Extracellular vesicle-mediated delivery of anti-miR-106b inhibits morphine-induced primary ciliogenesis in the brain

Rong Ma; Naseer A Kutchy; Zhongbin Wang; Guoku Hu

doi:10.1016/j.ymthe.2023.03.030

. 2023 Apr 3;31(5):1332–1345. doi: 10.1016/j.ymthe.2023.03.030

Extracellular vesicle-mediated delivery of anti-miR-106b inhibits morphine-induced primary ciliogenesis in the brain

Rong Ma ^1,^2,^∗, Naseer A Kutchy ^2,³, Zhongbin Wang ², Guoku Hu ^2,^∗∗

PMCID: PMC10188913 PMID: 37012704

Abstract

Repeated use of opioids such as morphine causes changes in the shape and signal transduction pathways of various brain cells, including astrocytes and neurons, resulting in alterations in brain functioning and ultimately leading to opioid use disorder. We previously demonstrated that extracellular vesicle (EV)-induced primary ciliogenesis contributes to the development of morphine tolerance. Herein, we aimed to investigate the underlying mechanisms and potential EV-mediated therapeutic approach to inhibit morphine-mediated primary ciliogenesis. We demonstrated that miRNA cargo in morphine-stimulated-astrocyte-derived EVs (morphine-ADEVs) mediated morphine-induced primary ciliogenesis in astrocytes. CEP97 is a target of miR-106b and is a negative regulator of primary ciliogenesis. Intranasal delivery of ADEVs loaded with anti-miR-106b decreased the expression of miR-106b in astrocytes, inhibited primary ciliogenesis, and prevented the development of tolerance in morphine-administered mice. Furthermore, we confirmed primary ciliogenesis in the astrocytes of opioid abusers. miR-106b-5p in morphine-ADEVs induces primary ciliogenesis via targeting CEP97. Intranasal delivery of ADEVs loaded with anti-miR-106b ameliorates morphine-mediated primary ciliogenesis and prevents morphine tolerance. Our findings bring new insights into the mechanisms underlying primary cilium-mediated morphine tolerance and pave the way for developing ADEV-mediated small RNA delivery strategies for preventing substance use disorders.

Keywords: morphine, extracellular vesicle, microRNA, primary cilium, intranasal, exosome, astrocyte, ciliogenesis, miR-106b, CEP97

Graphical abstract

Hu and colleagues investigated how morphine-induced ciliogenesis contributes to morphine tolerance. They found that miR-106b in morphine-stimulated extracellular vesicles mediates ciliogenesis by targeting CEP97. Moreover, intranasal delivery of ADEV-anti-miR-106b inhibits ciliogenesis and prevents morphine tolerance. This study highlights a potential therapeutic approach for preventing substance use disorders by targeting cilia.

Introduction

Abuse of opioids, such as morphine, has been reported to correlate with increased severity of central nervous system (CNS) disease via a number of potential mechanisms.¹^,²^,³^,⁴^,⁵ Both clinical and animal studies suggest that opioid-induced glial dysregulation is involved in the progressive degeneration of brain tissue in opioid abusers.⁶^,⁷^,⁸^,⁹^,¹⁰^,¹¹^,¹²^,¹³^,¹⁴^,¹⁵ Glial cells, such as astrocytes, play essential roles in brain homeostasis and functionality and are shown to be markedly activated by exposure to substances of abuse.¹⁴^,¹⁶

Extracellular vesicles (EVs), including microvesicles,¹⁷^,¹⁸ exosomes,¹⁹^,²⁰ and mitovesicles,²¹ are lipid-bound vesicles secreted by almost all types of cells into the extracellular space.²⁰^,²² EVs contain bioactive proteins, lipids, and DNA and RNA, involved in mediating communication among diverse cell types and tissues, including the CNS.²³^,²⁴^,²⁵ EVs deliver their cargo, such as microRNAs (miRNAs), to recipient cells and play crucial roles in various pathologies, including neuroinflammation in the context of substance abuse.²⁶^,²⁷^,²⁸^,²⁹^,³⁰^,³¹ Furthermore, due to their ability to efficiently shuttle small molecules between cells, EVs are emerging as promising therapeutic tools for treating many diseases.³²^,³³^,³⁴^,³⁵ Indeed, EVs have been used successfully to deliver small interfering RNAs (siRNAs) to astrocytes, neurons, and oligodendrocytes in mice.³⁶ Intranasal administration of EVs carrying the therapeutics is considered a preferred non-invasive method for rapidly delivering EV-encapsulated drug(s) into the brain.³⁷^,³⁸^,³⁹^,⁴⁰ Manipulating EV in vivo could thus be an effective strategy for the delivery of drugs to specific cells/organs.

