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Under stress, the heart undergoes extensive remodeling resulting in cardiac fibrosis and hypertrophy, ultimately contributing to chronic heart failure (CHF). Alterations in microRNA levels are associated with dysfunctional gene expression profiles involved in the pathogenesis of heart failure. We previously showed that myocardial infarction-induced microRNA-enriched extracellular vesicles (EVs) contribute to the reduction in antioxidant enzymes by targeting Nrf2 signaling in CHF. MicroRNA-27a (miRNA-27a) is the predominant microRNA contained in cardiac fibroblast-derived EVs contributing to oxidative stress along with hypertrophic gene expression in cardiomyocytes. In the present study, we observed that miRNA-27a passenger strand (miRNA-27a*) was markedly upregulated in the non-infarcted area of the left ventricle of rats with CHF and encapsulated into EVs and secreted into the circulation. Bioinformatic analysis revealed that PDZ and LIM domain 5 (PDLIM5) is one of the major targets of miRNA-27a*, playing a major role in cardiac structure and function, and potentially contributing to the progression of cardiac hypertrophy. Our in vivo data demonstrate that PDLIM5 is down-regulated in the progression of heart failure, accompanied with the upregulation of hypertrophic genes and consistent with alterations in miRNA-27a*. Moreover, exogenous administration of miRNA27a* mimics inhibit PDLIM5 translation in cardiomyocytes whereas a miRNA27a* inhibitor enhanced PDLIM5 expression. Importantly, we confirmed that infarcted hearts have higher abundance of miRNA-27a* in EVs compared to normal hearts and further demonstrated that cultured cardiac fibroblasts secrete miRNA27a*-enriched EVs into the extracellular space in response to Angiotensin II stimulation, which inhibited PDLIM5 translation, leading to cardiomyocyte hypertrophic gene expression. In vivo studies suggest that the administration of a miRNA-27a* inhibitor in CHF rats partially blocks endogenous miR-27a* expression, prevents hypertrophic gene expression and improves myocardial contractility. These findings suggest that cardiac fibroblast-secretion of miRNA27a*-enriched EVs may act as a paracrine signaling mediator of cardiac hypertrophy that has potential as a novel therapeutic target.

Alzheimer's disease (AD) is an age- and gender-associated brain disorder. Long noncoding RNAs (lncRNAs) have emerged as key regulators of brain development, homeostasis, and pathologies. Here, we used gene array data sets and bioinformatics analysis to identify differentially expressed age- and gender-associated lncRNAs in human AD brains. We found that the expressions of 16 age-associated and 13 gender-associated lncRNAs were dysregulated in AD brains. Notably, the expressions of age-associated lncRNAs-SNHG19 and LINC00672-were significantly correlated with Braak stage of AD, positively and negatively, respectively, whereas the expressions of gender-associated lncRNAs-RNF144A-AS1, LY86-AS1, and LINC00639-were negatively correlated with Braak stage of AD. Functional analysis suggests that the pathways involved in neurodegenerative diseases, synaptic vesicle cycle, and endocytosis were overly represented within age- and gender-associated lncRNA-correlating genes. The identification of age- and gender-associated lncRNAs and their differential expressions in the human AD brain provide potential targets for further experimental validation and mechanistic investigation, which could, in turn, pave the way for developing age- and gender-specific prevention and adjunctive therapeutic options for patients with AD.

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.

Long noncoding RNAs (lncRNAs) are large RNA transcripts that do not code for proteins but exert their effects in the form of RNA. To date many thousands of lncRNAs have been identified, their molecular functions and mechanisms of action however are largely unknown. The development of high-throughput experimental technologies, such as ChIRP (Chromatin isolation by RNA purification), CHART (Capture Hybridization Analysis of RNA Targets), RAP (RNA antisense purification), RIP (RNA Immunoprecipitation), CLIP (cross-linking and immunoprecipitation) and RNA pull-down, has led to a rapid expansion of lncRNA research and resulted in many publicly-available databases. This review provides an overview of the current methodologies available for discovering and investigating functions of lncRNAs in various human diseases. A comparison and application of these methods are also included. Finally, this paper surveys current databases containing annotations, interactome networks and functions of lncRNAs. The appropriate use of these methods and databases will provide not only high-resolution functional features of lncRNAs, but also enhance our understanding of the underlying mechanisms by which lncRNAs regulate a variety of biological processes.

In this study, Niu et al. suggest a role for pericytes in cocaine-mediated neuroinflammation. Exposure of pericytes to cocaine results in dose- and time-dependent release of CXCL10, which in turn leads to a concomitant induction of monocyte transmigration mediated by the σ-1R–Src–PDGFR-β–NF-κB axis., Cocaine is known to facilitate the transmigration of inflammatory leukocytes into the brain, an important mechanism underlying neuroinflammation. Pericytes are well-recognized as important constituents of the blood–brain barrier (BBB), playing a key role in maintaining barrier integrity. In the present study, we demonstrate for the first time that exposure of human brain vascular pericytes to cocaine results in enhanced secretion of CXCL10, leading, in turn, to increased monocyte transmigration across the BBB both in vitro and in vivo. This process involved translocation of σ-1 receptor (σ-1R) and interaction of σ-1R with c-Src kinase, leading to activation of the Src–PDGFR-β–NF-κB pathway. These findings imply a novel role for pericytes as a source of CXCL10 in the pericyte–monocyte cross talk in cocaine-mediated neuroinflammation, underpinning their role as active components of the innate immune responses.

