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. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: J Neuroimmune Pharmacol. 2019 Jul 20;15(3):390–399. doi: 10.1007/s11481-019-09864-z

Intranasal delivery of lincRNA-Cox2 siRNA loaded extracellular vesicles decreases lipopolysaccharide-induced microglial proliferation in mice

Ke Liao 1, Fang Niu 1, Raghubendra Singh Dagur 1, Mengfan He 2, Changhai Tian 3, Guoku Hu 1
PMCID: PMC6980430  NIHMSID: NIHMS1535239  PMID: 31325121

Abstract

Long non-coding RNAs (lncRNAs), including long intergenic non-coding RNAs (lincRNAs), play an important regulatory role in controlling various biological processes. Both in vitro and in vivo studies have demonstrated that lincRNA-Cox2 plays a global regulatory role in regulating the expression of immune genes. Extracellular vesicles (EVs) are cell-derived nanosized membrane vesicles that have gained increasing attention in recent years due to their ability to efficiently deliver therapeutics to specific target organs or cell types. In this study, we found that lincRNA-Cox2 controls the expression of a set of cell cycle genes in lipopolysaccharide (LPS)-stimulated microglial cells. Our in vitro study suggested that knocking down lincRNA-Cox2 reversed LPS-induced microglial proliferation. In addition, our in vivo study demonstrated that intranasally delivered lincRNA-Cox2-siRNA loaded EVs could reach the brain resulting in a significant decrease in the expression of lincRNA-Cox2 in the microglia. Importantly, lincRNA-Cox2-siRNA loaded EVs also decreased LPS-induced microglial proliferation in mice. These findings indicate that intranasal delivery of EV-loaded small RNA could be developed as therapeutics for treatment of a multitude of CNS disorders.

Keywords: lincRNA-Cox2, microglial proliferation, extracellular vesicle, intranasal delivery, LPS

Graphical Abstract

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Introduction

Under normal/physiological conditions, the number of microglial cells remains stable in the adult brain throughout life in mice and humans (Askew et al., 2017). However, the number of microglia is dramatically altered in several CNS diseases. For example, the number of microglia is increased by at least 40% in 5xfAD mice (a mouse model of Alzheimer's disease (AD)) compared to control mice (Spangenberg et al., 2016). Similarly, LPS treatment significantly increases the number of microglia both in vitro and in vivo (Monje et al., 2003). Interestingly, following depletion of microglial numbers in 5xfAD mice, there was a substantial recovery of contextual memory, and reversal of dendritic spine loss and subsequent neuronal loss (Spangenberg et al., 2016), thereby indicating that targeting microglial proliferation could be a therapeutic strategy for the treatment of neurodegenerative diseases.

Long non-coding RNAs (lncRNAs), including long intergenic non-coding RNAs (lincRNAs), are long non-coding regulatory RNAs that are greater than 200 nucleotides. Dysregulated lncRNAs have been shown to play a role in various neurodegenerative disorders, such as Alzheimer’s (Faghihi et al., 2008), Parkinson’s (Zhang et al., 2017) and Huntington’s (Lin et al., 2014) diseases. Previous studies have demonstrated the pivotal role of lincRNA-Cox2 in controlling the expression of inflammatory genes in phagocytic as well as epithelial cells (Carpenter et al., 2013; Hu et al., 2016; Tong et al., 2016; Elling et al., 2018; Xue et al., 2019). The role of lincRNA-Cox2 in microglial proliferation, however, remains unknown.

Extracellular vesicles (EVs) are cell-derived nanosized vesicles that play an important role in intercellular communication via their cargo mediators such as proteins, lipids, and RNAs that can be targeted to remote cells (Cocucci et al., 2009; Thery et al., 2009). Owing to their ability to efficiently shuttle small molecules between cells, EVs are gaining momentum as promising therapeutic tools for treatment of various neurological diseases. Indeed, EVs have been utilized successfully to deliver siRNAs (small interfering RNAs) to specific cell types in vivo in mice (van den Boorn et al., 2011). Intranasal administration of EVs is considered as a preferred noninvasive method for rapid delivery of EV-encapsulated drug(s) to the brain with selective uptake by microglial cells (Visweswaraiah et al., 2002; Lakhal and Wood, 2011; Zhuang et al., 2011; Grassin-Delyle et al., 2012). Manipulating EV-siRNAs could thus be an effective means to deliver therapeutics to specific target organs or cell types.

