Mechanism of human immunodeficiency virus-induced complement expression in astrocytes and neurons

The cerebral complement system is hypothesized to contribute to neurodegeneration in the pathogenesis of AIDS-associated neurological disorders. Our former results have shown that the human immunodeficiency virus (HIV) strongly induces the synthesis of complement factor C3 in astrocytes. This upregulation explains in vivo data showing elevated complement levels in the cerebrospinal fluid of patients with AIDS-associated neurological symptoms. Since inhibition of complement synthesis and activation in the brain may represent a putative therapeutic goal to prevent virus-induced damage, we analyzed in detail the mechanisms of HIV-induced modulation of C3 expression. HIV-1 increased the C3 levels in astrocyte culture supernatants from 30 to up to 400 ng/ml; signal transduction studies revealed that adenylate cyclase activation with upregulation of cyclic AMP is the central signaling pathway to mediate that increase. Furthermore, activity of protein kinase C is necessary for HIV induction of C3, since inhibition of protein kinase C by prolonged exposure to the phorbol ester tetradecanoyl phorbol acetate partly abolished the HIV effect. The cytokines tumor necrosis factor alpha and gamma interferon were not involved in mediating the HIV-induced C3 upregulation, since neutralizing antibodies had no effect. Besides whole HIV virions, the purified viral proteins Nef and gp41 are biologically active in upregulating C3, whereas Tat, gp120, and gp160 were not able to modulate C3 synthesis. Further experiments revealed that neurons were also able to respond on incubation with HIV with increased C3 synthesis, although the precise pattern was slightly different from that in astrocytes. This strengthens the hypothesis that HIV-induced complement synthesis represents an important mechanism for the pathogenesis of AIDS in the brain.     

Crystal structure of ubiquitin-like small archaeal modifier protein 1 (SAMP1) from Haloferax volcanii

HIV associated neurological disorders (HAND) is a common neurological complication in patients infected with HIV. The proinflammatory cytokines and chemokines produced by astrocytes play a pivotal role in neuroinflammatory processes in the brain and viral envelope gp120 has been implicated in this process. In view of increased levels of CCL5 observed in the CSF of HIV-1 infected patients, we studied the effects of gp120 on CCL5 expression in astrocytes and the possible mechanisms responsible for those effects. Transfection of the SVGA astrocyte cell line with a plasmid encoding gp120 resulted in a time-dependent increase in expression levels of CCL5 in terms of mRNA and protein by 24.6 ± 2.67- and 35.2 ± 6.1-fold, respectively. The fluorescent images showed localization of CCL5 in the processes of the astrocytes. The gp120-specific siRNA abrogated the gp120-mediated increase in CCL5 expression. We also explored a possible mechanism for the effects of gp120 on CCL5 expression. Using a specific inhibitor for the NF-κB pathway, we demonstrated that levels of gp120 induction of CCL5 expression can be abrogated by 44.6 ± 4.2% at the level of mRNA and 51.8 ± 5.0% at the protein level. This was further confirmed by knocking down NF-κB through the use of siRNA.     

Rapamycin attenuates the paraquat-induced pulmonary fibrosis through activating Nrf2 pathway

Oxidative stress is a key regulator of idiopathic pulmonary fibrosis. Paraquat (PQ)-induced pulmonary fibrosis seriously endangers people's health. Rapamycin has been reported to alleviate PQ-induced pulmonary fibrosis, but its underlying mechanism is unclear. The nuclear factor E2-related factor 2 (Nrf2) plays an important regulatory role in the antioxidant therapy of PQ-induced pulmonary fibrosis. In this study, we tried to confirm that rapamycin attenuates PQ-induced pulmonary fibrosis by regulating Nrf2 pathway. In vivo, we proved that rapamycin could inhibit the degree of PQ-induced oxidant stress as well as enhanced the expression of Nrf2. In vitro, rapamycin decreased the upregulated effects of cell death and apoptosis, fibrosis-related factors expression and fibroblast-to-myofibroblast transformation by PQ treatment. In vivo, rapamycin treatment reduced fibrosis degree and the expression of fibrosis-related factors in lung tissues of rat treated PQ. Furthermore, we also found that Nrf2 knockdown reduced the inhibitory effect of rapamycin on PQ-induced pulmonary fibrosis, as well as decreased Nrf2 transfer from the cytoplasm into the nucleus. Our findings demonstrated that the protective effect of rapamycin is associated with the activation of the Nrf2 pathway in pulmonary fibrosis induced by PQ poisoning.     