Background Morphine is used extensively in the clinical setting owing to its beneficial effects, such as pain relief; its therapeutic utility is limited because the prolonged use of morphine often results in tolerance and addiction. Astrocytes in the brain are a direct target of morphine action and play an essential role in the development of morphine tolerance. Primary cilia and the cilia-mediated sonic hedgehog (SHH) signaling pathways have been shown to play a role in drug resistance and morphine tolerance, respectively. Extracellular vesicles (EVs) play important roles as cargo-carrying vesicles mediating communication among cells and tissues. Methods C57BL/6N mice were administered morphine for 8 days to develop tolerance, which was determined using the tail-flick and hot plate assays. EVs were separated from astrocyte-conditioned media using either size exclusion chromatography or ultracentrifugation approaches, followed by characterization of EVs using nanoparticle tracking analysis for EV size distribution and number, Western blotting for EV markers, and electron microscopy for EV morphology. Astrocytes were treated with EVs for 24 hours, followed by assessing primary cilia by fluorescent immunostaining for primary cilia markers (ARL13B and acetylated tubulin). Results Morphine-tolerant mice exhibited an increase in primary cilia length and percentage of ciliated astrocytes. The levels of SHH protein were upregulated in morphine-stimulated astrocyte-derived EVs. SHH on morphine-stimulated astrocyte-derived EVs activated SHH signaling in astrocytes through primary cilia. Our in vivo study demonstrated that inhibition of either EV release or primary cilia prevents morphine tolerance in mice. Conclusions EV-mediated primary ciliogenesis contributes to the development of morphine tolerance.

Opioids, such as morphine, are the mainstay for the management of postsurgical pain. Over the last decade there has been a dramatic increase in deaths related to opioid overdose. While opioid abuse has been shown to result in increased neuroinflammation, mechanism(s) underlying this process, remain less understood. In recent years, microRNAs have emerged as key mediators of gene expression regulating both paracrine signaling and cellular crosstalk. MiRNAs constitute the extracellular vesicle (EV) cargo and can shuttle from the donor to the recipient cells. Exposure of human primary astrocytes to morphine resulted in induction and release of miR‐138 in the EVs isolated from conditioned media of cultured astrocytes. Released EVs were, in turn, taken up by the microglia, leading to activation of these latter cells. Interestingly, activation of microglia involved binding of the GUUGUGU motif of miR138 to the endosomal toll like receptor (TLR)7, leading, in turn, to cellular activation. These findings were further corroborated in vivo in wildtype mice wherein morphine administration resulted in increased microglial activation in the thalamus. In TLR7−/− mice on the other hand, morphine failed to induce microglial activation. These findings have ramifications for the development of EV‐loaded anti‐miRNAs as therapeutics for alleviating neuroinflammation in opioids abusers.

MicroRNA-124 (miR-124), a brain-enriched microRNA, is known to regulate microglial quiescence. Psychostimulants such as cocaine have been shown to activate microglia by downregulating miR-124, leading, in turn, to neuroinflammation. We thus rationalized that restoring the levels of miR-124 could function as a potential therapeutic approach for cocaine-mediated neuroinflammation. Delivering miRNA based drugs in the brain that are effective and less invasive, however, remains a major challenge in the field. Herein we engineered extracellular vesicles (EVs) and loaded them with miR-124 for delivery in the brain. Approach involved co-transfection of mouse dendritic cells with Dicer siRNA and RVG-Lamp2b plasmid to deplete endogenous miRNAs and for targeting the CNS, respectively. Mouse primary microglia (mPm) were treated with purified engineered EVs loaded with either Cy5-miR-124 or Cy5-scrambled miRNA oligos in the presence or absence of cocaine followed by assessing EV uptake and microglial activation. In vivo studies involved pretreating mice intranasally with engineered EVs followed by cocaine injection (20 mg/kg, i.p.). mPm exposed to EV-miR-124 exhibited reduced expression of miR-124 targets – TLR4 and STAT3 as well as ERK-1/2 and Iba1. In cocaine administered mice, EV-Cy5-miR-124 delivered intranasally were detected in the CNS and significantly reduced the expression of inflammatory markers TLR4, MYD88, STAT3 and NF-kB p65 while also downregulating the microglial activation marker, Iba1. Collectively, these findings suggest that engineered EVs can deliver miR-124 into the CNS, thereby alleviating cocaine-mediated microglial activation. Manipulating EV miRNAs can thus be envisioned as an efficient means for delivery of RNA-based therapeutics to target organs.

Almost all types of cells release extracellular vesicles (EVs) into the extracellular space. EVs such as exosomes and microvesicles are membrane-bound vesicles ranging in size from 30 to 1000 nm in diameter. Under normal conditions, EVs mediate cell to cell as well as inter-organ communication via the shuttling of their cargoes which include RNA, DNA and proteins. Under pathological conditions, however, the number, size and content of EVs are found to be altered and have been shown to play crucial roles in disease progression. Emerging studies have demonstrated that EVs are involved in many aspects of viral infection-mediated neurodegenerative diseases. In the current review, we will describe the interactions between EV biogenesis and the release of virus particles while also reviewing the role of EVs in various viral infections, such as HIV-1, HTLV, Zika, CMV, EBV, Hepatitis B and C, JCV, and HSV-1. We will also discuss the potential uses of EVs and their cargoes as biomarkers and therapeutic vehicles for viral infections.

