Transcriptional Transactivation Research Articles

Levetiracetam prevents neurophysiological changes and preserves cognitive function in the HIV-1 TAT transgenic mouse model of HIV-associated neurocognitive disorder.

HIV-associated neurocognitive disorder (HAND) affects nearly half of the 39 million people living with HIV. HAND symptoms range from subclinical cognitive impairment to dementia; the mechanisms that underlie HAND remain unclear and there is no treatment. The HIV-protein transactivator of transcription (TAT) is thought to contribute to HAND because it persists in the CNS and elicits neurotoxicity in animal models. Network hyperexcitability is associated with accelerated cognitive decline in neurodegenerative disorders. Here, we show that the antiepileptic drug levetiracetam (LEV) attenuated aberrant excitatory synaptic transmission, protected synaptic plasticity, reduced seizure susceptibility, and preserved cognition in inducible TAT (iTAT) transgenic male mice. iTAT mice had an increased frequency of spontaneous excitatory postsynaptic currents in hippocampal slice recordings and impaired long-term potentiation, a form of synaptic plasticity that underlies learning and memory. Two-week administration of LEV by osmotic minipump prevented both impairments. Kainic acid administered to iTAT mice induced a higher maximum behavioral seizure score, longer seizure duration, and a shorter latency to first seizure, consistent with a lower seizure threshold. LEV treatment prevented these in vivo signs of hyperexcitability. Lastly, in the Barnes maze, iTAT mice required more time to reach the goal, committed more errors, and received lower cognitive scores relative to iTAT mice treated with LEV. Thus, TAT expression drives functional deficits, suggesting a causative role in HAND. As LEV not only prevented aberrant synaptic activity in iTAT mice, but also prevented cognitive dysfunction, it may provide a promising pharmacological approach to the treatment of HAND. Significance Statement Around half of people living with HIV also suffer from HIV-associated neurocognitive disorder (HAND), for which there is no treatment. The HIV protein TAT causes toxicity that is thought to contribute to HAND. Here, we show that an antiepileptic drug, levetiracetam (LEV), prevented synaptic and cognitive impairments that develop in a TAT-expressing mouse. LEV is widely used to treat seizures and is well-tolerated in humans, including those with HIV. This study supports further investigation of LEV-treatment for neuroprotection in HAND.

Inclusion of TAT and NLS sequences in lipopeptide molecules generates homogenous nanoparticles for gene delivery applications

PurposesThe objective of this study is to develop a versatile gene carrier based on lipopeptides capable of delivering genetic material into target cells with minimal cytotoxicity. MethodsTwo lipopeptide molecules, palmitoyl-CKKHH and palmitoyl-CKKHH-YGRKKRRQRRR-PKKKRKV, were synthesized using solid phase peptide synthesis and evaluated as transfection agents. Physicochemical characterization of the lipopeptides included a DNA shift mobility assay, particle size measurement, and transmission electron microscopy (TEM) analysis. Cytotoxicity was assessed in CHO-K1 and HepG2 cells using the MTT assay, while transfection efficiency was determined by evaluating the expression of the green fluorescent protein-encoding gene. ResultsOur findings demonstrate that the lipopeptides can bind, condense, and shield DNA from DNase degradation. The inclusion of the YGRKKRRQRRR sequence, a transcription trans activator, and the PKKKRKV sequence, a nuclear localization signal, imparts desirable properties. Lipopeptide-based TAT-NLS/DNA nanoparticles exhibited stability for up to 20 days when stored at 6–8 °C, displaying uniformity with a compact size of approximately 120 nm. Furthermore, the lipopeptides exhibited lower cytotoxicity compared to the poly-L-lysine. Transfection experiments revealed that protein expression mediated by the lipopeptide occurred at a charge ratio ranging from 4.0 to 8.0. ConclusionThese results indicate that the lipopeptide, composed of a palmitoyl alkyl chain and TAT and NLS sequences, can efficiently condense and protect DNA, form stable and uniform nanoparticles, and exhibit promising characteristics as a potential gene carrier with minimal cytotoxicity.

Effects of SRI-32743, a Novel Quinazoline Structure-Based Compound, on HIV-1 Tat and Cocaine Interaction with Norepinephrine Transporter.

