Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 11, pp. 1970-1984 © Pleiades Publishing, Ltd., 2024.

1970

REVIEW

Extracellular Vesicles as Potential Biomarkers

in Addictive Disorders

Vsevolod V. Severtsev

1,2,a

*, Margarita A. Pavkina

, Nikolay N. Ivanets

Maria A. Vinnikova

1,3

, and Alexander A. Yakovlev

4,5

Sechenov First Moscow State Medical University, Ministry of Health of the Russian Federation,

119048 Moscow, Russia

Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine,

Federal Medical-Biological Agency of the Russian Federation, 143007 Moscow, Russia

Moscow Scientific and Practical Center of Narcology, Moscow Healthcare Department, 109390 Moscow, Russia

Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 117485 Moscow, Russia

Research and Clinical Center for Neuropsychiatry, Moscow Healthcare Department, 115419 Moscow, Russia

e-mail: severtsevmed@gmail.com

Received April 22, 2024

Revised August 3, 2024

Accepted August 9, 2024

Abstract— Small extracellular vesicles(sEVs) and their role in mental and addictive disorders are extremely prom-

ising research areas. Because of their small size, sEVs can pass through the blood–brain barrier. Themembrane

of sEVs contain proteins that protect them against destruction by the organism’s immune system. Due to these

properties, sEVs circulating in the blood can be used as potential biomarkers of processes occurring in the brain.

Exposure to psychoactive substances in  vitro and in  vivo affects sEV biogenesis and significantly alters the

amount of sEVs and chemical composition of their cargo. Based on the published reports, sEVs carry numerous

potential biomarkers of addictive pathologies, although the diagnostic significance of these markers still has to

be evaluated. A large body of evidence indicates that psychoactive substances influence Rab family GTPases,

Toll-like receptors, complement system components, and cytokines. In some studies, the effect of psychoactive

substances on sEVs was found to be sex-dependent. It has become commonly accepted that sEVs are involved

in the regulation of neuroinflammation and interaction between glial cells and neurons, as well as between

peripheral cells and cells of the central nervous system. Here, we formulated a hypothesis on the existence of

two mechanisms/stages involved in the effect of psychoactive substances on sEVs: the “fast” mechanism that

provides neuroplasticity, and the “slow” one, resulting from the impaired biogenesis of sEVs and formation

of aberrant vesicles.

DOI: 10.1134/S0006297924110117

Keywords: small extracellular vesicles, exosomes, biomarkers in psychiatry, addictology, fundamental mecha-

nisms of addictive disorders, dependence syndrome

Abbreviations: DA, dopamine; DR, dopamine receptor; EV, extracellular vesicle; MVB, multivesicular body; ESCRT, endo-

somal sorting complex required for transport; PAS,psychoactive substance; sEV, small extracellular vesicle; TLR,toll-like

receptors.

* To whom correspondence should be addressed.

INTRODUCTION

Addictive disorders are among the most pressing

problem of modern society. According to the World

Health Organization, the prevalence of alcohol use dis-

orders is 14.8% among men and 3.5% among women

in Europe and 11.5% among men and 5.1% among

women in the United States. In Russia, 1.2 million

people were under medical supervision for the alco-

hol dependence syndrome and alcohol abuse in 2020.

EXTRACELLULAR VESICLES IN ADDICTIVE DISORDERS 1971

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Based on the information provided by the United Na-

tions Organization, in 2021, there were ~296 million

drug users and ~40 million people with substance use

disorders worldwide  [1]. In Russia, the number of

patients under medical supervision for the addiction

syndrome and drug abuse reached 341,000 in 2020 [2].