Gastric epithelial cells (GES-1) stimulated by Helicobacter pylori (H. pylori) would affect the expression of related genes and induce the immune response of the cells. Abnormal methylation of DNA was one of the main causes. The aim of this study was to investigate phosphoinositol-3-kinase adaptor protein 1(PIK3AP1), which was screened from the chip data as an immune gene candidate to against the inflammatory response of cells caused by H. pylori infection. PIK3AP1 plays a key role in PI3K/AKT signaling pathway. The gene chip analysis and experimental results confirmed that PIK3AP1 expression was downregulated and PIK3AP1 promoter was hypermethylated after H. pylori stimulation in GES-1 cells. Meanwhile, the expression level of PIK3AP1 was significantly upregulated after 5-aza-dc treatment, and its expression was higher after 5-aza-dc and H. pylori co-treatment than that of H. pylori treatment but lower than that of 5-aza-dc treatment. Therefore, hypermethylation was the main reason for the down-regulation of PIK3AP1 after H. pylori stimulation. In addition, the intervention of PIK3AP1 inhibited the expression of downstream gene AKT, and suppressing the expression of the immunoinflammatory gene IL-6 in GES-1 cells. Furthermore, the intervention of PIK3AP1 would promote cell proliferation. In summary, hypermethylation of the PIK3AP1 promoter was accompanied by reduction of the expression level of PIK3AP1 in GES-1 cells by H. pylori stimulation. The expression of PIK3AP1, AKT, and IL-6 genes was positively correlated, Meanwhile, the PIK3AP1 can affect the proliferation of GES-1 cells. These results would be helpful to understand the innate immune response function of PIK3AP1 to pathogenic bacterial infection in the stomach.

Long intergenic noncoding RNAs (lincRNAs) are long noncoding transcripts (>200 nt) from the intergenic regions of annotated protein-coding genes. We report here that the lincRNA gene lincRNA-Tnfaip3, located at mouse chromosome 10 proximal to the tumor necrosis factor α-induced protein 3 (Tnfaip3) gene, is an early-primary response gene controlled by nuclear factor-κB (NF-κB) signaling in murine macrophages. Functionally, lincRNA- Tnfaip3 appears to mediate both the activation and repression of distinct classes of inflammatory genes in macrophages. Specifically, induction of lincRNA-Tnfaip3 is required for the transactivation of NF-κB-regulated inflammatory genes in response to bacterial LPSs stimulation. LincRNA-Tnfaip3 physically interacts with the high-mobility group box 1 (Hmgb1), assembling a NF-κB/Hmgb1/lincRNA-Tnfaip3 complex in macrophages after LPS stimulation. This resultant NF-κB/Hmgb1/lincRNA-Tnfaip3 complex can modulate Hmgb1-associated histone modifications and, ultimately, transactivation of inflammatory genes in mouse macrophages in response to microbial challenge. Therefore, our data indicate a new regulatory role of NF-κB-induced lincRNA-Tnfaip3 to act as a coactivator of NF-κB for the transcription of inflammatory genes in innate immune cells through modulation of epigenetic chromatin remodeling.—Ma, S., Ming, Z., Gong, A.-Y., Wang, Y., Chen, X., Hu, G., Zhou, R., Shibata, A., Swanson, P. C., Chen, X.-M. A long noncoding RNA, LincRNA-Tnfaip3, acts as a coregulator of NF-κB to modulate inflammatory gene transcription in mouse macrophages.

Although combination antiretroviral therapy (cART) has improved the health of millions of those living with HIV-1 (Human Immunodeficiency Virus, Type 1), the penetration into the central nervous system (CNS) of many such therapies is limited, thereby resulting in residual neurocognitive impairment commonly referred to as NeuroHIV. Additionally, while cART has successfully suppressed peripheral viremia, cytotoxicity associated with the presence of viral Transactivator of transcription (Tat) protein in tissues such as the brain, remains a significant concern. Our previous study has demonstrated that both HIV-1 Tat as well as opiates such as morphine, can directly induce synaptic alterations via independent pathways. Herein, we demonstrate that exposure of astrocytes to HIV-1 protein Tat mediates the induction and release of extracellular vesicle (EV) microRNA-7 (miR-7) that is taken up by neurons, leading in turn, to downregulation of neuronal neuroligin 2 (NLGN2) and ultimately to synaptic alterations. More importantly, we report that these impairments could be reversed by pretreatment of neurons with a neurotrophic factor platelet-derived growth factor-CC (PDGF-CC). Graphical Abstract.