The primary cilium is a singular cellular organelle that detects and transmits various extracellular cues to regulate diverse cellular processes during development, homeostasis, and disease pathogenesis.⁴¹^,⁴²^,⁴³ The activation of opioid receptors has been associated with the development of several brain disorders, such as addiction, and is also involved in regulating ciliogenesis. Studies have revealed that activating opioid receptors promotes primary ciliogenesis,⁴⁴ leading to the lateral diffusion of opioid receptors from the plasma membrane to primary cilia.⁴⁵^,⁴⁶ Primary ciliogenesis is precisely controlled in a temporally and spatially specific manner. CEP97 (centrosomal protein 97) and CCP110 (centrosomal protein 110) are required for cilia formation,⁴⁷^,⁴⁸ but overexpression of either CEP97 or CCP110 suppresses primary ciliogenesis.⁴⁹^,⁵⁰ Dysregulation of primary cilia and cilia signaling pathways are associated with various diseases, including cancer, aging, and Alzheimer’s disease.⁴³^,⁵¹ This study aimed to examine the underlying mechanisms by which EV-mediated primary ciliogenesis contributes to the development of opioid tolerance.

Our previous study demonstrated that morphine-ADEV-induced activation of primary ciliary signaling plays a role in morphine tolerance.⁵² Herein, we further demonstrated that miR-106b is upregulated in morphine-ADEVs, which induces primary ciliogenesis via targeting CEP97 in the recipient astrocytes. Moreover, we also demonstrated that intranasal delivery of EVs loaded with anti-miR-106b could reverse primary ciliogenesis in the astrocytes of morphine-administered mice.

Results

EVs released from morphine-stimulated astrocytes induce primary ciliogenesis in the recipient astrocytes

We previously demonstrated that morphine-ADEVs activate the primary cilia signaling pathway.⁵² Herein, we examined whether ADEVs play a role in morphine-induced primary ciliogenesis in astrocytes. We pretreated human primary astrocytes with EV release inhibitor - GW4869 (2 μM) for 1 h and then stimulated the cells with morphine for 24 h. As shown in Figures 1A–1C, inhibition of EV release blocked morphine-induced primary ciliogenesis in astrocytes without affecting cell viability (Figure S1A). We next separated EVs from conditioned media of astrocytes using the differential ultracentrifugation procedure and characterized EVs by transmission electron microscopy (TEM), ZetaView nanoparticle tracking analysis, and western blot for signature EV markers. As shown in Figures 1D–1F, EVs isolated from astrocytes ranged from 40 to 160 nm in diameter and expressed EV markers. Morphine stimulation increased the release of EVs from astrocytes (Figure 1G). We next treated astrocytes with either control-ADEVs or morphine-ADEVs, followed by staining for acetylated α-tubulin . As shown in Figures 1H–1J, morphine-ADEVs promoted primary ciliogenesis in astrocytes and had no impact on the viability of the cells (Figure S1B).

Morphine induces primary ciliogenesis through the release of extracellular vesicles (EVs) in astrocytes

(A) Immunostaining for α-tubulin in human primary astrocytes pretreated with EV release inhibitor – GW4869 for 1 h followed by morphine exposure for an additional 24 h. (B and C) Graphical representation of cilia length (B) and percentage of ciliated cells (C) as shown in (A) (n > 10 micrographs from three independent experiments). (D) Representative electron micrograph of EVs isolated from astrocyte culture. (E) Size distribution of EVs isolated from astrocyte cell culture by ZetaView. (F) Western blotting characterization of EVs purified from astrocyte-conditioned media via differential ultracentrifugation (UC). Calnexin, an endoplasmic reticular protein, was not present in the EV samples. (G) Number of EVs isolated from control and morphine-exposed astrocytes. (H) Immunostaining for acetylated α-tubulin (green) in human primary astrocytes stimulated with either control-ADEVs or morphine-ADEVs (100 EVs/cell) for 24 h. Boxed areas in the images under low magnification are enlarged and shown in the next panels on the right. (I and J) Graphical representation of cilia length (I) and percentage of ciliated cells (J) (n > 10 micrographs from three independent experiments). All experiments were done at least three independent times. Data are shown as mean ± SEM. ∗∗∗∗p < 0.0001.

CEP97 inhibits morphine-ADEV-mediated primary ciliogenesis in astrocytes

To validate the role of CEP97 in morphine-ADEV-mediated primary ciliogenesis, we transfected astrocytes with CLIP-CEP97 plasmid for 24 h followed by exposure cells to either control-ADEVs or morphine-ADEVs for an additional 24 h. CLIP-CEP97 was induced with doxycycline and then labeled with TMR substrate. As shown in Figures 2A–2C and as expected, morphine-ADEVs failed to induce primary ciliogenesis in CEP97 overexpressing astrocytes compared with CLIP-CEP97-negative astrocytes stimulated with morphine-ADEVs.