In the current study, we demonstrated that knockdown of lincRNA-Cox2 reversed the expression of LPS-induced cell cycle genes in mouse primary microglia resulting in the inhibition of microglial proliferation in vitro. Moreover, we also demonstrated that intranasal delivery of lincRNA-Cox2-siRNA could reverse LPS-induced microglial proliferation in mice.

Materials and Methods

Animals

All animal procedures were performed in strict accordance with the protocols approved by the Institutional Animal Care and Use Committee at the University of Nebraska Medical Center (UNMC). C57BL/6N WT mice were purchased from Charles River Laboratories (Wilmington, MA, USA) and housed under conditions of constant temperature and humidity on a 14-hr light and 10-hr dark cycle, with lights on at 7:00 a.m. Food and water were available ad libitum.

Mouse primary cell culture

Mouse primary microglia cells were obtained from 1- to 3-day-old C57BL/6N newborn pups, as described previously (Skaper et al., 2012). After digestion and dissociation of the dissected brain cortices in Hank’s buffered salt solution supplemented with trypsin (0.25%), mixed glial cultures were prepared by resuspending the cell suspension in DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Cells were plated at 20 × 106 cells/flask density onto 75-cm2 cell culture flasks. The cell medium was replaced on the 3rd day, and after the first medium change, macrophage colony-stimulating factor (M-CSF; 0.25 ng/mL; PeproTech, Rocky Hill, NJ, USA) was added to the flasks to promote microglial proliferation. The confluent mixed glial cultures (~ 10 d) were then subjected to shaking at 37°C at 220 × g for 2 h to promote microglial detachment from the flasks. The cell medium, comprising the detached microglia cells, was collected from each flask and centrifuged at 1000 × g for 5 min. The collected cells were plated onto 6-well cell culture plates (5 × 105 cells per well) for all ensuing experiments. Microglial purity was evaluated by immunocytochemistry using antibody specific for Iba-1(FUJIFILM Wako Chemicals U.S.A. Corporation, cat#: 019–19741) and used if >95% pure.

Mouse primary astrocytes were prepared from whole brains of post-natal (1- to 3-day-old) C57BL/6N mice and plated on poly-D-lysine pre-coated cell culture flasks containing DMEM (10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin). The cells were grown in a humidified atmosphere of 5% CO2/95% air at 37°C. When the astrocytes reached confluence, they were passaged by trypsinization and plated at a density of 106 cells/well on 24-well culture plates in a final volume of 1 mL of DMEM and grown in a humidified atmosphere of 5% CO2/95% air at 37°C. Immunocytochemical analyses demonstrated that the cultures were comprised of >95% glial fibrillary acidic protein (GFAP)-positive astrocytes.

BV-2 cell culture

The BV-2-immortalized cell line was generously provided by Dr. Sanjay Maggirwar (University of Rochester Medical Center, Rochester, NY, USA). Cells were grown and routinely maintained in DMEM (Invitrogen, 11995–065) with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen, 16000–044) at 37 °C and 5% CO2 and used up to 20 passages.

Small interfering RNA (siRNA) transfection

The following siRNA, obtained from Integrated DNA Technologies, Inc. (IDT), were used in this study: control siRNA (Control siRNA sense strand: 5’-UAAGGCUAUGAAGAGAUACUU-3' and Control siRNA antisense strand: 5’- GUAUCUCUUCAUAGCCUUAUU-3') and lincRNA-Cox2 siRNA (lincRNA-Cox2 siRNA sense strand: 5’-GCCCUAAUAAGUGGGUUGUUU-3' and lincRNA-Cox2 siRNA antisense strand: 5’-ACAACCCACUUAUUAGGGCUU-3ɹ). Cells were transfected with 40nM control- or lincRNA-Cox2 siRNA using lipofectamine RNAiMAX (Invitrogen) in serum-free Opti-MEM according to the manufacturer’s instructions.