Prolonged exposure to HIV-1 transactivator of transcription (Tat) protein dysregulates monoamine transmission, a physiological change implicated as a key factor in promoting neurocognitive disorders among people living with HIV. We have demonstrated that in vivo expression of Tat in Tat transgenic mice decreases dopamine uptake through both dopamine transporter (DAT) and norepinephrine transporter (NET) in the prefrontal cortex. Further, our novel allosteric inhibitor of monoamine transporters, SRI-32743, has been shown to attenuate Tat-inhibited dopamine transport through DAT and alleviates Tat-potentiated cognitive impairments. The current study reports the pharmacological profiles of SRI-32743 in basal and Tat-induced inhibition of human NET (hNET) function. SRI-32743 exhibited less affinity for hNET binding than desipramine, a classical NET inhibitor, but displayed similar potency for inhibiting hDAT and hNET activity. SRI-32743 concentration-dependently increased hNET affinity for [3H]DA uptake but preserved the Vmax of dopamine transport. SRI-32743 slowed the cocaine-mediated dissociation of [3H]Nisoxetine binding and reduced both [3H]DA and [3H]MPP+ efflux but did not affect d-amphetamine-mediated [3H]DA release through hNET. Finally, we determined that SRI-32743 attenuated a recombinant Tat1-86-induced decrease in [3H]DA uptake via hNET. Our findings demonstrated that SRI-32743 allosterically disrupts the recombinant Tat1-86-hNET interaction, suggesting a potential treatment for HIV-infected individuals with concurrent cocaine abuse.

Germ line ERG haploinsufficiency defines a new syndrome with cytopenia and hematological malignancy predisposition

AbstractThe genomics era has facilitated the discovery of new genes that predispose individuals to bone marrow failure (BMF) and hematological malignancy (HM). We report the discovery of ETS-related gene (ERG), a novel, autosomal dominant BMF/HM predisposition gene. ERG is a highly constrained transcription factor that is critical for definitive hematopoiesis, stem cell function, and platelet maintenance. ERG colocalizes with other transcription factors, including RUNX1 and GATA2, on promoters or enhancers of genes that orchestrate hematopoiesis. We identified a rare heterozygous ERG missense variant in 3 individuals with thrombocytopenia from 1 family and 14 additional ERG variants in unrelated individuals with BMF/HM, including 2 de novo cases and 3 truncating variants. Phenotypes associated with pathogenic germ line ERG variants included cytopenias (thrombocytopenia, neutropenia, and pancytopenia) and HMs (acute myeloid leukemia, myelodysplastic syndrome, and acute lymphoblastic leukemia) with onset before 40 years. Twenty ERG variants (19 missense and 1 truncating), including 3 missense population variants, were functionally characterized. Thirteen potentially pathogenic ETS domain missense variants displayed loss-of-function characteristics, thereby disrupting transcriptional transactivation, DNA binding, and/or nuclear localization. Selected variants overexpressed in mouse fetal liver cells failed to drive myeloid differentiation and cytokine-independent growth in culture and to promote acute erythroleukemia when transplanted into mice, concordant with these being loss-of-function variants. Four individuals displayed somatic genetic rescue by copy neutral loss of heterozygosity. Identification of predisposing germ line ERG variants has clinical implications for patient and family diagnoses, counseling, surveillance, and treatment strategies, including selection of bone marrow donors and cell or gene therapy.

Co-delivery of retinoic acid and miRNA by functional Au nanoparticles for improved survival and CT imaging tracking of MSCs in pulmonary fibrosis therapy

Mesenchymal stem cells (MSCs) have emerged as promising candidates for idiopathic pulmonary fibrosis (IPF) therapy. Increasing the MSC survival rate and deepening the understanding of the behavior of transplanted MSCs are of great significance for improving the efficacy of MSC-based IPF treatment. Therefore, dual-functional Au-based nanoparticles (Au@PEG@PEI@TAT NPs, AuPPT) were fabricated by sequential modification of cationic polymer polyetherimide (PEI), polyethylene glycol (PEG), and transactivator of transcription (TAT) penetration peptide on AuNPs, to co-deliver retinoic acid (RA) and microRNA (miRNA) for simultaneously enhancing MSC survive and real-time imaging tracking of MSCs during IPF treatment. AuPPT NPs, with good drug loading and cellular uptake abilities, could efficiently deliver miRNA and RA to protect MSCs from reactive oxygen species and reduce their expression of apoptosis executive protein Caspase 3, thus prolonging the survival time of MSC after transplantation. In the meantime, the intracellular accumulation of AuPPT NPs enhanced the computed tomography imaging contrast of transplanted MSCs, allowing them to be visually tracked in vivo. This study establishes an Au-based dual-functional platform for drug delivery and cell imaging tracking, which provides a new strategy for MSC-related IPF therapy.

