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
1
, Nikolay N. Ivanets
1
,
Maria A. Vinnikova
1,3
, and Alexander A. Yakovlev
4,5
1
Sechenov First Moscow State Medical University, Ministry of Health of the Russian Federation,
119048 Moscow, Russia
2
Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine,
Federal Medical-Biological Agency of the Russian Federation, 143007 Moscow, Russia
3
Moscow Scientific and Practical Center of Narcology, Moscow Healthcare Department, 109390 Moscow, Russia
4
Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 117485 Moscow, Russia
5
Research and Clinical Center for Neuropsychiatry, Moscow Healthcare Department, 115419 Moscow, Russia
a
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. Themembrane
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
inthe 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
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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
2+
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 etal. [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 phosphodiesterase4B
(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
inutero 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 12days 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]
SEVERTSEV et al.1978
BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024
Fig. 3. Hypothesis on the sEV involvement in the pathogenesis of substance use disorders proposed by the authors of this
review. The figure shows a system composed of astrocyte (on the left), microglia (at the top), and neuron (on the right). Below
the dividing line, there are fast changes: (1)synaptogenesis induced by the first effects of PAS on the neuron triggers the re-
lease of sEVs containing cargo that reduces the activity of proinflammatory factors and “requests” for synaptogenesis support
from the astrocyte; (2) in response to the “request” from the neuron, astrocyte releases sEVs containing anti-inflammatory
factors and factors controlling synaptogenesis; (3)this provides consolidation of neural connections and reduction of microg-
lial activity; (4)in parallel, PAS impairs cell metabolism, leading to the accumulation of calcium and reactive oxygen species
in the cell. Above the dividing line, there are “slow changes”: (i) dysregulation of energy metabolism and ionic imbalance
lead to impaired vesicle biogenesis; (ii)as a result, production of proinflammatory factors and pathological proteins increases;
(iii) this leads to the activation of microglia, reduced synaptogenesis, and development of neurophysiological impairments.
ΔFosB,delta-FosB protein; BDNF,brain-derived neurotrophic factor; cAMP,cyclic adenosine monophosphate; GluR,glutamate
receptor; NMDAR, N-methyl-D-aspartate receptor; mt-HK, mitochondrial hexokinase; ROS, reactive oxygen species.
with the total score on the Hamilton Depression Rat-
ing Scale in patients with methamphetamine addic-
tion but not heroin addiction. Researchers believe
that these microRNAs may have clinical application
as diagnostic and prognostic biomarkers in various
psychopathological states [74].
The study involving 37 methamphetamine-depen-
dent patients and 35 healthy volunteers of compara-
ble age and sex demonstrated a steady decrease in
the concentration of miR-137 responsible for neuro-
genesis and maturation of neurons in sEVs from the
peripheral blood of patients with methamphetamine
withdrawal syndrome [75]. Investigation of the in-
flammatory response in 20 patients with heroin de-
pendence abstaining for 7-14 days (early remission),
21 patients abstaining for approximately a year (pro-
longed remission), and 38 healthy volunteers demon-
strated that early remission was characterized by
the upregulated transcription of chemokines PF4 and
PPBP. Prolonged remission was accompanied by an in-
crease in the amount of RNAs coding for CD74, HLA-E,
SELENOH-205, RPL18-207, ABCA7, and RIG-1-like re-
ceptor [76]. Exosomes in the blood of these patients
contained long noncoding RNAs that can also be in-
volved in the activation of proinflammatory response
in patients with heroin dependence. In another study,
brain-derived sEVs from patients with the cannabinoid
dependence syndrome formed at the early age, con-
tained increased amounts of properdin and SHANK1,
proteins involved in the regulation of inflammation
and synaptogenesis [77].