Real-time PCR

To determine the expression of mouse GAPDH, lincRNA-Cox2, Mki67, Slfn1, Msh2, Cdkn3, Cdc6, Cdc25c and Birc5, cDNA was synthesized using a Verso cDNA kit (AB-1453/B; Thermo Fisher Scientific) according to the manufacturer’s instructions. Real-time PCR was performed using SYBR Green ROX qPCR Master Mix (QIAGEN, 330510). The primers were as followings: mouse GAPDH: 5’-TGCACCACCAACTGCTTAGC-3’ and 5’-ATGCCAGTGAGCTTCCCGTT-3’; mouse lincRNA-Cox2: 5’-AGTATGGGATAACCAGCTGAGGT-3’ and 5’-GAATGCTGAGAGTGGGAGAAATAG-3’; mouse Mki67: 5’-CAGGCTCCGTACTTTCCAATTC-3’ and 5’-TGCTTTGCTGCATTCCGA-3’; mouse Slfn1: 5’-GCTAGCTCGCAGTTGACTTCA-3’ and 5’-AAGTGTGATCCCTCCTGCATT-3’; mouse Msh2: 5’-CCTCCAGGCATGCTTGTGTT-3’ and 5’-CGATTTGGGCCATGAGTACAA-3’; mouse Cdkn3: 5’-GCTGCTGGGAAATCATGGA-3’ and 5’-TCGAAGGCTGTCTATGGCTTG-3’; mouse Cdc6: 5’-TTGCTCAGGAGATTGGTCGG-3’ and 5’-CAGCTGATCCATCTCGTCCAA-3’; mouse Cdc25c: 5’-AAAGGTGTGTGTGCTGCCAA-3’ and 5’-TTAAGGCTCCCAGGATGTGG-3’; and mouse Birc5: 5’-ACCCGATGACAACCCGATA-3’ and 5’-TCTTGGCTCTCTGTCTGTCCA-3’. The comparative cycle threshold (Ct) method (2^ΔΔCt) was used to calculate the relative level of gene expression. The Ct values were normalized to GAPDH, which served as an internal control.

Immunofluorescence

Cells cultured on slides or coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature followed by permeabilization with 0.3% Triton X-100 in PBS followed by incubation with H2O2 for 10 min. Sections were incubated with a blocking buffer containing 10% NGS in PBS for 1 h at room temperature followed by addition of rabbit anti-Iba-1(FUJIFILM Wako Chemicals U.S.A. Corporation, cat#: 019–19741), rat anti-BrdU (abcam, cat#: ab6326) or sheep anti-Ki67 antibodies(R&D Systems, cat#: AF7649) and incubated overnight at 4°C. Primary Abs were labeled with secondary Abs conjugated to the fluorescent probes, and nuclei were labeled with DAPI. Slides were covered with a coverslip with ProLong Gold antifade reagent (Invitrogen) and allowed to dry for 24 h at room temperature. Images were captured with a 20X objective.

EV Isolation, characterization and transfection

EV isolation and characterization procedures followed the recent MISEV 2018 guidelines (Thery et al., 2018). For EV isolation, to avoid bovine exosome and lipoprotein contamination (Simonsen, 2019), astrocytes were washed with PBS and cultured in serum-free medium for 24h followed by EV isolation using differential ultracentrifugation as described previously (Hu et al., 2013; Hu et al., 2018; Yang et al., 2018). In brief, serum-free conditioned media were harvested, centrifuged at 1,000 × g for 10 min to eliminate cells, and again spun at 10,000 × g for 30 min, followed by filtration through a 0.22-μm filter to remove cell debris. EVs were pelleted by ultracentrifugation (Beckman Ti70 rotor; Beckman Coulter, Brea, CA, USA) at 100,000 × g for 70 min. Purified EVs were subjected to characterization for signature exosomal markers by western blot, morphology by transmission electron microscopy (TEM) as well as number and size by ZetaView nanoparticle tracking analysis (Particle Metrix, Mebane NC) as we described previously (Hu et al., 2018; Yang et al., 2018).

