The HIV-1 Transcriptional Program: From Initiation to Elongation Control.

A large body of work in the last four decades has revealed the key pillars of HIV-1 transcription control at the initiation and elongation steps. Here, I provide a recount of this collective knowledge starting with the genomic elements (DNA and nascent TAR RNA stem-loop) and transcription factors (cellular and the viral transactivator Tat), and later transitioning to the assembly and regulation of transcription initiation and elongation complexes, and the role of chromatin structure. Compelling evidence support a core HIV-1 transcriptional program regulated by the sequential and concerted action of cellular transcription factors and Tat to promote initiation and sustain elongation, highlighting the efficiency of a small virus to take over its host to produce the high levels of transcription required for viral replication. I summarize new advances including the use of CRISPR-Cas9, genetic tools for acute factor depletion, and imaging to study transcriptional dynamics, bursting and the progression through the multiple phases of the transcriptional cycle. Finally, I describe current challenges to future major advances and discuss areas that deserve more attention to both bolster our basic knowledge of the core HIV-1 transcriptional program and open up new therapeutic opportunities.

A helping HAND: therapeutic potential of MAGL inhibition against HIV-1-associated neuroinflammation.

Background: Human immunodeficiency virus (HIV) affects nearly 40 million people globally, with roughly 80% of all people living with HIV receiving antiretroviral therapy. Antiretroviral treatment suppresses viral load in peripheral tissues but does not effectively penetrate the blood-brain barrier. Thus, viral reservoirs persist in the central nervous system and continue to produce low levels of inflammatory factors and early viral proteins, including the transactivator of transcription (Tat). HIV Tat is known to contribute to chronic neuroinflammation and synaptodendritic damage, which is associated with the development of cognitive, motor, and/or mood problems, collectively known as HIV-associated neurocognitive disorders (HAND). Cannabinoid anti-inflammatory effects are well documented, but therapeutic utility of cannabis remains limited due to its psychotropic effects, including alterations within brain regions encoding reward processing and motivation, such as the nucleus accumbens. Alternatively, inhibiting monoacylglycerol lipase (MAGL) has demonstrated therapeutic potential through interactions with the endocannabinoid system. Methods: The present study utilized a reward-related operant behavioral task to quantify motivated behavior in female Tat transgenic mice treated with vehicle or MAGL inhibitor MJN110 (1 mg/kg). Brain tissue was collected to assess dendritic injury and neuroinflammatory profiles, including dendritic microtubule-associated protein (MAP2ab) intensity, microglia density, microglia morphology, astrocyte density, astrocytic interleukin-1ß (IL-1ß) colocalization, and various lipid mediators. Results: No significant behavioral differences were observed; however, MJN110 protected against Tat-induced dendritic injury by significantly upregulating MAP2ab intensity in the nucleus accumbens and in the infralimbic cortex of Tat(+) mice. No or only minor effects were noted for Iba-1+ microglia density and/or microglia morphology. Further, Tat increased GFAP+ astrocyte density in the infralimbic cortex and GFAP+ astrocytic IL-1ß colocalization in the nucleus accumbens, with MJN110 significantly reducing these measures in Tat(+) subjects. Lastly, selected HETE-related inflammatory lipid mediators in the striatum were downregulated by chronic MJN110 treatment. Conclusions: These findings demonstrate anti-inflammatory and neuroprotective properties of MJN110 without cannabimimetic behavioral effects and suggest a promising alternative to cannabis for managing neuroinflammation.

Targeted degradation of hexokinase 2 for anti­inflammatory treatment in acute lung injury.

Acute lung injury (ALI) is an acute inflammatory lung disease associated with both innate and adaptive immune responses. Hexokinase 2 (HK2) is specifically highly expressed in numerous types of inflammation­related diseases and models. In the present study in vitro and in vivo effects of targeted degradation of HK2 on ALI were explored. The degradation of HK2 by the targeting peptide TAT (transactivator of transcription protein of HIV­1)­ataxin 1 (ATXN1)­chaperone­mediated autophagy­targeting motif (CTM) was demonstrated by ELISA and western blotting in vitro and in vivo. The inhibitory effects of TAT­ATXN1­CTM on lipopolysaccharide (LPS)­induced inflammatory responses were examined using ELISAs. The therapeutic effects of TAT­ATXN1­CTM on LPS­induced ALI were examined via histological examination and ELISAs in mice. 10 µM TAT­ATXN1­CTM administration decreased HK2 protein expression and the secretion of proinflammatory cytokines (TNF­α and IL­1ß) without altering HK2 mRNA expression in LPS­treated both in vitro and in vivo, while pathological lung tissue damage and the accumulation of leukocytes, neutrophils, macrophages and lymphocytes in ALI were also significantly suppressed by 10 µM TAT­ATXN1­CTM treatment. TAT­ATXN1­CTM exhibited anti­inflammatory activity in vitro and decreased the severity of ALI in vivo. HK2 degradation may represent a novel therapeutic approach for ALI.