CB2 Receptor Agonists Protect Human Dopaminergic Neurons against Damage from HIV-1 gp120

Despite the therapeutic impact of anti-retroviral therapy, HIV-1-associated neurocognitive disorder (HAND) remains a serious threat to AIDS patients, and there currently remains no specific therapy for the neurological manifestations of HIV-1. Recent work suggests that the nigrostriatal dopaminergic area is a critical brain region for the neuronal dysfunction and death seen in HAND and that human dopaminergic neurons have a particular sensitivity to gp120-induced damage, manifested as reduced function (decreased dopamine uptake), morphological changes, and reduced viability. Synthetic cannabinoids inhibit HIV-1 expression in human microglia, suppress production of inflammatory mediators in human astrocytes, and there is substantial literature demonstrating the neuroprotective properties of cannabinoids in other neuropathogenic processes. Based on these data, experiments were designed to test the hypothesis that synthetic cannabinoids will protect dopaminergic neurons against the toxic effects of the HIV-1 protein gp120. Using a human mesencephalic neuronal/glial culture model, which contains dopaminergic neurons, microglia, and astrocytes, we were able to show that the CB₁/CB₂ agonist WIN55,212-2 blunts gp120-induced neuronal damage as measured by dopamine transporter function, apoptosis and lipid peroxidation; these actions were mediated principally by the CB₂ receptor. Adding supplementary human microglia to our cultures enhances gp120-induced damage; WIN55,212-2 is able to alleviate this enhanced damage. Additionally, WIN55,212-2 inhibits gp120-induced superoxide production by purified human microglial cells, inhibits migration of human microglia towards supernatants generated from gp120-stimulated human mesencephalic neuronal/glial cultures and reduces chemokine and cytokine production from the human mesencephalic neuronal/glial cultures. These data suggest that synthetic cannabinoids are capable of protecting human dopaminergic neurons from gp120 in a variety of ways, acting principally through the CB₂ receptors and microglia.     

Up-regulation of the neuronal nicotinic receptor α7 by HIV glycoprotein 120: potential implications for HIV-associated neurocognitive disorder

Approximately 30-50% of the >30 million HIV-infected subjects develop neurological complications ranging from mild symptoms to dementia. HIV does not infect neurons, and the molecular mechanisms behind HIV-associated neurocognitive decline are not understood. There are several hypotheses to explain the development of dementia in HIV(+) individuals, including neuroinflammation mediated by infected microglia and neuronal toxicity by HIV proteins. A key protein associated with the neurological complications of HIV, gp120, forms part of the viral envelope and can be found in the CSF of infected individuals. HIV-1-gp120 interacts with several receptors including CD4, CCR5, CXCR4, and nicotinic acetylcholine receptors (nAChRs). However, the role of nAChRs in HIV-associated neurocognitive disorder has not been investigated. We studied the effects of gp120(IIIB) on the expression and function of the nicotinic receptor α7 (α7-nAChR). Our results show that gp120, through activation of the CXCR4 chemokine receptor, induces a functional up-regulation of α7-nAChRs. Because α7-nAChRs have a high permeability to Ca(2+), we performed TUNEL staining to investigate the effects of receptor up-regulation on cell viability. Our data revealed an increase in cell death, which was blocked by the selective antagonist α-bungarotoxin. The in vitro data are supported by RT-PCR and Western blot analysis, confirming a remarkable up-regulation of the α7-nAChR in gp120-transgenic mice brains. Specifically, α7-nAChR up-regulation is observed in mouse striatum, a region severely affected in HIV(+) patients. In summary, CXCR4 activation induces up-regulation of α7-nAChR, causing cell death, suggesting that α7-nAChR is a previously unrecognized contributor to the neurotoxicity associated with HIV infection.     