EVs were then transfected with siRNA using Exo-Fect Exosome Transfection Reagent (SBI; System Biosciences, Palo Alto, CA) according to the manufacturer’s instructions. Briefly, EVs (8 × 107) were incubated with Exo-Fect solution (10 μL), and 20 pmol lincRNA-Cox2 siRNA or control siRNA in 150 μL PBS at 37 °C for 10 mins. To purify the transfected EVs, 30 μl ExoQuick-TC was added and incubated on ice for additional 30 min followed by centrifugation at 14,000 rpm for 3 min to remove free siRNA, and Free Exo-Fect. The transfected EV pellet was then resuspended in 300 μL PBS.

PKH26 labeling of EVs

Purified EVs were labeled with PKH26 dye (Sigma-Aldrich, St. Louis, MO), following the manufacturer’s protocol. To eliminate excess dye (Puzar Dominkus et al., 2018; Simonsen, 2019), the labeled EVs were subjected to OptiPrep (Sigma-Aldrich) density gradient ultracentrifugation as described previously (DeMarino et al., 2018; Pleet et al., 2018). Briefly, labeled EVs or PBS mixed with PKH26 (served as a negative control) were layered on the top of a 5% to 60% OptiPrep gradient followed by ultracentrifugation at 100,000 × g for 18 h at 4 °C. The labeled EVs were then further washed with PBS, ultracentrifuged at 100,000× g for 70 min and carefully resuspended in PBS.

RNA in situ hybridization

Custom Stellaris® FISH Probes were designed against mouse lincRNA-Cox2 by utilizing the Stellaris® FISH Probe Designer (Biosearch Technologies, Inc., Petaluma, CA) available online at www.biosearchtech.com/stellarisdesigner. The formalin-fixed paraffin-embedded tissue sections were hybridized with the Stellaris FISH Probe set labeled with 670 Dye (Biosearch Technologies, Inc.), following the manufacturer’s instructions available online at www.biosearchtech.com/stellarisprotocols. Briefly, formalin-fixed paraffin-embedded tissue sections were deparaffinized and rehydrated using a decreasing percentage of ethanol. Antigen retrieval was performed by boiling the slides in 0.01-M Tris-EDTA buffer, pH 9, for 40 min. Brain sections were permeabilized with 0.1% Triton X-100 in 1X PBS for 10 minutes at room temperature, followed by blocking with 10% goat serum for 1h. After blocking, the sections were incubated with Iba-1 (1:500) primary antibody for 1 h at room temperature. Secondary Alexa Fluor 594 goat anti-mouse (Invitrogen, cat#: A-11032) and Fluor Alexa Fluor 488 goat anti-chicken(Invitrogen, cat#: A-11039) were added for 1h, followed by hybridization as per Stellaris® FISH manufacturer’s protocol and hereafter, the slides were mounted in Prolong gold anti-fade reagent with DAPI (Invitrogen). Fluorescent images were acquired using Z1 inverted microscope (Carl Zeiss, Thornwood, NY, USA) and analyzed using the AxioVs 40 Version 4.8.0.0 software (Carl Zeiss MicroImaging GmbH).

Cell proliferation assays

Mouse primary microglial cells were seeded in the 96-well plate with the density of 5000 cells / well. Cell proliferation assays were performed after transfecting siRNA and LPS treatment. Cell proliferation was assessed using the CyQUANT™ Cell Proliferation Assay Kit (Invitrogen) or Cell Counting Kit-8 (Sigma-Aldrich) according to the manufacturer’s instructions.

Statistical analyses

All the data were expressed as mean ± SEM, and appropriate statistical significance was determined based on the experimental strategy using GraphPad Prism version 6.01. The precise statistical analyses and experimental designs, including tests performed, exact p values, and sample sizes, are provided with the results describing each figure, or within the legend of each figure. Nonparametric Kruskal-Wallis one-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance between multiple groups, and unpaired Student’s t-test was used to compare between two groups.

