Tat Expression Research Articles

Chronic methamphetamine exposure exerts few effects on the iTat mouse model of HIV, but blocks Tat expression-induced slowed reward retrieval

Human immunodeficiency virus (HIV) continues to infect millions worldwide, negatively impacting neurobehavioral function. Further understanding of the combined effects of HIV and methamphetamine use is crucial, as methamphetamine use is prevalent in people with HIV. The HIV-associated protein Tat may contribute to cognitive dysfunction, modeled preclinically in mice using doxycycline (DOX)-inducible Tat expression (iTat). Tat may exert its effects on cognitive function via disruption of the dopamine transporter, similar to the action of methamphetamine. Additionally, Tat and methamphetamine both decrease interneuron populations, including those expressing calbindin. It is important to understand the combined effects of Tat and methamphetamine in preclinical models of HIV infection. Here, we used iTat transgenic mice and a chronic binge regimen of methamphetamine exposure to determine their combined impact on reward learning and motivation. We also measured calbindin expression in behavior-relevant brain regions. Before induction with DOX, iTat mice exhibited no differences in behavior. Chronic methamphetamine exposure before Tat induction impaired initial reward learning but did not affect motivation. Furthermore, DOX-induced Tat expression did not alter behavior, but slowed latencies to retrieve rewards. This effect of Tat, however, was not observed in methamphetamine-treated mice, indicative of a potential protective effect. Finally, Tat expression was associated with an increase in calbindin-expressing cells in the VTA, while methamphetamine exposure did not alter calbindin numbers. These findings may indicate a protective role of methamphetamine in HIV neuropathology, which in turn may help in our understanding of why people with HIV use methamphetamine at disproportionately higher rates.

Persistent sensory changes and sex differences in transgenic mice conditionally expressing HIV-1 Tat regulatory protein

HIV-associated sensory neuropathies (HIV-SN) are prevalent in >50% of patients aged over 45 years many of which report moderate to severe chronic pain. Previous preclinical studies have investigated the mechanisms by which HIV-1 causes sensory neuropathies and pain-like behaviors. The aim of the present study is to delineate the role of chronic HIV-1 trans-activator of transcription protein (Tat) exposure in the development of neuropathy in mice. The temporal effects of conditional Tat expression on the development of hypersensitivity to mechanical (von Frey filaments) and thermal (heat or cold) stimuli were tested in male and female mice that transgenically expressed HIV-1 Tat in a doxycycline-inducible manner. Inducing Tat expression produced an allodynic response to mechanical or cold (but not heat) stimuli that respectively persisted for at least 23-weeks (mechanical hypersensitivity) or at least 8-weeks (cold hypersensitivity). Both allodynic states were greater in magnitude among females, compared to males, and mechanical increased hypersensitivity progressively in females over time. Acute morphine or gabapentin treatment partly attenuated allodynia in males, but not females. Irrespective of sex, Tat reduced intraepidermal nerve fiber density, the mean amplitude of sensory nerve action potentials (but not conductance), engagement in some pain-related ethological behaviors (cage-hanging and rearing), and down-regulated PPAR-α gene expression in lumbar spinal cord while upregulating TNF-α expression in dorsal root ganglion. Taken together, these data reveal fundamental sex differences in mechanical and cold hypersensitivity in response to Tat and demonstrate the intractable nature in female mice to current therapeutics. Understanding the role of Tat in these pathologies may aid the design of future therapies aimed at mitigating the peripheral sensory neuropathies that accompany neuroHIV.

Biologically synthesized CuO nanoparticles induce physiological, metabolic, and molecular changes in the hazel cell cultures.