Although the terms “addiction” and “dependence”

are not fully equivalent, the term “addictive disor-

ders” has become more and more accepted in mod-

ern lexicon, substituting the more traditional term

“dependence diseases”. Addiction, or dependence, is

a chronic progredient disease that manifests itself as a

pleasure-seeking behavior or cravings for a psychoac-

tive substance (PAS), despite obvious negative conse-

quences for an individual[3]. Addictive disorders are

recognized as such based on the presence of common

clinical symptoms manifested as specific features in

the interaction between an individual and an object

of addictive behavior  [3,  4]. It is currently believed

that a significant role in the pathophysiology of ad-

dictive disorders belong to alterations in signaling in

the reward and stress response brain circuitries, in

particular, in their architectonics, caused by morpho-

logical changes associated with the PAS-induced neu-

roplasticity [3].

Secretion of small extracellular vesicles (sEVs) is

an important element of intercellular communication,

beside the well-studied neurotransmitter systems  [5].

sEVs are cell-derived vesicles of approximately hun-

dreds of nanometers in diameter that are limited by

the lipid bilayer and carry molecules of biological or-

igin. Previously, it had been generally believed that

sEVs are used by cells to get rid of unnecessary sub-

stances, but this point of view has now lost its popu-

larity. The two well-studied types of sEVs – exosomes

and ectosomes – are secreted by almost all body cells

and can be detected in almost any biological fluid.

Ectosomes and exosomes differ in biogenesis but have

highly similar functional and structural properties.

The classification of these vesicles is poorly devel-

oped  [6]; for the sake of convenience, vesicles small-

er than 200  nm are considered sEVs and those larger

than 200  nm are considered large EVs [6].

An interest in sEVs is due to the following proper-

ties of these particles. The markers on the sEV mem-

brane are recognized by macrophages, which allows

sEVs to enter tissues and persist for a relatively long

period of time without being destroyed by the im-

mune system  [7]. sEVs are able to penetrate through

the histohematic barriers, including the blood–brain

barrier  [8]. Due to these two properties, sEVs are pres-

ent in all biological fluids, from which they can be

isolated for analysis. These features also determine an

increasing interest in sEVs in the context of addictive

and psychiatric disorders. In this review, we focused

our attention on the studies of sEVs that might shed

light on specific processes in the pathogenesis of ad-

dictive disorders and can potentially be used in solv-

ing various clinical challenges.

EXTRACELLULAR VESICLES

Biogenesis of extracellular vesicles (EVs). Bio-

genesis of sEVs has been described in detail in  [9].

Exosomes are products of cell endolysosomal system.

Primary endosomes mature to become, first, second-

ary endosomes and then multivesicular bodies(MVBs).

After MVB fusion with the plasma membrane, intra-

luminal vesicles become exosomes  [9]. In another

pathway, MVBs fuse with lysosomes followed by the

degradation of their content. The fate of MVBs largely

depends on small GTPases of the Rab family located

on the MVB membrane. The presence of Rab27 on the

membrane results in the MVB fusion with the plasma

membrane and secretion of exosomes  [10], while the

presence of Rab7 targets MVBs to the fusion with ly-

sosomes and degradation of their cargo  [11] (Fig.  1).

However, depending on the conditions and type of

cells, regulation of intracellular transport of MVBs

may involve other members of the Rab family [12].

Ectosomes are formed when a section of the

plasma membrane and some part of the cytoplasm

directly bud off the cell [9]. Previously, ectosomes had

been called microvesicles, or oncosomes. The process

of ectosome secretion includes, first, formation of a

microdomain containing lipids and proteins in the cell

plasma membrane, and then budding of this section

into the extracellular space [9]. There are several spe-

cific pathways of ectosome secretion, but to a large

extent, the mechanisms of biogenesis and secretion

of ectosomes and exosomes overlap [13].

Formation of sEVs involves diverse cellular ma-

chinery. The main specialized structure participating

inthe formation and transport of intracellular vesicles

is ESCRT (endosomal sorting complex required for trans-

port)  [14] consisting of four components (ESCRT0-3),

which sequentially bind to the sites on the plasma or

endosomal membranes, recruit some specialized pro-

teins, and finally, cause the newly formed vesicle to

bud off  [15]. In addition to this mechanism, vesicle

formation involves the syndecan–syntenin–ALIX mo-

lecular cascade  [16], tetraspanins  [17], changes in the

actin cytoskeleton  [18], neutral sphingomyelinase  [19],

ceramide  [20], and arrestin domain-containing pro-

tein (ARRDC1)  [21]. Vesicle packaging involves sever-

al post-translational protein modifications, which are

well-known due to their role in other intracellular pro-

cesses. The best-studied of them is ubiquitination  [22].