Helicobacter pylori ( H. pylori ) is a spiral shaped gram-negative bacterium that induces immune responses in the gastric mucosa. Toll-like receptors (TLRs) play important roles in mediating inflammatory cytokines by recognition of conserved molecular patterns on bacteria. Changes in the expression of toll-like receptor (TLR) 2, TLR4 and the relative inflammatory cytokines were analyzed in normal gastric epithelial GES-1 cells following treatment with H. pylori or Escherichia coli lipopolysaccharide ( E. coli LPS) in order to investigate the contribution of TLRs in recognizing and mediating the inflammatory response to H. pylori , and study the differences in TLRs’ performance between H. pylori and E. coli . Specific inhibitors for the declines in TLR2 and TLR4 were also employed. The results showed that H. pylori infection increased TLR2 expression in GES-1 cells, but TLR4 remained unchanged regardless of H. pylori or TLR2 small interfering RNA treatment. Furthermore, the secretion of cyclooxygenase-2 (COX-2) induced by H. pylori was inhibited by declines in TLR2, but not in TLR4. In conclusion, TLR2 plays an even more important role than TLR4 not only in recognizing H. pylori , but also in the induction of inflammatory cytokines initiated by H. pylori . However, both TLR2 and TLR4 are necessary in mediating the inflammatory response to E. coli LPS.

Diminished adult neurogenesis is known to play a key role in the pathogenesis of diverse neurodegenerative disorders such as HIV-associated neurological disorders (HAND). Cocaine, often abused by HIV-infected patients, has been suggested to worsen HIV-associated CNS disease. Mounting evidence also indicates that HIV infection can lead not only to neuronal dysfunction or loss, but can also negatively impact neurogenesis, resulting in generation of fewer adult neural progenitor cells (NPCs) in the dentate gyrus of the hippocampus, brain area critical for memory and learning. The crucial role of platelet-derived growth factor-BB (PDGF-BB) in providing tropic support for the neurons as well as in promoting NPC proliferation has been demonstrated by us previously. However, whether PDGF-BB regulates neuronal differentiation especially in the context of HAND and drug abuse remains poorly understood. In this study, we demonstrate that pretreatment of rat hippocampal NPCs with PDGF-BB restored neuronal differentiation that had been impaired by HIV Tat and cocaine. To further study the intracellular mechanism(s) involved in this process, we examined the role of transient receptor potential canonical (TRPC) channels in mediating neuronal differentiation in the presence of PDGF-BB. TRPC channels are Ca2+-permeable, nonselective cationic channels that elicit a variety of physiological functions. Parallel but distinct ERK, Akt signaling pathways with downstream activation of CREB were found to be critical for neuronal differentiation. Pharmacological blocking of TRPC channels resulted in suppression of PDGF-mediated differentiation and PDGF-BB-induced activation of ERK and Akt, culminating also to inhibition of PDGF-induced activation of CREB. Taken together, these findings underpin the role of TRPC channel as a novel target regulating cell differentiation mediated by PDGF-BB. This finding could have implications for development of therapeutic interventions aimed at restoration of Tat and cocaine-mediated impairment of neurogenesis in drug abusing HAND patients.

Increased life expectancy of patients diagnosed with HIV in the current era of antiretroviral therapy is unfortunately accompanied with the prevalence of HIV-associated neurocognitive disorders (HANDs) and risk of comorbidities such as Alzheimer-like pathology. HIV-1 transactivator of transcription (Tat) protein has been shown to induce the production of toxic neuronal amyloid protein and also enhance neurotoxicity. The contribution of astrocytes in Tat-mediated amyloidosis remains an enigma. We report here, in simian immunodeficiency virus (SIV)+ rhesus macaques and patients diagnosed with HIV, brain region–specific up-regulation of amyloid precursor protein (APP) and Aβ (40 and 42) in astrocytes. In addition, we find increased expression of β-site cleaving enzyme (BACE1), APP, and Aβ in human primary astrocytes (HPAs) exposed to Tat. Mechanisms involved up-regulation of hypoxia-inducible factor (HIF-1α), its translocation and binding to the long noncoding RNA (lncRNA) BACE1‐antisense transcript (BACE1-AS), resulting, in turn, in the formation of the BACE1-AS/BACE1 RNA complex, subsequently leading to increased BACE1 protein, and activity and generation of Aβ-42. Gene silencing approaches confirmed the regulatory role of HIF-1α in BACE1-AS/BACE1 in Tat-mediated amyloidosis. This is the first report implicating the role of the HIF-1α/lncRNABACE1-AS/BACE1 axis in Tat-mediated induction of astrocytic amyloidosis, which could be targeted as adjunctive therapies for HAND-associated Alzheimer-like comorbidity., This study shows that astrocytes contribute to the Alzheimer-like pathology seen in the brains of individuals with HIV, and that this is regulated by the transcription factor HIF-1α and the lncRNA BACE1-AS and can contribute to HIV-associated neurocognitive disorders.

The blood-brain barrier (BBB) is a tight barrier that is critical for preventing the entry of pathogens and small molecules into the brain. HIV protein Tat (Tat) is known to disrupt the tight junctions of the BBB. Autophagy is an intracellular process that involves degradation and recycling of damaged organelles to the lysosome and has recently been implicated in the BBB disruption. The role of autophagy in Tat-mediated BBB disruption, however, remains elusive. Herein we hypothesized that Tat induces endothelial autophagy resulting in decreased expression of the tight junction protein ZO-1 leading to breach of the BBB. In this study, we demonstrated that exposure of human brain microvessel endothelial cells (HBMECs) to Tat resulted in induction of autophagy in a dose- and time-dependent manner, with upregulation of BECN1/Beclin 1, ATG5 and MAP1LC3B proteins and a concomitant downregulation of the tight junction protein ZO-1 ultimately leading to increased endothelial cell monolayer paracellular permeability in an in vitro BBB model. Pharmacological and genetic inhibition of autophagy resulted in the abrogation of Tat-mediated induction of MAP1LC3B with a concomitant restoration of tight junction proteins, thereby underscoring the role of autophagy in Tat-mediated breach of the BBB. Additionally, our data also demonstrated that Tat-mediated induction of autophagy involved PELI1/K63-linked ubiquitination of BECN1. Increased autophagy and decreased ZO-1 was further recapitulated in microvessels isolated from the brains of HIV Tg26 mice as well as the frontal cortex of HIV-infected autopsied brains. Overall, our findings identify autophagy as an important mechanism underlying Tat-mediated disruption of the BBB.

