Mitigation of cocaine-mediated mitochondrial damage,defective mitophagy and microglial activation by superoxide dismutase mimetics

ABSTRACT

Although cocaine exposure has been shown to potentiate neuroinflammation by upregulating glial activation in the brain, the role of mitophagy in this process remains an enigma. In the present study, we sought to examine the role of impaired mitophagy in cocaine-mediated activation of microglia and to determine the ameliorative potential of superoxide dismutase mimetics in this context. Our findings demonstrated that exposure of mouse primary microglial cells (mPMs) to cocaine resulted in decreased mitochondrial membrane potential, that was accompanied by increased expression of mitophagy markers, PINK1 and PRKN. Exposure of microglia to cocaine also resulted in increased expression of DNM1L and OPTN with a concomitant decrease in the rate of mitochondrial oxygen consumption as well as impaired mitochondrial functioning. Additionally, in the presence of cocaine, microglia also exhibited upregulated expression of autophagosome markers, BECN1, MAP1LC3B-II, and SQSTM1. Taken together, these findings suggested diminished mitophagy flux and accumulation of mitophagosomes in the presence of cocaine. These findings were further confirmed by imaging techniques such as transmission electron microscopy and confocal microscopy. Cocaine-mediated activation of microglia was further monitored by assessing the expression of the microglial marker (ITGAM) and the inflammatory cytokine (Tnf, Il1b, and Il6) mRNAs. Pharmacological, as well as gene-silencing approaches aimed at blocking both the autophagy/mitophagy and SIGMAR1 expression, underscored the role of impaired mitophagy in cocaine-mediated activation of microglia. Furthermore, superoxide dismutase mimetics such as TEMPOL and MitoTEMPO were shown to alleviate cocaine-mediated impaired mitophagy as well as microglial activation.

Abbreviations: 3-MA: 3-methyladenine; Δψm: mitochondrial membrane potential; ACTB: actin, beta; AIF1: allograft inflammatory factor 1; ATP: adenosine triphosphate; BAF: bafilomycin A₁; BECN1: beclin 1, autophagy related; CNS: central nervous system; DNM1L: dynamin 1 like; DMEM: Dulbecco modified Eagle medium; DAPI: 4,6-Diamidino-2-phenylindole; DRD2: dopamine receptor D2; ECAR: extracellular acidification rate; FBS: fetal bovine serum; FCCP: Trifluoromethoxy carbonylcyanide phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IL1B: interleukin 1, beta; IL6: interleukin 6; ITGAM: integrin subunit alpha M; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mPMs: mouse primary microglial cells; MRC: maximal respiratory capacity; NFKB: nuclear factor kappa B; NLRP3: NLR family pyrin domain containing 3; NTRK2: neurotrophic receptor tyrosine kinase 2; OCR: oxygen consumption rate; OPTN: optineurin; PBS: phosphate buffered saline; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TNF: tumor necrosis factor     

Vitamin D limits inflammation-linked microRNA expression in adipocytes in vitro and in vivo: A new mechanism for the regulation of inflammation by vitamin D

Inflammation of adipose tissue is believed to be a contributing factor to many chronic diseases associated with obesity. Vitamin D (VD) is now known to limit this metabolic inflammation by decreasing inflammatory marker expression and leukocyte infiltration in adipose tissue. In this study, we investigated the impact of VD on microRNA (miR) expression in inflammatory conditions in human and mouse adipocytes, using high-throughput methodology (miRNA PCR arrays). Firstly, we identified three miRs (miR-146a, miR-150, and miR-155) positively regulated by TNFα in human adipocytes. Interestingly, the expression of these miRs was strongly prevented by 1,25(OH)₂D preincubation. These results were partly confirmed in 3T3-L1 adipocytes (for miR-146a and miR-150). The ability of VD to control the expression of these miRs was confirmed in diet-induced obese mice: the levels of the three miRs were increased following high fat (HF) diet in epididymal white adipose tissue and reduced in HF diet fed mice supplemented with VD. The involvement of NF-κB signaling in the induction of these miRs was confirmed in vitro and in vivo using aP2-p65 transgenic mice. Finally, the ability of VD to deactivate NF-κB signaling, via p65 and IκB phosphorylation inhibition in murine adipocyte, was observed and could constitute a driving molecular mechanism. This study demonstrated for the first time that VD modulates the expression of miRs in adipocytes in vitro and in adipose tissue in vivo through its impact on NF-κB signaling pathway, which could represent a new mechanism of regulation of inflammation by VD.     