The utilization of plant extracts in nanoparticle (NP) synthesis has been suggested as a nature-friendly method and an efficient alternative to the conventional approaches such as physical and chemical methods. Taxol is a valuable medicinal compound, and hazelnut has been suggested as one of the sustainable resources for producing this metabolite. In the present research, copper oxide (CuO) nanoparticles (NPs) were biologically synthesized by utilizing hazelnut leaf extracts. FTIR, XRD, EDAX, DLS, and SEM analyses were used for characterizing and confirming the synthesized NPs. The effect of biosynthesized CuO NPs (10 and 90ppm), para-aminobenzoic acid (PABA) (20ppm), and CuSO4 (10ppm) on the cell viability, biochemical properties, expression of TAT and GGPPS genes, and accumulation of taxol and baccatin III in hazelnut cell cultures was investigated. The results indicated that biosynthesized CuO NPs significantly influenced the cell viability, amount of ROS, antioxidant capacity, lipid peroxidation, secondary metabolite production, and expression pattern of the genes engaged in the biosynthesis pathway of taxanes in the C. avellana L. cells. The cytotoxicity of CuO NPs to cells was dose dependent and increased with increasing its concentration, as evidenced by a decline in the survival rate and cell membrane integrity. Furthermore, the utilization of 10ppm CuSO4 caused more toxicity in the cells than the same concentration of CuO NPs. This result could be attributed to the fact that plant extracts components act as a coating for the NPs and reduce their toxicity. Treatment of the cell cultures with CuO (10ppm) + PABA (20ppm) and CuO (10ppm) induced the highest radical scavenging activity. The activity of antioxidant enzymes was increased with increasing the copper oxide NPs level from 10 to 90ppm. Contrariwise, the cell's survival rate, radical scavenging activity, and amount of secondary metabolites were significantly reduced in the higher levels of copper oxide NPs (90ppm) compared to the 10ppm. The combined utilization of 10ppm copper oxide NPs and 20ppm PABA considerably stimulated the TAT and GGPPS genes expression and produced the highest amount of taxol and baccatin III. KEY POINTS: • CuO NPs were biologically synthesized using the hazel leaf extracts and confirmed by FTIR, XRD, EDAX, DLS, and SEM analyses. • CuO NPs significantly affected the amount of ROS, antioxidant capacity, and lipid peroxidation in C. avellana L. cells. • Treatment of the hazel cells with CuO NPs increased the production of secondary metabolites including taxol and baccatin III and expression of thegenes involved in taxol and baccatin III biosynthesis (TAT and GGPPS).

Age-related neuroendocrine, cognitive, and behavioral co-morbidities are promoted by HIV-1 Tat expression in male mice.

In the U.S. about half of the HIV-infected individuals are aged 50 and older. In men living with HIV, secondary hypogonadism is common and occurs earlier than in seronegative men, and its prevalence increases with age. While the mechanisms(s) are unknown, the HIV-1 trans-activator of transcription (Tat) protein disrupts neuroendocrine function in mice partly by dysregulating mitochondria and neurosteroidogenesis. We hypothesized that conditional Tat expression in middle-aged male transgenic mice [Tat(+)] would promote age-related comorbidities compared to age-matched controls [Tat(−)]. We expected Tat to alter steroid hormone milieu consistent with behavioral deficits. Middle-aged Tat(+) mice had lower circulating testosterone and progesterone than age-matched controls and greater circulating corticosterone and central allopregnanolone than other groups. Young Tat(+) mice had greater circulating progesterone and estradiol-to-testosterone ratios. Older age or Tat exposure increased anxiety-like behavior (open field; elevated plus-maze), increased cognitive errors (radial arm water maze), and reduced grip strength. Young Tat(+), or middle-aged Tat(−), males had higher mechanical nociceptive thresholds than age-matched counterparts. Steroid levels correlated with behaviors. Thus, Tat may contribute to HIV-accelerated aging.

HIV Tat and cocaine interactively alter genome-wide DNA methylation and gene expression and exacerbate learning and memory impairments.

SUMMARYCocaine use is a major comorbidity of HIV-associated neurocognitive disorder (HAND). In this study, we show that cocaine exposure worsens the learning and memory of doxycycline-inducible and brain-specific HIV Tat transgenic mice (iTat) and results in 14,838 hypermethylated CpG-related differentially methylated regions (DMRs) and 15,800 hypomethylated CpG-related DMRs, which are linked to 52 down- and 127 upregulated genes, respectively, in the hippocampus of iTat mice. These genes are mostly enriched at the neuronal function-, cell morphology-, and synapse formation-related extracellular matrix (ECM) receptor-ligand interaction pathway and mostly impacted in microglia. The accompanying neuropathological changes include swollen dendritic spines, increased synaptophysin expression, and diminished glial activation. We also find that sex (female) and age additively worsen the behavioral and pathological changes. These findings together indicate that chronic cocaine and long-term Tat expression interactively contribute to HAND, likely involving changes of DNA methylation and ECM receptor-ligand interactions.

Effect of Zinc Oxide nanoparticle on physiological characteristics, rosmarinic acid production and expression of TAT and 4-Cl genes in Lemongrass (Melissa officinalis L.)

