Extracellular vesicles—Mediators of opioid use disorder?

Opioid use disorder (OUD) is a growing health emergency in the United States leading to an epidemic of overdose deaths. OUD is recognized as an addictive brain disorder resulting in psychological, cognitive and behavioural dysfunction. These observed clinical dysfunctions are a result of cellular changes that occur in the brain. Derangements in inflammation, neurogenesis and synaptic plasticity are observed in the brains of OUD patients. The mechanisms of these derangements are unclear; however, extracellular vesicles (EVs), membrane bound particles containing protein, nucleotides and lipids are currently being investigated as agents that invoke these cellular changes. The primary function of EVs is to facilitate intercellular communication by transfer of cargo (protein, nucleotides and lipids) between cells; however, changes in this cargo have been observed in models of OUD suggesting that EVs may be agents promoting the observed cellular derangements. This review summarizes evidence that altered cargo of EVs, specifically protein and miRNA, in models of OUD promote impairments in neurons, astrocytes and microglial cells. These findings support the premise that opioids alter EVs to detrimentally affect neuro‐cellular function resulting in the observed addictive, psychological and neurocognitive deficits in OUD patients.


| INTRODUCTION
Opioid use disorder (OUD) is a major health problem in the United States leading to significant morbidity and mortality. 1Lethal and non-lethal overdoses have increased over the past decade, primarily due to the use of synthetic opioids, specifically fentanyl. 2,3D is a chronic brain disorder that evokes severe and painful withdrawal symptoms when attempts are made to stop using the drug.
The mechanisms of OUD affecting the brain are not well understood; however, it is well accepted that prolonged opioid use causes structural changes and neurocognitive deficits. 4,57][8][9] Dendritic connections and synaptic plasticity are impaired, and increased apoptosis is present in the nucleus accumbens, a part of the brain that is involved in mediating motivational and emotional process and also in the hippocampus, the centre of learning and memory. 10,11These cellular impairments may alter the brain in such a manner, which produces addictive behaviour and psychological, cognitive and behavioural dysfunction.The mediator of these changes are unknown, but research is beginning to focus on the role of extracellular vesicles (EVs) or more specifically, exosomes, small EVs (sEVs).
EVs are a heterogeneous group of membrane containing structures ranging in size from 30 to 1000 nm in size, which are released by a variety of cells. 12,13The mechanism of release includes outward budding of the plasma membrane or inward budding of the endosomal membrane resulting in multi-vesicular bodies. 12Several subclasses of EVs exist comprising of exosomes, micro-particles and apoptotic bodies.5][16] The primary function of exosomes is to facilitate intercellular communication by transferring their cargo, proteins, nucleotides (DNA, mRNA, microRNA [miR] and long non-coding RNA) and lipids, to other cells.The cargo most studied has been miRs, as they have the power to influence gene expression in an exponential fashion due to their numerous targets.
Exosomes have the ability to migrate across the blood brain barrier (BBB) enabling them to be biomarkers and mediating factors of neurological and other physiological events. 17,18Thus, exosomes released into the peripheral blood circulation could cross the BBB and deliver its contents to target cerebrovascular and parenchymal brain cells.
Conversely, exosomes released by neurons, astrocytes and glial cells could cross the BBB and influence distant organs.This review will focus on role that EVs and exosomes have in the pathogenesis and physiological functions of OUD.

