; Y.X. and M.C. collected data; X.W. wrote the paper.
We thank two/three anonymous reviewers for excellent criticism of the article.
This work was supported by grants from the National Natural Science Foundation of China (No. 81801069 to Yu Hu and No. 81500925 to Xiong Wang).
The authors declare no conflict of interest.