Acute Administration of HIV-1 Tat Protein Drives Glutamatergic Alterations in a Rodent Model of HIV-Associated Neurocognitive Disorders.

HIV-1-associated neurocognitive disorders (HAND) are a major comorbidity of HIV-1 infection, marked by impairment of executive function varying in severity. HAND affects nearly half of people living with HIV (PLWH), with mild forms predominating since the use of anti-retroviral therapies (ART). The HIV-1 transactivator of transcription (Tat) protein is found in the cerebrospinal fluid of patients adherent to ART, and its administration or expression in animals causes cognitive symptoms. Studies of Tat interaction with the N-methyl-D-aspartate receptor (NMDAR) suggest that glutamate toxicity contributes to Tat-induced impairments. To identify changes in regional glutamatergic circuitry underlying cognitive impairment, we injected recombinant Tat86 or saline to medial prefrontal cortex (mPFC) of male Sprague-Dawley rats. Rats were assessed with behavioral tasks that involve intact functioning of mPFC including the novel object recognition (NOR), spatial object recognition (SOR), and temporal order (TO) tasks at 1 and 2 postoperative weeks. Following testing, mPFC tissue was collected and analyzed by RT-PCR. Results showed Tat86 in mPFC-induced impairment in SOR, and upregulation of Grin1 and Grin2a transcripts. To further understand the mechanism of Tat toxicity, we assessed the effects of full-length Tat101 on gene expression in mPFC by RNA sequencing. The results of RNAseq suggest that glutamatergic effects of Tat86 are maintained with Tat101, as Grin2a was upregulated in Tat101-injected tissue, among other differentially expressed genes. Spatial learning and memory impairment and Grin2a upregulation suggest that exposure to Tat protein drives adaptation in mPFC, altering the function of circuitry supporting spatial learning and memory.

Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for Sensitive Analysis of Residual Protein Tat Bh1-101 in Lentiviral Vectors for Gene Therapy.

Lentiviral (LV) vector-based gene therapy is gaining popularity for treating a wide range of diseases. Various LV vectors are being developed for transducing cells in cellular gene therapy at St. Jude. Some LV vectors are produced using stable 293T packaging cell lines, which includes gag-pol-rev-tat and virus-glycoprotein. Transactivating factor (transactivator of transcription [Tat]) is a regulatory protein that drastically increases the efficiency of lentiviral transcription. Residual analysis of Tat is critical for gene vector quality and safety. In this work, we developed a highly sensitive liquid chromatography-tandem mass spectrometry method for analysis of residual Tat in Lentivirus as an alternative to enzyme-linked immunosorbent assay. Residual Tat in LV can be accurately quantified with high specificity with a limit of detection of 0.3 ng/mL.

Endolysosome iron restricts Tat-mediated HIV-1 LTR transactivation by increasing HIV-1 Tat oligomerization and ß-catenin expression.

HIV-1 transactivator of transcription (Tat) protein is required for HIV-1 replication, and it has been implicated in the pathogenesis of HIV-1-associated neurocognitive disorder (HAND). HIV-1 Tat can enter cells via receptor-mediated endocytosis where it can reside in endolysosomes; upon its escape from these acidic organelles, HIV-1 Tat can enter the cytosol and nucleus where it activates the HIV-1 LTR promoter. Although it is known that HIV-1 replication is affected by the iron status of people living with HIV-1 (PLWH), very little is known about how iron affects HIV-1 Tat activation of the HIV-1 LTR promoter. Because HIV-1 proteins de-acidify endolysosomes and endolysosome de-acidification affects subcellular levels and actions of iron, we tested the hypothesis that the endolysosome pool of iron is sufficient to affect Tat-induced HIV-1 LTR transactivation. Ferric (Fe3+) and ferrous (Fe2+) iron both restricted Tat-mediated HIV-1 LTR transactivation. Chelation of endolysosome iron with deferoxamine (DFO) and 2-2 bipyridyl, but not chelation of cytosolic iron with deferiprone and deferasirox, significantly enhanced Tat-mediated HIV-1 LTR transactivation. In the presence of iron, HIV-1 Tat increasingly oligomerized and DFO prevented the oligomerization. DFO also reduced protein expression levels of the HIV-1 restriction agent beta-catenin in the cytosol and nucleus. These findings suggest that DFO increases HIV-1 LTR transactivation by increasing levels of the more active dimeric form of Tat relative to the less active oligomerized form of Tat, increasing the escape of dimeric Tat from endolysosomes, and/or reducing beta-catenin protein expression levels. Thus, intracellular iron might play a significant role in regulating HIV-1 replication, and these findings raise cautionary notes for chelation therapies in PLWH.