| NEURODEGENERATION AND sEV IN OUD
Clinically, OUD is associated with impaired cognitive function, with deficits in attention, learning and memory. 19,20At the cellular level, microglial activation and BBB disruption have been observed in opioid drug abusers, suggesting an OUD mediated neurovascular inflammatory response. 9,21,22In addition, histopathology studies have shown increased deposition of hyperphosphorylated tau in multiple brain regions of opioid drug abusers with hyperphosphorylated tau accumulating at a much faster rate when compared to age-matched controls. 23This finding of hyperphosphorylated tau in opioid drug abusers is similar to that found in Alzheimer's disease.Tau is abnormally hyperphosphorylated in Alzheimer's disease, at least three-fold greater than normal tau, leading to the formation of neurofibrillary tangles, a histopathological hallmark of Alzheimer's and other neurodegenerative diseases. 24The density of neurofibrillary tangles in the neocortex correlates with dementia. 25These studies suggest that opioid drug abusers show evidence of early Alzheimer's disease pathology, suggesting that neurovascular inflammation contributes to the observed cognitive impairment in OUD.
7][28][29] The cargo of exosomes isolated from patients with OUD vary significantly when compared to non-OUD patients. 26These variations provide circumstantial evidence to the observed cellular and behavioural changes that occur in OUD patients.A longitudinal comprehensive 3-year opioid use study was performed by Kumar et al., testing the effect of oxycodone SA (short-acting) on brain cell (neuron, astrocytes and microglia) exosomes isolated from the plasma of Cynomolgus monkeys. 28The study consisted of six monkeys (10-13 years old), three of which were administered oxycodone for 3 years, and three control monkeys.At the end of the 3 year period, brain MRIs were performed on each monkey, which demonstrated a significant decrease in the volume of grey matter in the frontal, and parietal lobes of the oxycodone SA group.Exosomes were isolated from plasma of each group and further partitioned into brain cell specific groups by antibody labelling magnetic bead technique selection using the L1 cell adhesion molecule for neurons (NE), glutamate aspartate transporter for astrocytes (AE), and transmembrane protein119 for microglial cells (ME).1][32][33][34] Their results demonstrated that neuron derived exosomal cargo correlated with the observed decrease in grey matter of the oxycodone SA group.
Neurodegenerative protein biomarkers neurofilament light (NFL) chain protein, α-synuclein and Aβ1-42 were detected in exosomes from all plasma partitioned groups (NE, AE and ME) in the oxycodone SA group, which were significantly correlated with the observed volume grey matter decrease in the oxycodone SA group.To assess a pro-inflammatory stress response, cultured astrocytes were treated with neuron-derived exosomes to determine if the glucocorticoid receptor would translocate to the nucleus.Their data robustly showed that NE exosomes from the oxycodone SA group promoted glucocorticoid receptor translocation to the nucleus confirming that NE exosomes isolated from the oxycodone SA group elicited a stress/ inflammatory response in cultured astrocytes.In addition to protein cargo, miRNA cargo also demonstrated significant changes between control monkeys and monkeys treated with oxycodone SA. let 7a-5p miRNA was significantly increased in the NE and ME exosome oxycodone SA groups.Elevated expression of let 7a-5p miRNA has been observed in C. elegans models of Parkinson's disease, while reduced expression is correlated with reduced levels of the neurodegenerative marker α-synuclein. 33,35,36MiRNA-16-5p, a miRNA that may prevent amyloid beta-injury, was decreased in all NE, AE and ME exosomes in the oxycodone SA group, suggesting a correlation with results found in the serum of Alzheimer's disease patients. 37,38Collectively, results from this important non-human primate study provide evidence that exosomes isolated from opioid users contain proteins and miRNAs that may contribute to the neurodegeneration and dementia that are observed in long-term opioid use.

