Adult Human Brain Tissue Cultures to Study NeuroHIV.

HIV-associated neurocognitive disorders (HAND) persist under antiretroviral therapy as a complex pathology that has been difficult to study in cellular and animal models. Therefore, we generated an ex vivo human brain slice model of HIV-1 infection from surgically resected adult brain tissue. Brain slice cultures processed for flow cytometry showed >90% viability of dissociated cells within the first three weeks in vitro, with parallel detection of astrocyte, myeloid, and neuronal populations. Neurons within brain slices showed stable dendritic spine density and mature spine morphologies in the first weeks in culture, and they generated detectable activity in multi-electrode arrays. We infected cultured brain slices using patient-matched CD4+ T-cells or monocyte-derived macrophages (MDMs) that were exposed to a GFP-expressing R5-tropic HIV-1 in vitro. Infected slice cultures expressed viral RNA and developed a spreading infection up to 9 days post-infection, which were significantly decreased by antiretrovirals. We also detected infected myeloid cells and astrocytes within slices and observed minimal effect on cellular viability over time. Overall, this human-centered model offers a promising resource to study the cellular mechanisms contributing to HAND (including antiretroviral toxicity, substance use, and aging), infection of resident brain cells, and new neuroprotective therapeutics.

Alprazolam Prompts HIV-1 Transcriptional Reactivation and Enhances CTL Response Through RUNX1 Inhibition and STAT5 Activation.

The HIV-1 pandemic is a significant challenge to the field of medicine. Despite advancements in antiretroviral (ART) development, 38 million people worldwide still live with this disease without a cure. A significant barrier to the eradication of HIV-1 lies in the persistently latent pool that establishes early in the infection. The "shock and kill" strategy relies on the discovery of a latency-reversing agent (LRA) that can robustly reactivate the latent pool and not limit immune clearance. We have found that a benzodiazepine (BDZ), that is commonly prescribed for panic and anxiety disorder, to be an ideal candidate for latency reversal. The BDZ Alprazolam functions as an inhibitor of the transcription factor RUNX1, which negatively regulates HIV-1 transcription. In addition to the displacement of RUNX1 from the HIV-1 5'LTR, Alprazolam potentiates the activation of STAT5 and its recruitment to the viral promoter. The activation of STAT5 in cytotoxic T cells may enable immune activation which is independent of the IL-2 receptor. These findings have significance for the potential use of Alprazolam in a curative strategy and to addressing the neuroinflammation associated with neuroHIV-1.

ß-Arrestin-dependent ERK signaling reduces anxiety-like and conditioned fear-related behaviors in mice.

G protein-coupled receptors (GPCRs) are implicated in the regulation of fear and anxiety. GPCR signaling involves canonical G protein pathways but can also engage downstream kinases and effectors through scaffolding interactions mediated by ß-arrestin. Here, we investigated whether ß-arrestin signaling regulates anxiety-like and fear-related behavior in mice in response to activation of the GPCR δ-opioid receptor (δOR or DOR). Administration of ß-arrestin-biased δOR agonists to male C57BL/6 mice revealed ß-arrestin 2-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the dorsal hippocampus and amygdala and ß-arrestin 1-dependent activation of ERK1/2 in the nucleus accumbens. In mice, ß-arrestin-biased agonist treatment was associated with reduced anxiety-like and fear-related behaviors, with some overlapping and isoform-specific input. In contrast, applying a G protein-biased δOR agonist decreased ERK1/2 activity in all three regions as well as the dorsal striatum and was associated with increased fear-related behavior without effects on baseline anxiety. Our results indicate a complex picture of δOR neuromodulation in which ß-arrestin 1- and 2-dependent ERK signaling in specific brain subregions suppresses behaviors associated with anxiety and fear and opposes the effects of G protein-biased signaling. Overall, our findings highlight the importance of noncanonical ß-arrestin-dependent GPCR signaling in the regulation of these interrelated emotions.

Morphine exposure exacerbates HIV-1 Tat driven changes to neuroinflammatory factors in cultured astrocytes.