Results

LPS-induced lincRNA-Cox2 regulates the expression of cell proliferation genes

Transcriptional profiling analysis has demonstrated a broad regulatory role for lincRNA-Cox2 in the inflammatory response to LPS stimulation in both macrophages and microglia (Carpenter et al., 2013; Hu et al., 2016). Additionally, further analysis of the profiling data set, produced by the Agilent SurePrint G3 Mouse Gene Expression Microarray (G4852A), indicated that knockdown of lincRNA-Cox2 significantly reversed LPS-mediated alterations of the cell cycle gene expression in BV-2 cells (Figure 1A). These findings were further validated in mouse primary microglial cells transfected with either control- or lincRNA-Cox2-siRNA for 24h, followed by exposure of cells to either PBS or LPS for 4h, and subsequently assessed for the expression of indicated genes by qPCR. As shown in Figure 1B, lincRNA-Cox2 was significantly upregulated in LPS stimulated microglia compared with control microglia. As expected, the expression of lincRNA-Cox2 was significantly downregulated in microglia transfected with lincRNA-Cox2-siRNA compared with control siRNA group. As shown in Figures 1C-1I, in mouse primary microglia exposed to LPS, there was significantly altered expression of cell cycle genes compared with control cells. Interestingly, knockdown of lincRNA-Cox2 significantly reversed the expression of cell cycle genes in LPS stimulated microglia compared with microglia transfected with scrambled siRNA. These results thus underpin the role of lincRNA-Cox2 as a key modulator of LPS-mediated microglial proliferation.

Figure 1. Knockdown of lincRNA-Cox2 reverses the expression of LPS-induced cell cycle genes in microglia.

Figure 1.

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(A) Heat map analysis of microarray data showing hierarchical clustering of lincRNA-Cox2-regulated cell cycle genes in LPS stimulated BV-2 cells. (B) Mouse primary microglial cells were transfected with lincRNA-Cox2-siRNA for 24 h, followed by exposure of cells to LPS for 4 h, and subsequently assessed for the expression of lincRNA-Cox2 by qPCR, and relative gene expression was calculated from Ct values using a 2^ΔΔCt method. (C) - (I) Validation of representative microarray data by qPCR for genes indicated in mouse primary microglia (Also see (B)). The relative amounts of mRNA for each of the indicated genes were determined by qPCR, and relative gene expression was calculated from Ct values using a 2^ΔΔCt method. Fold-changes were normalized to control siRNA of untreated sample. Data are mean ± SEM of at least three independent experiments. Nonparametric Kruskal-Wallis one-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance between multiple groups. *p < 0.05; * *p < 0.01, * * *p < 0.001 versus control. #p < 0.05, # #p < 0.01, # # #p < 0.001 versus control-siRNA + LPS group.

Knockdown of lincRNA-Cox2 reverses LPS-induced microglial proliferation

We next sought to validate the role of lincRNA-Cox2 in LPS-induced microglial proliferation. For this, mouse primary microglial cells were transfected with either control- or lincRNA-Cox2-siRNA for 24h followed by exposure of cells to LPS for an additional 24h and then to BrdU incorporation for 30 min. Cells were then fixed and stained with anti-Iba-1, and anti-BrdU antibodies, and analyzed by confocal microscopy. As shown in Figure 2A, mouse primary microglia exhibited a low rate of basal proliferation under normal conditions. Treatment with LPS, however, resulted in a marked increase in the microglial division. Similarly, exposure of cells to LPS also resulted in an increased numbers of microglia expressing the cell division protein Ki67 (Figure 2B). Interestingly, when microglial cells transfected with lincRNA-Cox2-siRNA were exposed to LPS, there were decreased numbers of BrdU positive (Figure 2A) and Ki67 positive (Figure 2B) microglia compared with that in LPS-stimulated cells transfected with control-siRNA. The finding that knockdown of lincRNA-Cox2 decreased LPS-induced microglial proliferation was further confirmed using CyQuant (Figure 2C) and CCK-8 (Figure 2D) assays.

Figure 2. Knockdown of lincRNA-Cox2 reverses LPS-induced microglial proliferation.

Figure 2.