In other words, cells utilize many intracellular mech-

anisms for sEV biogenesis and secretion, instead

of a particular specialized pathway. The mechanisms

SEVERTSEV et al.1972

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Fig.  1. Primary endosome matures and becomes secondary endosome and then an MVB. After the fusion of the Rab27-

containing MVB with the plasma membrane, intralumenal vesicles become exosomes. The alternative pathway involves the

fusion of the Rab7-containing MVBs with lysosomes and degradation of their cargo.

of formation and secretion of ectosomes and exosomes

are largely the same. However, the heterogeneity of

sEV population should be taken into consideration,

since even a single cell can secrete sEVs by different

mechanisms depending on its functional state.

The role of vesicles in cell-cell signaling. Re-

cently, there has been a growing body of evidence

that sEVs perform various important functions in a

body. In particular, it was suggested that cells use sEVs

to exchange information. This hypothesis has been

confirmed in may studies, so that some researchers

even speak about a special “vesicular signaling sys-

tem”  [23]. The idea that sEVs can act as mediators of

cell–cell communication was formulated for the first

time in the 1990s  [24] based on the studies of infor-

mation exchange between immune cells. It was found

that antigen-presenting cells secrete major histocom-

patibility complex  II proteins as component of exo-

somes. Although this process is rather slow, it allows

antigen-presenting cells to present antigens to T  cells.

Most examples of sEV-mediated communication

have been obtained in brain cells. Moreover, from the

structural point of view, it is easy to find evidence that

brain sEVs form a neurotransmitter system, which, on

one hand, is indisputably different from the classical

neurotransmitter systems, but on the other hand,

shares undeniable similarity with them  [25]. sEVs se-

creted by oligodendrocytes stimulated axonal trans-

port in the adjacent neurons  [26]. Astrocyte-derived

sEVs affected the spontaneous activity of neurons  [27],

significantly modified their induced activity  [28], and

altered the synaptic machinery morphology in these

cells  [29]. Neurons secrete sEVs that affect the func-

tions of other neurons  [30] and surrounding endothe-

lial cells  [31]. The studies of vesicle-mediated commu-

nication in the brain draw a similar picture: brain

cells use sEVs to help neurons perform their functions;

non-neuronal cells use sEVs to provide trophic support

to neurons[32,  33]. However, in the case of long-term

brain pathologies, sEVs can exert a deleterious effect

on neurons [34].

The existence of sEV-mediated information ex-

change between body cells is currently beyond any

doubt. However, the mechanisms of targeted delivery

of sEVs, their internalization, and intracellular sort-

ing have been studied rather poorly. So far, there are

single works on the role of specific molecules in the

recognition of sEVs by cells and on the role of particu-

lar component of sEVs in realization of their functions,

which might be due to the fact that such experiments

are technically difficult, and the capabilities of tech-

nologies used to study vesicles are often insufficient.

However, these obstacles do not prevent the develop-

ment of sEV-based diagnostic tests and even thera-

pies [35].

Exosome- and ectosome-based diagnostics.

Recent studies have opened new prospects for the

practical use of exosomes and ectosomes. Diagnos-

tics remains the main area requiring sEV application,

although multiple attempts have been made to use

these vesicles in therapy. Circulating sEVs can pene-

trate through the blood–brain barrier, while sEVs of

the neuronal and glial origin are found in the pe-

ripheral blood. The latter allows to monitor changes

EXTRACELLULAR VESICLES IN ADDICTIVE DISORDERS 1973

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Fig.  2. The role of sEV signaling in the pathogenesis of PAS-induced disorders. BDNF, brain-derived neurotrophic factor;

LTD, long-term depression; LTP, long-term potentiation; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxalopropionic acid; NMDA,

N-methyl-D-aspartate; Dyn, dynorphin; CRH, corticotropin-releasing hormone; DA, dopamine.

occurring in the brain based on the composition of

brain-derived EVs isolated from the peripheral blood.

