NRF2 Antagonizes HIV-1 Tat and Methamphetamine-Induced BV2 Cell Ferroptosis by Regulating SLC7A11.

Methamphetamine (METH) and HIV-1 lead to oxidative stress and their combined effect increases the risk of HIV-associated neurocognitive disorder (HAND), which may be related to the synergistic ferroptotic impairment in microglia. Ferroptosis is a redox imbalance cell damage associated with iron overload that is linked to the pathogenic processes of METH and HIV-1. NRF2 is an antioxidant transcription factor that plays a protective role in METH and HIV-1-induced neurotoxicity, but its mechanism has not been fully elucidated. To explore the role of ferroptosis in METH abuse and HIV-1 infection and the potential role of NRF2 in this process, we conducted METH and HIV-1 Tat exposure models using the BV2 microglia cells. We found that METH and HIV-1 Tat reduced the expression of ferroptotic protein GPX4 and the cell viability and enhanced the expression of P53 and the level of ferrous iron, while the above indices were significantly improved with pretreatment of ferrostatin-1. In addition, NRF2 knockdown accelerated METH and HIV-1 Tat-induced BV2 cell ferroptosis accompanied by decreased expression of SLC7A11. On the contrary, NRF2 stimulation significantly increased the expression of SLC7A11 and attenuated ferroptosis in cells. In summary, our study indicates that METH and HIV-1 Tat synergistically cause BV2 cell ferroptosis, while NRF2 antagonizes BV2 cell ferroptotic damage induced by METH and HIV-1 Tat through regulation of SLC7A11. Overall, this study provides potential therapeutic strategies for the treatment of neurotoxicity caused by METH and HIV-1 Tat, providing a theoretical basis and new targets for the treatment of HIV-infected drug abusers.

HIV Tat Protein Induces Myocardial Fibrosis Through TGF-ß1-CTGF Signaling Cascade: A Potential Mechanism of HIV Infection-Related Cardiac Manifestations.

Human immunodeficiency virus (HIV) infection is a risk factor of cardiovascular diseases (CVDs). HIV-infected patients exhibit cardiac dysfunction coupled with cardiac fibrosis. However, the reason why HIV could induce cardiac fibrosis remains largely unexplored. HIV-1 trans-activator of transcription (Tat) protein is a regulatory protein, which plays a critical role in the pathogenesis of various HIV-related complications. In the present study, recombinant Tat was administered to mouse myocardium or neonatal mouse cardiac fibroblasts in different doses. Hematoxylin-eosin and Masson's trichrome staining were performed to observe the histological changes of mice myocardial tissues. EdU staining and MTS assay were used to evaluate the proliferation and viability of neonatal mouse cardiac fibroblasts, respectively. Real-time PCR and western blot analysis were used to detect CTGF, TGF-ß1, and collagen I mRNA and protein expression levels, respectively. The results showed that Tat promoted the occurrence of myocardial fibrosis in mice. Also, we found that Tat increased the proliferative ability and the viability of neonatal mouse cardiac fibroblasts. The protein and mRNA expression levels of TGF-ß1 and CTGF were significantly upregulated both in Tat-treated mouse myocardium and neonatal mouse cardiac fibroblasts. However, co-administration of TGF-ß inhibitor abrogated the enhanced expression of collagen I induced by Tat in neonatal mouse cardiac fibroblasts. In conclusion, Tat contributes to HIV-related cardiac fibrosis through enhanced TGF-ß1-CTGF signaling cascade.

Antipruritic Effect of Nalbuphine, a Kappa Opioid Receptor Agonist, in Mice: A Pan Antipruritic.