هدف: هدف از این مطالعه بررسی تاثیر نانو­اکسید­ روی بر خصوصیات رشدی و فیزیولوژیکی، میزان تولید رزمارینیک­اسید و بیان ژن­های کلیدی در مسیر بیوسنتزی این ترکیب در گیاه بادرنجبویه است.مواد و روش‏ها: در این مطالعه، دانه ­رست­های 30 روزه بادرنجبویه با غلظت­های صفر، 06/0 و 12/0 میلی‌گرم بر لیتر نانوذره‌­اکسید­ روی تیمار شدند و سپس پارامترهای رشدی، میزان رنگیزه­های فتوسنتزی، پرولین، گلایسین بتائین، پروتئین، فعالیت آنزیم­های آنتی ­اکسیدانتی و میزان تولید رزمارینیک­اسید مورد بررسی قرار گرفت. هم‌چنین بیان ژن‌های کلیدی در مسیر بیوسنتزی رزمارینیک­اسید با روش Real-time PCR بررسی شد.نتایج: نتایج حاکی از این است که تیمار نانو­ذره ‌اکسید­روی باعث افزایش رنگیزه­های فتوسنتزی کلروفیلی، کاروتنوئیدها و فعالیت آنتی ­اکسیدانتی این گیاه شد. هم‌چنین بیش‌ترین میزان پرولین و گلایسین بتائین نیز تحت غلظت 12/0 میلی‌گرم بر لیتر این تیمار به‌دست آمد. هم‌چنین نانوذره‌اکسید­ روی باعث افزایش بیان ژن­های مسیر بیوسنتزی رزمارینیک­اسید (TAT و 4-Cl) و در نتیجه افزایش مقدار این ترکیب شد.نتیجه‏گیری: براساس نتایج این تحقیق نانوذره‌ اکسیدروی می­تواند بر مراحل رشدی و فیزیولوژیکی گیاه بادرنجبویه اثر گذاشته و بنابراین می­توان از آن به‌منظور افزایش تولید رزمارینیک­اسید استفاده کرد.

Inhibitory Neurotransmission Is Sex-Dependently Affected by Tat Expression in Transgenic Mice and Suppressed by the Fatty Acid Amide Hydrolase Enzyme Inhibitor PF3845 via Cannabinoid Type-1 Receptor Mechanisms.

(1) Background. The endocannabinoid (eCB) system, which regulates physiological and cognitive processes, presents a promising therapeutic target for treating HIV-associated neurocognitive disorders (HAND). Here we examine whether upregulating eCB tone has potential protective effects against HIV-1 Tat (a key HIV transactivator of transcription) protein-induced alterations in synaptic activity. (2) Methods. Whole-cell patch-clamp recordings were performed to assess inhibitory GABAergic neurotransmission in prefrontal cortex slices of Tat transgenic male and female mice, in the presence and absence of the fatty acid amide hydrolase (FAAH) enzyme inhibitor PF3845. Western blot and mass spectrometry analyses assessed alterations of cannabinoid receptor and enzyme protein expression as well as endogenous ligands, respectively, to determine the impact of Tat exposure on the eCB system. (3) Results. GABAergic activity was significantly altered upon Tat exposure based on sex, whereas the effectiveness of PF3845 to suppress GABAergic activity in Tat transgenic mice was not altered by Tat or sex and involved CB1R-related mechanisms that depended on calcium signaling. Additionally, our data indicated sex-dependent changes for AEA and related non-eCB lipids based on Tat induction. (4) Conclusion. Results highlight sex- and/or Tat-dependent alterations of GABAergic activity and eCB signaling in the prefrontal cortex of Tat transgenic mice and further increase our understanding about the role of FAAH inhibition in neuroHIV.

EXPRESSION AND ACTIVITY OF AMINOTRANSFERASES IN THE LIVER OF STREPTOZOTOCIN-DIABETIC RATS: THE EFFECT OF MIFEPRISTONE.