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(A) Mouse primary microglial cells were transfected with lincRNA-Cox2-siRNA for 24 h, followed by exposure of cells to LPS (20ng/mL) for an additional 24 h and to BrdU incorporation for 30 min, and subsequently fixed, stained with anti-Iba-1, and anti-BrdU antibodies, and analyzed by confocal microscopy. (B) Mouse primary microglial cells were transfected with lincRNA-Cox2-siRNA for 24 h, followed by exposure of cells to LPS for an additional 24 h, and subsequently fixed, stained with anti-Iba-1, and anti-Ki67 antibodies, and analyzed by confocal microscopy. (C, D) Mouse primary microglial cells were transfected with lincRNA-Cox2-siRNA for 24 h, followed by exposure of cells to LPS for an additional 24 h. Cell proliferation was evaluated by (C) CyQuant assay and (D) Cell Counting Kit-8 (CCK-8) assay. Data are mean ± SEM of at least three independent experiments. Nonparametric Kruskal-Wallis one-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance between multiple groups. *p < 0.05; * * *p < 0.001 versus control. #p < 0.05, # #p < 0.01 versus control-siRNA + LPS group. ns, not significant.

EVs can be used for the delivery of siRNA

Our recent study has demonstrated that intranasal delivery of lincRNA-Cox2-siRNA-loaded astrocyte-derived EVs could restore microglial function in morphine-administrated mice (Hu et al., 2018). Herein, we sought to investigate whether intranasal delivery of lincRNA-Cox2-siRNA-loaded astrocyte-derived EVs could decrease microglial proliferation in LPS-administrated mice. To this end, we first sought to isolate EVs from conditioned media of mouse primary astrocytes using a differential ultracentrifugation procedure. Purified EVs were characterized by western blot for signature exosomal markers - Alix, CD63, and TSG101 as well as the endoplasmic reticulum protein - Calnexin (non-exosomal protein). As shown in Figure 3A, immunoblotting of the EV lysates revealed the presence of exosomal markers Alix, CD63, and TSG101 rather than Calnexin. Purified EVs were also characterized by transmission electron microscopy (TEM). As shown in Figure 3B, TEM images showed spherical-shaped EVs ranging in size from 40–100 nm in diameter. Additionally, the number and size distribution of EVs were further validated by ZetaView, which showed a reasonable yield of EVs with the expected size [~ 100nm; Figure 3C]. To test whether EVs can be used for the delivery of siRNA, we first transfected EVs with cy5 labeled small RNAs using Exo-Fect followed by purification with ExoQuick and treated mouse primary microglial cells with the cy5-RNA loaded EVs for 20 minutes followed by immunostaining and confocal microscopy. As shown in Figure 3D, EVs efficiently delivered small RNAs into microglial cells, however, in absence of EVs, labeled RNAs were not present in the microglia. To further examine the efficiency of EV-mediated siRNA delivery, we treated mouse primary microglial cells with EVs loaded with lincRNA-Cox2-siRNA for 24h followed by assessment for the expression of lincRNA-Cox2 using qPCR. As shown in Figure 3E, EV-mediated delivery of lincRNA-Cox2-siRNA resulted in significant downregulation of lincRNA-Cox2 in mouse primary microglia. These results are consistent with previous report (Aqil et al., 2019) and thus suggest that EVs can be used as delivery vehicles of siRNA.

Figure 3. EVs can be used for the delivery of siRNA.

Figure 3.

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(A) EVs were characterized by western blot for exosomal markers —Alix, CD63, TSG101. Calnexin (non-exosomal protein) was used as negative control. (B) Representative transmission electron microscopy (TEM) observation of EVs, Scale bar= 100nm. (C) Size and particle distribution plots of isolated EVs from cell culture by ZetaView nanosight tracking analysis (NTA). EVs are visualized in the inset image - a screenshot of a video generated from the ZetaView NTA showing light scattering of EVs. (D) EVs loaded with Cy5 labeled small RNA were taken up by mouse primary microglia. (E) Mouse primary microglial cells were treated with lincRNA-Cox2-siRNA loaded EVs for 24h followed by assessment for the expression of lincRNA-Cox2 by qPCR. Data are mean ± SEM of six independent experiments. Significant differences (two-tailed t-test), ****p < 0.0001; ns, not significant.