Antipruritic effects of kappa opioid receptor (KOR) agonists have been shown in rodent models of acute and chronic scratching (itchlike behavior). Three KOR agonists, nalfurafine, difelikefalin, and nalbuphine, are in clinical studies for antipruritic effects in chronic itch of systemic and skin diseases. Nalfurafine (in Japan) and difelikefalin (in the USA) were approved to be used in the treatment of chronic itch in hemodialysis patients. The FDA-approved nalbuphine has been used in clinic for over 40 years, and it is the only narcotic agonist that is not scheduled. We aimed to study (a) antiscratch activity of nalbuphine against TAT-HIV-1 protein (controls HIV transcription)-, deoxycholic acid (DCA, bile acid)-, and chloroquine (CQ)-induced scratching in a mouse model of acute itch; and (b) whether the effect of nalbuphine is produced via KORs. First, dose-responses were developed for pruritogens. Mice were pretreated with nalbuphine (0.3-10 mg/kg) and then a submaximal dose of pruritogens were administered and the number of scratching bouts was counted. To study if the antiscratch effect of nalbuphine is produced via KOR, we used KOR knock out mice and pharmacologic inhibition of KORs using nor-binaltorphimine, a KOR antagonist. For this aim, we used CQ as a pruritogen. We found that: (a) TAT-HIV-1 protein elicits scratching in a dose-dependent manner; (b) nalbuphine inhibits scratching induced by TAT-HIV-1, DCA, and CQ dose-dependently; and (c) nalbuphine inhibits scratching induced by CQ through KORs. In conclusion, nalbuphine inhibits scratching elicited by multiple pruritogens.

Activation of α7 nicotinic acetylcholine receptor ameliorates HIV-associated neurology and neuropathology.

HIV-associated neurocognitive disorders (HAND) in the era of combination antiretroviral therapy are primarily manifested as impaired behaviours, glial activation/neuroinflammation and compromised neuronal integrity, for which there are no effective treatments currently available. In the current study, we used doxycycline-inducible astrocyte-specific HIV Tat transgenic mice (iTat), a surrogate HAND model, and determined effects of PNU-125096, a positive allosteric modulator of α7 nicotinic acetylcholine receptor (α7 nAChR) on Tat-induced behavioural impairments and neuropathologies. We showed that PNU-125096 treatment significantly improved locomotor, learning and memory deficits of iTat mice while inhibited glial activation and increased PSD-95 expression in the cortex and hippocampus of iTat mice. Using α7 nAChR knockout mice, we showed that α7 nAChR knockout eliminated the protective effects of PNU-125096 on iTat mice. In addition, we showed that inhibition of p38 phosphorylation by SB239063, a p38 MAPK-specific inhibitor exacerbated Tat neurotoxicity in iTat mice. Last, we used primary mouse cortical individual cultures and neuron-astrocytes co-cultures and in vivo staining of iTat mouse brain tissues and showed that glial activation was directly involved in the interplay among Tat neurotoxicity, α7 nAChR activation and the p38 MAPK signalling pathway. Taken together, these findings demonstrated for the first time that α7 nAChR activation led to protection against HAND and suggested that α7 nAChR modulator PNU-125096 holds significant promise for development of therapeutics for HAND.

GPR18 drives FAAH inhibition-induced neuroprotection against HIV-1 Tat-induced neurodegeneration.

Human immunodeficiency virus type 1 (HIV-1) is known to provoke microglial immune responses which likely play a paramount role in the development of chronic neuroinflammatory conditions and neuronal damage related to HIV-1 associated neurocognitive disorders (HAND). In particular, HIV-1 Tat protein is a proinflammatory neurotoxin which predisposes neurons to synaptodendritic injury. Drugs targeting the degradative enzymes of endogenous cannabinoids have shown promise in reducing inflammation with minimal side effects in rodent models. Considering that markers of neuroinflammation can predict the extent of neuronal injury in HAND patients, we evaluated the neurotoxic effect of HIV-1 Tat-exposed microglia following blockade of fatty acid amid hydrolyze (FAAH), a catabolic enzyme responsible for degradation of endocannabinoids, e.g. anandamide (AEA). In the present study, cultured murine microglia were incubated with Tat and/or a FAAH inhibitor (PF3845). After 24 h, cells were imaged for morphological analysis and microglial conditioned media (MCM) was collected. Frontal cortex neuron cultures (DIV 7-11) were then exposed to MCM, and neurotoxicity was assessed via live cell calcium imaging and staining of actin positive dendritic structures. Results demonstrate a strong attenuation of microglial responses to Tat by PF3845 pretreatment, which is indicated by 1) microglial changes in morphology to a less proinflammatory phenotype using fractal analysis, 2) a decrease in release of neurotoxic cytokines/chemokines (MCP-1/CCL2) and matrix metalloproteinases (MMPs; MMP-9) using ELISA/multiplex assays, and 3) enhanced production of endocannabinoids (AEA) using LC/MS/MS. Additionally, PF3845's effects on Tat-induced microglial-mediated neurotoxicity, decreased dysregulation of neuronal intracellular calcium and prevented the loss of actin-positive staining and punctate structure in frontal cortex neuron cultures. Interestingly, these observed neuroprotective effects appeared to be independent of cannabinoid receptor activity (CB1R & CB2R). We found that a purported GPR18 antagonist, CID-85469571, blocked the neuroprotective effects of PF3845 in all experiments. Collectively, these experiments increase understanding of the role of FAAH inhibition and Tat in mediating microglial neurotoxicity in the HAND condition.