Despite antiretroviral therapy human immunodeficiency virus type-1 (HIV-1) infection results in neuroinflammation of the central nervous system that can cause HIV-associated neurocognitive disorders (HAND). The molecular mechanisms involved in the development of HAND are unclear, however, they are likely due to both direct and indirect consequences of HIV-1 infection and inflammation of the central nervous system. Additionally, opioid abuse in infected individuals has the potential to exacerbate HIV-comorbidities, such as HAND. Although restricted for productive HIV replication, astrocytes (comprising 40-70% of all brain cells) likely play a significant role in neuropathogenesis in infected individuals due to the production and response of viral proteins. The HIV-1 protein Tat is critical for viral transcription, causes neuroinflammation, and can be secreted from infected cells to affect uninfected bystander cells. The Wnt/ß-catenin signaling cascade plays an integral role in restricting HIV-1 infection in part by negatively regulating HIV-1 Tat function. Conversely, Tat can overcome this negative regulation and inhibit ß-catenin signaling by sequestering the critical transcription factor TCF-4 from binding to ß-catenin. Here, we aimed to explore how opiate exposure affects Tat-mediated suppression of ß-catenin in astrocytes and the downstream modulation of neuroinflammatory genes. We observed that morphine can potentiate Tat suppression of ß-catenin activity in human astrocytes. In contrast, Tat mutants deficient in secretion, and lacking neurotoxic effects, do not affect ß-catenin activity in the presence or absence of morphine. Finally, morphine treatment of astrocytes was sufficient to reduce the expression of genes involved in neuroinflammation. Examining the molecular mechanisms of how HIV-1 infection and opiate exposure exacerbate neuroinflammation may help us inform or predict disease progression prior to HAND development.

Benzodiazepines Drive Alteration of Chromatin at the Integrated HIV-1 LTR.

Antiretroviral therapy (ART) lowers human immunodeficiency virus type 1 (HIV-1) viral load to undetectable levels, but does not eliminate the latent reservoir. One of the factors controlling the latent reservoir is transcriptional silencing of the integrated HIV-1 long terminal repeat (LTR). The molecular mechanisms that control HIV-1 transcription are not completely understood. We have previously shown that RUNX1, a host transcription factor, may play a role in the establishment and maintenance of HIV-1 latency. Prior work has demonstrated that inhibition of RUNX1 by the benzodiazepine (BDZ) Ro5-3335 synergizes with suberanilohydroxamic acid (SAHA) to activate HIV-1 transcription. In this current work, we examine the effect of RUNX1 inhibition on the chromatin state of the integrated HIV-1 LTR. Using chromatin immunoprecipitation (ChIP), we found that Ro5-3335 significantly increased the occupancy of STAT5 at the HIV-1 LTR. We also screened other BDZs for their ability to regulate HIV-1 transcription and demonstrate their ability to increase transcription and alter chromatin at the LTR without negatively affecting Tat activity. These findings shed further light on the mechanism by which RUNX proteins control HIV-1 transcription and suggest that BDZ compounds might be useful in activating HIV-1 transcription through STAT5 recruitment to the HIV-1 LTR.

Real-time visualization of chromatin modification in isolated nuclei.

Chromatin modification is traditionally assessed in biochemical assays that provide average measurements of static events given that the analysis requires components from many cells. Microscopy can visualize single cells, but the cell body and organelles can hamper staining and visualization of the nucleus. Normally, chromatin is visualized by immunostaining a fixed sample or by expressing exogenous fluorescently tagged proteins in a live cell. Alternative microscopy tools to observe changes of endogenous chromatin in real-time are needed. Here, we isolated transcriptionally competent nuclei from cells and used antibody staining without fixation to visualize changes in endogenous chromatin. This method allows the real-time addition of drugs and fluorescent probes to one or more nuclei while under microscopy observation. A high-resolution map of 11 endogenous nuclear markers of the histone code, transcription machinery and architecture was obtained in transcriptionally active nuclei by performing confocal and structured illumination microscopy. We detected changes in chromatin modification and localization at the single-nucleus level after inhibition of histone deacetylation. Applications in the study of RNA transcription, viral protein function and nuclear architecture are presented. This article has an associated First Person interview with the first author of the paper.

Targeted inhibition of ANKRD1 disrupts sarcomeric ERK-GATA4 signal transduction and abrogates phenylephrine-induced cardiomyocyte hypertrophy.

AIMS: Accumulating evidence suggest that sarcomere signalling complexes play a pivotal role in cardiomyocyte hypertrophy by communicating stress signals to the nucleus to induce gene expression. Ankyrin repeat domain 1 (ANKRD1) is a transcriptional regulatory protein that also associates with sarcomeric titin; however, the exact role of ANKRD1 in the heart remains to be elucidated. We therefore aimed to examine the role of ANKRD1 in cardiomyocyte hypertrophic signalling. METHODS AND RESULTS: In neonatal rat ventricular myocytes, we found that ANKRD1 is part of a sarcomeric signalling complex that includes ERK1/2 and cardiac transcription factor GATA4. Treatment with hypertrophic agonist phenylephrine (PE) resulted in phosphorylation of ERK1/2 and GATA4 followed by nuclear translocation of the ANKRD1/ERK/GATA4 complex. Knockdown of Ankrd1 attenuated PE-induced phosphorylation of ERK1/2 and GATA4, inhibited nuclear translocation of the ANKRD1 complex, and prevented cardiomyocyte growth. Mice lacking Ankrd1 are viable with normal cardiac function. Chronic PE infusion in wild-type mice induced significant cardiac hypertrophy with reactivation of the cardiac fetal gene program which was completely abrogated in Ankrd1 null mice. In contrast, ANKRD1 does not play a role in haemodynamic overload as Ankrd1 null mice subjected to transverse aortic constriction developed cardiac hypertrophy comparable to wild-type mice. CONCLUSION: Our study reveals a novel role for ANKRD1 as a selective regulator of PE-induced signalling whereby ANKRD1 recruits and localizes GATA4 and ERK1/2 in a sarcomeric macro-molecular complex to enhance GATA4 phosphorylation with subsequent nuclear translocation of the ANKRD1 complex to induce hypertrophic gene expression.