Interventions that suppress hepatic gluconeogenesis from amino acids may be useful for improving glycemic control in diabetic patients. It was shown that administration of glucocorticoid receptor antagonist Mifepristone (MIF) leads to variously pronounced changes in the alanine-, aspartate-, tyrosine- aminotransferases (ALT, AST, TAT) activity in the liver of experimental animals. It has been suggested that this selective effect of MIF may be related to differences in the expression of the corresponding genes. The aim of the study was to investigate the gene expression and activity of ALT, AST and TAT in the liver of rats with streptozotocin-related diabetes (StD) under the long-term oral MIF administration. Male Wistar rats (n=48) with StD under the 10-days oral MIF administration were used. It was measured the activity of ALT, AST, TAT enzymes and relative expression of this genes in the liver of experimental animals. In rats with StD the gene expression of all three studied aminotransferases in the liver was statistically significantly increased and their activity was increased as well. MIF administration did not change the studied genes expression and enzymes activity to healthy rats and caused a decrease in expression of ALT and AST genes and activity of these enzymes to rats with StD. However, the expression of the TAT gene and the activity of this enzyme in the liver of rats with StD increased upon MIF administration in comparison with animals with StD. The introduction of MIF against the background of StD reduces the expression of genes and the activity of ALT and AST in the liver, what determine the transamination of amino acids to include them in gluconeogenesis, but increases the expression of genes and the activity of TAT, what determine the inclusion of tyrosine in the biogenic amines synthesis. The mechanisms of such selectivity require further study.

Transcriptomic identification of HBx-associated hub genes in hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignancies around the world. Among the risk factors involved in liver carcinogenesis, hepatitis B virus (HBV) X protein (HBx) is considered to be a key regulator in hepatocarcinogenesis. Whether HBx promotes or protects against HCC remains controversial, therefore exploring new HBx-associated genes is still important.MethodsHBx was overexpressed in HepG2, HepG2.2.15 and SMMC-7721 cell lines, primary mouse hepatocytes and livers of C57BL/6N mice. High-throughput RNA sequencing profiling of HepG2 cells with HBx overexpression and related differentially-expressed genes (DEGs), pathway enrichment analysis, protein-protein interaction networks (PPIs), overlapping analysis were conducted. In addition, Gene Expression Omnibus (GEO) and proteomic datasets of HBV-positive HCC datasets were used to verify the expression and prognosis of selected DEGs. Finally, we also evaluated the known oncogenic role of HBx by oncogenic array analysis.ResultsA total of 523 DEGs were obtained from HBx-overexpressing HepG2 cells. Twelve DEGs were identified and validated in cells transiently transfected with HBx and three datasets of HBV-positive HCC transcription profiles. In addition, using the Kaplan-Meier plotter database, the expression levels of the twelve different genes were further analyzed to predict patient outcomes.ConclusionAmong the 12 identified HBx-associated hub genes, HBV-positive HCC patients expressing ARG1 and TAT showed a good overall survival (OS) and relapse-free survival (RFS). Thus, ARG1 and TAT expression could be potential prognostic markers.

Silicon and nitric oxide synergistically modulate the production of essential oil and rosmarinic acid in Salvia officinalis under Cu stress.

The individual impact of silicon (Si) and nitric oxide (NO) on secondary metabolism in several plant species has been reported, but their combined effect has never been evaluated yet. Therefore, in this study, single and combined impacts of NO and Si on the biosynthesis of rosmarinic acid (RA) and essential oil (EO) content in leaves of Salvia officinalis were investigated under both non-stress and Cu stress conditions. The results indicated that high Cu concentration decreased biomass and the content of polyphenols, but elevated electrolyte leakage, while lower Cu concentrations, especially 200μM Cu, increased the content of polyphenols, EO, and antioxidant capacity in leaves of S. officinalis. The foliar application of sodium silicate (1mM Si) and sodium nitroprusside (200μM SNP as a NO donor) alone and particularly in combination improved shoot dry biomass, restored chlorophyll and carotenoids, increased EO content, the amounts of flavonoids, and phenolic compounds especially RA, and enhanced antioxidant capacity in the leaves of S. officinalis under both non-stress and Cu stress conditions. Copper treatment increased NO content, upregulated expression of PAL, TAT, and RAS genes, and enhanced phenylalanine ammonia-lyase activity in the leaves, which were responsible for improving the production of phenolic compounds, particularly rosmarinic acid. Foliar spraying with Si and SNP intensified these attributes. All responses were more pronounced when NO and Si were simultaneously applied under Cu stress. These findings suggest that NO and Si synergistically modulate secondary metabolism through upregulation of related gene expression and enzyme activities under both non-stress and Cu stress conditions.

Monoacylglycerol Lipase Inhibitor MJN110 Reduces Neuronal Hyperexcitability, Restores Dendritic Arborization Complexity, and Regulates Reward-Related Behavior in Presence of HIV-1 Tat.