miR‐124 Contributes to the functional maturity of microglia

During early development of the central nervous system (CNS), a subset of yolk‐sac derived myeloid cells populate the brain and provide the seed for the microglial cell population, which will self‐renew throughout life. As development progresses, individual microglial cells transition from a phagocytic amoeboid state through a transitional morphing phase into the sessile, ramified, and normally nonphagocytic microglia observed in the adult CNS under healthy conditions. The molecular drivers of this tissue‐specific maturation profile are not known. However, a survey of tissue resident macrophages identified miR‐124 to be expressed in microglia. In this study, we used transgenic zebrafish to overexpress miR‐124 in the mpeg1 expressing yolk‐sac‐derived myeloid cells that seed the microglia. In addition, a systemic sponge designed to neutralize the effects of miR‐124 was used to assess microglial development in a miR‐124 loss‐of‐function environment. Following the induction of miR‐124 overexpression, microglial motility and phagocytosis of apoptotic cells were significantly reduced. miR‐124 overexpression in microglia resulted in the accumulation of residual apoptotic cell bodies in the optic tectum, which could not be achieved by miR‐124 overexpression in differentiated neurons. Conversely, expression of the miR‐124 sponge caused an increase in the motility of microglia and transiently rescued motility and phagocytosis functions when activated simultaneously with miR‐124 overexpression. This study provides in vivo evidence that miR‐124 activity has a key role in the development of functionally mature microglia. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 507–518, 2016     

MiR-410 Is Overexpressed in Liver and Colorectal Tumors and Enhances Tumor Cell Growth by Silencing FHL1 via a Direct/Indirect Mechanism

FHL1 is an important tumor-suppressor that is downregulated in multiple tumors by unknown mechanisms. We demonstrated that miR-410 specifically targets the 3′UTR of FHL1. Furthermore, using DNA bisulfite modification and sequencing experiments, we demonstrated that the FHL1 promoter is hypermethylated in cancer cells. FHL1 methylation is increased upon miR-410 expression, suggesting that the regulation of FHL1 by miR-410 occurs by a dual mechanism. Using chromatin immunoprecipitation assays, we observed that miR-410 overexpression results in the increased binding of DNMT3A at the FHL1 promoter, which could explain how miR-410 regulates FHL1 methylation. Importantly, in vitro and in vivo results suggest that miR-410 may have oncogenic properties. Furthermore, both miR-410 and DNMT3A are upregulated in clinical human liver and colorectal tumors cancers. Our results suggest that miR-410 may function as an oncomiR and are consistent with its key function in regulating FHL1 in certain digestive system cancers.     

microRNA-34b/c on chromosome 11q23 is aberrantly methylated in chronic lymphocytic leukemia