Drugs of Abuse,Dopamine, and HIV-Associated Neurocognitive Disorders/HIV-Associated Dementia

Although the incidence of HIV-associated dementia (HAD) has declined, HIV-associated neurocognitive disorders (HAND) remain a significant health problem despite use of highly active antiretroviral therapy. In addition, the incidence and/or severity of HAND/HAD are increased with concomitant use of drugs of abuse, such as cocaine, marijuana, and methamphetamine. Furthermore, exposure to most drugs of abuse increases brain levels of dopamine, which has been implicated in the pathogenesis of HIV. This review evaluates the potential role of dopamine in the potentiation of HAND/HAD by drugs of abuse. In the brain, multiplication of HIV in infected macrophages/microglia could result in the release of HIV proteins such as gp120 and Tat, which can bind to and impair dopamine transporter (DAT) functions, leading to elevated levels of dopamine in the dopaminergic synapses in the early asymptomatic stage of HIV infection. Exposure of HIV-infected patients to drugs of abuse, especially cocaine and methamphetamine, can further increase synaptic levels of dopamine via binding to and subsequently impairing the function of DAT. This accumulated synaptic dopamine can diffuse out and activate adjacent microglia through binding to dopamine receptors. The activation of microglia may result in increased HIV replication as well as increased production of inflammatory mediators such as tumor necrosis factor (TNF)-alpha and chemokines. Increased HIV replication can lead to increased brain viral load and increased shedding of HIV proteins, gp120 and Tat. These proteins, as well as TNF-alpha, can induce cell death of adjacent dopaminergic neurons via apoptosis. Autoxidation and metabolism of accumulated synaptic dopamine can lead to generation of reactive oxygen species (hydrogen peroxide), quinones, and semiquinones, which can also induce apoptosis of neurons. Increased cell death of dopaminergic neurons can eventually lead to dopamine deficit that may exacerbate the severity and/or accelerate the progression of HAND/HAD.     

Neuroinflammation-Induced Interactions between Protease-Activated Receptor 1 and Proprotein Convertases in HIV-Associated Neurocognitive Disorder

The proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1β [IL-1β]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg₄₁↓ (where the down arrow indicates the cleavage location), and potentially by PCs at Arg₄₁XXXXArg₄₆↓. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV infectivity. Additionally, a proximity ligation assay revealed that furin and PC7 interact with PAR1. Reciprocally, increased furin expression reduces the plasma membrane abundance of PAR1 by trapping it in the trans-Golgi network. Furthermore, soluble PC5A/PACE4 can target/disarm cell surface PAR1 through cleavage at Arg₄₆↓. PACE4/PC5A decreased calcium mobilization induced by thrombin stimulation. Our data reveal a new PC-PAR1-interaction pathway, which offsets the effects of HIV-induced neuroinflammation, viral infection, and potentially the development of HAND.     

细胞间粘附分子5（ICAM-5）在HIV相关神经损伤中的保护性作用

为探究细胞间粘附分子5 (intercellular adhesion molecule 5,ICAM-5)在HIV相关神经认知损伤中的作用,用ELISA法测定HIV感染者脑脊液样本和体外动物神经细胞培养体系中可溶性细胞间粘附分子5(ICAM-5s)的含量|蛋白印迹法检测ICAM-5蛋白表达|免疫荧光法观察神经细胞形态学变化|用CytoTox 96非放射性细胞毒性实验检测神经细胞死亡率.抗ICAM-5单克隆抗体Cy3标记的免疫荧光染色结果显示,ICAM-5可在神经元细胞的胞体和突起表达,且经HIV神经毒性蛋白gp120 500pmol/L处理的神经细胞平均突起长度显著小于无gp120处理的对照组|体外神经细胞培养体系中,gp120+基质金属蛋白酶3（MMP3）实验组的ICAM-5s含量显著高于gp120组,且前者神经元细胞的死亡率高于后者|在 HIV感染者中,HIV相关神经认知障碍（HIV associated neurocognitive disorder,HAND）患者脑脊液中ICAM 5s的水平显著高于认知功能正常的患者.结果表明,ICAM-5可能具有标记神经细胞突起的潜能,但其确切性有待进一步实验验证|ICAM-5与HIV相关神经认知功能损伤相关,具有潜在的神经元细胞保护作用.     