miRNA cargo in morphine-ADEVs mediates primary ciliogenesis in astrocytes via downregulating CEP97

miRNA cargo in EVs can be taken up by recipient cells and plays a vital role in the latter cells.⁵³ CEP97 is a negative regulator of ciliogenesis and dysregulation of CEP97 has been implicated in various ciliopathies.⁴⁹ We found that miRNA cargo in morphine-ADEVs could be involved in morphine-mediated primary ciliogenesis via targeting CEP97 in astrocytes and contribute to morphine tolerance. To confirm, we transfected astrocytes with either Dicer-siRNA (to block all miRNA formation) or scrambled siRNA for 24 h, followed by morphine stimulation for an additional 24 h. Knockdown of DICER1 in cells and depletion of miRNAs in ADEVs was confirmed by western blot and qPCR, respectively (Figures S1C–S1E). miRNA-depleted-ADEVs were isolated from the conditioned media and incubated with astrocytes for 24 h, followed by immunostaining for CEP97 and acetylated α-tubulin. As shown in Figures 3A and 3B, morphine-ADEVs decreased the expression of CEP97 in astrocytes compared with control-ADEVs. ADEVs isolated from Dicer-siRNA transfected astrocytes stimulated with morphine failed to downregulate CEP97 compared with morphine-ADEVs isolated from scrambled siRNA transfected cells. Concurrently, ADEVs isolated from Dicer-siRNA transfected astrocytes stimulated with morphine failed to increase the length and frequency of primary cilia compared with ADEVs isolated from morphine-stimulated astrocytes transfected with scrambled siRNA oligos in astrocytes (Figures 3A, 3C, and 3D). These results underpin the pivotal role of miRNA cargo in ADEVs and CEP97 in morphine-mediated primary ciliogenesis in astrocytes.

Morphine-induced miRNA cargo in ADEVs mediates downregulation of CEP97 and primary ciliogenesis in astrocytes

(A) Immunostaining for CEPP97 (green) and acetylated α-tubulin (pink) in human primary astrocytes exposed to Ctrl-ADEVs or morphine-ADEVs isolated from human primary astrocytes transfected with Dicer-siRNA/siRNA-Ctrl for 24 h, followed by morphine exposure for an additional 24 h. Boxed areas in the images under low magnification are enlarged and shown in the next panels on the right. (B) Graphical representation of the CEP97 mean intensity (arbitrary units) as shown in (A). (C and D) Graphical representation of cilia length (C) and percentage of ciliated cells (D) (n > 10 micrographs). Data are shown as mean ± SEM. ∗∗∗∗p < 0.0001.

miR-106b-5p in morphine-ADEVs induces primary ciliogenesis via targeting CEP97 in astrocytes

We previously identified a set of upregulated miRNAs in morphine-ADEVs, including miR-106b-5p using RNA sequencing (RNA-seq).⁵⁴ Targetscan analysis predicted CEP97 is a conserved target of miR-106b-5p (Figure 4A). We validated the upregulation of miR-106-5p in morphine-ADEVDs by qPCR (Figure 4B). To examine whether miR-106b is enriched inside or associated with the surface of EVs, we treated EVs with RNase A in the presence or absence of Triton X-100 (Figure 4B). miR-106 was insensitive to RNase A digestion but was degraded following detergent solubilization. These findings confirmed that miR-106 is localized in the lumen of EVs. To examine the specificity of miRNA functionality in the recipient astrocytes, it was important to first examine the delivery of miRNA into these cells. We applied a co-culture system, as illustrated in Figure 4C. Astrocytes in the transwell insert were transfected with TSG101-Cherry plasmid (to label EVs) and with either scrambled Alex488N-miRNA or Alex488N-miR-106b-5p for 24 h and then cultured with bottom astrocytes for an additional 24 h. Expression of CEP97 and primary cilia in the bottom astrocytes was assessed by immunostaining for CEP97 and acetylated α-tubulin, respectively. A blank transwell insert with no cells served as a negative control to exclude the possibility of direct transfection of the plasmid and labeled miRNA into the bottom cells. As shown in Figures 4D–4F, miRNA containing ADEVs released from transfected cells in the transwell insert were taken up by the bottom cells. Interestingly, miR-106b⁺ cells showed downregulation of CEP97 and increased primary cilia length and percentage of ciliated cells compared with miR-control⁺ cells (Figures 4E, 4G, and 4H).

ADEVs loaded with anti-miR-106b inhibit morphine-induced primary ciliogenesis in astrocytes

EVs have not only been recognized for their role as communication vesicles delivering cargo to neighboring/distant cells but have also been implicated as conduits for efficient delivery of RNA drugs into the brain.³⁸^,⁵⁵ We first examined the therapeutic potential of ADEVs loaded with anti-miR-106b. We treated human primary astrocytes with morphine and/or ADEVs loaded with anti-miR-106b. Real-time PCR results suggest that morphine stimulation upregulated the expression of miR-106b in Control-ADEV-treated astrocytes but failed to induce miR-106 in astrocytes treated with EVs loaded with anti-miR-106b (Figure 5A). Importantly, morphine stimulation failed to induce primary ciliogenesis in astrocytes treated with EVs loaded with anti-miR-106b (Figures 5B–5D) without disrupting the viability of the cells (Figure S1F).