Molecular Insight into the Effect of HIV-TAT Protein on Amyloid-β Peptides

Molecular Insight into the Effect of HIV-TAT Protein on Amyloid-β Peptides

Neuronal expression of herpes simplex virus-1 VP16 protein induces pseudorabies virus escape from silencing and reactivation.

Alpha herpesvirus (α-HV) particles enter their hosts from mucosal surfaces and efficiently maintain fast transport in peripheral nervous system (PNS) axons to establish infections in the peripheral ganglia. The path from axons to distant neuronal nuclei is challenging to dissect due to the difficulty of monitoring early events in a dispersed neuron culture model. We have established well-controlled, reproducible, and reactivateable latent infections in compartmented rodent neurons by infecting physically isolated axons with a small number of viral particles. This system not only recapitulates the physiological infection route but also facilitates independent treatment of isolated cell bodies or axons. Consequently, this system enables study not only of the stimuli that promote reactivation but also the factors that regulate the initial switch from productive to latent infection. Adeno-associated virus (AAV)-mediated expression of herpes simplex-1 (HSV-1) VP16 alone in neuronal cell bodies enabled the escape from silencing of incoming pseudorabies virus (PRV) genomes. Furthermore, the expression of HSV VP16 alone reactivated a latent PRV infection in this system. Surprisingly, the expression of PRV VP16 protein supported neither PRV escape from silencing nor reactivation. We compared transcription transactivation activity of both VP16 proteins in primary neurons by RNA sequencing and found that these homolog viral proteins produce different gene expression profiles. AAV-transduced HSV VP16 specifically induced the expression of proto-oncogenes including c-Jun and Pim2. In addition, HSV VP16 induces phosphorylation of c-Jun in neurons, and when this activity is inhibited, escape of PRV silencing is dramatically reduced.IMPORTANCEDuring latency, alpha herpesvirus genomes are silenced yet retain the capacity to reactivate. Currently, host and viral protein interactions that determine the establishment of latency, induce escape from genome silencing or reactivation are not completely understood. By using a compartmented neuronal culture model of latency, we investigated the effect of the viral transcriptional activator, VP16 on pseudorabies virus (PRV) escape from genome silencing. This model recapitulates the physiological infection route and enables the study of the stimuli that regulate the initial switch from a latent to productive infection. We investigated the neuronal transcriptional activation profiles of two homolog VP16 proteins (encoded by HSV-1 or PRV) and found distinct gene activation signatures leading to diverse infection outcomes. This study contributes to understanding of how alpha herpesvirus proteins modulate neuronal gene expression leading to the initiation of a productive or a latent infection.

Reprofiling synthetic glucocorticoid-induced leucine zipper fusion peptide as a novel and effective hair growth promoter.

Hair is a biofilament with unique multi-dimensional values. In human, in addition to physiologic impacts, hair loss and hair related disorders can affect characteristic features, emotions, and social behaviors. Despite significant advancement, there is a dire need to explore alternative novel therapies with higher efficacy, less side effects and lower cost to promote hair growth to treat hair deficiency. Glucocorticoid-induced leucine zipper (GILZ) is a protein rapidly induced by glucocorticoids. Studies from our group and many others have suggested that a synthetic form of GILZ, TAT-GILZ, a fusion peptide of trans-activator of transcription and GILZ, can function as a potent regulator of inflammatory responses, re-establishing and maintaining the homeostasis. In this study, we investigate whether TAT-GILZ could promote and contribute to hair growth. For our pre-clinical model, we used 9-12 week-old male BALB/c and nude (athymic, nu/J) mice. We applied TAT-GILZ and/or TAT (vehicle) intradermally to depilated/hairless mice. Direct observation, histological examination, and Immunofluorescence imaging were used to assess the effects and compare different treatments. In addition, we tested two current treatment for hair loss/growth, finasteride and minoxidil, for optimal evaluation of TAT-GILZ in a comparative fashion. Our results showed, for the first time, that synthetic TAT-GILZ peptide accelerated hair growth on depilated dorsal skin of BALB/c and induced hair on the skin of athymic mice where hair growth was not expected. In addition, TAT-GILZ was able to enhance hair follicle stem cells and re-established the homeostasis by increasing counter inflammatory signals including higher regulatory T cells and glucocorticoid receptors. In conclusion, our novel findings suggest that reprofiling synthetic TAT-GILZ peptide could promote hair growth by increasing hair follicle stem cells and re-establishing homeostasis.