Methamphetamine and inflammatory cytokines increase neuronal Na+/K+-ATPase isoform 3: relevance for HIV associated neurocognitive disorders

Methamphetamine (METH) abuse in conjunction with human immunodeficiency virus (HIV) exacerbates neuropathogenesis and accelerates neurocognitive impairments in the central nervous system (CNS), collectively termed HIV Associated Neurocognitive Disorders (HAND). Since both HIV and METH have been implicated in altering the synaptic architecture, this study focused on investigating alterations in synaptic proteins. Employing a quantitative proteomics approach on synaptosomes isolated from the caudate nucleus from two groups of rhesus monkeys chronically infected with simian immunodeficiency virus (SIV) differing by one regimen, METH treatment, we identified the neuron specific Na(+)/K(+)-ATPase alpha 1 isoform 3 (ATP1A3) to be up regulated after METH treatment, and validated its up regulation by METH in vitro. Further studies on signaling mechanisms revealed that the activation of ATP1A3 involves the extracellular regulated kinase (ERK) pathway. Given its function in maintaining ionic gradients and emerging role as a signaling molecule, changes in ATP1A3 yields insights into the mechanisms associated with HAND and interactions with drugs of abuse.     

p62/SQSTM1 protects against cisplatin-induced oxidative stress in kidneys by mediating the cross talk between autophagy and the Keap1-Nrf2 signalling pathway

Acute kidney injury (AKI) is a major kidney disease associated with poor clinical outcomes. Oxidative stress is predominantly involved in the pathogenesis of AKI. Autophagy and the Keap1-Nrf2 signalling pathway are both involved in the oxidative-stress response. However, the cross talk between these two pathways in AKI remains unknown. Here, we found that autophagy is upregulated during cisplatin-induced AKI. In contrast with previous studies, we observed a marked increase in p62. We also found that p62 knockdown reduces autophagosome formation and the expression of LC3II. To explore the cross talk between p62 and the Keap1-Nrf2 signalling pathway, HK-2 cells were transfected with siRNA targeting Nrf2, and we found that Nrf2 knockdown significantly reduced cisplatin-induced p62 expression. Moreover, p62 knockdown significantly decreased the protein expression of Nrf2, as well as Heme Oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase l (NQO1), whereas the expression of kelch-like ECH-associated protein 1 (Keap1) was upregulated. These results indicate that p62 creates a positive feedback loop in the Keap1-Nrf2 signalling pathway. Finally, we examined the role of p62 in cell protection during cisplatin-induced oxidative stress, and we found that p62 silencing in HK-2 cells increases apoptosis and reactive oxygen species (ROS) levels, which further indicates the protective role of p62 under oxidative stress and suggests that the cytoprotection 62 mediated is in part by regulating autophagic activity or the Keap1-Nrf2 signalling pathway. Taken together, our results have demonstrated a reciprocal regulation of p62, autophagy and the Keap1-Nrf2 signalling pathway under oxidative stress, which may be a potential therapeutic target against AKI.     

Nutritional strategies to modulate inflammation and oxidative stress pathways via activation of the master antioxidant switch Nrf2

The nuclear factor E2-related factor 2 (Nrf2) plays an important role in cellular protection against cancer, renal, pulmonary, cardiovascular and neurodegenerative diseases where oxidative stress and inflammation are common conditions. The Nrf2 regulates the expression of detoxifying enzymes by recognizing the human Antioxidant Response Element (ARE) binding site and it can regulate antioxidant and anti-inflammatory cellular responses, playing an important protective role on the development of the diseases. Studies designed to investigate how effective Nrf2 activators or modulators are need to be initiated. Several recent studies have shown that nutritional compounds can modulate the activation of Nrf2–Keap1 system. This review aims to discuss some of the key nutritional compounds that promote the activation of Nrf2, which may have impact on the human health.     

转录因子Nrf2在肝脏疾病中的作用

NF-E2相关因子2(nuclear erythroid 2-related factor 2,Nrf2)是一种能调节肝脏中大量解毒和抗氧化防御基因表达的重要转录因子.氧化应激与各种形式的肝损伤有密切的关系.Nrf2由亲电体压力或氧化应激激活,并通过结合抗氧化反应元件(antioxidant response element,ARE)诱导其靶基因,从而对细胞产生保护作用.因此,Nrf2通路在肝脏疾病中的作用已被深入研究.多种动物模型研究结果表明,Nrf2通路通过靶基因表达,在对抗病毒性肝炎、药物性肝损伤、酒精性肝病、非酒精性脂肪肝及肝癌方面表现出了不同的生物功能.根据Nrf2及其信号通路在对抗肝损伤中产生保护作用的相关文献,本文综述并讨论了其作为治疗肝损伤的药物作用靶点方面可能的应用前景.