HIV-1 Tat-Induced Astrocytic Extracellular Vesicle miR-7 Impairs Synaptic Architecture.

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.

Inhibition of the Dead Box RNA Helicase 3 Prevents HIV-1 Tat and Cocaine-Induced Neurotoxicity by Targeting Microglia Activation.

HIV-1 Associated Neurocognitive Disorder (HAND) is a common and clinically detrimental complication of HIV infection. Viral proteins, including Tat, released from infected cells, cause neuronal toxicity. Substance abuse in HIV-infected patients greatly influences the severity of neuronal damage. To repurpose small molecule inhibitors for anti-HAND therapy, we employed MOLIERE, an AI-based literature mining system that we developed. All human genes were analyzed and prioritized by MOLIERE to find previously unknown targets connected to HAND. From the identified high priority genes, we narrowed the list to those with known small molecule ligands developed for other applications and lacking systemic toxicity in animal models. To validate the AI-based process, the selective small molecule inhibitor of DDX3 helicase activity, RK-33, was chosen and tested for neuroprotective activity. The compound, previously developed for cancer treatment, was tested for the prevention of combined neurotoxicity of HIV Tat and cocaine. Rodent cortical cultures were treated with 6 or 60 ng/ml of HIV Tat and 10 or 25 µM of cocaine, which caused substantial toxicity. RK-33 at doses as low as 1 µM greatly reduced the neurotoxicity of Tat and cocaine. Transcriptome analysis showed that most Tat-activated transcripts are microglia-specific genes and that RK-33 blocks their activation. Treatment with RK-33 inhibits the Tat and cocaine-dependent increase in the number and size of microglia and the proinflammatory cytokines IL-6, TNF-α, MCP-1/CCL2, MIP-2, IL-1α and IL-1ß. These findings reveal that inhibition of DDX3 may have the potential to treat not only HAND but other neurodegenerative diseases. Graphical Abstract RK-33, selective inhibitor of Dead Box RNA helicase 3 (DDX3) protects neurons from combined Tat and cocaine neurotoxicity by inhibition of microglia activation and production of proinflammatory cytokines.

HIV and Alzheimer's disease: complex interactions of HIV-Tat with amyloid ß peptide and Tau protein.

In patients infected with the human immunodeficiency virus (HIV), the HIV-Tat protein may be continually produced despite adequate antiretroviral therapy. As the HIV-infected population is aging, it is becoming increasingly important to understand how HIV-Tat may interact with proteins such as amyloid ß and Tau which accumulate in the aging brain and eventually result in Alzheimer's disease. In this review, we examine the in vivo data from HIV-infected patients and animal models and the in vitro experiments that show how protein complexes between HIV-Tat and amyloid ß occur through novel protein-protein interactions and how HIV-Tat may influence the pathways for amyloid ß production, degradation, phagocytosis, and transport. HIV-Tat may also induce Tau phosphorylation through a cascade of cellular processes that lead to the formation of neurofibrillary tangles, another hallmark of Alzheimer's disease. We also identify gaps in knowledge and future directions for research. Available evidence suggests that HIV-Tat may accelerate Alzheimer-like pathology in patients with HIV infection which cannot be impacted by current antiretroviral therapy.

Chronic Intrahippocampal Infusion of HIV-1 Neurotoxic Proteins: A Novel Mouse Model of HIV-1 Associated Inflammation and Neural Stem Cell Dysfunction.