Despite the success of combination antiretroviral therapy (cART), there is increased prevalence of HIV-associated neurocognitive disorders (HAND) in HIV-1-infected individuals on cART, which poses a major health care challenge. Adding further complexity to this long-term antiretroviral use is the comorbidity with drugs of abuse such as morphine, cocaine, and methamphetamine, which can in turn, exacerbate neurologic and cognitive deficits associated with HAND. Furthermore, HIV proteins, such as the transactivator of transcription (Tat) and the envelope protein (gp120), as well as antiretrovirals themselves can also contribute to the progression of neurodegeneration underlying HAND. In the field of NeuroHIV and drug addiction, EVs hold the potential to serve as biomarkers of cognitive dysfunction, targets of therapy, and as vehicles for therapeutic delivery of agents that can ameliorate disease pathogenesis. Based on the success of a previous Satellite Symposium in 2015 at the ISEV meeting in Washington, experts again expanded on their latest research findings in the field, shedding light on the emerging trends in the field of Extracellular Vesicle (EV) biology in NeuroHIV and drug abuse. The satellite symposium sought to align experts in the fields of NeuroHIV and drug abuse to share their latest insights on the role of EVs in regulating neuroinflammation, neurodegeneration, peripheral immune response, and HIV latency in HIV-infected individuals with or without the comorbidity of drug abuse.

Chronic neuroinflammation still remains a common underlying feature of HIV-infected patients on combined anti-retroviral therapy (cART). Previous studies have reported that despite near complete suppression of virus replication by cART, cytotoxic viral proteins such as HIV trans-activating regulatory protein (Tat) continue to persist in tissues such as the brain and the lymph nodes, thereby contributing, in part, to chronic glial activation observed in HIV-associated neurological disorders (HAND). Understanding how the glial cells cross talk to mediate neuropathology is thus of paramount importance. MicroRNAs (miR) also known as regulators of gene expression, have emerged as key paracrine signaling mediators that regulate disease pathogenesis and cellular crosstalk, through their transfer via the extracellular vesicles (EV). In the current study we have identified a novel function of miR-9, that of mediating microglial migration. We demonstrate that miR-9 released from Tat-stimulated astrocytes can be taken up by microglia resulting in their migratory phenotype. Exposure of human astrocytoma (A172) cells to HIV Tat resulted in induction and release of miR-9 in the EVs, which, was taken up by microglia, leading in turn, increased migration of the latter cells, a process that could be blocked by both an exosome inhibitor GW4869 or a specific target protector of miR-9. Furthermore, it was also demonstrated that EV miR-9 mediated inhibition of the expression of target PTEN, via its binding to the 3’UTR seed sequence of the PTEN mRNA, was critical for microglial migration. To validate the role of miR-9 in this process, microglial cells were treated with EVs loaded with miR-9, which resulted in significant downregulation of PTEN expression with a concomitant increase in microglial migration. These findings were corroborated by transfecting microglia with a specific target protector of PTEN, that blocked miR-9-mediated downregulation of PTEN as well as microglial migration. In vivo studies wherein the miR-9 precursor-transduced microglia were transplanted into the striatum of mice, followed by assessing their migration in response to a stimulus administered distally, further validated the role of miR-9 in mediating microglial migration. Collectively, our findings provide evidence that glial crosstalk via miRs released from EVs play a vital role in mediating disease pathogenesis and could provide new avenues for development of novel therapeutic strategies aimed at dampening neuropathogenesis.

Salmonella enteritidis infection occurs in enterogenous diseases, such as gastroenteritis and parenteral focal infection, which often involve inflammation of intestinal epithelial cells. The nuclear factor kappa B (NF-κB) pathway participates in the innate immune response to many gram-negative pathogenic bacteria and initiates inflammation in epithelial cells. KH-type splicing regulatory protein (KSRP) is a multi-domain RNA-binding protein that recruits the exosome-containing mRNA degradation complex to mRNAs coding for inflammatory response factors. However, it remains unclear whether KSRP is regulated by NF-κB signaling pathway in response to S. enteritidis infection and affects the development of inflammation. Accordingly, in this study, we investigated the role of KSRP in mediating the response to S. enteritidis in Caco-2 cells. The data revealed that S. enteritidis infection decreased KSRP expression, which was suppressed by blocking the NF-κB pathway. Additionally, S. enteritidis infection significantly increased the expression of inducible nitric oxide synthase and cyclooxygenase-2. Overexpression of KSRP reduced the expression levels of inflammatory factors in Caco-2 cells. KSRP was regulated by the NF-κB signaling pathway and participated in mediating the innate immune response to S. enteritidis infection in Caco-2 cells, and KSRP acted as a negative regulator of inflammatory gene expression.