TAT-Conjugated NDUFS8 Can Be Transduced into Mitochondria in a Membrane-Potential-Independent Manner and Rescue Complex I Deficiency.

NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) is a nuclear-encoded core subunit of human mitochondrial complex I. Defects in NDUFS8 are associated with Leigh syndrome and encephalomyopathy. Cell-penetrating peptide derived from the HIV-1 transactivator of transcription protein (TAT) has been successfully applied as a carrier to bring fusion proteins into cells without compromising the biological function of the cargoes. In this study, we developed a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFS8 defects. Two fusion proteins (TAT-NDUFS8 and NDUFS8-TAT) were exogenously expressed and purified from Escherichia coli for transduction of human cells. In addition, similar constructs were generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFS8 and NDUFS8-TAT were delivered into mitochondria and correctly processed. Interestingly, the mitochondrial import of TAT-containing NDUFS8 was independent of mitochondrial membrane potential. Treatment with TAT-NDUFS8 not only significantly improved the assembly of complex I in an NDUFS8-deficient cell line, but also partially rescued complex I functions both in the in-gel activity assay and the oxygen consumption assay. Our current findings suggest the considerable potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency.

HIV-1 Transactivator of Transcription (Tat) Co-operates With AP-1 Factors to Enhance c-MYC Transcription.

HIV-1 infection often leads to the development of co-morbidities including cancer. Burkitt lymphoma (BL) is one of the most over-represented non-Hodgkin lymphoma among HIV-infected individuals, and displays a highly aggressive phenotype in this population group, with comparatively poorer outcomes, despite these patients being on anti-retroviral therapy. Accumulating evidence indicates that the molecular pathogenesis of HIV-associated malignancies is unique, with components of the virus playing an active role in driving oncogenesis, and in order to improve patient prognosis and treatment, a better understanding of disease pathobiology and progression is needed. In this study, we found HIV-1 Tat to be localized within the tumor cells of BL patients, and enhanced expression of oncogenic c-MYC in these cells. Using luciferase reporter assays we show that HIV-1 Tat enhances the c-MYC gene promoter activity and that this is partially mediated via two AP-1 binding elements located at positions -1128 and -1375 bp, as revealed by mutagenesis experiments. We further demonstrate, using pull-down assays, that Tat can exist within a protein complex with the AP-1 factor JunB, and that this complex can bind these AP-1 sites within the c-MYC promoter, as shown by in vivo chromatin immunoprecipitation assays. Therefore, these findings show that in HIV-infected individuals, Tat infiltrates B-cells, where it can enhance the expression of oncogenic factors, which contributes toward the more aggressive disease phenotype observed in these patients.

Potent Antibacterial Activity of Synthetic Peptides Designed from Salusin-ß and HIV-1 Tat(49-57).

Salusin-ß is an endogenous bioactive peptide that was identified in a human full-length enriched cDNA library using bioinformatics analyses. In our previous study, we found that synthetic salusin-ß exhibits antibacterial activity against only Gram-positive microorganisms such as Staphylococcus aureus NBRC 12732. Salusin-ß has an ability to depolarize the cytoplasmic membrane of this bacterium, and this phenomenon may be linked to the antibacterial activity of this peptide. A cell-penetrating peptide (CPP), human immunodeficiency virus (HIV)-1 transactivator of transcription (Tat) (49-57) is a short cationic peptide that can traverse cell membranes. In this report, synthetic peptide conjugates of salusin-ß and HIV-1 Tat(49-57) showed potent antibacterial activities against both Gram-positive Staphylococcus aureus NBRC 12732 and Gram-negative Escherichia coli NBRC 12734. The synthetic peptides also depolarized the cytoplasmic membrane of Escherichia coli NBRC 12734 as well as Staphylococcus aureus NBRC 12732. These results suggested that HIV-1 Tat(49-57) is a protein transduction domain or CPP that changes the interaction mode between salusin-ß and the cell membrane of Escherichia coli NBRC 12734. By binding to HIV-1 Tat(49-57), salusin-ß showed a broad antibacterial spectrum regardless of whether the target was a Gram-positive or Gram-negative bacterium.