While current therapeutic strategies for people living with human immunodeficiency virus type 1 (HIV-1) suppress virus replication peripherally, viral proteins such as transactivator of transcription (Tat) enter the central nervous system early upon infection and contribute to chronic inflammatory conditions even alongside antiretroviral treatment. As demand grows for supplemental strategies to combat virus-associated pathology presenting frequently as HIV-associated neurocognitive disorders (HAND), the present study aimed to characterize the potential utility of inhibiting monoacylglycerol lipase (MAGL) activity to increase inhibitory activity at cannabinoid receptor-type 1 receptors through upregulation of 2-arachidonoylglycerol (2-AG) and downregulation of its degradation into proinflammatory metabolite arachidonic acid (AA). The MAGL inhibitor MJN110 significantly reduced intracellular calcium and increased dendritic branching complexity in Tat-treated primary frontal cortex neuron cultures. Chronic MJN110 administration in vivo increased 2-AG levels in the prefrontal cortex (PFC) and striatum across Tat(+) and Tat(–) groups and restored PFC N-arachidonoylethanolamine (AEA) levels in Tat(+) subjects. While Tat expression significantly increased rate of reward-related behavioral task acquisition in a novel discriminative stimulus learning and cognitive flexibility assay, MJN110 altered reversal acquisition specifically in Tat(+) mice to rates indistinguishable from Tat(–) controls. Collectively, our results suggest a neuroprotective role of MAGL inhibition in reducing neuronal hyperexcitability, restoring dendritic arborization complexity, and mitigating neurocognitive alterations driven by viral proteins associated with latent HIV-1 infection.

The RNA-Binding Proteins SRP14 and HMGB3 Control HIV-1 Tat mRNA Processing and Translation During HIV-1 Latency.

HIV-1 Tat protein is essential for virus production. RNA-binding proteins that facilitate Tat production may be absent or downregulated in resting CD4+ T-cells, the main reservoir of latent HIV in people with HIV (PWH) on antiretroviral therapy (ART). In this study, we examined the role of Tat RNA-binding proteins on the expression of Tat and control of latent and productive infection. Affinity purification coupled with mass spectrometry analysis was used to detect binding partners of MS2-tagged tat mRNA in a T cell-line model of HIV latency. The effect of knockdown and overexpression of the proteins of interest on Tat transactivation and translation was assessed by luciferase-based reporter assays and infections with a dual color HIV reporter virus. Out of the 243 interactions identified, knockdown of SRP14 (Signal Recognition Particle 14) negatively affected tat mRNA processing and translation as well as Tat-mediated transactivation, which led to an increase in latent infection. On the other hand, knockdown of HMGB3 (High Mobility Group Box 3) resulted in an increase in Tat transactivation and translation as well as an increase in productive infection. Footprinting experiments revealed that SRP14 and HMGB3 proteins bind to TIM-TAM, a conserved RNA sequence-structure in tat mRNA that functions as a Tat IRES modulator of tat mRNA. Overexpression of SRP14 in resting CD4+ T-cells from patients on ART was sufficient to reverse HIV-1 latency and induce virus production. The role of SRP14 and HMGB3 proteins in controlling HIV Tat expression during latency will be further assessed as potential drug targets.

Inflammatory response within the nervous system in Tat induced HIV-associated sensory neuropathy

Abstract HIV sensory neuropathy (HIV-SN) affects 30–50% of patients infected with HIV. The role of Tat in HIV-SN has not been studied extensively. Previously, using the doxycycline (DOX) inducible HIV-1 Tat transgenic (iTat) mice, we showed that Tat expression induced neuropathy-like behaviors. Here, we further investigated the role of inflammatory responses within dorsal root ganglia (DRG) and lumbar spinal cord in the development of peripheral neuropathy. To induce Tat expression, DOX was given to iTat mice daily (i.p.) for 14 days. Lumbar spinal cord and DRG tissues were collected at days 0 (no injection), 3, 7, 14, 21, 28 and 35 following the initial injection of DOX. Vehicle control mice received pH-matched phosphate-buffered saline injections. qRT-PCR was performed with DRG tissues. Tat expression induced either transient or progressive upregulation of RNA levels of CCL2, CCL3, CCL4, CCL5, CXCL10, and IL-1beta. Lumbar spinal cord tissues underwent NanoString RNA analysis using a panel of 200+ inflammation-related genes. The patterns of the changes of these genes over time were clustered via the Short Time-series Expression Miner program. Out of the 4 patterns identified, the most significant one was a cluster of genes that were downregulated over time upon Tat induction. Gene ontology term enrichment analysis of this gene cluster via the PantherGo tool further identified a set of genes (IL-6, MAPK2K4, IL-10, HRAS1, BCL2L1, RHOA, JUN, and CREB1) that prevent/inhibit neuronal cell death/apoptosis. In all, both upregulation of selected pro-inflammatory cytokines within DRG, and down regulation of inflammation-related, neuronal survival genes within the spinal cord, could contribute to Tat-mediated peripheral neuropathy.