A commonly deleted region in chronic lymphocytic leukemia (CLL) is the 11q22–23 region, which encompasses the ATM gene. Evidence suggests that tumor suppressor genes other than ATM are likely to be involved in CLL with del(11q). A microRNA (miR) cluster including the miR-34b and miR-34c genes is located, among other genes, within the commonly deleted region (CDR) at 11q. Interestingly, these miRs are part of the TP53 network and have been shown to be epigenetically regulated. In this study, we investigated the expression and methylation status of these miRs in a well-characterized cohort of CLL, including cases with/without 11q-deletion. We show that the miR-34b/c promoter was aberrantly hypermethylated in a large proportion of CLL cases (48%, 25/52 cases). miR-34b/c expression correlated inversely to DNA methylation (P = 0.003), and presence of high H3K37me3 further suppressed expression regardless of methylation status. Furthermore, increased miR-34b/c methylation inversely correlated with the presence of 11q-deletion, indicating that methylation and del(11q) independently silence these miRs. Finally, 5-azacytidine and trichostatin A exposure synergistically increased the expression of miR-34b/c in CLL cells, and transfection of miR-34b or miR-34c into HG3 CLL cells significantly increased apoptosis. Altogether, our novel data suggest that miR-34b/c is a candidate tumor suppressor that is epigenetically silenced in CLL.     

Cocaine-mediated microglial activation involves the ER stress-autophagy axis

Cocaine abuse leads to neuroinflammation, which, in turn, contributes to the pathogenesis of neurodegeneration associated with advanced HIV-1 infection. Autophagy plays important roles in both innate and adaptive immune responses. However, the possible functional link between cocaine and autophagy has not been explored before. Herein, we demonstrate that cocaine exposure induced autophagy in both BV-2 and primary rat microglial cells as demonstrated by a dose- and time-dependent induction of autophagy-signature proteins such as BECN1/Beclin 1, ATG5, and MAP1LC3B. These findings were validated wherein cocaine treatment of BV-2 cells resulted in increased formation of puncta in cells expressing either endogenous MAP1LC3B or overexpressing GFP-MAP1LC3B. Specificity of cocaine-induced autophagy was confirmed by treating cells with inhibitors of autophagy (3-MA and wortmannin). Intriguingly, cocaine-mediated induction of autophagy involved upstream activation of 2 ER stress pathways (EIF2AK3- and ERN1-dependent), as evidenced by the ability of the ER stress inhibitor salubrinal to ameliorate cocaine-induced autophagy. In vivo validation of these findings demonstrated increased expression of BECN1, ATG5, and MAP1LC3B-II proteins in cocaine-treated mouse brains compared to untreated animals. Increased autophagy contributes to cocaine-mediated activation of microglia since pretreatment of cells with wortmannin resulted in decreased expression and release of inflammatory factors (TNF, IL1B, IL6, and CCL2) in microglial cells. Taken together, our findings suggest that cocaine exposure results in induction of autophagy that is closely linked with neuroinflammation. Targeting autophagic proteins could thus be considered as a therapeutic strategy for the treatment of cocaine-related neuroinflammation diseases.     

MircoRNA-29a in Astrocyte-derived Extracellular Vesicles Suppresses Brain Ischemia Reperfusion Injury via TP53INP1 and the NF-κB/NLRP3 Axis

Brain ischemia reperfusion injury (BIRI) is defined as a series of brain injury accompanied by inflammation and oxidative stress. Astrocyte-derived extracellular vesicles (EVs) are importantly participated in BIRI with involvement of microRNAs (miRs). Our study aimed to discuss the functions of miR-29a from astrocyte-derived EVs in BIRI treatment. Thus, astrocyte-derived EVs were extracted. Oxygen and glucose deprivation (OGD) cell models and BIR rat models were established. Then, cell and rat activities and pyroptosis-related protein levels in these two kinds of models were detected. Functional assays were performed to verify inflammation and oxidative stress. miR-29a expression in OGD cells and BIR rats was measured, and target relation between miR-29a and tumor protein 53-induced nuclear protein 1 (TP53INP1) was certified. Rat neural function was tested. Astrocyte-derived EVs improved miR-29a expression in N9 microglia and rat brains. Astrocyte-derived EVs inhibited OGD-induced injury and inflammation in vitro, reduced brain infarction, and improved BIR rat neural functions in vivo. miR-29a in EVs protected OGD-treated cells and targeted TP53INP1, whose overexpression suppressed the protective function of EVs on OGD-treated cells. miR-29a alleviated OGD and BIRI via downregulating TP53INP1 and the NF-κB/NLRP3 pathway. Briefly, our study demonstrated that miR-29a in astrocyte-derived EVs inhibits BIRI by downregulating TP53INP1 and the NF-κB/NLRP3 axis.