Morphine failed to induce primary ciliogenesis in astrocytes treated with ADEVs loaded with anti-miR-106b

(A) Real-time PCR analysis of miR-106b-5p in astrocytes treated with EVs loaded with anti-miR-106b and/or morphine for 24 h. (B) Immunostaining for acetylated α-tubulin (green) in human primary astrocytes stimulated with ADEVs loaded with anti-miR-106b and/or morphine. Boxed areas in the images under low magnification are enlarged and shown in the next panels on the right. (C and D) Graphical representation of cilia length (C) and percentage of ciliated cells (D) (n > 10 micrographs from three independent experiments). All experiments were done at least three independent times. Data are shown as mean ± SEM. ∗p < 0.05 versus control.

Intranasal delivery of ADEVs loaded with anti-miR-106b decreases levels of miR-106b in brain-derived EVs isolated from morphine-administered mice

Next, we labeled ADEVs with DsRed dye, then washed the EVs using Optiprep gradient ultracentrifugation to eliminate free DsReddye and labeled contaminants. Negative control EVs were subjected to three liquid nitrogen freeze-thaw and sonication cycles followed by Optiprep gradient ultracentrifugation to eliminate free dsRed dye and labeled contaminants. Mice were intranasally administered dsRed-labeled ADEVs, and 4 and 24 h later, animals were transcardially perfused with ice-cold PBS, followed by tissue harvesting and monitoring for delivery efficacy and biodistribution using the IVIS imaging system. As shown in Figure 6A, labeled EVs were primarily localized in the brain, liver, and kidney following 4 h and 24 h of EV administration. These results suggest that ADEVs could be used as delivery vesicles for RNA drugs to the brain.

Intranasal delivery of ADEVs loaded with anti-miR-106b reduces miR-106b in brain-derived EVs

(A) ADEVs were labeled with dsRed and subjected to gradient ultracentrifuge to get rid of free dsRed dye. DsRed-labeled EVs were intranasally delivered to mice. Mice were killed 4 and 24 h posttreatment, and the harvested organs were imaged using a Xenogen IVIS 200 imager. A scale of the radiance efficiency is presented on the right. (B) Real-time PCR analysis of miR-106b-5p in the brain. (C) Representative electron micrograph of EVs isolated from brain tissue. (D) Size distribution of brain-derived EVs by ZetaView. (E) Western blotting characterization of brain-derived EVs. Calnexin, an endoplasmic reticular protein, was not present in the EV samples. (F) Number of brain-derived EVs. (G) Real-time PCR analysis of miR-106b-5p in brain-derived EVs. All experiments were done at least three independent times. Data are shown as mean ± SEM. ∗∗∗∗p < 0.0001 versus control; ∗∗p < 0.005 versus control; ####p < 0.0001 versus morphine; ###p < 0.0005 versus morphine; ##p < 0.005 versus morphine.

We intranasally delivered ADEVs loaded with anti-miR-106b, followed by injecting morphine 30 min later into mice for 5 days. As shown in Figure 6B, intranasal delivery of ADEVs loaded with anti-miR-106b inhibited the expression of miR-106b in the brain of mice administrated morphine. Brain-derived EVs were isolated using the Optiprep gradient ultracentrifugation method, as described previously.⁵⁶ Brain-derived EVs were characterized using TEM, ZetaView, and western blot. As shown in Figures 6C and 6D, the size distribution of the brain-derived EVs ranges from 50 to 250 nm, with a peak at ∼120 nm. There were no significant changes in the expressions of TSG101 and CD9 in the brain-derived EVs isolated from morphine-administrated mice compared with saline-treated mice (Figures 6E and S2). There were significantly increased numbers of brain-derived EVs isolated from morphine-administered mice compared with those isolated from saline mice, and inhibition of miR-106b did not alter morphine-induced EV release (Figure 6F). However, inhibition of miR-106b decreased the expression of miR-106b in brain-derived EVs from morphine-administered mice (Figure 6G).

Intranasal delivery of ADEVs loaded with anti-miR-106b ameliorates morphine-mediated primary ciliogenesis in astrocytes and prevents morphine tolerance

Our previous study demonstrated that primary cilia inhibition prevented morphine tolerance.⁵² We sought to examine whether block miR-106b could inhibit primary ciliogenesis and prevent morphine tolerance. In this study, mice were administered either saline or repeated escalating doses of morphine three times a day for 4 days. ADEVs loaded with anti-miR-106b were administered 30 min before the first injection of morphine for all days. The tail-flick assay was used to evaluate the development of morphine tolerance on day 5. As shown in Figure 7A, treatment with ADEV-anti-miR-106b significantly suppressed morphine tolerance. Finally, intranasal delivery of ADEVs loaded with anti-miR-106b significantly attenuated primary ciliogenesis in the astrocytes of morphine-administered mice (Figures 7B–7D).

Intranasal delivery of ADEVs loaded with anti-miR-106b ameliorates morphine-mediated primary ciliogenesis in astrocytes

(A) Cumulative dose-response curves for morphine-induced antinociceptive effects in the tail-flick test. (B) Representative confocal images of immunofluorescent staining on cryosections (30 μm thick) by ARL13B, a primary cilium marker (pink), GFAP, an astrocyte marker (green), in the brain of morphine-administered mice to identify primary cilia that protrude from GFAP positive astrocytes. (C, D) Graphical representation of cilia length (C) and percentage of ciliated cells (D) (n > 10 micrographs from three independent experiments). All experiments were done at least three independent times. Data are shown as mean ± SEM. ∗∗∗∗p < 0.0001 versus control; ####p < 0.0001 versus morphine.