Upstream open reading frame in 5'-untranslated region reduces titin mRNA translational efficiency.

Titin is the largest known protein and a critical determinant of myofibril elasticity and sarcomere structure in striated muscle. Accumulating evidence that mRNA transcripts are post-transcriptionally regulated by specific motifs located in the flanking untranslated regions (UTRs) led us to consider the role of titin 5'-UTR in regulating its translational efficiency. Titin 5'-UTR is highly homologous between human, mouse, and rat, and sequence analysis revealed the presence of a stem-loop and two upstream AUG codons (uAUGs) converging on a shared in frame stop codon. We generated a mouse titin 5'-UTR luciferase reporter construct and targeted the stem-loop and each uAUG for mutation. The wild-type and mutated constructs were transfected into the cardiac HL-1 cell line and primary neonatal rat ventricular myocytes (NRVM). SV40 driven 5'-UTR luciferase activity was significantly suppressed by wild-type titin 5'-UTR (∼ 70% in HL-1 cells and ∼ 60% in NRVM). Mutating both uAUGs was found to alleviate titin 5'-UTR suppression, while eliminating the stem-loop had no effect. Treatment with various growth stimuli: pacing, PMA or neuregulin had no effect on titin 5'-UTR luciferase activity. Doxorubicin stress stimuli reduced titin 5'-UTR suppression, while H2O2 had no effect. A reported single nucleotide polymorphism (SNP) rs13422986 at position -4 of the uAUG2 was introduced and found to further repress titin 5'-UTR luciferase activity. We conclude that the uAUG motifs in titin 5'-UTR serve as translational repressors in the control of titin gene expression, and that mutations/SNPs of the uAUGs or doxorubicin stress could alter titin translational efficiency.

Mitochondrial fusion protein MFN2 interacts with the mitostatin-related protein MNS1 required for mouse sperm flagellar structure and function.

BACKGROUND: Cilia and the sperm flagellum share many structural properties. Meiosis-specific nuclear structural 1 (MNS1) is a recently characterized protein that is abundantly expressed in post-meiotic spermatids and is required for proper flagellar and motile cilia formation. To explore the possible functions of MNS1, we performed a BLAST search and determined it is homologous to the conserved domain pfam13868, exemplified by mitostatin. This protein interacts with mitofusin 2 (MFN2), a protein that participates in regulating mitochondrial associations to subcellular organelles. We hypothesized that an association between MFN2 and MNS1 in the sperm is involved in flagellar biogenesis and function. RESULTS: In the studies reported here, MFN2 was found in murine reproductive and somatic tissues high in ciliary content while MNS1 was present as two closely migrating bands in reproductive tissues. Interestingly, mitostatin was also present in reproductive tissues. Similar to Mns1 and mitostatin, Mfn2 was expressed in the testis as detected by RT-PCR. In addition, Mfn2 and Mns1 decreased in expression from pachytene spermatocytes to condensing spermatids as assessed by quantitative RT-PCR. Co-immunoprecipitation demonstrated an association between MFN2 and MNS1 in spermatogenic cells. Indirect immunofluorescence indicated that MFN2 and MNS1 co-localized to the sperm flagellum in freshly collected cauda epididymal sperm. MFN2 associated with the midpiece while MNS1 was present throughout the sperm tail in caput and cauda epididymal sperm. In spermatogenic cells, MFN2 was seen in the mitochondria, and MNS1 was present throughout the cell cytoplasm. MFN2 and MNS1 were present in detergent-resistant flagellar structures of the sperm. CONCLUSIONS: These results demonstrate that MFN2 and MNS1 are present in spermatogenic cells and are an integral part of the sperm flagellum, indicating they play a role in flagellar biogenesis and/or function.

Adenine nucleotide metabolism and a role for AMP in modulating flagellar waveforms in mouse sperm.