Role of Divalent Cations in HIV-1 Replication and Pathogenicity.

Divalent cations are essential for life and are fundamentally important coordinators of cellular metabolism, cell growth, host-pathogen interactions, and cell death. Specifically, for human immunodeficiency virus type-1 (HIV-1), divalent cations are required for interactions between viral and host factors that govern HIV-1 replication and pathogenicity. Homeostatic regulation of divalent cations' levels and actions appear to change as HIV-1 infection progresses and as changes occur between HIV-1 and the host. In people living with HIV-1, dietary supplementation with divalent cations may increase HIV-1 replication, whereas cation chelation may suppress HIV-1 replication and decrease disease progression. Here, we review literature on the roles of zinc (Zn2+), iron (Fe2+), manganese (Mn2+), magnesium (Mg2+), selenium (Se2+), and copper (Cu2+) in HIV-1 replication and pathogenicity, as well as evidence that divalent cation levels and actions may be targeted therapeutically in people living with HIV-1.

TAT-dextran-mediated mitochondrial transfer enhances recovery from models of reperfusion injury in cultured cardiomyocytes.

Acute myocardial infarction is a leading cause of death among single organ diseases. Despite successful reperfusion therapy, ischaemia reperfusion injury (IRI) can induce oxidative stress (OS), cardiomyocyte apoptosis, autophagy and release of inflammatory cytokines, resulting in increased infarct size. In IRI, mitochondrial dysfunction is a key factor, which involves the production of reactive oxygen species, activation of inflammatory signalling cascades or innate immune responses, and apoptosis. Therefore, intercellular mitochondrial transfer could be considered as a promising treatment strategy for ischaemic heart disease. However, low transfer efficiency is a challenge in clinical settings. We previously reported uptake of isolated exogenous mitochondria into cultured cells through co-incubation, mediated by macropinocytosis. Here, we report the use of transactivator of transcription dextran complexes (TAT-dextran) to enhance cellular uptake of exogenous mitochondria and improve the protective effect of mitochondrial replenishment in neonatal rat cardiomyocytes (NRCMs) against OS. TAT-dextran-modified mitochondria (TAT-Mito) showed a significantly higher level of cellular uptake. Mitochondrial transfer into NRCMs resulted in anti-apoptotic capability and prevented the suppression of oxidative phosphorylation in mitochondria after OS. Furthermore, TAT-Mito significantly reduced the apoptotic rates of cardiomyocytes after OS, compared to simple mitochondrial transfer. These results indicate the potential of mitochondrial replenishment therapy in OS-induced myocardial IRI.

In vivo uterine local gene delivery system using TAT-displaying bionanocapsules.

BACKGROUND: The uterus is an organ that is directly accessible via the transvaginal route, whereas the drug delivery system and the gene delivery system (GDS) for the uterus are very limited, even in animal models. In the present study, we optimized a bionanocapsule (BNC) comprising a hepatitis B virus envelope L-protein particle, for which a structurally similar particle has been used as an immunogen of a conventional HB vaccine worldwide for more than 30 years, as a local uterine GDS using a mouse model. METHODS: To display various antibodies for re-targeting to different cells other than hepatic cells, the pre-S1 region of BNC was replaced with a tandem form of the protein A-derived immunoglobulin G Fc-interacting region (Z domain, ZZ-BNC). To induce strong cell adhesion after local administration into the uterine cavity, ZZ-BNC was modified with a transactivator of transcription (TAT) peptide. RESULTS: Gene transfer using TAT-modified ZZ-BNC is approximately 5000- or 18-fold more efficient than the introduction of the same dose of naked DNAs or the use of the cationic liposomes, respectively. TAT-modified ZZ-BNC was rapidly eliminated from the uterus and had no effect on the pregnancy rate, litter size or fetal growth. CONCLUSIONS: TAT-modified ZZ-BNC could be a useful GDS for uterine endometrial therapy via local uterine injection.