The most impressive results for the clinical appli-

cation of sEVs have been obtained in the diagnostics

of cancer. This area has been studied quite extensively,

and some of the findings are currently tested in clini-

cal trials. The very first studies of sEVs in oncology re-

sulted in the identification of surface proteins in sEVs

derived from cancer cells [36], so these proteins have

been investigated as potential biomarkers for cancer

diagnostics. The functions of sEVs produced by cancer

cell have also been studied. Thus, it was shown that

sEVs derived from cancer cells suppressed immunity in

premetastatic niches promoting further metastasis[37].

The therapeutic applications of sEVs are large-

ly restricted to using stem cell-derived sEVs for the

treatment of diseases  [38]. Currently developed ap-

proaches are aimed at the isolation and culturing of

stem cells with the purpose of collecting the maxi-

mum amount of sEVs  [39]. The obtained vesicles can

be preserved and used when necessary for therapy or

pathology prevention.

sEVs IN ADDICTIVE DISORDERS

In psychiatry in general and in addiction stud-

ies in particular, the leading role in the pathogenesis

of addition is attributed to the functional changes in

the metabolism of neurotransmitters. It is believed

that a generalized mechanism of neural response to

PASs proceeds as follows  [3]. PAS affects a cell and

alters the functioning of one of the neurotransmitter

systems. The activation of the cognate receptors in-

duces a signaling cascade that triggers the synthesis

of appropriate molecules. These molecules cause long-

term changes in neurons, thus ensuring learning and

memory processes, including those occurring in re-

sponse to substance use (intoxication and tolerance)

and anticipation of substance use (cravings). These

changes are preserved and maintained at the molec-

ular level due to the neuroplasticity associated with

the substance use  [40,  41]. Current studies confirm the

fundamental role of glial cells in neuroplasticity.

It is possible that sEVs serve as a link between the

well-studied processes involved in the pathogenesis

of addictive disorders (Fig.  2). sEVs act as transport

vehicles for the efficient targeted delivery of required

molecules because, due to the presence of specific sur-

face proteins, they can reach the “addressees” directly,

without affecting nontarget cells.

One of the functions of sEVs is involvement in the

release of signaling molecules by astrocytes, resulting

in long-term changes in neurons[28]. The second func-

tion is activation of microglial cells to provide pruning

or other reactive changes  [42]. The third function is

sending messages to peripheral tissues  [43].

At the same time, interactions between the neu-

rons and glia are largely provided by ATP-dependent

processes and depend on the ionic balance in the ex-

tracellular matrix  [44]. It was found that astrocytes are

involved in the formation of long-term memory in rats,

as activation of gamma-aminobutyric acid(GABA) and

acetylcholine receptors (and associated G  proteins) in

astrocytes affects the long-term memory consolidation

through the long-term changes in the Ca

levels and

modulation of the cFos expression  [45]. It is known

that activation of dopamine receptors (DRs) leads to

the long-term changes in neural stem cells  [46]. More-

over, the sensitivity to dopamine(DA) was found to be

altered in neural stem cells of patients with psychiat-

ric disorders compared to cells from mentally healthy

individuals. Assis-de-Lemos etal.  [46] have shown that

the DA-induced changes involve mitochondrial hex-

okinase (mt-HK), an enzyme essential for cell energy

metabolism. Therefore, DA may act as a regulator of

energy metabolism in neurons, the sensitivity of ener-

gy metabolism to DA being dependent on genetic pre-

disposition. Hence, impaired energy metabolism might

be an important component in the pathophysiological

mechanism of addictive disorders. Energy metabolism

is also directly related to the sEV biogenesis, suggesting

its association with changes in the sEV signaling in the

pathology of addictive disorders.