Intranasal delivery of lincRNA-Cox2-siRNA loaded EVs decreased LPS-induced microglial proliferation in mice

We next sought to test the biodistribution of intranasally administered EVs. Mice were administered EVs labeled with the PKH26-dye intranasally once a day for four days at which time animals were transcardially perfused with ice-cold PBS, followed by tissue harvesting and assessing biodistribution using in vivo imaging system (IVIS). As shown in Figure 4A, PKH26+ EVs were primarily localized in the lungs, brain, gut, liver, and heart following 4-day EV administration. Furthermore, as we reported previously (Hu et al., 2018) and as shown in Figure 4B, immunostaining of the brain sections of mice demonstrated the presence of PKH26+/Iba1+ microglia in the brain. Next, mice were intranasally administered lincRNA-Cox2-siRNA-loaded EVs, once a day for four consecutive days. On day 2, mice received a single intraperitoneal (i.p.) injection of LPS (5mg/kg) or an equivalent volume of saline. Mice also received injections of BrdU (dissolved in 0.9% NaCl, 100 mg/kg body weight, Sigma-Aldrich) for 3 consecutive days (day2-day4). One day after the last BrdU application, animals were subjected to transcardial perfusion with ice-cold PBS, and brains were processed for RNA fluorescent in situ hybridization (FISH) and immunohistochemistry. As shown in Figure 4C, there was a significant increase in the expression of lincRNA-Cox2 in the microglia of LPS-administered mice compared with that in saline control mice. As expected, intranasal delivery of lincRNA-Cox2-siRNA loaded EVs significantly decreased LPS-induced expression of lincRNA-Cox2 in vivo. Interestingly, there was a significant increase in the number of BrdU+/Iba-1+ microglia in LPS-administrated mice compared with that in saline-injected controls, whereas intranasal delivery of lincRNA-Cox2-siRNA loaded EVs significantly decreased LPS-induced microglial proliferation in mice (Figure 4D and 4E), suggesting EV-mediated lincRNA-Cox2-siRNA delivery inhibits LPS-induced microglial proliferation in the brain.

Figure 4. Intranasal delivery of lincRNA-Cox2-siRNA loaded EVs decreases LPS-induced microglial proliferation in mice.

Figure 4.

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(A) Biodistribution of PKH26 labeled EVs in the tissues of mice using IVIS (n=3). (B) Immunostaining of the brain sections of mice administered PKH26-labeled EVs for the microglial marker Iba1 (green). Representative micrographs are shown (original magnification ×40). Scale bar represents 10 μm. (C) Fluorescent images of mouse brain section triple-labeled for lincRNA-Cox2 and microglial cells (Iba-1) with DAPI. (D) Fluorescent images of Iba-1, BrdU, and DAPI in the mouse thalamus. (E) Quantification of Iba-1 and BrdU cofluorescence of sections as in (C).Scale bar represents 25μm in (C) and (D). Data are mean ± SEM of at least three independent experiments. Nonparametric Kruskal-Wallis one-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance between multiple groups. *p < 0.05 versus control. #p < 0.05 versus control-siRNA + LPS group.

Discussion

Multiple lines of evidence have demonstrated that lincRNA-Cox2 regulates distinct sets of genes, such as immune genes, in phagocytic cells (Carpenter et al., 2013; Hu et al., 2016; Elling et al., 2018; Xue et al., 2019). More importantly, the regulatory role of lincRNA-Cox2 in controlling immune genes was also observed in multiple models of lincRNA-Cox2-deficient mice (Elling et al., 2018). Herein, we reveal that lincRNA-Cox2 regulates microglial proliferation by controlling the expression of a set of cell cycle genes. Loss-of-function analyses suggested that knocking down lincRNA-Cox2 reversed LPS-induced microglial proliferation in vitro and in vivo, and importantly, EV-based delivery of lincRNA-Cox2-siRNA shows the therapeutic potentials for neurodegenerative disorders.