Aurora kinase B, playing a vital, important role in mitosis, is frequently detected to be overexpressed in many cancer cell lines and various tumor tissues, including prostatic carcinoma. Given the essential function of Aurora kinase B in mitosis and its association with tumorigenesis, it might be a drug target for prostatic carcinoma treatment. In our study, short hairpin RNA targeting Aurora kinase B was cloned into a pGPU6 plasmid vector and then transfected into human prostatic carcinoma cells. The expression level of Aurora kinase B was verified by reverse transcription-polymerase chain reaction and Western blot. At the same time, cell apoptosis was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, fluorescent staining, and flow cytometric analysis. Furthermore, prostate carcinoma cells were injected into mice to establish a tumor xenograft model. Previous studies have shown the effect of pGPU6-shAURKB plasmid on tumor growth in a prostate carcinoma xenogenic implantation model. From the study, we knew that the Aurora kinase B was significantly downregulated in prostate carcinoma cells, and cell apoptosis was also detected higher in treated groups than that in control groups. Moreover, in the prostate carcinoma xenogenic implantation model, compared with the control groups, the tumor growth was inhibited about 78.7% in the pGPU6-shAURKB plasmid–treated group, and cell apoptosis in the experimental group was notably higher than that in control groups. The average duration of tumor-bearing mice was prolonged to about 35 days. The results of experiment indicated that specific knockdown of Aurora kinase B led to prostate carcinoma cells apoptosis and inhibited tumor growth. Our data clearly confirmed that specific knockdown of Aurora kinase B expression by vector-based short hairpin RNA/liposome may be a potential new approach to treat human prostatic carcinoma.

Abuse of psychostimulants, such as cocaine, has been shown to be closely associated with complications of the lung, such as pulmonary hypertension, edema, increased inflammation, and infection. However, the mechanism by which cocaine mediates impairment of alveolar epithelial barrier integrity that underlies various pulmonary complications has not been well determined. Herein, we investigate the role of cocaine in disrupting the alveolar epithelial barrier function and the associated signaling cascade. Using the combinatorial electric cell-substrate impedance sensing and FITC-dextran permeability assays, we demonstrated cocaine-mediated disruption of the alveolar epithelial barrier, as evidenced by increased epithelial monolayer permeability with a concomitant loss of the tight junction protein zonula occludens-1 (Zo-1) in both mouse primary alveolar epithelial cells and the alveolar epithelial cell line, L2 cells. To dissect the signaling pathways involved in this process, we demonstrated that cocaine-mediated induction of permeability factors, platelet-derived growth factor (PDGF-BB) and vascular endothelial growth factor, involved reactive oxygen species (ROS)-dependent induction of hypoxia-inducible factor (HIF)-1α. Interestingly, we demonstrated that ROS-dependent induction of another transcription factor, nuclear factor erythroid-2-related factor-2, that did not play a role in cocaine-mediated barrier dysfunction. Importantly, this study identifies, for the first time, that ROS/HIF-1α/PDGF-BB autocrine loop contributes to cocaine-mediated barrier disruption via amplification of oxidative stress and downstream signaling. Corroboration of these cell culture findings in vivo demonstrated increased permeability of the alveolar epithelial barrier, loss of expression of Zo-1, and a concomitantly increased expression of both HIF-1α and PDGF-BB. Pharmacological blocking of HIF-1α significantly abrogated cocaine-mediated loss of Zo-1. Understanding the mechanism(s) by which cocaine mediates barrier dysfunction could provide insights into the development of potential therapeutic targets for cocaine-mediated pulmonary hypertension.

In the study we made use of DOTAP (1,2-dioleoyl-3-trimethylammonium), DOPE (1,2-dioleoyl-snglycero-3-phosphoethanolamine) and PEG-PE (polyethylene glycol- polyethylene) to make cationic PEG-liposomes by ultrasonic dispersion method. The plasmid pGPU6 combined with cationic PEG-liposomes or Liopofectamin 2000 was used to transfect PC3 cells to judge the transfection efficiency. HE staining showed that the pGUP6-shAurora B plasmid/liposomes complex could significantly inhibit tumor growth in mice tumor model. The results indicated that there was no remarkable difference between the homemade liposomes and Lipofectamin 2000 after transfection, with transfection efficiency over 80%. And the homemade liposomes also had high transfection efficiency in vivo. No significant side effects were observed on weight, coat condition, behavior or appetite and the life span of mice treated with pGPU6-shAurora B were extended. Beyond that, there were no differences in mortality or in pathological changes to the heart, liver, spleen, lungs and kidneys among all the mice.

Cai et al. demonstrate that morphine exposure dysregulates synaptic balance in the hippocampus. This effect involves a novel pathway involving ROS, ER stress, and autophagy and can be rescued by PDGF., Our findings suggest that morphine dysregulates synaptic balance in the hippocampus, a key center for learning and memory, via a novel signaling pathway involving reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and autophagy. We demonstrate in this study that exposure of morphine to hippocampal neurons leads to a reduction in excitatory synapse densities with a concomitant enhancement of inhibitory synapse densities via activation of the μ opioid receptor. Furthermore, these effects of morphine are mediated by up-regulation of intracellular ROS from NADPH oxidase, leading, in turn, to sequential induction of ER stress and autophagy. The detrimental effects of morphine on synaptic densities were shown to be reversed by platelet-derived growth factor (PDGF), a pleiotropic growth factor that has been implicated in neuroprotection. These results identify a novel cellular mechanism involved in morphine-mediated synaptic alterations with implications for therapeutic interventions by PDGF.