SEVERTSEV et al.1974

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

In recent decades, the genetic basis of many psy-

chiatric disorders has become the research focus of

the scientific community. The most significant asso-

ciations with addictive disorders have been demon-

strated for the polymorphisms of genes coding for

FTO (fat mass and obesity-associated protein), type  2

dopamine receptor (DRD2), and phosphodiesterase4B

(PDE4B) [47]. Gene expression is regulated by epigen-

etic mechanisms, such as post-translational modifica-

tion of histones, DNA methylation, and expression of

microRNAs (noncoding RNAs). Regulation of gene ex-

pression by noncoding RNA is closely related to sEVs,

since these RNAs are among the most common cargo

molecules carried by sEVs  [48]. The role of sEVs in the

dendritic spine density and number of DRs was con-

firmed in the studies of mice exposed to oxycodone

inutero and during the postnatal period. It was found

that an increase in the miR-504 content in brain sEVs

led to a decrease in the dendritic spine density and

upregulation of the DRD1 and DRD2 expression  [49].

Predisposition to addiction to a great extent depends

on the genetically determined features of CNS func-

tioning. Thus, the genetic profile of 9 to 10-year-old

substance-naive children with hereditary predisposi-

tion for addiction and mental disorders corresponded

to a profile with a high polygenic risk of addiction  [47].

It was found in animal models that epigenetic changes

can persist in germ cells [50].

sEVs might be also important for the reproduc-

tive functions and preservation of epigenetic chang-

es in the progeny. In particular, Lyu et  al.  [51] have

shown that addition of exosomes from the semen of

cocaine users and HIV-positive patients to cultured

monocytes led to the cell adhesion, cytoskeleton reor-

ganization, and chemotactic migration  [51]. The role

of sEVs in the semen has been poorly studied; they

have been investigated mostly as putative markers of

prostate cancer.

PASs affect many aspects of sEV biogenesis, as

well as their size, number, and internalization. Thus,

the number of fluorescently labeled neuronal exo-

somes internalized by microglial cells and astrocytes

in the nucleus accumbens of mice self-administering

cocaine was much smaller than in the controls [52].

The same team of authors had demonstrated that

exosomes regulate the activity of the GLT1 glutamate

transporter by transporting miR-124-3p from neurons

to astrocytes[53]. The study conducted in mouse liver

tissue, cultured cell, and human tissue samples has

shown that exposure of hepatocytes to the increased

levels of alcohol upregulated expression of miR-155,

resulting in the suppression of autophagy and signifi-

cant increase in the exosome production[54]. Incuba-

tion of microglial BV-2 cells with ethanol for 24 and

72 h decreased the number of exosomes depending

on the duration of exposure, as well as increased the

content of Rab7 and reduced the content of CD63[55].

Self-administration of nicotine for 22 days by female

rats increased the average size of sEVs (a similar ef-

fect was observed in male rats but did not reach the

level of significance). Female rats also demonstrat-

ed an increase in the expression of genes involved

in the ESCRT-dependent and independent pathways.

In addition, there were multiple changes in the com-

position of protein cargo transported by sEVs  [56].

Astrocyte-derived sEVs carrying miR-106b-5p[57] and

sonic hedgehog(SHH) protein[58] affected ciliogenesis

in astrocytes with a developed tolerance to morphine,

indicating involvement of sEVs in the morphological

changes of CNS cells.

The above data suggest that by impairing sEV bio-

genesis, PASs cause the appearance of aberrant sEVs

that act on nontarget cells and organs or affect the

target cells in an unwanted manner or with an un-

wanted strength. Immune system is one of the body

systems negatively affected by PAS. In particular, the

pathogenesis of PAS-induced changes includes, inter

alia, direct effect of dopamine on the expression of

inflammatory factors in immune cells, activity of im-

mune cells, and even modulation of T  cell differenti-

ation  [59]. Many studies have demonstrated that exo-

somes participate in cell–cell interactions as carriers

of inflammatory mediators. Ibáñez et  al.  [60] found that

changes in the content of exosomes isolated from cul-

tured astrocytes exposed to ethanol for 24  h included

an increase in the amounts of microRNAs (miR-146a)

and inflammation-associated proteins (COX-2), as

well as upregulated expression of inflammation-relat-

ed genes (IL-1B, Traf6, Mapk14, Foxo3). However, the

most important finding was that these changes were

absent in the exosomes of astrocytes deficient by

the Toll-like receptor  4 (TLR4)  [60]. On the other hand,

there are data that cocaine increases the content of

miR-124, resulting in the reduced expression of TLR4

and other proinflammatory proteins [61].