Kodidela et al. [78] have studied the groups of
HIV-positive alcohol and tobacco users and found sig-
nificant differences in the properdin concentration
EXTRACELLULAR VESICLES IN ADDICTIVE DISORDERS 1979
BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024
in the sEVs from healthy volunteers and HIV-posi-
tive smokers and in the hemopexin concentration in
sEVs from healthy subjects and HIV-positive alcohol
users [78]. The same research team has shown that
concentrations of GFAP and L1CAM in sEVs circulat-
ing in the peripheral blood of HIV-positive alcohol
and tobacco users were higher compared to those in
healthy volunteers [79]. They also revealed differenc-
es between the levels of interleukins and chemokines
in the plasma and exosomes of HIV-positive smokers
and drinkers and HIV patients who abstained from
alcohol and tobacco. However, this study was carried
out in a small sample (15 subjects only), which sig-
nificantly complicates interpretation of the obtained
results. Moreover, some of the studied cytokines have
not been detected at all [80]. Table 1 summarizes all
potential biomarkers described above.
The therapeutic potential of exosomes in sub-
stance use disorders has been demonstrated in a num-
ber of studies. For example, exosomes isolated from
mesenchymal stem cells reduced alcohol intake, signs
of oxidative stress, neuroinflammation, activation of
astrocytes, and density of microglia and promoted
GLT1 expression in the nucleus accumbens in rats
[81]. Mellado et al. [82] have shown that intravenous
administration of exosomes isolated from mesenchy-
mal stem cells prevented neuroinflammation devel-
opment in young mice by decreasing the activity of
related genes, as well as reduced myelin and synapse
damage, which had a positive effect on cognitive func-
tions [82]. Intranasal administration of sEVs loaded
with lincRNA-Cox2-siRNA suppressed proliferation of
microglia induced by bacterial lipopolysaccharide[42].
Therefore, the effects of PASs on the sEV-medi-
ated signaling are extremely versatile and involve
signaling microRNAs present in sEVs and associated
with the regulation of inflammation, alterations in the
biogenesis of exosomes by microglial cells responsible
for immune response, and increase in the content of
pathological proteins. Unfortunately, most of the ob-
tained data cannot be fully extrapolated onto clinical
practice and leave a lot of room for speculation.
Based on the available data, we have formulated
a hypothesis on the existence of two stages of sEV-me-
diated cellular response to PASs (Fig. 3). The first “fast”
stage involves the binding of PAS to the cell recep-
tors in the CNS and triggering of molecular cascades,
eventually leading to changes in the biogenesis and
loading of sEVs. These changes might underlie brain
neuroplasticity, i.e., learning and memory.
The second “slow” stage might be associated with
the accumulation of inflammation-like changes and
impaired cell metabolism. At this stage, sEVs play the
role of major modulators of inflammation, immune
response activation, and, probably, sensitization of
immune cells to their own antigens. These changes
might be involved in the formation of cognitive defi-
cit and impaired impulse control associated with the
long-term use of PASs.
CONCLUSIONS
Based on the accumulated data, the studies of
exosomes and other small extracellular vesicles in
addictive disorders are a highly promising research
area. The key advantage of vesicles freely circulating
in the peripheral blood over other biological markers
is their ability to penetrate through the blood–brain
barrier. It will be reasonable to assume that detailed
investigation of the role of sEVs in pathophysiological
processes underlying addictive disorders will result
in the discovery of multifunctional biomarkers. First,
identification of specific differences between sEVs in
healthy people and individuals with a history of ad-
diction can ensure early diagnostics and detection of
risk groups. Second, elucidation of changes in the pro-
cesses involving sEVs against the background of estab-
lished addiction can lead to the detection of targets for
developing new therapeutic techniques. Third, moni-
toring sEVs might make allow to study the response
to therapy not only based on clinical signs but also
at the molecular level.
Contributions. V.V.S., A.A.Y., and M.A.V. developed
concept and analyzed the available literature; V.V.S.
and A.A.Y. wrote the manuscript; A.A.Y., V.V.S., and
M.A.P. prepared figures; N.N.I. and M.A.V. edited the
manuscript.
Funding. This work was supported by ongoing in-
stitutional funding. No additional grants to carry out
or direct this particular research were obtained.
Ethics declarations. This work does not contain
any studies involving human and animal subjects.
The authors of this work declare that they have no
conflicts of interest.
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