HIV-1 infection causes chronic neuroinflammation resulting in cognitive decline associated with diminution of survival of neural stem cells (NSC). In part, this is attributable to production of toxic viral proteins (gp120 and tat) by infected cells in the brain that can activate microglia. Here, we evaluated a novel model for HIV-1 neuropathogenesis by direct administration of viral proteins into the hippocampus. Chronic administration of either HIV-1 gp120 or tat over 14 days significantly decreased NSC proliferation, survival and neuroblast formation (by 32-37%) within the hippocampal subgranular zone as detected by doublecortin/BrdU or Ki67-positive cells. Intrahippocampal administration of gp120 or tat induced microglial activation within the hippocampus as determined by increases in microglial number and increases in the volume of the microglia (2.5-3-fold, evaluated by double IBA-1/CD68 staining). We further assessed inflammatory responses within the hippocampus by RNAseq and Ingenuity Pathway Analysis. There was a significant mRNA upregulation of numerous inflammatory mediators including Il1b, Icam1, Il12a, Ccl2, and Ccl4. These data suggest that chronic administration induces a prolonged inflammatory state within the hippocampus that negatively affects NSC survival potentially leading to cognitive dysfunction. Graphical Abstract.

The attenuation of HIV-1 Tat-induced neurotoxicity by Salvianic acid A and Danshen granule.

The neurotoxicity of HIV-1 Tat protein contributes significantly to the pathogenesis of HAND, and hence the attractive therapeutic strategies focusing on Tat-induced neurotoxicity are warranted. Salvia miltiorrhiza have been known to antioxidant property and neuroprotective effects. The Danshen granule is the pharmaceutical dosage forms of Salvia miltiorrhiza and Salvianic acid A is an essential chemical constituent of Salvia miltiorrhiza. However, the protective effects of Salvianic acid A and Danshen granule on Tat-induced neurotoxicity remain unknown. Here, we found that Salvianic acid A and Danshen granule remarkable inhibited Tat-induced cell death, blocked LDH release and rescued dendritic spine loss. Furthermore, Salvianic acid A and Danshen granule significantly ameliorates Tat-induced intracellular ROS and MDA production, attenuates cell apoptosis. In addition, Salvianic acid A and Danshen granule pretreatment obviously increases antioxidant enzymatic activity of CAT, SOD and GSH-Px and inhibits apoptotic pathways. In conclusion, this study demonstrated that Salvianic acid A and Danshen granule provides substantial neuroprotection against Tat-induced neurotoxicity, which may be new therapeutic agent in Tat induced HAND or neurodegenerative diseases.

Dysregulation of Neuronal Cholesterol Homeostasis upon Exposure to HIV-1 Tat and Cocaine Revealed by RNA-Sequencing.

HIV-1 Tat protein is released from HIV-1-infected cells and can enter non-permissive cells including neurons. Tat disrupts neuronal homeostasis and may contribute to the neuropathogenesis in people living with HIV (PLWH). The use of cocaine by PLWH exacerbates neuronal dysfunction. Here, we examined the mechanisms by which Tat and cocaine facilitate alterations in neuronal homeostatic processes. Bioinformatic interrogation of the results from RNA deep sequencing of rat hippocampal neurons exposed to Tat alone indicated the dysregulation of several genes involved in lipid and cholesterol metabolism. Following exposure to Tat and cocaine, the activation of cholesterol biosynthesis genes led to increased levels of free cholesterol and cholesteryl esters in rat neurons. Results from lipid metabolism arrays validated upregulation of several processes implicated in the biogenesis of ß-amyloid and Alzheimer's disease (AD), including sterol o-acyltransferase 1/acetyl-coenzyme A acyltransferase 1 (SOAT1/ACAT1), sortilin-related receptor L1 (SORL1) and low-density lipoprotein receptor-related protein 12 (LRP12). Further studies in Tat-treated primary neuronal cultures and brain tissues from HIV-1 transgenic mice as well as SIV-infected macaques confirmed elevated levels of SOAT1/ACAT 1 proteins. Our results offer novel insights into the molecular events involved in HIV and cocaine-mediated neuronal dysfunction that may also contribute to neuropathogenic events associated with the development of AD.

HIV-1 Tat and methamphetamine co-induced oxidative cellular injury is mitigated by N-acetylcysteine amide (NACA) through rectifying mTOR signaling.