In a large-scale study by Chand et al.  [62], ma-

caques and rats self-administering methamphetamine

exhibited numerous changes in exosomes, including

increase in their size from 100-200  nm to 50-500  nm,

as well as upregulation of genes involved in the func-

tioning of the ESCRT Alix complex. The authors also

observed an increase in the content of exosomal sur-

face proteins (Alix, TSG101, HSP70, and CD63), which

they interpreted as changes in the ratio between par-

ticular subtypes of exosomes. The level of miR-29a also

increased but only after a long-term exposure of cul-

tured cells to methamphetamine and only in sEVs (but

not in large EVs). The authors suggested that miR-29a

acts through the activation of the TLR7 signaling and

increase in the production of proinflammatory cyto-

kines (IL-1β and TNFα) and CCR5 and CXCR3 recep-

tors in the microglia [62]. Importantly, TLR4 and TLR7

EXTRACELLULAR VESICLES IN ADDICTIVE DISORDERS 1975

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

not only participate in the inflammatory response, but

are also involved in the activity of endosomal system,

which relates their functions to the sEV biogenesis.

It was shown that in cultured cells, alpha-synu-

clein can be transported from neurons to astrocytes

with the involvement of exosomes [63]. In monkeys

self-administering oxycodone for three years, the con-

tent of neurofilament light chains in the exosomes de-

rived from neurons and astroglia and isolated from

the blood and the content of alpha-synulcein in the

exosomes derived from neurons and isolated from

blood were significantly increased [64]. This increase

correlated with the reduced volume of frontal and

parietal lobes. Rather surprisingly, the size of exo-

somes isolated from the blood of animals chronically

exposed to oxycodone was 133 nm, i.e., significantly

exceeded the size of exosomes in the blood of con-

trol animals (102  nm). The concentrations of several

microRNAs in the exosomes derived from neurons,

astroglia, and microglia differed in the control and

chronically narcotized animals  [64]. Sil et  al.  [65] ob-

served an increase in the beta-amyloid level in the

astrocytes of the frontal cortex and basal ganglia of

morphine-dependent macaques. They reproduced this

effect in cultured human astrocytes, confirming an in-

crease in the number of beta-amyloid-carrying sEVs,

as well as upregulation of expression of proinflamma-

tory mediators [65]. Moreover, addition of exosomes

derived from neurons, astroglia, and microglia to cul-

tured monocytes induced a proinflammatory response.

Because exosomes of chronically narcotized animals

differed from the control vesicles in many parameters,

the authors suggested that circulating brain-derived

vesicles can be indicators of the severity of neurode-

generation in the brain, as well as some other pro-

cesses. These parameters included microRNA levels,

proinflammatory potential, and even vesicle size, i.e.,

characteristics seemingly unrelated to the biological

effect of exosomes. In general, this study suggested

that exosomes circulating in the blood can be sourc-

es of information about a wide variety of processes

occurring in the brain during chronic narcotization.

The relationship between sEVs detected in the pe-

ripheral blood and neuroinflammation has been con-

firmed experimentally. When exosomes derived from

the plasma of mice injected with Escherichia coli li-

popolysaccharide were intravenously administered

into intact mice, the latter demonstrated upregulated

expression of many inflammatory factors, as well as

microgliosis and astrogliosis [66].