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

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. 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. 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. These findings demonstrate anti-inflammatory and neuroprotective properties of MJN110 without cannabimimetic behavioral effects and suggest a promising alternative to cannabis for managing neuroinflammation.

Delivery of AKT1 phospho-forms to human cells reveals differential substrate selectivity.

Protein kinase B (AKT1) is a serine/threonine kinase that regulates fundamental cellular processes, including cell survival, proliferation, and metabolism. AKT1 activity is controlled by two regulatory phosphorylation sites (Thr308, Ser473) that stimulate a downstream signaling cascade through phosphorylation of many target proteins. At either or both regulatory sites, hyperphosphorylation is associated with poor survival outcomes in many human cancers. Our previous biochemical and chemoproteomic studies showed that the phosphorylated forms of AKT1 have differential selectivity toward peptide substrates. Here, we investigated AKT1-dependent activity in human cells, using a cell-penetrating peptide (transactivator of transcription, TAT) to deliver inactive AKT1 or active phospho-variants to cells. We used enzyme engineering and genetic code expansion relying on a phosphoseryl-transfer RNA (tRNA) synthetase (SepRS) and tRNASep pair to produce TAT-tagged AKT1 with programmed phosphorylation at one or both key regulatory sites. We found that all TAT-tagged AKT1 variants were efficiently delivered into human embryonic kidney (HEK 293T) cells and that only the phosphorylated AKT1 (pAKT1) variants stimulated downstream signaling. All TAT-pAKT1 variants induced glycogen synthase kinase (GSK)-3α phosphorylation, as well as phosphorylation of ribosomal protein S6 at Ser240/244, demonstrating stimulation of downstream AKT1 signaling. Fascinatingly, only the AKT1 variants phosphorylated at S473 (TAT-pAKT1S473 or TAT-pAKT1T308,S473) were able to increase phospho-GSK-3β levels. Although each TAT-pAKT1 variant significantly stimulated cell proliferation, cells transduced with TAT-pAKT1T308 grew significantly faster than with the other pAKT1 variants. The data demonstrate differential activity of the AKT1 phospho-forms in modulating downstream signaling and proliferation in human cells.

NF-κB1 deficiency promotes macrophage-derived adrenal tumors but decreases neurofibromas in HTLV-I LTR-Tax transgenic mice.

Human T-cell leukemia virus type I (HTLV-I) is an oncogenic virus whose infection can cause diverse diseases, most notably adult T-cell leukemia/lymphoma (ATL or ATLL), an aggressive and fatal malignancy of CD4 T cells. The oncogenic ability of HTLV-I is mostly attributed to the viral transcriptional transactivator Tax. Tax alone is sufficient to induce specific tumors in mice depending on the promotor used to drive Tax expression, thereby being used to understand HTLV-I tumorigenesis and model the tumor types developed in Tax transgenic mice. Tax exerts its oncogenic role predominantly by activating the cellular transcription factor NF-κB. Here, we report that genetic deletion of NF-κB1, the prototypic member of the NF-κB family, promotes adrenal medullary tumors but suppresses neurofibromas in mice with transgenic Tax driven by the HTLV-I Long Terminal Repeat (LTR) promoter. The adrenal tumors are derived from macrophages. Neoplastic macrophages also infiltrate the spleen and lymph nodes, causing splenomegaly and lymphadenopathy in mice. Nevertheless, the findings could be human relevant, because macrophages are important target cells of HTLV-I infection and serve as a virus reservoir in vivo. Moreover, the spleen, lymph nodes and adrenal glands are the most common sites of tumor cell infiltration in HTLV-I-infected patients. These data provide new mechanistic insights into the complex interaction between Tax and NF-κB, therefore improving our understanding of HTLV-I oncogenic pathogenesis. They also expand our knowledge and establish a new animal model of macrophage neoplasms and adrenal tumors.