Methamphetamine (Meth) is an addictive psychostimulant whose abuse is intimately linked to increased risks for HIV-1 infection. Converging lines of evidence indicate that Meth also aggravates the symptoms of HIV-associated neurocognitive disorders (HAND), though the underlying mechanisms remain poorly understood. By using the lipophilic antioxidant N-acetylcysteine amide (NACA) as an interventional agent, we examined the roles of oxidative stress in autophagy and apoptosis induced by HIV-Tat (the transactivator of transcription), Meth or their combined treatment in human SH-SY5Y neuroblastoma cells and in the rat striatum. Oxidative stress was monitored in terms of the production of intracellular reactive oxygen species (ROS) and antioxidant reserves including glutathione peroxidase (GPx) and Cu,Zn-superoxide dismutase (SOD). NACA significantly reduced the level of ROS and restored GPx and SOD to levels comparable to that of normal control, implying a cytoprotective effect of NACA against oxidative stress elicited by Tat- and/or Meth. Protein expression of mammalian target of rapamycin (mTOR) was measured in SH-SY5Y cells and in the rat striatum to further explore the underlying mechanism of NACA protect against oxidative stress. The results support a beneficial effect of NACA in vivo and in vitro through rectification of the mTOR signaling pathway. Collectively, our study shows that NACA protects against Meth and/or Tat-induced cellular injury in vitro and in the rat striatum in vivo by attenuating oxidative stress, apoptosis and autophagy, at least in part, via modulation of mTOR signaling.

Neuroprotective effects of fatty acid amide hydrolase catabolic enzyme inhibition in a HIV-1 Tat model of neuroAIDS.

The HIV-1 transactivator of transcription (Tat) is a neurotoxin involved in the pathogenesis of HIV-1 associated neurocognitive disorders (HAND). The neurotoxic effects of Tat are mediated directly via AMPA/NMDA receptor activity and indirectly through neuroinflammatory signaling in glia. Emerging strategies in the development of neuroprotective agents involve the modulation of the endocannabinoid system. A major endocannabinoid, anandamide (N-arachidonoylethanolamine, AEA), is metabolized by fatty acid amide hydrolase (FAAH). Here we demonstrate using a murine prefrontal cortex primary culture model that the inhibition of FAAH, using PF3845, attenuates Tat-mediated increases in intracellular calcium, neuronal death, and dendritic degeneration via cannabinoid receptors (CB1R and CB2R). Live cell imaging was used to assess Tat-mediated increases in [Ca2+]i, which was significantly reduced by PF3845. A time-lapse assay revealed that Tat potentiates cell death while PF3845 blocks this effect. Additionally PF3845 blocked the Tat-mediated increase in activated caspase-3 (apoptotic marker) positive neurons. Dendritic degeneration was characterized by analyzing stained dendritic processes using Imaris and Tat was found to significantly decrease the size of processes while PF3845 inhibited this effect. Incubation with CB1R and CB2R antagonists (SR141716A and AM630) revealed that PF3845-mediated calcium effects were dependent on CB1R, while reduced neuronal death and degeneration was CB2R-mediated. PF3845 application led to increased levels of AEA, suggesting the observed effects are likely a result of increased endocannabinoid signaling at CB1R/CB2R. Our findings suggest that modulation of the endogenous cannabinoid system through inhibition of FAAH may be beneficial in treatment of HAND.

Critical Role of Beclin1 in HIV Tat and Morphine-Induced Inflammation and Calcium Release in Glial Cells from Autophagy Deficient Mouse.

We previously showed that autophagy is an important component in human immunodeficiency virus (HIV) replication and in the combined morphine-induced neuroinflammation in human astrocytes and microglia. Here we further studied the consequences of autophagy using glial cells of mice partially lacking the essential autophagy gene Atg6 (Beclin1) exposed to HIV Tat and morphine. Tat is known to cause an inflammatory response, increase calcium release, and possibly interact with autophagy pathway proteins. Following Tat exposure, autophagy-deficient (Becn1+/-) glial cells had significantly and consistently reduced levels in the pro-inflammatory cytokine IL-6 and the chemokines RANTES and MCP-1 when compared to Tat-treated cells from control (C57BL/6J) mice, suggesting an association between the inflammatory effects of Tat and Beclin1. Further, differences in RANTES and MCP-1 secretion between C57BL/6J and Becn1+/- glia treated with Tat and morphine also suggest a role of Beclin1 in the morphine-induced enhancement. Analysis of autophagy maturation by immunoblot suggests that Beclin1 may be necessary for Tat, and to a lesser extent morphine-induced arrest of the pathway as demonstrated by accumulation of the adaptor protein p62/SQSTM1 in C57BL/6J glia. Calcium release induced by Tat alone or in combination with morphine in C57BL/6J glia was significantly reduced in Becn1+/- glia while minimal interactive effect of Tat with morphine in the production of reactive oxygen or nitrogen species was detected in glia derived from Becn1+/- or C57BL/6J. Overall, the data establish a role of Beclin1 in Tat and morphine-mediated inflammatory responses and calcium release in glial cells and support the notion that autophagy mediates Tat alone and combined morphine-induced neuropathology.