Caobi et  al.  [67] discovered dose-dependent changes

in the content of inflammation-regulating microRNAs

(miR-627-5p, miR-378e, miR-150-5p, miR-1290) in the

exosomes produced by peripheral blood mononucle-

ar cells isolated from healthy doors and then infect-

ed with HIV and exposed to morphine. For example,

the level of miR-1290 was 12 times higher compared

to the control [67]. Microglial exosomes isolated post

mortem from the hypothalamus of rats exposed dai-

ly to alcohol were characterized by the increased

levels of apoptotic factors, such as the complement

protein C1q, membrane attack complex, and reac-

tive oxygen species  [68]. Cocaine impaired biogene-

sis and altered composition of exosomes, as well as

affected expression of exosomal proteins by cultured

microglial cell  [69]. This psychostimulant also altered

intracellular expression of small GTPases of the Rab

family, presumably affecting intracellular vesicle

transport. These alterations in the intracellular traffic,

e.g., changes in the proportion of MVBs fusing with

lysosomes for subsequent degradation, might have

caused an observed decrease in the exosome secretion.

The authors also revealed that cocaine decreased the

viability of microglial cells. In another study, the con-

tent of ganglioside GD1a in brain-derived sEVs isolated

from mice exposed to cocaine for 12days increased in

sEVs obtained from male (but not female) mice, sug-

gesting the existence of sex differences in the effect

of cocaine on the lipid composition of sEV [70].

Evaluation of microRNAs in human extracellular

vesicles and experiments in mice have demonstrated

that alcohol intoxication decreased the levels of anti-

inflammatory microRNAs (miR-146a-5p, miR-21-5p,

miR-182-5p) in plasma EVs from women and female

mice, but increased their content in EVs isolated

from males. In the cerebral cortex of female mice,

ethanol downregulated the levels of miR-146a-5p and

miR-21-5p, while simultaneously promoting expres-

sion of inflammatory target genes (Traf6, Stat3, and

Camk2a)  [71]. In a small study (6 patients with alcohol

dependence and 6 healthy volunteers), 254 differen-

tially expressed (149 upregulated and 105 downreg-

ulated) circular RNAs were identified in the plasma

exosomes; one of them, hsa-circ-0004771, was suggest-

ed as a biomarker of alcohol dependence severity [72].

Chen et al. [73] examined 140 male patients that

had been treated at the Kunming Medical University

from January 2018 to October 2019: 60 patients with

heroin addiction, 60  patients with methamphet-

amine addiction, and 20  healthy controls. The authors

showed that the levels of exosomal hsa-miR-451a and

hsa-miR-21a have a prognostic capacity (AUC, 0.966

and 0.861) for the heroin and methamphetamine use,

respectively. An increased content of hsa-miR-744-5p

was associated with the acute phase of withdrawal

syndrome and positively correlated with the serotonin

levels in patients with heroin and methamphetamine

addiction [73]. The authors also found a relationship

between miR-92a-3p, miR-363-3p, miR-16-5p, and miR-

129-5p and the total score on the Hamilton Anxiety

Rating Scale. The levels of miR-92a-3p, miR-363-3p,

miR-16-5p, and miR-129-5p significantly correlated

SEVERTSEV et al.1976

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Table  1. Potential biomarkers in sEVs

Source Potential marker Associated process References

Rats administered with

oxycodone for 14 days

miR-504

changes in the expression

of dopamine receptors DRD1

and DRD2

[49]

Semen of HIV-positive

drug users

reverse transcriptase,

HIV p24 antigen, benzoylecgonine

HIV progression

and cocaine use

[51]

Mice self-administering

cocaine

impaired internalization

of exosomes

impairment

of neuronal–glial signaling

[52]

Mouse brain preparation miR-124-3p

increased activity

of astrocytic glutamate

transporter

[53]

Cultured mouse and human

liver cells exposed

to ethanol

miR-155

reduced autophagy

in hepatocytes and increased

number of exosomes

[54]

Cultured microglial cells

exposed to ethanol

increased concentration of Rab7;

decreased concentration of CD63

impaired biogenesis

of exosomes

[55]