Microglia are the professional phagocytes of the brain and play an essential role in maintaining homeostasis within the brain (Kettenmann et al., 2011). Under pathological conditions, microglia undergo a series of morphological and functional changes, including proliferation, morphological alterations, migration and phagocytosis (Wolf et al., 2017). Microglial proliferation is one of the very early events in the development of various CNS diseases. Although microglia are considered as neuroprotective cells of the CNS, in a mouse model of AD, chronic microglial elimination rescued dendritic spine loss and prevented neuronal loss while also reducing overall neuroinflammation (Spangenberg et al., 2016). Similarly, to study the role of microglia in experimental autoimmune encephalomyelitis, Heppner et al. generated CD11b-HSV-TK mice as a model of microglial paralysis. These mice express a lineage-restricted Herpes virus thymidine kinase (HSV-TK) suicide gene driven by the CD11b promoter, so that administration of ganciclovir will lead to microglial paralysis. Using this model, the authors have demonstrated that microglial paralysis resulted in delayed onset, reduced clinical signs and a strong reduction of CNS inflammation (Heppner et al., 2005). Microglia could thus be considered as promising targets for therapeutic intervention (Cartier et al., 2014). Herein, we identified a novel role of lincRNA-Cox2 in controlling microglial proliferation. We also demonstrated that knockdown of lincRNA-Cox2 reduced LPS-induced microglial proliferation both in vitro and in vivo. Of note, knockdown of lincRNA-Cox2 significantly altered the expression of cell cycle genes in unstimulated microglial cells compared with control-siRNA-transfected cells (Figure 1), which is consistent with previous reports (Carpenter et al., 2013; Hu et al., 2016), and suggests that endogenous basal levels of lincRNA-Cox2 are essential for controlling the transcription of other genes, such as cell cycle and inflammatory genes. However, knockdown of lincRNA-Cox2 did not significantly affect microglial proliferation in unstimulated cells (Figure 2), indicating there is litter or no microglial proliferation under normal conditions.

The bioavailability and innate biological nature of EVs make them promising vehicles for drug delivery. Indeed, Alvarez-Erviti et al. (Alvarez-Erviti et al., 2011) have successfully delivered GAPDH-siRNA via intravenously injected EVs specifically to neurons, microglia, and oligodendrocytes in the brain, resulting in a tissue-specific gene knockdown. In addition, intranasal administration of EVs is considered as an effective and less invasive method for drug delivery into the brain (Visweswaraiah et al., 2002; Lakhal and Wood, 2011; Zhuang et al., 2011; Grassin-Delyle et al., 2012). We have recently developed an EV-based strategy of in vivo siRNA delivery into the CNS (Hu et al., 2018). In the current study, after determining the roles of lincRNA-Cox2 in LPS-mediated microglial proliferation in vitro, we applied a similar approach to deliver EVs loaded with lincRNA-Cox2-siRNA to LPS-administered mice. We found that lincRNA-Cox2-siRNA-loaded EVs could reach the brain and be taken up by microglia resulting in a significant decrease of LPS-induced lincRNA-Cox2 expression and microglial proliferation in LPS-administered mice compared with control mice.

In conclusion, our findings suggest that lincRNA-Cox2 is a key regulator of cell cycle genes in microglia, and LPS-dysregulated microglial proliferation can be ameliorated by the knockdown of lincRNA-Cox2 via intranasal delivery of lincRNA-Cox2-siRNA loaded EVs. These findings could shed light on the development of EV-loaded RNA drug target(s) as therapeutics for a multitude of CNS disorders involving microglial deficits.

Acknowledgments

This work was supported by grants DA042704, DA046831, MH112848, and DA043138 from the National Institutes of Health (NIH). The support of the Nebraska Center for Substance Abuse Research is acknowledged. The project described was also supported by the NIH, National Institute of Mental Health, 2P30MH062261. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We would like to thank Dr. Shilpa Buch for highly useful comments and suggestions that greatly improved the manuscript. Also, we are grateful to Shannon Callen for comments.

Footnotes

Publisher's Disclaimer: This Author Accepted Manuscript is a PDF file of a an unedited peer-reviewed manuscript that has been accepted for publication but has not been copyedited or corrected. The official version of record that is published in the journal is kept up to date and so may therefore differ from this version.

Conflicts of Interest

The authors have no conflicts of interest.

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