     

Dl‐3‐n‐butylphthalide attenuates acute inflammatory activation in rats with spinal cord injury by inhibiting microglial TLR4/NF‐κB signalling

In this study, we examined the neuroprotective effects and anti‐inflammatory properties of Dl‐3‐n‐butylphthalide (NBP) in Sprague‐Dawley (SD) rats following traumatic spinal cord injury (SCI) as well as microglia activation and inflammatory response both in vivo and in vitro. Our results showed that NBP improved the locomotor recovery of SD rats after SCI an significantly diminished the lesion cavity area of the spinal cord, apoptotic activity in neurons, and the number of TUNEL‐positive cells at 7 days post‐injury. NBP inhibited activation of microglia, diminished the release of inflammatory mediators, and reduced the upregulation of microglial TLR4/NF‐κB expression at 1 day post‐injury. In a co‐culture system with BV‐2 cells and PC12 cells, NBP significantly reduced the cytotoxicity of BV‐2 cells following lipopolysaccharide (LPS) stimulation. In addition, NBP reduced the activation of BV‐2 cells, diminished the release of inflammatory mediators, and inhibited microglial TLR4/NF‐κB expression in BV‐2 cells. Our findings demonstrate that NBP may have neuroprotective and anti‐inflammatory properties in the treatment of SCI by inhibiting the activation of microglia via TLR4/NF‐κB signalling.     

miR-124在抑郁症中的作用机制

抑郁症是世界范围内最常见的精神疾病之一,发病机制复杂,研究仍处于探索阶段。微RNA（miRNA）作为表观遗传机制的重要调控因子,在抑郁症的发生发展中起着重要作用。miR-124是神经系统中表达最丰富的miRNA之一,参与了神经元分化、小胶质细胞激活等生物事件。近年研究表明,miR-124在抑郁症患者和动物模型中表达异常,并参与了病理生理机制。然而,miR-124的表达异常情况及其相关机制的研究结果是较复杂的、甚至矛盾的。故本文对此进行梳理,总结了miR-124在抑郁症中的研究进展。     

Activation of glucagon-like peptide-1 receptor in microglia attenuates neuroinflammation-induced glial scarring via rescuing Arf and Rho GAP adapter protein 3 expressions after nerve injury

Rationale: The neuroinflammation is necessary for glial group initiation and clearance of damaged cell debris after nerve injury. However, the proinflammatory polarization of excessive microglia amplifies secondary injury via enhancing cross-talk with astrocytes and exacerbating neurological destruction after spinal cord injury (SCI). The glucagon-like peptide-1 receptor (GLP-1R) agonist has been previously shown to have a neuroprotective effect in neurodegeneration, whereas its potency in microglial inflammation after SCI is still unknown.Methods: The effect and mechanism of GLP-1R activation by exendin-4 (Ex-4) were investigated in in vitro cultured glial groups and in vivo in SCI mice. Alterations in the gene expression after GLP-1R activation in inflammatory microglia were measured using mRNA sequencing. The microglial polarization, neuroinflammatory level, and astrocyte reaction were detected by using western blotting, flow cytometry, and immunofluorescence. The recoveries of neurological histology and function were also observed using imaging and ethological examinations.Results: GLP-1R activation attenuated microglia-induced neuroinflammation by reversing M1 subtypes to M2 subtypes in vitro and in vivo. In addition, activation of GLP-1R in microglia blocked production of reactive astrocytes. We also found less neuroinflammation, reactive astrocytes, corrected myelin integrity, ameliorated histology, and improved locomotor function in SCI mice treated with Ex-4. Mechanistically, we found that Ex-4 rescued the RNA expression of Arf and Rho GAP adapter protein 3 (ARAP3). Knockdown of ARAP3 in microglia reversed activation of RhoA and the pharmacological effect of Ex-4 on anti-inflammation in vitro.Conclusion: Ex-4 exhibited a previously unidentified role in reducing reactive astrocyte activation by mediation of the PI3K/ARAP3/RhoA signaling pathway, by neuroinflammation targeting microglia, and exerted a neuroprotective effect post-SCI, implying that activation of GLP-1R in microglia was a therapeutical option for treatment of neurological injury.     