Protein Kinase C Beta II Inhibitor Conjugated with Myristic Acid and Trans-Activator of Transcription improves intracellular delivery and cardiovascular protection in porcine myocardial ischemia/reperfusion injury

Introduction: While timely reperfusion of oxygenated blood is critical during resuscitation of ischemic myocardium, oxidative stress during reperfusion still leads to ischemia/reperfusion (I/R) injury and ultimately, cardiomyocyte death. The major sources of oxidative stress targeted in this study are NADPH oxidase (NOX-2) and mitochondria, which are both activated by protein kinase C beta II (PKCβII). In previous studies with an ex-vivo rat heart I/R model, Myristic Acid (Myr) and Trans-Activator Transcription (Tat; YGRKKRRQRRR) conjugated PKCβII inhibitor (Myr-Tat-PKCβII-; N-Myr-Tat-CC-SLNPEWNET) showed cardioprotective effects and decreased infarct size. In this study, we hypothesized that Myr-Tat-PKCβII- will offer cardioprotective effects in comparison with a scrambled peptide control (Myr-Tat-PKCβII-scram; N-Myr-Tat-CC-WNPESLNTE) in an in-vivo porcine myocardial I/R model. Methods: Regional I(1 hour)/R(3 hours) was induced in Male Yorkshire pigs (38-50kg) by inflating a balloon to occlude the Left Anterior Descending Artery (LAD) at the level of the second diagonal branch (supplies 40% of anterior left ventricle) and then deflating it to allow for reperfusion. Then, a bolus of either Myr-Tat-PKCβII- or Myr-Tat-PKCβII-scram (20ng/kg; ~100pmol in blood) was immediately administered into the LAD. Cardiac function and injury were measured by monitoring changes in ejection fraction (EF) and through routine measurements of serum creatine phosphokinase (CPK), troponin I, and myoglobin, respectively. After I/R, the hearts were stained with 1% Evans Blue dye to identify the area at risk (AR) and 1% triphenyltetrazolium chloride to determine the area of necrosis (AN). Infarct size was then quantified (AN/AR) and all data was analyzed via Student’s t-test. Results: Analysis showed that Myr-Tat-PKCβII- significantly restored EF to within 1.40.7% of baseline compared to controls which only restored EF to within 6.42.1% (p<0.05) of baseline EF. Myr-Tat-PKCβII- showed a significant increase in serum myoglobin levels at 1 hr of reperfusion (1362.1 83.4 μg/mL, n=4) compared to scrambled control (202.1197.3 μg/mL, n=3 p<0.05). Myr-Tat-PKCβII- reduced infarct size to 10.0±2.8%; n=4; compared to scrambled control hearts (28.5±8.3%; n=6; p<0.05). CPK and Troponin I levels were comparable in both groups. These results suggest that Myr-Tat-PKCβII- can help prevent cardiac injury when given immediately after an ischemic event. Conclusions: Data from ex-vivo rat heart I/R model, coupled with data from this in-vivo porcine myocardial I/R model indicate the effcacy of Myr-Tat-PKCβII- in preventing oxidative stress-induced myocardial I/R injury when given at the beginning of reperfusion. These findings suggest that Myr-Tat-PKCβII- could be useful in the clinical setting when administered immediately after cardiac resuscitation. Future studies include the treatment of rat cardiomyocyte cells with Myr-Tat-PKCβII- prior to 24 hr hypoxia/24 hr reoxygenation, followed by cell viability assays compared to untreated control. This additional data can help determine the optimal dose to use in a 12 week survival study using the same porcine myocardial I/R protocol. Funding: This study was funded by the Philadelphia College of Osteopathic Medicine, Department of Biomedical Sciences, Division of Research, and the Center for Chronic Diseases of Aging, the National Heart, Lung & Blood Institute at NIH (grant # 1R43HL160338-01) and Young Therapeutics, LLC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Co-regulatory Proteins Integrate Metabolic Signals to Regulate ESRRG Transcriptional Transactivation in Stomach Parietal Cell Differentiation