In vivo proton magnetic resonance spectroscopy detection of metabolite abnormalities in aged Tat-transgenic mouse brain.

Most individuals living with HIV in the USA are over 45 years old and are vulnerable to the combined effects of HIV and aging. Antiretroviral therapies reduce HIV morbidity and mortality but do not prevent HIV trans-activator of transcription (Tat) protein expression or development of HIV-associated neurocognitive disorder (HAND), which may be caused by Tat. Tat-transgenic (Tat-tg) mice are used to study Tat's effects, typically after transgene induction with doxycycline. However, uninduced Tat-tg mice experience transgene leak and model aspects of HAND when aged, including neuroinflammation. We used in vivo 9.4-tesla proton magnetic resonance spectroscopy to compare neurochemistry in aged versus young female and male uninduced Tat-tg mice. Aged Tat-tg mice demonstrated measurable tat mRNA brain expression and had lower medial prefrontal cortex (MPFC) GABA, glutamate, and taurine levels and lower striatal GABA and taurine levels. Females had lower MPFC glutathione and taurine and lower striatal taurine levels. Brain testosterone levels were negatively correlated with age in aged males but not females. Aged mice had cortical abnormalities not previously reported in aged wild-type mice including lower MPFC GABA and taurine levels. As glutathione and taurine levels reflect inflammation and oxidative stress, our data suggest that Tat may exacerbate these processes in aged Tat-tg mice. However, additional studies in controls not expressing Tat are needed to confirm this point and to deconvolve individual effects of age and Tat expression. Sex steroid hormone supplements, which counter climacteric effects, increase taurine levels, and reduce inflammation and oxidative stress, could attenuate some of the brain abnormalities we identified in aged Tat-tg mice.

HIV-1 Tat and Morphine Differentially Disrupt Pyramidal Cell Structure and Function and Spatial Learning in Hippocampal Area CA1: Continuous versus Interrupted Morphine Exposure.

About half the people infected with human immunodeficiency virus (HIV) have neurocognitive deficits that often include memory impairment and hippocampal deficits, which can be exacerbated by opioid abuse. To explore the effects of opioids and HIV on hippocampal CA1 pyramidal neuron structure and function, we induced HIV-1 transactivator of transcription (Tat) expression in transgenic mice for 14 d and co-administered time-release morphine or vehicle subcutaneous implants during the final 5 d (days 9–14) to establish steady-state morphine levels. Morphine was withheld from some ex vivo slices during recordings to begin to assess the initial pharmacokinetic consequences of opioid withdrawal. Tat expression reduced hippocampal CA1 pyramidal neuronal excitability at lower stimulating currents. Pyramidal cell firing rates were unaffected by continuous morphine exposure. Behaviorally, exposure to Tat or high dosages of morphine impaired spatial memory Exposure to Tat and steady-state levels of morphine appeared to have largely independent effects on pyramidal neuron structure and function, a response that is distinct from other vulnerable brain regions such as the striatum. By contrast, acutely withholding morphine (from morphine-tolerant ex vivo slices) revealed unique and selective neuroadaptive shifts in CA1 pyramidal neuronal excitability and dendritic plasticity, including some interactions with Tat. Collectively, the results show that opioid-HIV interactions in hippocampal area CA1 are more nuanced than previously assumed, and appear to vary depending on the outcome assessed and on the pharmacokinetics of morphine exposure.

Restoration of KCC2 Membrane Localization in Striatal Dopamine D2 Receptor-Expressing Medium Spiny Neurons Rescues Locomotor Deficits in HIV Tat-Transgenic Mice.