Rat brain

males: cyclin Y, GABA-A γ2-long

isoform, ATP-citrate synthase,

dolichol-phosphate mannosyltrans-

ferase subunit 1, mesoderm-specific

transcript homologue protein;

females: aquaporin-1, ezrin,

phosphatidylinositol 5-phosphate

4-kinase type 2α

various regulatory functions [56]

Cultured astrocytes from

mice with the developed

tolerance to morphine

miR-106b-5p

activation of ciliogenesis

through the expression

of CEP97

[57]

Cultured astrocytes from

mice with the developed

tolerance to morphine

Sonic hedgehog (SHH) protein

increased number

of astrocytes with a cilium;

increase in the cilium length

[58]

Cultured astrocytes

exposed to ethanol

COX-2; IL-1B, Traf6, Mapk14,

and Foxo3 genes;

miR-146a

TLR4-mediated

proinflammatory changes

[60]

Macaques and rats

self-administering

methamphetamine

Alix, TSG101, HSP70, and CD63;

miR-29a;

change in the size of exosomes

alteration in EV biogenesis,

proinflammatory changes

in microglia

[62]

SH-SY5Y cells exposed

to methamphetamine

α-synuclein cognitive deficit [63]

Neuron- and astroglia-

derived exosomes isolated

from the blood of monkeys

self-administering

oxycodone for three years

α-synuclein,

neurofilament light chains

cognitive deficit [64]

Astrocytes obtained from

the brain preparation

of macaques administered

with morphine

toxic forms of amyloid cognitive deficit [65]

EXTRACELLULAR VESICLES IN ADDICTIVE DISORDERS 1977

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Table 1 (cont.)

Source Potential marker Associated process References

HIV-infected peripheral

blood monocytes exposed

to morphine

miR-627-5p, miR-378e, miR-150-5p,

miR-1290

numerous putative

signaling pathways associated

with the modulation

of inflammation

[67]

Cultured microglial cells

exposed to ethanol

complement C1q protein

activation

of proinflammatory pathways

[68]

Cultured microglial cells

expression of small GTPases

of the Rab family

sEV biogenesis [69]

Brain of mice administered

with cocaine

increase in the GD1a ganglioside

content in males

BDNF regulation [70]

EVs from human

and mouse females

and males after

exposure to ethanol

in female mice and women:

downregulated expression

of miR-146a-5p, miR-21-5p,

miR-182-5p, miR-146a-5p, miR-21-5p;

upregulated expression of Traf6,

Stat3, and Camk2a genes

proinflammatory response [71]

Patients with alcohol

dependence syndrome

hsa-circ-0004771

modulation of various

cellular processes

[72]

Patients with heroin

and methamphetamine

dependence syndrome;

acute phase of heroin

and methamphetamine

withdrawal syndrome

differences between the

addicts and control subjects

in the contents of hsa-miR-451a

and hsa-miR-21a and in the content

of hsa-miR-744-5p during acute

phase of withdrawal syndrome

modulation of various

cellular processes

[73]

Plasma of patients

with heroin

and methamphetamine

addiction

miR-16-5p, miR-129-5p, miR-363-3p,

miR-92a-3p

synaptogenesis, cell adhesion,

focal contacts, and major

histocompatibility complex

class II

[74]

Patients with

methamphetamine

withdrawal syndrome

miR-137 regulation of cell functions [75]

Patients with heroin

dependence during early

(7-14 days) and prolonged

(about a year) remission

during early remission:

chemokines PF4 and PPBP;

during prolonged remission:

increased levels of mRNAs

for CD74, HLA-E, SELENOH-205,

RPL18-207, ABCA7, RIG-1-like

receptor

immunity [76]

Plasma of patients

with cannabinoid

dependence

properdin, SHANK1

regulation of inflammation

and synaptogenesis

[77]

Plasma of HIV-positive

alcohol and tobacco users

hemopexin, properdin regulation of inflammation [78]

sEVs isolated from the blood

of HIV-positive alcohol

and tobacco users

GFAP and L1CAM

proteins identifying

the origin of sEVs

[79]