Protective Effects of Ginsenoside Rb1 against Blood-Brain Barrier Damage Induced by Human Immunodeficiency Virus-1 Tat Protein and Methamphetamine in Sprague-Dawley Rats.

Although antiretroviral therapy has helped to improve the lives of individuals infected with human immunodeficiency virus 1 (HIV-1), these patients are often still afflicted with HIV-1-associated neurocognitive disorders, which can lead to neurocognitive impairment and even dementia, and continue to hamper their quality of life. Methamphetamine abuse in HIV-1 patients poses a potential risk for HIV-associated neurocognitive disorders, because methamphetamine and HIV-1 proteins such as transactivator of transcription can synergistically damage the blood-brain barrier (BBB). In this study, we aimed to examine the effects of methamphetamine and HIV-1 Tat protein on the blood-brain barrier function and to determine whether ginsenoside Rb1 (GsRb1) plays a role in protecting the BBB. Sprague-Dawley rats were divided into four groups. The experimental groups received methamphetamine and HIV-1 Tat protein or both and the control group received saline or GsRb1 pretreatment. Oxidative stress-related factors, tight junction (TJ) proteins, blood-brain barrier permeability, and morphological changes were recorded in each group. The results showed that the group treated with Methamphetamine[Formula: see text]Tat showed a significant change at the ultrastructural level and in the levels of oxidative stress-related factors, TJ proteins, and BBB permeability, suggesting that the BBB function was severely damaged by HIV-1 Tat and methamphetamine synergistically. However, malondialdehyde levels and BBB permeability were lower and the oxidative stress-related factors superoxide dismutase and glutathione were higher in the GsRb1-treated group than in the Methamphetamine[Formula: see text]Tat-treated group, indicating that GsRb1 can protect the BBB against the toxic effects of HIV-1 Tat and methamphetamine. These results show that GsRb1 may offer a potential therapeutic option for patients with HIV-associated neurocognitive disorders or other neurodegenerative diseases.

HIV-1 Tat-induced diarrhea is improved by the PPARalpha agonist, palmitoylethanolamide, by suppressing the activation of enteric glia.

BACKGROUND: Diarrhea is a severe complication in HIV-1-infected patients with Trans-activator of transcription (HIV-1 Tat) protein being recognized as a major underlying cause. Beside its direct enterotoxic effects, Tat protein has been recently shown to affect enteric glial cell (EGC) activity. EGCs regulate intestinal inflammatory responses by secreting pro-inflammatory molecules; nonetheless, they might also release immune-regulatory factors, as palmytoilethanolamide (PEA), which exerts anti-inflammatory effects by activating PPARα receptors. We aimed at clarifying whether EGCs are involved in HIV-1 Tat-induced diarrhea and if PEA exerts antidiarrheal activity. METHODS: Diarrhea was induced by intracolonic administration of HIV-1 Tat protein in rats at day 1. PEA alone or in the presence of peroxisome proliferator-activated receptor (PPAR) antagonists was given intraperitoneally from day 2 to day 7. S100B, iNOS, NF-kappaB, TLR4 and GFAP expression were evaluated in submucosal plexi, while S100B and NO levels were measured in EGC submucosal plexi lysates, respectively. To verify whether PEA effects were PPARα-mediated, PPARα-/- mice were also used. After 7 days from diarrhea induction, endogenous PEA levels were measured in submucosal plexi homogenates deriving from rats and PPARα-/- mice. RESULTS: HIV-1 Tat protein induced rapid onset diarrhea alongside with a significant activation of EGCs. Tat administration significantly increased all hallmarks of neuroinflammation by triggering TLR4 and NF-kappaB activation and S100B and iNOS expression. Endogenous PEA levels were increased following HIV-1 Tat exposure in both wildtype and knockout animals. In PPARα-/- mice, PEA displayed no effects. In wildtype rats, PEA, via PPARα-dependent mechanism, resulted in a significant antidiarrheal activity in parallel with marked reduction of EGC-sustained neuroinflammation. CONCLUSIONS: EGCs mediate HIV-1 Tat-induced diarrhea by sustaining the intestinal neuroinflammatory response. These effects are regulated by PEA through a selective PPARα-dependent mechanism. PEA might be considered as an adjuvant therapy in HIV-1-induced diarrhea.