People infected with HIV (PWH) are highly susceptible to striatal and hippocampal damage. Motor and memory impairments are common among these patients, likely as behavioral manifestations of damage to these brain regions. GABAergic dysfunction from HIV infection and viral proteins such as transactivator of transcription (Tat) have been well documented. We recently demonstrated that the neuron specific Cl− extruder, K+ Cl− cotransporter 2 (KCC2), is diminished after exposure to HIV proteins, including Tat, resulting in disrupted GABAAR-mediated hyperpolarization and inhibition. Here, we utilized doxycycline (DOX)-inducible, GFAP-driven HIV-1 Tat transgenic mice to further explore this phenomenon. After two weeks of Tat expression, we found no changes in hippocampal KCC2 levels, but a significant decrease in the striatum that was associated with hyperlocomotion in the open field assay. We were able to restore KCC2 activity and baseline locomotion with the KCC2 enhancer, CLP290. Additionally, we found that CLP290, whose mechanism of action has yet to be described, acts to restore phosphorylation of serine 940 resulting in increased KCC2 membrane localization. We also examined neuronal subpopulation contributions to the noted effects and found significant differences. Dopamine D2 receptor-expressing medium spiny neurons (MSNs) were selectively vulnerable to Tat-induced KCC2 loss, with no changes observed in dopamine D1 receptor-expressing MSNs. These results suggest that disinhibition/diminished hyperpolarization of dopamine D2 receptor-expressing MSNs can manifest as increased locomotion in this context. They further suggest that KCC2 activity might be a therapeutic target to alleviate motor disturbances related to HIV.

Astrocyte HIV-1 Tat Differentially Modulates Behavior and Brain MMP/TIMP Balance During Short and Prolonged Induction in Transgenic Mice

Despite effective antiretroviral therapy (ART), mild forms of HIV-associated neurocognitive disorders (HAND) continue to afflict approximately half of all people living with HIV (PLWH). As PLWH age, HIV-associated inflammation perturbs the balance between brain matrix metalloproteinases (MMPs) and their tissue inhibitors of metalloproteinases (TIMPs), likely contributing to neuropathogenesis. The MMP/TIMP balance is associated with cognition, learning, and memory, with TIMPs eliciting neuroprotective effects. Dysregulation of the MMP/TIMP balance was evident in the brains of PLWH where levels of TIMP-1, the inducible family member, were significantly lower than non-infected controls, and MMPs were elevated. Here, we evaluated the MMP/TIMP levels in the doxycycline (DOX)-induced glial fibrillary acidic protein promoter-driven HIV-1 transactivator of transcription (Tat) transgenic mouse model. The HIV-1 protein Tat is constitutively expressed by most infected cells, even during ART suppression of viral replication. Many studies have demonstrated indirect and direct mechanisms of short-term Tat-associated neurodegeneration, including gliosis, blood-brain barrier disruption, elevated inflammatory mediators and neurotoxicity. However, the effects of acute vs. prolonged exposure on Tat-induced dysregulation remain to be seen. This is especially relevant for TIMP-1 as expression was previously shown to be differentially regulated in human astrocytes during acute vs. chronic inflammation. In this context, acute Tat expression was induced with DOX intraperitoneal injections over 3 weeks, while DOX-containing diet was used to achieve long-term Tat expression over 6 months. First, a series of behavior tests evaluating arousal, ambulation, anxiety, and cognition was performed to examine impairments analogous to those observed in HAND. Next, gene expression of components of the MMP/TIMP axis and known HAND-relevant inflammatory mediators were assessed. Altered anxiety-like, motor and/or cognitive behaviors were observed in Tat-induced (iTat) mice. Gene expression of MMPs and TIMPs was altered depending on the duration of Tat expression, which was independent of the HIV-associated neuroinflammation typically implicated in MMP/TIMP regulation. Collectively, we infer that HIV-1 Tat-mediated dysregulation of MMP/TIMP axis and behavioral changes are dependent on duration of exposure. Further, prolonged Tat expression demonstrates a phenotype comparable to asymptomatic to mild HAND manifestation in patients.

HIV-1 Tat Dysregulates the Hypothalamic-Pituitary-Adrenal Stress Axis and Potentiates Oxycodone-Mediated Psychomotor and Anxiety-Like Behavior of Male Mice.