The majority of human miRNA genes is transcribed by polymerase II and can be classified as class II genes similar to protein-coding genes. Whereas current research on miRNAs has focused on the physiological and pathological functions, the molecular mechanisms underlying their transcriptional regulation are largely unknown. We recently reported that lipopolysaccharide (LPS) alters mature miRNA expression profile in human biliary epithelial cells. In this study, we tested the role of transcription factor NF-kappaB in LPS-induced transcription of select miRNA genes. Of the majority of LPS-up-regulated mature miRNAs in cultured human biliary epithelial cells, potential NF-kappaB binding sites were identified in the putative promoter elements of their corresponding genes. Inhibition of NF-kappaB activation by SC-514, an IKK2 inhibitor, blocked LPS-induced up-regulation of a subset of pri-miRNAs, including pri-miR-17-92, pri-miR-125b-1, pri-miR-21, pri-miR-23b-27b-24-1, pri-miR-30b, pri-miR-130a and pri-miR-29a. Moreover, direct binding of NF-kappaB p65 subunit to the promoter elements of mir-17-92, mir-125b-1, mir-21, mir-23b-27b-24-1, mir-30b and mir-130a genes was identified by chromatin immunoprecipitation analysis and confirmed by the luciferase reporter assay. Thus, a subset of miRNA genes is regulated in human biliary epithelial cells through NF-kappaB activation induced by LPS, suggesting a role of the NF-kappaB pathway in the transcriptional regulation of miRNA genes.

Exosomes are membrane-enriched extracellular vesicles with a proposed diameter in the range of 30–100 nm. They are released during both normal homeostasis as well as under pathological conditions by most cell types. In recent years, there has been robust interest in the study of these vesicles as conduits for the delivery of information between cells in both analogous as well as disparate tissues. Their ability to transport specialized cargo including signaling mediators, proteins, messenger RNA and miRNAs characterizes these vesicles as primary facilitators of cell-to-cell communication and regulation. Exosomes have also been demonstrated to have important roles in the field of cancer biology and metastasis. More recently, their role in several neurodegenerative disorders has been gaining increased momentum as these particles have been shown to promote the spread of toxic factors such as amyloid beta and prions, adding further validity to their role as important regulators of disease pathogenesis. This review briefly summarizes current findings and thoughts on exosome biology in the context of neurodegenerative disorders and the manipulation of these particles for the development of potential therapeutic strategies.

Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. They are speculated to transfer molecules to neighboring or distant cells and modulate many physiological and pathological procedures. Exosomes released from the gastrointestinal epithelium to the basolateral side have been implicated in antigen presentation. Here, we report that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum. Release of exosomes involves activation of TLR4/IKK2 signaling through promoting the SNAP23-associated vesicular exocytotic process. Downregulation of let-7 family miRNAs by activation of TLR4 signaling increases SNAP23 expression, coordinating exosome release in response to C. parvum infection. Intriguingly, exosomes carry antimicrobial peptides of epithelial cell origin, including cathelicidin-37 and beta-defensin 2. Activation of TLR4 signaling enhances exosomal shuttle of epithelial antimicrobial peptides. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. Direct binding to the C. parvum sporozoite surface is required for the anti-C. parvum activity of released exosomes. Biliary epithelial cells also increase exosomal release and display exosome-associated anti-C. parvum activity following LPS stimulation. Our data indicate that TLR4 signaling regulates luminal exosome release and shuttling of antimicrobial peptides from the gastrointestinal epithelium, revealing a new arm of mucosal immunity relevant to antimicrobial defense., Author Summary Exosomes are secreted membranous nanovesicles produced by a variety of cells. Exosomes shuttle various molecules to transfer them to neighboring or distant cells, and have been implicated as mediators in cell-cell communications to modulate physiological and pathological procedures. Here, we report that luminal release of exosomal vesicles is an important component of Toll-like receptor 4 (TLR4)-associated gastrointestinal epithelial defense against infection by Cryptosporidium parvum, an obligate intracellular protozoan that infects gastrointestinal epithelial cells. Activation of TLR4 signaling in host epithelial cells following C. parvum infection promotes luminal release of epithelial exosomes and exosomal shuttling of antimicrobial peptides from the epithelium. By direct binding to the C. parvum surface, exosomal vesicles reveal anti-C. parvum activity. Activation of TLR4 signaling in epithelial cells after LPS stimulation also increases exosomal release and exosome-associated anti-C. parvum activity. Therefore, we speculate that TLR4-mediated exosome release may be relevant to innate mucosal immunity in general, representing a new target for therapeutic intervention for infectious diseases at the mucosal surface.