Arginine-rich membrane-permeable peptides are seriously toxic.

The membrane-permeable peptides (MPP) such as undecapeptides TAT (YGRKKRRQRRR) and CTP (YGRRARRRRRR) have been receiving much attention for delivering various kinds of low membrane-permeability materials in vitro and in vivo. We have successfully used MPP in carrying various proteins through blood-brain barrier (BBB) in treatment of many kinds of nervous diseases. However, people always concentrate their mind on the efficacy and the mechanism of permeation of the conjugates across BBB, but overlook the toxicity of the membrane-permeable peptide itself. Once we injected intravenously not very large amounts of gamma-aminobutyric acid-MPP (GABA-MPP) to the mice, to our great surprise, the mice died within seconds with seizure, whereas the GABA control mice well survived. Thus, the importance of the toxicity of MPPs and their conjugates comes into the field of our vision. The low LD50 values of arginine-rich TAT (27.244 mg kg-1 ) and CTP (21.345 mg kg-1 ) per se in mice indicate that they all fall within the range of highly toxic chemicals. Among the arginine-rich peptides, R11 (RRRRRRRRRRR), a peptide composed purely of arginine residues, has the lowest LD50 value (16.5 mg kg-1 ) and manifests the highest toxicity, whereas TD (ACSSSPSKHCG), a peptide without arginine residue, shows a much lower toxicity and higher survival rate in mice. The mass percentage of arginine-rich MPP in the conjugate is critically important, the mass radio of arginine in the MPP appears a linear correlation with the toxicity. Thus we conclude, the arginine-rich MPPs are more suitable for using in the macro-molecular conjugates, but not in the small-molecular one.

The use of electronic-neutral penetrating peptides cyclosporin A to deliver pro-apoptotic peptide: A possibly better choice than positively charged TAT.

Cell-penetrating peptides (CPPs) are increasingly important in transporting macromolecules across cell membranes, but their use remains confined to narrow clinical applications due to the systemic toxicity induced by their positive charges. Several newly discovered electronic neutral penetrating peptides are not attracting much attention because their penetrating capacity is normally far less powerful than cationic or amphiphilic CPPs. In this study, we found the electronic neutral cyclic peptide cyclosporin A (CsA) exhibited 5.6-fold and 19.1-fold stronger penetrating capacity, respectively, than two reported electronic neutral peptides PFVYLI (PFV) and pentapeptide VPTLQ (VPT) in MCF-7 human breast cancer cells. To systematically evaluate the efficiency and toxicity of CsA, we utilized CsA to deliver a membrane-impenetrable pro-apoptotic peptide (PAD) and compared this to the well-established cationic penetrating peptide TAT (RKKRRQRRR). By conjugating CsA to PAD, the internalization of PAD increased 2.2- to 4.7-fold in four different tumor cell lines, and that of CsA-PAD conjugate was significantly higher than TAT-PAD conjugate in MCF-7 and HeLa human cervical cancer cells. Cytotoxicity studies demonstrated that CsA-PAD exhibited a large increase in cell cytotoxicity compared to PAD in four different tumor cell lines, with the effect being similar or greater than the effect of TAT-PAD, depending upon the cell type. The mechanistic studies demonstrated that modifying CsA or TAT did not change the cytotoxicity mechanism of PAD, which occurred via mitochondrial membrane damage related to apoptosis. In vivo studies showed that CsA-PAD could achieve similar anti-tumor efficacy to TAT-PAD but with much lower systemic toxicity, especially to the heart and liver. In conclusion, our study demonstrates for the first time that the electronic-neutral penetrating peptide CsA can be used as a powerful tool to deliver peptide drugs with similar efficiency and less toxicity than the positively charged TAT peptide.