While most ATP, the main energy source driving sperm motility, is derived from glycolysis and oxidative phosphorylation, the metabolic demands of the cell require the efficient use of power stored in high-energy phosphate bonds. In times of high energy consumption, adenylate kinase (AK) scavenges one ATP molecule by transphosphorylation of two molecules of ADP, simultaneously yielding one molecule of AMP as a by-product. Either ATP or ADP supported motility of detergent-modeled cauda epididymal mouse sperm, indicating that flagellar AKs are functional. However, the ensuing flagellar waveforms fueled by ATP or ADP were qualitatively different. Motility driven by ATP was rapid but restricted to the distal region of the sperm tail, whereas ADP produced slower and more fluid waves that propagated down the full flagellum. Characterization of wave patterns by tracing and superimposing the images of the flagella, quantifying the differences using digital image analysis, and computer-assisted sperm analysis revealed differences in the amplitude, periodicity, and propagation of the waves between detergent-modeled sperm treated with either ATP or ADP. Surprisingly, addition of AMP to the incubation medium containing ATP recapitulated the pattern of sperm motility seen with ADP alone. In addition to AK1 and AK2, which we previously demonstrated are present in outer dense fibers and mitochondrial sheath of the mouse sperm tail, we show that another AK, AK8, is present in a third flagellar compartment, the axoneme. These results extend the known regulators of sperm motility to include AMP, which may be operating through an AMP-activated protein kinase.

Signaling in sperm: toward a molecular understanding of the acquisition of sperm motility in the mouse epididymis.

Sperm motility encompasses a wide range of events involving epididymal maturation and activation of biochemical pathways, most notably cyclic AMP (cAMP)-protein kinase A (PKA) activation. Following the discovery of guanine-nucleotide exchange factors (RAPGEFs), also known as exchange proteins activated by cAMP, we investigated the separate roles of PKA and RAPGEFs in sperm motility. RT-PCR showed the presence of Rapgef3, Rapgef4, and Rapgef5, as well as several known RAPGEF partner mRNAs, in spermatogenic cells. However, Rapgef3 and Rapgef4 appeared to be less abundant in condensing spermatids versus pachytene spermatocytes. Similarly, many of these proteins were detected by immunoblotting. RAPGEF5 was detected in germ cells and murine epididymal sperm. Indirect immunofluorescence localized SGK1, SGK3, AKT1 pT(308), and RAPGEF5 to the acrosome, while PDPK1 was found in the postacrosomal region. SGK3 was present throughout the tail, while PDPK1 and AKT1 pT(308) were in the midpiece. When motility was assessed in demembranated cauda epididymal sperm, addition of ATP and the selective ligand for RAPGEFs, 8-pCPT-2'-O-Me-cAMP, resulted in motility, but the sperm were unable to undergo hyperactivated-like motility. In contrast, when demembranated cauda epididymal sperm were incubated with ATP plus dibutyryl cAMP, sperm became motile and progressed to hyperactivated-like motility. However, no significant difference was observed when intact sperm were examined. GSK3 phosphorylation was altered in the presence of H89, a PKA inhibitor. Significantly, intact caput epididymal sperm became motile when incubated in the presence of extracellular ATP. These results provide evidence for a new pathway involved in endowing sperm with the capacity to swim.

Male mice express spermatogenic cell-specific triosephosphate isomerase isozymes.

Triosephosphate isomerase 1 (TPI1) is a member of the glycolytic pathway, which is a critical source of energy for motility in mouse sperm. By immunoblotting, we detected two male, germ line-specific TPI1 bands (Mr 33,400 and 30,800) as well as the somatic-type band (Mr 27,700). Although all three bands were observed in spermatogenic cells, somatic-type TPI1 disappeared from sperm during epididymal maturation. In vitro dephosphorylation analysis suggested that the two male, germ line-specific TPI1 bands were not the result of phosphorylation of the 27,700 Mr TPI1 band. The Mr 33,400; 30,800; and 27,700 TPI1 bands corresponded to the respective sizes of the proteins predicted to use the first, second, and third possible initiation codons of the Tpi1 cDNA. We performed immunofluorescence on epididymal sperm and determined that TPI1 specifically localized in the principal piece. The antibody staining was stronger in cauda epididymal sperm than in caput epididymal sperm, a finding consistent with the identification of TPI1 as a cauda epididymal sperm-enriched protein. Immunofluorescence with sodium dodecyl sulfate (SDS)-insoluble flagellar accessory structures showed a strong TPI1 signal only in the principal piece, indicating that TPI1 is a component of the fibrous sheath. Northern blot hybridization detected longer Tpi1 transcripts (1.56 kb) in mouse testis, whereas somatic tissues had shorter transcripts (1.32 kb). As there is only one triosephosphate isomerase gene in the mouse genome, we conclude that the three variants we see in sperm result from the use of alternative translation start codons in spermatogenic cells.