Cardiac involvement is a well-documented complication of human immunodeficiency virus-1 (HIV-1) infection. Previous studies have demonstrated increased adhesion of monocytes to human vascular endothelial cells in HIV-infected individuals. HIV Tat protein, which is the transactivator of transcription (Tat), plays a key role in activating endothelial cells. In the present study, we demonstrated that exposure of HUVECs to HIV Tat protein resulted in induced expression of cell adhesion molecules specifically ICAM-1, leading to increased adhesion of monocytes to the endothelium. This effect of Tat was mediated through activation of mitogen-activated protein kinases and downstream transcription factor NF-κB. Increased expression of ICAM-1 was regulated by microRNA (miRNA) miR-221 and to some extent by miR-222, both of which are known to target ICAM-1. Functional inhibition of the respective miRNAs with anti-miR oligonucleotides resulted in induction of ICAM-1 protein in HUVECs. Furthermore, Tat-stimulated regulation of ICAM-1 via miR-221/-222 involved the NF-kB-dependent pathway. Functional implication and specificity of up-regulated ICAM-1 was confirmed using the ICAM-1 neutralizing antibody in the in vitro cell adhesion assays. These findings were further confirmed in vivo using the HIV transgenic (Tg) rats. These animals not only demonstrated increased expression of ICAM-1 mRNA, with a concomitant reduction in the expression of miR-221 in the aorta and heart, but also had increased expression of the ICAM-1 protein that was predominantly in the endothelial cell layer. Taken together, these findings implicate that Tat-mediated induction of ICAM-1 expression plays a critical role in monocyte adhesion observed in HIV-1-associated cardiomyopathies.

The signaling pathways associated with the Toll-like receptors (TLRs) and nuclear factor-kappaB (NF-κB) are essential to pro-inflammatory cytokine and chemokine expression, as well as initiating innate epithelial immune responses. The TLR/NF-κB signaling pathways must be stringently controlled through an intricate network of positive and negative regulatory elements. MicroRNAs (miRNAs) are non-coding small RNAs that regulate the stability and/or translation of protein-coding mRNAs. Herein we report that miR-16 promotes NF-κB-regulated transactivation of the IL-8 gene by suppression of the silencing mediator for retinoid and thyroid hormone receptor (SMRT). LPS stimulation activated miR-16 gene transcription in human monocytes (U937) and biliary epithelial cells (H69) through MAPK-dependent mechanisms. Transfection of cells with the miR-16 precursor promoted LPS-induced production of IL-8, IL-6, and IL-1α, without a significant effect on their RNA stability. Instead, an increase in NF-κB-regulated transactivation of the IL-8 gene was confirmed in cells following transfection of miR-16 precursor. Importantly, miR-16 targeted the 3′-untranslated region of SMRT and caused translational suppression of SMRT. LPS decreased SMRT expression via upregulation of miR-16. Moreover, functional manipulation of SMRT altered NF-κB-regulated transactivation of LPS-induced IL-8 expression. These data suggest that miR-16 targets SMRT and modulates NF-κB-regulated transactivation of the IL-8 gene.

Cryptosporidium parvum is a protozoan parasite that infects the gastrointestinal epithelium and causes diarrheal disease worldwide. Innate epithelial immune responses are key mediators of the host's defense to C. parvum. MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level and are involved in regulation of both innate and adaptive immune responses. Using an in vitro model of human cryptosporidiosis, we analyzed C. parvum-induced miRNA expression in biliary epithelial cells (i.e., cholangiocytes). Our results demonstrated differential alterations in the mature miRNA expression profile in cholangiocytes following C. parvum infection or lipopolysaccharide stimulation. Database analysis of C. parvum-upregulated miRNAs revealed potential NF-κB binding sites in the promoter elements of a subset of miRNA genes. We demonstrated that mir-125b-1, mir-21, mir-30b, and mir-23b-27b-24-1 cluster genes were transactivated through promoter binding of the NF-κB p65 subunit following C. parvum infection. In contrast, C. parvum transactivated mir-30c and mir-16 genes in cholangiocytes in a p65-independent manner. Importantly, functional inhibition of selected p65-dependent miRNAs in cholangiocytes increased C. parvum burden. Thus, we have identified a panel of miRNAs regulated through promoter binding of the NF-κB p65 subunit in human cholangiocytes in response to C. parvum infection, a process that may be relevant to the regulation of epithelial anti-microbial defense in general., Author Summary MicroRNAs (miRNAs) are newly identified small non-coding RNAs that regulate gene expression at the posttranscriptional level. While much of our understanding of the cellular processes modulated by miRNAs has come from studies on development and tumorigenesis, the role of miRNAs in immune responses is now being gradually uncovered. Nevertheless, whether miRNA-mediated posttranscriptional gene regulation is involved in the fine-tuning of epithelial cell immune responses against pathogen infection remains undefined. Cryptosporidium parvum is a protozoan parasite that infects gastrointestinal epithelium. TLR/NF-κB-mediated innate immune responses by epithelial cells are critical to the host's defense to infection. Using an in vitro model of human cryptosporidiosis, we show here differential alterations in the miRNA expression profile in biliary epithelial cells following C. parvum infection. Promoter binding of NF-κB p65 subunit accounts for the upregulation of a panel of miRNA genes in cells infected by C. parvum. Importantly, functional inhibition of several NF-κB p65-dependent miRNAs in epithelial cells increases C. parvum infection burden. Our findings suggest that host epithelial cells activate NF-κB signaling to regulate miRNA expression in response to C. parvum infection. Moreover, NF-κB-mediated miRNA expression is involved in epithelial anti-microbial defense. Our study provides new insights into epithelial cell immunoregulation.