Inhibitory Control Deficits Associated with Upregulation of CB1R in the HIV-1 Tat Transgenic Mouse Model of Hand.

In the era of combined antiretroviral therapy, HIV-1 infected individuals are living longer lives; however, longevity is met with an increasing number of HIV-1 associated neurocognitive disorders (HAND) diagnoses. The transactivator of transcription (Tat) is known to mediate the neurotoxic effects in HAND by acting directly on neurons and also indirectly via its actions on glia. The Go/No-Go (GNG) task was used to examine HAND in the Tat transgenic mouse model. The GNG task involves subjects discriminating between two stimuli sets in order to determine whether or not to inhibit a previously trained response. Data reveal inhibitory control deficits in female Tat(+) mice (p = .048) and an upregulation of cannabinoid type 1 receptors (CB1R) in the infralimbic (IL) cortex in the same female Tat(+) group (p < .05). A significant negative correlation was noted between inhibitory control and IL CB1R expression (r = -.543, p = .045), with CB1R expression predicting 30% of the variance of inhibitory control (R2 = .295, p = .045). Furthermore, there was a significant increase in spontaneous excitatory postsynaptic current (sEPSC) frequencies in Tat(+) compared to Tat(-) mice (p = .008, across sexes). The increase in sEPSC frequency was significantly attenuated by bath application of PF3845, a fatty acid amide hydrolase (FAAH) enzyme inhibitor (p < .001). Overall, the GNG task is a viable measure to assess inhibitory control deficits in Tat transgenic mice and results suggest a potential therapeutic treatment for the observed deficits with drugs which modulate endocannabinoid enzyme activity. Graphical Abstract Results of the Go/No-Go operant conditioning task reveal inhibitory control deficits in female transgenic Tat(+) mice without significantly affecting males. The demonstrated inhibitory control deficits appear to be associated with an upregulation of cannabinoid type 1 receptors (CB1R) in the infralimbic (IL) cortex in the same female Tat(+) group.

TAT-Modified ω-Conotoxin MVIIA for Crossing the Blood-Brain Barrier.

As the first in a new class of non-opioid drugs, ω-Conotoxin MVIIA was approved for the management of severe chronic pains in patients who are unresponsive to opioid therapy. Unfortunately, clinical application of MVIIA is severely limited due to its poor ability to penetrate the blood-brain barrier (BBB), reaching the central nervous system (CNS). In the present study, we have attempted to increase MVIIA's ability to cross the BBB via a fusion protein strategy. Our results showed that when the TAT-transducing domain was fused to the MVIIA C-terminal with a linker of varied numbers of glycine, the MVIIA-TAT fusion peptide exhibited remarkable ability to cross the bio-membranes. Most importantly, both intravenous and intranasal administrations of MVIIA-TAT in vivo showed therapeutic efficacy of analgesia. Compared to the analgesic effects of intracerebral administration of the nascent MVIIA, these systemic administrations of MVIIA-TAT require higher doses, but have much prolonged effects. Taken together, our results showed that TAT conjugation of MVIIA not only enables its peripheral administration, but also maintains its analgesic efficiency with a prolonged effective time window. Intranasal administration also rendered the MVIIA-TAT advantages of easy applications with potentially reduced side effects. Our results may present an alternative strategy to improve the CNS accessibility for neural active peptides.

Locked Nucleic Acid Nanomicelle with Cell-Penetrating Peptides for Glutathione-Triggered Drug Release and Cell Fluorescence Imaging.

Herein, we constructed a multifunctional spherical nanomicelle drug delivery system to improve the efficiency of cell uptake. The paclitaxel (PTX)-locked nucleic acid (LNA) monomer and the carboxyfluorescein (FAM)-labeled DNA were mixed together to assemble and form a spherical nanomicelle that was functionalized with transactivator of transcription (TAT), a cell-penetrating peptides (CPPs). A bioreductively activated disulfide was used to link the hydrophobic PTX to the LNA, allowing the PTX to be released freely in the presence of glutathione (GSH) upon cell uptake. Based on magnetic separation, the synthetic process of PTX-LNA monomers avoids time-consuming and labor-intensive shortcomings. Cellular uptake of PTX-LNA-TAT nanomicelle and the drug release occur rapidly as proved by fluorescence microscopy and flow cytometry. The resulting nanomicelle was greater stability, monodisperse size, and the high therapeutic potential. Furthermore, the system can readily achieve detection of GSH in the cancer cells. The detection limit for commercial GSH determined was 1.0 × 10-9 M by using PTX/Fluorescein isothiocyanate (FITC)-LNA/black hole quencher 1 (BHQ-1) as a probe.