Human immunodeficiency virus (HIV) is associated with co-morbid affective and stress-sensitive neuropsychiatric disorders that may be related to dysfunction of the hypothalamic-pituitary-adrenal (HPA) stress axis. The HPA axis is perturbed in up to 46% of HIV patients, but the mechanisms are not known. The neurotoxic HIV-1 regulatory protein, trans-activator of transcription (Tat), may contribute. We hypothesized that HPA dysregulation may contribute to Tat-mediated interactions with oxycodone, a clinically-used opioid often prescribed to HIV patients. In transgenic male mice, Tat expression produced significantly higher basal corticosterone levels with adrenal insufficiency in response to a natural stressor or pharmacological blockade of HPA feedback, recapitulating the clinical phenotype. On acute exposure, HIV-1 Tat interacted with oxycodone to potentiate psychomotor and anxiety like-behavior in an open field and light-dark transition tasks, whereas repeated exposure sensitized stress-related psychomotor behavior and the HPA stress response. Pharmacological blockade of glucocorticoid receptors (GR) partially-restored the stress response and decreased oxycodone-mediated psychomotor behavior in Tat-expressing mice, implicating GR in these effects. Blocking corticotrophin-releasing factor (CRF) receptors reduced anxiety-like behavior in mice that were exposed to oxycodone. Together, these effects support the notion that Tat exposure can dysregulate the HPA axis, potentially raising vulnerability to stress-related substance use and affective disorders.

Effect of valproic acid on the hepatic differentiation of mesenchymal stem cells in 2D and 3D microenvironments.

Mesenchymal stem cells (MSCs) have multi-lineage differentiation potential which make them an excellent source for cell-based therapies. Histone modification is one of the major epigenetic regulations that play central role in stem cell differentiation. Keeping in view their ability to maintain gene expression essential for successful differentiation, it was interesting to examine the effects of valproic acid (VPA), a histone deacetylase inhibitor, in the hepatic differentiation of MSCs within the 3D scaffold. MSCs were treated with the optimized concentration of VPA in the 3D collagen scaffold. Analyses of hepatic differentiation potential of treated MSCs were performed by qPCR, immunostaining and periodic acid Schiffassay. Our results demonstrate that MSCs differentiate into hepatic-like cells when treated with 5mM VPA for 24h. The VPA-treated MSCs have shown significant upregulation in the expression of hepatic genes, CK-18 (P < 0.05), TAT (P < 0.01), and AFP (P < 0.001), and hepatic proteins, AFP (P < 0.05) and ALB (P < 0.01). In addition, acetylation of histones (H3 and H4) was significantly increased (P < 0.001) in VPA-pretreated cells. Further analysis showed that VPA treatment significantly enhanced (P < 0.01) glycogen storage, an important functional aspect of hepatic cells. The present study revealed the effectiveness of VPA in hepatic differentiation within the 3D collagen scaffold. These hepatic-like cells may have an extended clinical applicability in future for successful liver regeneration.

HIV-1 Tat promotes age-related cognitive, anxiety-like, and antinociceptive impairments in female mice that are moderated by aging and endocrine status.

Hypogonadism is a common comorbidity associated with HIV-1 that is more prevalent among infected individuals over the age of 45. The underlying mechanisms are unknown, but both combined antiretroviral therapeutics and HIV-1 proteins, such as trans-activator of transcription protein (Tat), dysregulate steroid-synthetic mechanisms including lipid storage/synthesis and mitochondrial function. Thus, Tat expression may accelerate age-related comorbidities partly by impairing endocrine function. Few studies exist of Tat-mediated behavioral deficits in aged animals and effects of endocrine status have not been investigated. Accordingly, we tested whether conditional Tat expression in aged (~ 1.5years old), female, Tat-transgenic [Tat(+)] mice increases anxiety-like behavior, impairs cognition, and augments mechanical allodynia, when compared to age-matched controls that do not express Tat protein [Tat(-)]. We further tested whether aged mice that maintained their endocrine status (pre-estropausal) were more resilient to Tat/age-related comorbidities than peri- or post-estropausal mice. Tat and endocrine aging status exerted separate and interacting effects that influenced anxiety-like and cognitive behaviors. Peri- and post-estropausal mice exhibited greater anxiety-like behavior in the elevated plus-maze and impaired learning in the radial arm water maze compared to pre-estropausal mice. Irrespective of estropause status, Tat(+) mice demonstrated impaired learning, reduced grip strength, and mechanical allodynia compared to Tat(-) mice. Tat exposure reduced circulating estradiol in post-estropausal mice and increased the estradiol-to-testosterone ratio in pre-estropausal mice. Changes in circulating estradiol, testosterone, and progesterone correlated with grip strength. Thus, endocrine status is an important factor in age-related anxiety, cognition, neuromuscular function, and allodynia that can be accelerated by HIV-1 Tat protein.