| MORPHINE, sEV AND THE ASTROCYTE
The astrocyte, the most abundant cell type in the brain, has recently gained attention with regard to its secretion of exosomes, or more broadly, sEVs. 39Astrocytes are supportive cells of the neuron, as the astrocytes secrete neurotrophic factors to regulate synaptogenesis, neuronal differentiation and promote neuronal survival. 40Additionally, astrocytes contribute to the formation and maintenance of the BBB. 41Similar to other CNS cells, astrocytes actively secrete exosomes and EVs, providing another area of investigation regarding the role opioids play in cognitive impairment, abuse and addiction clinically, and neuroinflammation at the cellular level. 42rphine is a commonly used opioid and its chronic usage results in cognitive decline. 43Astrocytes secrete significant amounts of EVs, so research has explored the role astrocyte EVs play in OUD.Sil et al. demonstrated significant up-regulation of amyloid isoforms, Aβ1-42 and AβmOC64 in astrocytes, identified by immunostaining with GFAP in the frontal cortex and basal ganglia of a rhesus macaque monkey model of chronic morphine usage. 29In addition, amyloid precursor protein (APP) and IL-1β protein were present in the frontal cortex and basal ganglia.Using an in vitro model of human primary astrocytes, they showed that morphine induced amyloidosis is dependent on HIF-1α, a known inducer of β-site cleaving enzyme (BACE1) enzyme, which cleaves Aβ from APP.Moreover, they found that astrocytes exposed to morphine secreted significant numbers of EVs that carried amyloid cargos, and when HiF-1α was silenced, the number of astrocyte derived EVs was reduced as well as the amount of amyloid cargo.
In vivo, when EVs were isolated from the brains of the rhesus macaque monkeys, varieties of amyloid cargoes were found, as well as IL-1β.Together, these findings suggest that morphine promotes induction of astrocyte amyloidosis via the HIF-1α/BACE1 pathway, and sEVs secreted from morphine-exposed astrocytes contain high amounts of amyloid along with proinflammatory cytokines.These released EVs could then promote widespread neuroinflammation and deposition of amyloid.
5][46] SHH signalling plays a role in cell maintenance in the adult; however, its abnormal activation results in human disease. 47ong with the increased production of SHH, astrocytes exposed to morphine have been shown to increase the length of cilia in primary astrocytes and increase the percentage of ciliated astrocytes.Cilia are important and understudied cell organelles that are implicated in human disease. 48Primary cilia are essential organelles on the cell surface that transmit external and extracellular signals to the cell.SHH signalling is transmitted to the cell by primary cilia. 49Ma et al. demonstrated that astrocytes exposed to morphine released EVs that upregulated production of SHH in bystander astrocytes, which in turn activated the SHH pathway and induced primary ciliogenesis in their mouse model of morphine tolerance. 46The resulting ciliogenesis generated more and longer cilia.They also showed that neighbouring astrocytes internalize these SHH containing EVs, initiating a chain reaction, which induces activation of the ciliary SHH pathway, resulting in morphine tolerance.These data indicate that morphine induces changes in EV cargo, cellular structure and activation of developmental pathways that are normally silent in adulthood.
Astrocyte derived EVs influence and communicate with other neighbouring cells. 29,50When astrocytes are exposed to morphine, they secrete EVs that influence the behaviour of microglial cells. 21,51[54] Hu et al., in a mechanistic study, showed that morphine induced astrocyte EVs are taken up by microglial cells, specifically, in endosomes, with subsequent activation of Toll-like receptor 7 (TLR-7). 27Moreover, astrocyte-EVs were found to increase the numbers of miRNAs, which was determined to be the mechanism by which TLR7 was activated.In recipient microglia, morphine induced astrocyte-EVs increased translocation of NF-ⱪB p65 from the cytoplasm into the nucleus, demonstrating the vital role of morphine-induced astrocyte-EVs in activating the TLR7/NF-ⱪB signalling pathway.Activation of this signalling pathway leads to upregulation of lincRNA-Cox2, a coregulator of transcription factors that control inflammatory responses in microglia.Interestingly, lincRNA-Cox2 downregulates several phagocytic genes, leading to impaired microglial phagocytosis. 27Additional studies regarding the mechanisms of neurodegeneration support the findings that impairment of microglial phagocytosis leads to accumulation of toxic metabolites, which may inhibit cellular function in the brain. 55,56Together, these studies indicate that morphine ultimately leads to impaired microglial function suggesting that the ensuing microglial phagocytic impairment provokes the decreased cognitive function observed in chronic morphine users.In addition, these data provide evidence that impaired microglial function is a shared finding among neurodegenerative diseases. 57ioid use worsens inflammatory diseases such as inflammatory bowel disease and sepsis. 58,59Opioid use alters the tight inflammatory balance that exists in the intestine.Ongoing exposure to foreign bacteria and food antigens produces a challenging inflammatory environment in the intestine, and a tightly controlled homeostasis is necessary to promote food absorption and to regulate inflammatory response.Zhang et al. demonstrated that morphine alters the intestinal EV cargo resulting in an abnormal inflammatory environment. 60ing a mouse and human intestinal organoid model, they compared EVs isolated from control and morphine treated intestinal organoids on various immune cells, mouse bone-marrow derived-macrophages, dendritic cells, microglial cells and human monocytes.These immune cells were stimulated with LPS and then were treated with control intestinal organoid EVs and morphine intestinal organoid EVs.The results showed that control EVs suppressed cytokine release in the immune cells, while morphine treated EVs were unable to suppress cytokine release.Moreover, the authors discovered that the miRNA cargo of the intestinal morphine EVs was significantly altered, specifically, let7c-5p levels were decreased while the levels of miR-186, -181a, and -302d were increased.Let 7c-5p targets IL-6, an early immune response, and TLR4, a receptor for LPS, suggesting the disturbed mechanism of the immune modulation of the intestinal EVs treated with morphine, is attributed to the reduced levels of let 7c-5p.
Therefore, the authors concluded that morphine alters the EVs secreted from intestinal cells to promote a dysfunctional immune response that is associated with inflammatory bowel diseases.Tables 1 and 2 summarize these studies into the altered EV cargo, protein and RNA, respectively.The tables list the opioid, the cell type that produces and receives the EVs, altered cargo and the main associated effects.