Neuronal Exosomal miRNA-dependent Translational Regulation of Astroglial Glutamate Transporter GLT1

Perisynaptic astrocytes express important glutamate transporters, especially excitatory amino acid transporter 2 (EAAT2, rodent analog GLT1) to regulate extracellular glutamate levels and modulate synaptic activation. In this study, we investigated an exciting new pathway, the exosome-mediated transfer of microRNA (in particular, miR-124a), in neuron-to-astrocyte signaling. Exosomes isolated from neuron-conditioned medium contain abundant microRNAs and small RNAs. These exosomes can be directly internalized into astrocytes and increase astrocyte miR-124a and GLT1 protein levels. Direct miR-124a transfection also significantly and selectively increases protein (but not mRNA) expression levels of GLT1 in cultured astrocytes. Consistent with our in vitro findings, intrastriatal injection of specific antisense against miR-124a into adult mice dramatically reduces GLT1 protein expression and glutamate uptake levels in striatum without reducing GLT1 mRNA levels. MiR-124a-mediated regulation of GLT1 expression appears to be indirect and is not mediated by its suppression of the putative GLT1 inhibitory ligand ephrinA3. Moreover, miR-124a is selectively reduced in the spinal cord tissue of end-stage SOD1 G93A mice, the mouse model of ALS. Subsequent exogenous delivery of miR-124a in vivo through stereotaxic injection significantly prevents further pathological loss of GLT1 proteins, as determined by GLT1 immunoreactivity in SOD1 G93A mice. Together, our study characterized a new neuron-to-astrocyte communication pathway and identified miRNAs that modulate GLT1 protein expression in astrocytes in vitro and in vivo.     

Mitochondria in activated microglia <Emphasis Type="Italic">in vitro</Emphasis>

In the CNS, microglia become activated, i.e. change their functional state and phenotype, in response to a wide variety of pathological stimuli. Since this activation is triggered at a very low threshold and at the same time remains territorially restricted, the spatial distribution of activated microglia can be used as a sensitive, generic measure of the anatomical localisation of ongoing disease processes. One protein complex, undetectable in resting microglia but highly up-regulated upon activation in vivo and in vitro, is the ‘peripheral benzodiazepine binding site’, as measured by binding of the isoquinoline derivate PK11195. Particularly numerous in the outer membrane of mitochondria, this binding site has also been referred to as the ‘mitochondrial benzodiazepine receptor’. The de novo expression of this receptor by activated microglia suggests that the process of activation may be associated with important qualitative changes in the state of mitochondria. Here, we provide confocal light- and electron microscopic evidence that the activation of microglia indeed entails conspicuous mitochondrial alterations. In cultured rat microglia stained with the fluorescent probe, JC-1, a sensitive indicator of mitochondrial membrane potential, we demonstrate that stimulation by bacterial lipopolysaccharide and interferon-γ increases the number of microglial mitochondrial profiles and leads to marked changes in their morphology. Prominent elongated, “needle-like” mitochondria are a characteristic feature of activated microglia in vitro. Electron microscopically, an abundance of abnormal profiles, including circular cristae or ring- and U-shaped membranes, are found. Our observations support the notion that the previously reported increase in microglial binding of PK11195, that labelled with carbon-11 ([¹¹C] (R)-PK11195) has clinical use for the visualisation of activated microglia in vivo by positron emission tomography, may at least in part relate to an increased number and altered functional state of microglial mitochondria.