Primary ciliogenesis in the astrocytes of opioid users

Finally, the findings were validated in human brain samples. We performed double immunofluorescence staining for ARL13B (a marker for primary cilia) and GFAP (a marker of astrocytes) on paraffin slices of human brain tissues (striatum) from either unaffected controls or opioid abuse. The frequency (ratio of ciliated cells) and length of primary cilia were significantly increased in astrocytes in the striatum of opioid users, compared with unaffected control subjects (Figure 8).

Increased frequency and length of primary cilia in the astrocytes of opioid abuses

(A) Representative confocal images of immunofluorescence staining on paraffin slices (8 μm thick) of human brain tissues (striatum) from either unaffected controls or opioid abuse by GFAP (red) and ARL13B (green). (B and C) Quantification of cilia length (B) and ciliated cells (C). n = 5 per group, 10 slices per group. Error bars represent SEM. ∗∗∗∗p < 0.0001 for unpaired Student’s t test. (D) Clinical Data Summary for Patients.

Discussion

The present study demonstrated that miR-106b is upregulated in the EVs released from morphine-stimulated astrocytes. miR-106b in morphine-ADEVs can be taken up by bystander astrocytes and induce primary ciliogenesis via downregulating CEP97. Our findings indicate that the upregulation of miR-106b in ADEVs contributes to the development of morphine tolerance via inducing primary ciliogenesis. Intranasal delivery of ADEVs loaded with anti-miR-106b inhibits primary ciliogenesis and prevents morphine tolerance in mice.

Abnormal ciliogenesis has been implicated in various human diseases, including neurodegenerative diseases and drug resistance.⁵⁷^,⁵⁸ A recent study demonstrated that the length of primary cilia and the percentage of ciliated cells are increased in drug-resistant cancer cells.⁵⁸ Our previous work demonstrated that pharmacologic inhibition of primary cilia prevented morphine tolerance in mice.⁵² It is well-known that morphine-induced opioid receptor internalization/desensitization contributes to morphine tolerance.⁵⁹^,⁶⁰^,⁶¹^,⁶²^,⁶³^,⁶⁴^,⁶⁵^,⁶⁶^,⁶⁷^,⁶⁸^,⁶⁹^,⁷⁰ Primary ciliogenesis plays a key role in orchestrating the trafficking of various membrane receptors.⁷¹^,⁷²^,⁷³ Thus, morphine-ADEV-induced primary ciliogenesis could 7lead to morphine tolerance via opioid receptor internalization/recycling impairment and desensitization, which warrants further investigation. In addition, studies have linked various G protein-coupled receptors (GPCRs), including opioid receptors, to primary ciliogenesis,⁴⁴^,⁴⁵^,⁴⁶^,⁷⁴ suggesting that other opioids may have a similar impact on ciliogenesis and opioid tolerance. Nevertheless, it is crucial to note that only a few opioid receptors, such as mu and delta opioid receptors, have been found to be associated with ciliogenesis.⁴⁴^,⁴⁵ Consequently, additional investigations are necessary to establish whether the results presented in the current study can be extended to other opioids. Moreover, cilia dysfunction has been implicated in various neurological disorders, including Alzheimer’s disease, ciliopathies, and certain types of brain cancer.⁴³^,⁷⁵^,⁷⁶ The findings reported in this study may have implications for developing new treatments for these disorders, as targeting cilia function may be a novel therapeutic approach. Additional research is required to ascertain the degree to which the results presented in this study are applicable to these conditions.

EVs are the secretory membrane-encapsulated vesicles that mediate intercellular communication by transferring bioactive molecules such as miRNAs to recipient cells.⁷⁷^,⁷⁸^,⁷⁹ miRNAs in ADEVs play an essential role in the brain under various conditions. EVs released from interleukin (IL)-1β and tumor necrosis factor (TNF)α-treated astrocytes contain miR-125a-5p and miR-16–5p and decreased dendritic growth, dendritic complexity, spike rates, and burst activity in neurons.⁸⁰ Our previous findings demonstrated that miR-7 in HIV TAT-stimulated astrocytes could be taken up by neurons and lead to synaptic injury.⁸¹ The current study demonstrated that morphine treatment increased the expression of miR-106b in ADEVs, which, when taken up, promoted primary ciliogenesis in astrocytes via targeting CEP97, indicating miRNA in ADEVs could contribute to the development of morphine tolerance. Of note, miR-106b is upregulated in several cancer types and is considered an oncogene, and thus could be a therapeutic target.⁸² The crucial involvement of miR-106b in the advancement and onset of various forms of cancer has been demonstrated, including lung, breast, and colorectal cancers, by targeting tumor suppressors such as p21, p53, and RB1, as well as genes involved in apoptosis, cell cycle regulation, and DNA damage response.⁶⁹^,⁸³^,⁸⁴^,⁸⁵^,⁸⁶^,⁸⁷ Furthermore, EVs can originate from cilia through a process of budding. Ciliary EVs can be generated at the ciliary tip, the axoneme, and the ciliary base.⁸⁸ It is worth exploring the possibility that morphine-triggered EV release could be due to the promotion of primary ciliogenesis by morphine in future investigations.