| OPIOIDS, EVs AND THE PRE-AND POST-NATAL PERIOD
Opioid exposure in the pre-or post-natal period is detrimental to neurological development. 61A suggested mechanism of injury is the alteration of EV cargo, which impacts synaptic function.Shahjin et al. used a rat model of development, which identified key-brain derived EV miRNA signatures for altered neurodevelopment in both in utero oxycodone exposure and post-natal oxycodone exposure. 62In utero exposure was accomplished by administration of oxycodone to the mother during mating and gestation, while post-natal administration was accomplished by breast-milk exposure.When compared to saline controls, brain derived EVs from the oxycodone group of both the in utero and post-natal group were significantly larger (>200 nm).The in utero group showed the greatest number of altered miRNAs when compared to controls, and these altered miRNAs were associated with depression at later stages of development.Specifically, miR-504 was upregulated, and it is associated with mental-retardation, bipolar disorder and depression, 63,64 and modulation of the dopamine receptor D1 (DRD1).Thus, the authors analysed the expression of this receptor in the synaptosomal fractions extracted from the cortices of the in utero group and they found that DRD1 was upregulated when compared to controls.DRD1 receptor regulates signalling mechanisms involved with dendritic spines. 65These findings all support the premise that the altered EVs influence brain development and promote subsequent neurological and psychiatric disease.
The downregulated miRs in the in utero oxycodone group when compared to controls included miR-30c-1, -384-5p and -195-5p.7][68] The authors also found changes in EV cargo in the post-natal exposure to oxycodone group, which can occur clinically due to maternal depression or treatment of post-operative pain. 62Upregulation of miRs-9277, -7977 and -451-5p was observed in the oxycodone group, while miRs-544-3p, -26a-2, let 7c-2, -190a-5p and -1306-5p were downregulated.Upregulation of miR-7977 is associated with myeloid neoplasms, while miR-451 is associated with impaired mitochondrial dynamics in relation to neurogenesis. 69,70Among the downregulated miRs, miR-26a-2 is associated with impaired long-term potentiation and paediatric brain tumours. 66,71As noted above, reduced let 7c-2 is associated with distorted modulation of inflammation.Finally, reduction of miR-190a is associated with downregulation of both excitatory and inhibitory neurotransmission pathways. 72,73These findings support the hypothesis that opioid exposure in the pre-or post-natal period alters brain-derived EV cargo, which may alter inflammatory response and impair neurodevelopment (Table 3).

CONFLICT OF INTEREST STATEMENT
None.
F I G U R E 1 Proposed mechanism of the role opioids have on astrocytes, neurons and microglia in altering the cargo of extracellular vesicles (EVs) secreted by these cells resulting in significant alteration in intercellular communication and the associated effects.Created with BioRender.com.

T A B L E 1
Protein.
Down regulation of neurotransmission pathwaysAbuse of opioids is a chronic worldwide problem, and advances in treatment may not progress until specific mechanisms of addiction and cognitive impairment are deduced.Investigating the mechanisms of addiction should be a primary focus as not all drug users become lifelong abusers.Reducing the use of opioids in medicine will encompass the use of non-opioid medications, cognitive therapy and more research into the mechanisms of pain at the cellular and molecular level.One mechanism that is under active investigation is the role that exosomes and EVs play in facilitating intercellular communication and how opioids alter the cargo of exosomes and EVs, resulting in significant alteration in intercellular communication (Figure1).This review summarizes significant findings of the changes that occur in exosomes and EVs when cells are exposed to opioids.These findings support the premise that opioids alter exosomes and EVs to detrimentally affect neurodevelopment, inflammation and neuro-cellular function, which may OUD.AUTHOR CONTRIBUTIONSStudy concept, research of articles, drafting of manuscript: Daniel C.Morris Critical revision of manuscript for important intellectual content and structure: Alex Zacharek, Zhenggang Zhang and Michael Chopp.