Acid-producing stomach parietal cells (PC) are metabolically active and energy demanding cells. Aberrant PC function and census leads to complications ranging from peptic ulcers to stomach adenocarcinoma. Effcient energy expenditure and catabolic metabolism are required for normal PC differentiation from progenitor cells. Here, our data show that the master metabolic transcriptional factor, estrogen-related receptor gamma ( ESRRG), governs murine and human PC differentiation. We are further investigating how known metabolically controlled co-regulatory proteins — Sirtuin 1 (SIRT1), Nuclear Receptor Subfamily 0 Group B Member 2 (NR0B2), and CREB/ATF BZIP Transcription Factor (CREBZF, or SMILE) — integrate stimuli to enable ESRRG transcriptional activation during PC emergence from precursors. Specifically, our lab has developed an acute and reversible PC ablation model to monitor PC recovery in which Simian diphtheria toxin receptor (DTR) expression under the Atp4b promoter targets PC-specific ablation by diphtheria toxin (DT). Additionally, we employ high-dose tamoxifen (HD TAM), which causes a more general gastric injury (independent of tamoxifen effects on estrogen) from PC death along with reactive spasmolytic polypeptide expressing metaplasia (SPEM) of remaining cells. Immunofluorescence, western blotting, and qRT-PCR revealed SIRT1 is upregulated before emergence of early PC clones post-DT and decreases later, suggesting positive regulation of early PC lineage-commitment from stem cells. During the same early time frame, factors NR0B2 and CREBZF decreased, suggesting contrasting roles. Administration of Resv to mice accelerated PC recovery after injury and expanded PCs even at homeostasis. ChIP and ongoing ChIP-seq and Cut&Run assays show ESRRG binds cis-regulatory elements of the metabolic genes and the co-regulatory proteins to promote PC lineage commitment in both mice and in a human gastric cell line in which stable ESRRG-GFP is expressed under a doxycycline-inducible promoter. In other ongoing work, we are generating mice with PC-specific Sirt1/ Nr0b2 ablation to further dissect PC regeneration dynamics and conducting gastric organoid experiments with NR0B2/SIRT1 pharmacological modulators to monitor PC differentiation effects. These organoid samples will be further subjected to epigenetic analysis (Cut&Run, ATACseq) to study chromatin landscape changes. We conclude that cellular metabolic integration by co-regulators enables ESRRG-mediated transactivation during PC differentiation, and SIRT1 promotes early PC lineage-commitment that might offer a promising pro-PC differentiation therapeutic strategy. This work is supported by funding from the NIH through the National Institute of Diabetes and Digestive and Kidney Diseases and the National Cancer Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TAT38 and TAT38 mimics potently inhibit adipogenesis by repressing C/EBPα, PPARγ, Pi-PPARγ, and SREBP1 expression

Antiretroviral therapy-naive people living with HIV possess less fat than people without HIV. Previously, we found that HIV-1 transactivator of transcription (TAT) decreases fat in ob/ob mice. The TAT38 (a.a. 20–57) is important in the inhibition of adipogenesis and contains three functional domains: Cys-ZF domain (a.a. 20–35 TACTNCYCAKCCFQVC), core-domain (a.a. 36–46, FITKALGISYG), and protein transduction domain (PTD)(a.a. 47–57, RAKRRQRRR). Interestingly, the TAT38 region interacts with the Cyclin T1 of the P-TEFb complex, of which expression increases during adipogenesis. The X-ray crystallographic structure of the complex showed that the Cys-ZF and the core domain bind to the Cyclin T1 via hydrophobic interactions. To prepare TAT38 mimics with structural and functional similarities to TAT38, we replaced the core domain with a hydrophobic aliphatic amino acid (from carbon numbers 5 to 8). The TAT38 mimics with 6-hexanoic amino acid (TAT38 Ahx (C6)) and 7-heptanoic amino acid (TAT38 Ahp (C7)) inhibited adipogenesis of 3T3-L1 potently, reduced cellular triglyceride content, and decreased body weight of diet-induced obese (DIO) mice by 10.4–11 % in two weeks. The TAT38 and the TAT38 mimics potently repressed the adipogenic transcription factors genes, C/EBPα, PPARγ, and SREBP1. Also, they inhibit the phosphorylation of PPARγ. The TAT peptides may be promising candidates for development into a drug against obesity or diabetes.

Transactivator of Transcription (Tat)-Induced Neuroinflammation as a Key Pathway in Neuronal Dysfunction: A Scoping Review