A GluN2B-Selective NMDAR Antagonist Reverses Synapse Loss and Cognitive Impairment Produced by the HIV-1 Protein Tat.

HIV-associated neurocognitive disorder (HAND) affects approximately half of HIV-infected patients. Loss of synaptic connections is a hallmark of many neurocognitive disorders, including HAND. The HIV-1 protein transactivator of transcription (Tat) disrupts synaptic connections both in vitro and in vivo and has been linked to impaired neurocognitive function in humans. In vitro studies have shown that ifenprodil, an antagonist selective for GluN2B-containing NMDARs, reverses synapse loss when applied after Tat. Here, we tested the hypothesis that Tat-induced loss and ifenprodil-mediated rescue of synaptic spines in vivo would predict impairment and rescue of cognitive function. Using intracranial multiphoton imaging, we found that infusion of 100 ng of HIV-1 Tat into the lateral ventricle of yellow fluorescent protein-expressing transgenic mice produced a 17 ± 1% loss of dendritic spines in layer 1 of retrosplenial cortex. Repeated imaging of the same dendrites over 3 weeks enabled longitudinal experiments that demonstrated sustained spine loss after Tat infusion and transient rescue after ifenprodil administration (10 mg/kg, i.p.). Parallel trace fear conditioning experiments showed that spine loss predicted learning deficits and that the time course of ifenprodil-induced rescue of spine density correlated with restoration of cognitive function. These results show for the first time that, during exposure to an HIV-1 neurotoxin in vivo, alteration of GluN2B-containing NMDAR signaling suppresses spine density and impairs learning. Pharmacological inhibition of these NMDARs rescued spines and restored cognitive function. Drugs that rescue synapses may improve neurocognitive function in HAND.SIGNIFICANCE STATEMENT Synaptodendritic damage correlates with cognitive decline in HIV-associated neurocognitive disorder (HAND) patients. We developed an in vivo imaging approach for longitudinal tracking of spine density that enabled correlation of synaptic changes with behavioral outcomes in a model of HAND. We show for the first time that spine loss after exposure to an HIV-1 protein can be reversed pharmacologically and that loss and recovery of dendritic spines predict impairment and restoration of cognitive function, respectively. Therefore, synapse loss, the hallmark of cognitive decline in HAND, is reversible. Drugs that restore spine density may have broad application for improving cognitive function during the early phases of neurodegenerative diseases.

Endocannabinoids exert CB1 receptor-mediated neuroprotective effects in models of neuronal damage induced by HIV-1 Tat protein.

In the era of combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) is considered a chronic disease that specifically targets the brain and causes HIV-1-associated neurocognitive disorders (HAND). Endocannabinoids (eCBs) elicit neuroprotective and anti-inflammatory actions in several central nervous system (CNS) disease models, but their effects in HAND remain unknown. HIV-1 does not infect neurons, but produces viral toxins, such as transactivator of transcription (Tat), that disrupt neuronal calcium equilibrium and give rise to synaptodendritic injuries and cell death, the former being highly correlated with HAND. Consequently, we tested whether the eCBs N-arachidonoylethanolamine (anandamide/AEA) and 2-arachidonoyl-glycerol (2-AG) offer neuroprotective actions in a neuronal culture model. Specifically, we examined the neuroprotective actions of these eCBs on Tat excitotoxicity in primary cultures of prefrontal cortex neurons (PFC), and whether cannabinoid receptors mediate this neuroprotection. Tat-induced excitotoxicity was reflected by increased intracellular calcium levels, synaptodendritic damage, neuronal excitability, and neuronal death. Further, upregulation of cannabinoid 1 receptor (CB1R) protein levels was noted in the presence of HIV-1 Tat. The direct application of AEA and 2-AG reduced excitotoxic levels of intracellular calcium and promoted neuronal survival following Tat exposure, which was prevented by the CB1R antagonist rimonabant, but not by the CB2R antagonist AM630. Overall, our findings indicate that eCBs protect PFC neurons from Tat excitotoxicity in vitro via a CB1R-related mechanism. Thus, the eCB system possesses promising targets for treatment of neurodegenerative disorders associated with HIV-1 infection.