Reduced intraepidermal nerve fibre density, glial activation, and sensory changes in HIV type-1 Tat-expressing female mice: involvement of Tat during early stages of HIV-associated painful sensory neuropathy.

INTRODUCTION: HIV infection is associated with chronic pain states, including sensory neuropathy, which affects greater than 40% of patients. OBJECTIVES AND METHODS: To determine the impact of HIV-Tat induction on nociceptive behaviour in female mice conditionally expressing HIV Tat1-86 protein through a doxycycline (DOX)-driven glial fibrillary acidic protein promoter, intraepidermal nerve fibre density and immune cell activation in the dorsal root ganglion (DRG) and spinal cord were assessed by immunohistochemistry. Mice were assessed for mechanical and thermal sensitivity for 9 weeks using von-Frey and Hargreaves tests. RESULTS: Intraepidermal nerve fibre density was significantly reduced after 6 weeks of Tat induction, similar to sensory neuropathy seen in clinical HIV infection. Tat induction through DOX caused a significant reduction in paw withdrawal thresholds in a time-dependent manner starting the 4th week after Tat induction. No changes in paw withdrawal latencies were seen in Tat(-) control mice lacking the tat transgene. Although reductions in paw withdrawal thresholds increased throughout the study, no significant change in spontaneous motor activity was observed. Spinal cord (cervical and lumbar), DRG, and hind paw skin were collected at 8 days and 6 weeks after Tat induction. HIV-Tat mRNA expression was significantly increased in lumbar DRG and skin samples 8 days after DOX treatment. Tat induced a significant increase in the number of Iba-1 positive cells at 6 weeks, but not after 8 days, of exposure. No differences in glial fibrillary acidic protein immunoreactivity were observed. CONCLUSION: These results suggest that Tat protein contributes to painful HIV-related sensory neuropathy during the initial stages of the pathogenesis.

TAT-PEP Enhanced Neurobehavioral Functional Recovery by Facilitating Axonal Regeneration and Corticospinal Tract Projection After Stroke.

Paired immunoglobulin-like receptor B (PirB) has been identified as a new receptor for myelin-associated inhibitory (MAI) proteins, which may play important role in axonal regeneration and corticospinal tract (CST) projection associated with neurobehavioral function recovery after stroke. Here, we found that the expression of PirB was increased in the cortical penumbra from 1 to 28 days after transient focal cerebral ischemic reperfusion of rats. Then, transactivator of transcription-PirB extracellular peptide (TAT-PEP) was generated that might block the interactions between MAIs and PirB. The results showed that TAT-PEP displayed high affinity for MAIs and ameliorated their inhibitory effect on neurite growth. Furthermore, TAT-PEP can widely distribute in the penumbra after intraperitoneal injection. Then, we found that TAT-PEP enhanced neurite growth and alleviated growth cone collapse after oxygen glucose deprivation (OGD) injury. In addition, TAT-PEP promoted long-term neurobehavioral functional recovery through enhancing axonal regeneration and CST projection. Finally, the observations demonstrated that POSH/RhoA/growth-associated protein 43 (GAP43) as PirB-associated downstream signaling molecules played important role in neurobehavioral functional recovery after stroke. Moreover, the underlying mechanism associated with TAT-PEP-mediated promoting axonal regeneration and CST projection was by intervening in the expression of POSH, RhoA, and GAP43. These studies suggest that TAT-PEP may represent an attractive therapeutic strategy against stroke.

TAT-Ngn2 Enhances Cognitive Function Recovery and Regulates Caspase-Dependent and Mitochondrial Apoptotic Pathways After Experimental Stroke.