EVs have been proposed to deliver therapeutics, including small RNAs, for treating various diseases.²² Intranasal administration of EVs is a non-invasive method for delivering EV-encapsulated drug(s) to the brain.⁵⁴^,⁸⁹^,⁹⁰^,⁹¹ Studies have shown that miRNAs such as let-7,⁹² miR-378a-3p,⁹³ and miR-219,⁹⁴ regulate opioid tolerance via targeting mu-opioid receptors or brain-derived neurotrophic factor. Herein we showed that intranasal delivery of ADEVs loaded with anti-miR-106b downregulated levels of miR-106b inhibited primary ciliogenesis in astrocytes and prevented morphine tolerance in mice.

We recognize the constraints associated with relying on a single gauge of opioid tolerance in this study. The tail-flick test was used to measure thermal nociception. While this test is a commonly used measure of opioid tolerance,⁹⁵^,⁹⁶ other behavioral tests may provide complementary information about opioid tolerance and its underlying mechanisms. For example, mechanical or chemical nociception tests⁹⁷^,⁹⁸ may provide additional insights into the development of opioid tolerance.

Our findings suggest that miRNA cargo in ADEVs mediate morphine-induced primary ciliogenesis in astrocytes. Our in vitro results showed that CEP97 is a target of miR-106b and is a negative regulator of primary ciliogenesis. Our in vivo findings demonstrated that intranasal delivery of ADEVs loaded with anti-miR-106b decreased the expression of miR-106b in astrocytes and ADEVs, inhibited primary ciliogenesis, and prevented the development of tolerance in morphine-administered mice. Our findings bring new insights into the mechanisms underlying primary cilium-mediated morphine tolerance and pave the way for developing ADEV-mediated small RNA delivery strategies for preventing and treating substance use disorders.

Materials and methods

Animals

Male C57BL/6 mice, aged 6–8 weeks, were acquired from Charles River Laboratories, Inc. (Wilmington, MA, USA) and bred in the animal facility of the University of Nebraska Medical Center (UNMC) in conditions that included controlled temperature and humidity, a 12-h light/dark cycle (lights turned on at 07:00 AM), and unlimited access to food and water. The Institutional Animal Care and Use Committee at UNMC reviewed and approved all animal protocols.

Intranasal delivery of EVs

Mice were anesthetized in an anesthesia chamber and placed in a supine position in an anesthesia chamber. EVs (20 μg/100 μL) in saline were administered intranasally as drops with a small pipette every 2 min into alternating sides of the nasal cavity for 10 min. To determine the tissue distribution of EVs in vivo, we harvested various tissues and monitored for efficiency of delivery and biodistribution using a Xenogen IVIS 200 imager.

Morphine analgesia and tolerance

The tail-flick test (LE7106 analgesia-meter, Panlab Harvard, MA, USA) was used to evaluate the analgesic response to morphine at 30 min after each morphine injection. Mice were habituated to the tail-flick device for 2 min before each test session. A halogen lamp was focused on the tail, and the withdrawal reflex time was determined by a photocell. Tail-flick latency was assessed both before and 1 day after morphine administration. Cutoff time of 10 s for the tail-flick test was followed to avoid injury to the mice. The antinociceptive response was expressed as a percentage of maximal possible effect (MPE), where MPE % = (test latency − baseline latency) × 100/(cutoff latency-baseline latency).

Cell cultures

Human primary astrocytes were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and were cultured in astrocyte medium (ScienCell). Human primary astrocytes at passage 10 or less were used in this study. Cells were deprived of serum for 24 h before the treatment.

Reagents and brain tissues

Morphine was obtained from R&D Systems Inc. (Minneapolis, MN, USA). Lipofectamine 2000 and miRNA primers were purchased from Life Technologies. pEBTetDBl-CLIP-Cep97 was a gift from Kai Johnsson (Addgene plasmid #136850; http://n2t.net/addgene:136850; RRID:Addgene_136850). miR-106b inhibitor (Catalog number: 4427975, Assay ID MH10067) and DICER1 Silencer were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). CLIP-Cell TMR-Star was purchased from BioLabs. Human tissue was obtained from the NIH NeuroBioBank.