AbstractThe activity of HIV-1 and its viral proteins within the central nervous system (CNS) is responsible for a wide array of neuropathological effects, resulting in a spectrum of neurocognitive deficits defined as HIV-associated neurocognitive disorders (HAND). Amongst the various viral proteins, the transactivator of transcription (Tat) remains detectable even with effective antiretroviral therapy (ART) and suppressed viremia, highlighting the significance of this protein in the modern ART era. Tat has been extensively researched in both fundamental and clinical settings due to its role in neuroinflammation, neuronal damage, and neurocognitive impairment amongst people living with HIV (PLHIV). To date, numerous fundamental studies have explored Tat-induced neuroinflammation. However, there is no clear consensus on the most frequently studied inflammatory markers or the consistency in the levels of these Tat-induced inflammatory marker levels across different studies. Therefore, we conducted a scoping review of studies investigating Tat-induced neuroinflammation. We conducted searches in PubMed, Scopus, and Web of Science databases using a search protocol tailored specifically to adhere to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses for scoping reviews (PRISMA-ScR) guidelines. From the 22 included studies, findings suggest that the HIV-1 Tat protein amplifies levels of neuroinflammatory markers. Amongst the vast array of inflammatory markers explored in the included studies, consistent results point to higher levels of CCL2, IL-6, IL-8, and TNF-α in primary cells and cell lines exposed to or transfected with HIV-1 Tat. These markers are regulated by key inflammatory pathways, such as the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol 3-kinase (PI3K) pathway, the p38 MAPK pathway, and nuclear factor-kB (NF-kB). Furthermore, Tat has been shown to induce neuronal apoptosis, both directly and indirectly. With regards to study designs, utilizing full-length Tat101 at concentrations ranging from 100 to 1000 ng/ml and durations of 24 and 48 h appears optimal for investigating Tat-induced neuroinflammation. In this context, we highlight specific inflammatory markers and pathways that are potentially pivotal in Tat-induced neuroinflammation and subsequent neuronal damage. A deeper investigation into these markers and pathways is crucial to better understand their roles in the development of HAND.

Nanoscale detection and real-time monitoring of free radicals in a single living cell under the stimulation of targeting moieties using a nanodiamond quantum sensor

Intracellular radicals play important roles in cell signaling and regulation of growth factors, cytokines, transcription, apoptosis, and immunomodulation, among others. To gain a more comprehensive understanding of their biological functions from a spatio-temporal pers­pective, there is a great need for nanoscale sensitive tools that allow real-time detection of these reactive species. Currently, intracellular radical probes are based on chemical reactions that could significantly alter radical levels during detection. Due to the excellent bio­compatibility and favorable photophysical properties of nitrogen-vacancy (NV–) centers in fluorescent nanodiamonds (fNDs), the fNDs can serve as a powerful and chemically inert nanotool for intracellular radical detection. In this study, a positively charged nanogel (NG) coating was prepared to prevent the precipitation of fNDs and promote cellular internalization. After internalization of nanodiamond-nanogels (fND-NGs), different stimulators, namely somatostatin (SST), triphenylphosphonium (TPP), and trans-activator of transcription (TAT) peptide, which are widely used cell- or organelle-targeting ligands in medicine, drug delivery, and diagnostics, were applied to stimulate the cells. In parallel, the intracellular radical changes under stimulation of SST, TPP, and TAT ligands were monitored by fND-NGs in a home-built optically detected magnetic resonance (ODMR) microscope. Our method allows for detecting intracellular radicals in-situ and monitoring their real-time changes during incubation with the targeting ligands in a single living cell. We believe that our method will provide insights into the generation of radical stress in cells, which could improve our fundamental understanding of the pharmacology and signaling pathways of widely used cell- and organelle-targeting ligands associated with free radicals.

SP1 transcriptionally activates HTR2B to aggravate traumatic spinal cord injury by shifting microglial M1/M2 polarization.

Spinal cord injury (SCI) can result in structural and functional damage to the spinal cord, which may lead to loss of limb movement and sensation, loss of bowel and bladder control, and other complications. Previous studies have revealed the critical influence of trans-acting transcription factor 1 (SP1) in neurological pathologies, however, its role and mechanism in SCI have not been fully studied. The study was performed using mouse microglia BV2 stimulated using lipopolysaccharide (LPS) and male adult mice subjected to spinal hitting. Western blotting was performed to detect protein expression of SP1, 5-hydroxytryptamine (serotonin) receptor 2B (HTR2B), BCL2-associated x protein (Bax), B-cell lymphoma-2 (Bcl-2), inducible nitric oxide synthase (iNOS), clusters of differentiation 86 (CD86), Arginase 1 (Arg-1) and clusters of differentiation 206 (CD206). Cell viability and apoptosis were analyzed by MTT assay and TUNEL assay. mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-4 (IL-4) and tumor necrosis factor-β (TNF-β) were quantified by quantitative real-time polymerase chain reaction. The association of SP1 and HTR2B was identified by chromatin immunoprecipitation assay and dual-luciferase reporter assay. HE staining assay was performed to analyze the pathological conditions of spinal cord tissues. LPS treatment induced cell apoptosis and inhibited microglia polarization from M1 to M2 phenotype, accompanied by an increase of Bax protein expression and a decrease of Bcl-2 protein expression, however, these effects were relieved after SP1 silencing. Mechanism assays revealed that SP1 transcriptionally activated HTR2B in BV2 cells, and HTR2B knockdown rescued LPS-induced effects on BV2 cell apoptosis and microglial M1/M2 polarization. Moreover, SP1 absence inhibited BV2 cell apoptosis and promoted microglia polarization from M1 to M2 phenotype by decreasing HTR2B expression. SCI mouse model assay further showed that SP1 downregulation could attenuate spinal hitting-induced promoting effects on cell apoptosis of spinal cord tissues and microglial M1 polarization. SP1 transcriptionally activated HTR2B to aggravate traumatic SCI by shifting microglial M1/M2 polarization.