Neurogenin-2 (Ngn2) is a basic helix-loop-helix (bHLH) transcription factor that contributes to the identification and specification of neuronal fate during neurogenesis. In our previous study, we found that Ngn2 plays an important role in alleviating neuronal apoptosis, which may be viewed as an attractive candidate target for the treatment of cerebral ischemia. However, novel strategies require an understanding of the function and mechanism of Ngn2 in mature hippocampal neurons after global cerebral ischemic injury. Here, we found that the expression of Ngn2 decreased in the hippocampus after global cerebral ischemic injury in mice and in primary hippocampal neurons after oxygen glucose deprivation (OGD) injury. Then, transactivator of transcription (TAT)-Ngn2, which was constructed by fusing a TAT domain to Ngn2, was effectively transported and incorporated into hippocampal neurons after intraperitoneal (i.p.) injection and enhanced cognitive functional recovery in the acute stage after reperfusion. Furthermore, TAT-Ngn2 alleviated hippocampal neuronal damage and apoptosis, and inhibited the cytochrome C (CytC) leak from the mitochondria to the cytoplasm through regulating the expression levels of brain-derived neurotrophic factor (BDNF), phosphorylation tropomyosin-related kinase B (pTrkB), Bcl-2, Bax and cleaved caspase-3 after reperfusion injury in vivo and in vitro. These findings suggest that the downregulation of Ngn2 expression may have an important role in triggering brain injury after ischemic stroke and that the neuroprotection of TAT-Ngn2 against stroke might involve the modulation of BDNF-TrkB signaling that regulates caspase-dependent and mitochondrial apoptotic pathways, which may be an attractive therapeutic strategy for cerebral ischemic injury.

Recombinant neuroglobin ameliorates early brain injury after subarachnoid hemorrhage via inhibiting the activation of mitochondria apoptotic pathway.

Neuroglobin (Ngb) overexpression is considered as an intrinsic neuroprotective response. Therefore, exogenous Ngb increased in brain tissues has become a promising therapeutic strategy for neurological diseases. Previous studies demonstrated that transactivator of transcription (TAT) protein transduction domain was able to mediate synthetic Ngb entrance into neurons, and then protected brain from hypoxia-ischemic injury. However, the role of recombinant Ngb on early brain injury following subarachnoid hemorrhage (SAH) has not been elucidated. The objectives of this study were to investigate the expression of endogenous Ngb in brain using a rabbit model of SAH, and to verify whether TAT-Ngb fusion protein could be delivered into brain parenchyma, as well as to explore the neuroprotective effect of Ngb and its possible mechanisms. We found that Ngb expressions were up regulated in the transcript and protein levels in a similar time dependent manner after SAH as compared to the sham group. Moreover, TAT-Ngb fusion protein was successfully generated and transferred into brain neurons. Compared with the saline- and Ngb-treated group, neuronal viabilities and neurological outcomes were significantly improved 72 h post-SAH in the TAT-Ngb-treated group. Likewise, anti-apoptotic Bcl-2 protein was also elevated obviously. Conversely, pro-apoptotic factors including caspase 3, caspase 9 and Bax were greatly decreased after TAT-Ngb treatment. Our results suggest that Ngb plays a neuroprotective effect in rabbits suffering from SAH possibly through inhibiting the SAH-induced activation of mitochondria apoptotic pathway. Furthermore, TAT-mediated Ngb delivery into brain may be a promising therapeutic approach.

Expression of an RNA glycosidase inhibits HIV-1 transactivation of transcription.

HIV-1 (human immunodeficiency virus) transcription is primarily controlled by the virally encoded Tat (transactivator of transcription) protein and its interaction with the viral TAR (transcription response element) RNA element. Specifically, binding of a Tat-containing complex to TAR recruits cellular factors that promote elongation of the host RNA polymerase engaging the viral DNA template. Disruption of this interaction halts viral RNA transcription. In the present study, we investigated the effect of pokeweed antiviral protein (PAP), an RNA glycosidase (EC#: 3.2.2.22) synthesized by the pokeweed plant (Phytolacca americana), on transcription of HIV-1 mRNA. We show that co-expression of PAP with a proviral clone in culture cells resulted in a Tat-dependent decrease in viral mRNA levels. PAP reduced HIV-1 transcriptional activity by inhibiting Tat protein synthesis. The effects of PAP expression on host factors AP-1 (activator protein 1), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B-cells) and specificity protein 1, which modulate HIV-1 transcription by binding to the viral LTR (5'-long terminal repeat), were also investigated. Only AP-1 showed a modest JNK pathway-dependent increase in activity in the presence of PAP; however, this activation was not sufficient to significantly enhance transcription from a partial viral LTR containing AP-1 binding sites. Therefore, the primary effect of PAP on HIV-1 transcription is to reduce viral RNA synthesis by decreasing the abundance of Tat. These findings provide a mechanistic explanation for the observed decrease in viral RNAs in cells expressing PAP and contribute to our understanding of the antiviral effects of this plant protein.