EV preparation

The isolation of EVs from conditioned media and brain tissues was performed according to previously published guidelines.⁹⁹ As previously described, EVs were separated from conditioned media (FBS depleted) of cell cultures using the differential centrifugations method.¹⁰⁰ Specifically, the conditioned media was collected, subjected to centrifugation at 1,000 × g for 10 min, and then sequentially spun at 10,000 × g for 30 min. The supernatant was passed through 0.22-μm filters, and then subjected to ultracentrifugation at 100,000 × g for 70 min. The Optiprep gradient ultracentrifugation method was used to separate EVs from brain tissues, as described previously.⁵⁶ Iodixanol fractions 2–7 (from top to bottom) were collected, pooled, and resuspended in sterile 1x PBS, followed by ultracentrifugation at 100,000 × g for 70 min to pellet brain-derived EVs. The resulting EV pellets were resuspended in phosphate-buffered saline (PBS) for further analysis. EV markers, including TSG101 and CD9, were detected using the western blotting assay. The number and size distribution of EVs were analyzed using ZetaView Particle Metrix, as previously reported.⁵⁴^,¹⁰¹

Cell viability assay

To assess cell viability, the CyQUANT Assay Kit (Invitrogen, CA, USA) was used, as previously described.⁸⁹ Briefly, cells were plated in 96-well plates at a density of 5,000 cells per well and treated as per the experimental design. After freezing the cells in the microplate at −70°C for 1 h, the plates were thawed at room temperature, and 200 μL of the CyQUANT GR dye/cell-lysis buffer was added to each well, followed by incubation for 2–5 min at room temperature. The plates were then read at excitation at 480 nm and emission at 520 nm. The test cells were compared with their respective control cells, which were considered 100% viable. Data were presented as mean ± SEM of six replicates.

Western blotting

As described previously,⁸⁹ EVs were lysed using the Mammalian Cell Lysis kit (Sigma-Aldrich, St. Louis, MO, USA). The proteins were separated using a sodium dodecyl sulfate (SDS)-polyacrylamide gel and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked with 5% nonfat dry milk, 0.05% Tween 20 in Tris-buffered saline (TBS, 150 mM NaCl, 10 mM Tris-HCl, pH 8) (TTBS) for 1 h at room temperature (RT). The primary antibody was incubated with the membrane in 1% nonfat milk overnight at 4°C. Primary antibodies specific for TSG101 (1:1,000; Proteintech; Rosemont, IL, USA), CD9 (1:1,000; Abcam, Cambridge, MA, USA), DICER1 (1:1,000; ThermoFisher, Waltham, MA, USA), horseradish peroxidase (HRP)-conjugated Beta Actin (1:10,000; Proteintech) and CALNEXIN (1:1000; Proteintech) were used in this study. The following day, the membrane was washed three times with TTBS for 10 min each, followed by incubation with secondary antibody-alkaline phosphatase-conjugated to goat anti-mouse/rabbit immunoglobulin (Ig)G (1:10,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 1 h at RT. The membrane was rinsed three times with TTBS for 10 min each and then visualized with West Chemiluminescent Substrate (Thermo Fisher Scientific). The experiments were performed at least three times, and the figures display representative blots.

RNA isolation and TaqMan MicroRNA assays

RNA was purified using the Quick-RNA MiniPrep Kit (ZYMO Research, CA, USA) according to the manufacturer’s instructions. TaqMan MicroRNA Reverse Transcription Kit (PN 4366596) and TaqMan Universal PCR Master Mix (PN 4324018) were used for miRNA measurement. The expression levels of miR-106b were calculated by normalizing to U6 small nuclear RNA (snRNA) as reported previously.⁸¹^,¹⁰²^,¹⁰³

Immunostaining

Mice were perfused intracardially with 1 X PBS, followed by 4% paraformaldehyde. After dissection, brains were fixed in 4% paraformaldehyde overnight at 4°C and then immersed in a 30% sucrose/PBS solution until they sank. Afterward, the brains were embedded in optimal cutting temperature compound and sliced into 30-μm sections using a CM1950 Leica cryostat. Brain slices were mounted on glass slides (Fisher Scientific, Waltham, MA, USA) and stored at −80°C until use. Brain slides were stained with antibody specific for GFAP (1:1,500; Abcam) and ARL13B (1:80; Proteintech) overnight at 4°C. On the following day, the sections were washed thrice with PBS for 3 min each and then incubated with Alexa Fluor 488, 549 and/or 647-conjugated secondary antibodies (Invitrogen, San Diego, CA, USA) for 1 h at RT. Following a final washing with PBS, the slides or coverslips were mounted with mounting medium (Prolong Gold Antifade Reagent; Invitrogen, Grand Island, NY, USA) and kept in the dark overnight at RT. Fluorescent images were captured using a Z1 inverted microscope with a ×20/0.75 objective, and the resulting images were processed using ZEN 2.5 (blue edition; Zeiss) and Adobe Photoshop CC 2019 (version 20.0.0) software. Image analysis was performed using ImageJ software (1.52a, NIH, USA).