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.

Negative lipid membranes enhance the adsorption of TAT-decorated elastin-like polypeptide micelles

A cell-penetrating peptide (CPP) is a short amino-acid sequence capable of efficiently translocating across the cellular membrane of mammalian cells. However, the potential of CPPs as a delivery vector is hampered by the strong reduction of its translocation efficiency when it bears an attached molecular cargo. To overcome this problem, we used previously developed diblock copolymers of elastin-like polypeptides (ELPBCs), which we end functionalized with TAT (transactivator of transcription), an archetypal CPP built from a positively charged amino acid sequence of the HIV-1 virus. These ELPBCs self-assemble into micelles at a specific temperature and present the TAT peptide on their corona. These micelles can recover the lost membrane affinity of TAT and can trigger interactions with the membrane despite the presence of a molecular cargo. Herein, we study the influence of membrane surface charge on the adsorption of TAT-functionalized ELP micelles onto giant unilamellar vesicles (GUVs). We show that the TAT-ELPBC micelles show an increased binding constant toward negatively charged membranes compared to neutral membranes, but no translocation is observed. The affinity of the TAT-ELPBC micelles for the GUVs displays a stepwise dependence on the lipid charge of the GUV, which, to our knowledge, has not been reported previously for interactions between peptides and lipid membranes. By unveiling the key steps controlling the interaction of an archetypal CPP with lipid membranes, through regulation of the charge of the lipid bilayer, our results pave the way for a better design of delivery vectors based on CPPs.

Acute Effects of Monoacylglycerol Lipase Inhibitor ABX1431 on Neuronal Hyperexcitability, Nociception, Locomotion, and the Endocannabinoid System in HIV-1 Tat Male Mice.

Background: Evidence suggests that monoacylglycerol lipase (MAGL) inhibitors can potentially treat HIV symptoms by increasing the concentration of 2-arachidonoylglycerol (2-AG). We examined a selective MAGL inhibitor ABX1431 in the context of neuroHIV. Methods: To assess the effects of ABX1431, we conducted in vitro and in vivo studies. In vitro calcium imaging on frontal cortex neuronal cultures was performed to evaluate the role of ABX1431 (10, 30, 100 nM) on transactivator of transcription (Tat)-induced neuronal hyperexcitability. Following in vitro experiments, in vivo experiments were performed using Tat transgenic male mice. Mice were treated with 4 mg/kg ABX1431 and assessed for antinociception using tail-flick and hot plate assays followed by locomotor activity. After the behavioral experiments, their brains were harvested to quantify endocannabinoids (eCB) and related lipids through mass spectrometry, and cannabinoid type-1 and -2 receptors (CB1R and CB2R) were quantified through western blot. Results: In vitro studies revealed that adding Tat directly to the neuronal cultures significantly increased intracellular calcium concentration, which ABX1431 completely reversed at all concentrations. Preincubating the cultures with CB1R and CB2R antagonists showed that ABX1431 exhibited its effects partially through CB1R. In vivo studies demonstrated that acute ABX1431 increased overall total distance traveled and speed of mice regardless of their genotype. Mass spectrometry and western blot analyses revealed differential effects on the eCB system based on Tat expression. The 2-AG levels were significantly upregulated following ABX1431 treatment in the striatum and spinal cord. Arachidonic acid (AA) was also upregulated in the striatum of vehicle-treated Tat(+) mice. No changes were noted in CB1R expression levels; however, CB2R levels were increased in ABX1431-treated Tat(-) mice only. Conclusion: Findings indicate that ABX1431 has potential neuroprotective effects in vitro partially mediated through CB1R. Acute treatment of ABX1431 in vivo shows antinociceptive effects, and seems to alter locomotor activity, with upregulating 2-AG levels in the striatum and spinal cord.