Cells cultured on coverslips were fixed in 4% formaldehyde in PBS for 10 min at RT. Afterward, the coverslips were rinsed thrice with PBS for 3 min each, permeabilized with 0.2% Triton X-100 for 5 min, rinsed thrice again, and then blocked in 10% goat serum in PBS for 1 h at RT. Afterward, the coverslips were incubated with CEP97 (1:80; Proteintech) and/or acetylated α-tubulin (1:1000, Sigma-Aldrich) overnight at 4°C. On the following day, the coverslips were washed three times with PBS for 3 min each and then incubated with Alexa Fluor 488 and/or 647–conjugated secondary antibodies (Invitrogen, San Diego, CA, USA) for 1 h at RT. Following a final washing with PBS, the slides or coverslips were mounted with mounting medium (Prolong Gold Antifade Reagent, Invitrogen, Grand Island, NY, USA), and fluorescent images were captured using a Z1 inverted microscope with a ×20/0.75 objective. The resulting images were processed using ZEN 2.5 (blue edition; Zeiss) and Adobe Photoshop CC 2019 (version 20.0.0) software, and analyzed using ImageJ software (1.52a, NIH, USA). Cell and cilia numbers were quantified by counting DAPI-stained nuclei and fluorescence-labeled cilia, respectively, using binary images in ImageJ and the particle analysis plugin. The length of the cilia was measured in pixels using the straight-line tool in ImageJ and recorded through the "Analyze > Measure" function. To obtain the conversion factor of μm/pixel, the length in μm was divided by the length in pixels.

Transmission electron microscopy

EVs were subjected to treatment with 2% paraformaldehyde and 2% glutaraldehyde in 0.1M PBS. Then, 3 μL of the treated EVs were carefully placed onto a 200-mesh formvar-coated copper grid, left to adsorb for 5 min, and processed with standard uranyl acetate staining. The grid was subsequently washed with PBS three times and left to semi-dry for 2 min at RT before being observed under an FEI Tecnai G2 Spirit transmission electron microscope.

Statistical analysis

Statistical analyses were performed using a two-tailed Student’s t test for comparing two groups, and one-way ANOVA with post hoc Tukey’s multiple comparisons for comparing multiple groups. The significance level was set at p < 0.05. The figures were created using GraphPad Prism version 9.31 for Windows (GraphPad Software, San Diego, CA, USA).

Acknowledgments

This work was supported by startup funds from University of Nebraska Medical Center and the National Institutes of Health (NIH) grants R21DA046831, R21DA042704, R01DA043138, and R01MH112848. The project described was also supported by NIH grant 2P30MH062261. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We are grateful to Drs. Shilpa Buch and Ke Liao, and Mr. Shannon Callen for their outstanding technical assistance. We thank the Nebraska Center for Substance Abuse Research (NCSAR) for its support. We also thank Tom Bargar and Nicholas Conoan of the Electron Microscopy Core Facility (EMCF) at the University of Nebraska Medical Center for technical assistance. The EMCF is supported by state funds from the Nebraska Research Initiative (NRI) and the University of Nebraska Foundation, and institutionally by the Office of the Vice Chancellor for Research.

Author contributions

R.M, N.K., Z.W., and G.H. designed and performed the experiments; collected, analyzed, and discussed the data; and drafted, revised and approved the final manuscript.

Declaration of interests

The authors declare no competing interests.

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.ymthe.2023.03.030.

Contributor Information

Rong Ma, Email: marong@hust.edu.cn.

Guoku Hu, Email: guoku.hu@unmc.edu.

Supplemental information

Document S1. Figures S1 and S2

mmc1.pdf^{(794.1KB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(6.3MB, pdf)}

Data availability statement

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1 and S2

mmc1.pdf^{(794.1KB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(6.3MB, pdf)}

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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PERMALINK

Extracellular vesicle-mediated delivery of anti-miR-106b inhibits morphine-induced primary ciliogenesis in the brain

Rong Ma

Naseer A Kutchy

Zhongbin Wang

Guoku Hu

Abstract

Graphical abstract

Introduction

Results

EVs released from morphine-stimulated astrocytes induce primary ciliogenesis in the recipient astrocytes

Figure 1.

CEP97 inhibits morphine-ADEV-mediated primary ciliogenesis in astrocytes

Figure 2.

miRNA cargo in morphine-ADEVs mediates primary ciliogenesis in astrocytes via downregulating CEP97

Figure 3.

miR-106b-5p in morphine-ADEVs induces primary ciliogenesis via targeting CEP97 in astrocytes

Figure 4.

ADEVs loaded with anti-miR-106b inhibit morphine-induced primary ciliogenesis in astrocytes

Figure 5.

Intranasal delivery of ADEVs loaded with anti-miR-106b decreases levels of miR-106b in brain-derived EVs isolated from morphine-administered mice

Figure 6.

Intranasal delivery of ADEVs loaded with anti-miR-106b ameliorates morphine-mediated primary ciliogenesis in astrocytes and prevents morphine tolerance

Figure 7.

Primary ciliogenesis in the astrocytes of opioid users

Figure 8.

Discussion

Materials and methods

Animals

Intranasal delivery of EVs

Morphine analgesia and tolerance

Cell cultures

Reagents and brain tissues

EV preparation

Cell viability assay

Western blotting

RNA isolation and TaqMan MicroRNA assays

Immunostaining

Transmission electron microscopy

Statistical analysis

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Contributor Information

Supplemental information

Data availability statement

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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