ß-catenin regulates HIV latency and modulates HIV reactivation.

Latency is the main obstacle towards an HIV cure, with cure strategies aiming to either elicit or prevent viral reactivation. While these strategies have shown promise, they have only succeeded in modulating latency in a fraction of the latent HIV reservoir, suggesting that the mechanisms controlling HIV latency are not completely understood, and that comprehensive latency modulation will require targeting of multiple latency maintenance pathways. We show here that the transcriptional co-activator and the central mediator of canonical Wnt signaling, ß-catenin, inhibits HIV transcription in CD4+ T cells via TCF-4 LTR binding sites. Further, we show that inhibiting the ß-catenin pathway reactivates HIV in a primary TCM cell model of HIV latency, primary cells from cART-controlled HIV donors, and in CD4+ latent cell lines. ß-catenin inhibition or activation also enhanced or inhibited the activity of several classes of HIV latency reversing agents, respectively, in these models, with significant synergy of ß-catenin and each LRA class tested. In sum, we identify ß-catenin as a novel regulator of HIV latency in vitro and ex vivo, adding new therapeutic targets that may be combined for comprehensive HIV latency modulation in HIV cure efforts.

Discovery of circulating blood biomarkers in patients with and without Modic changes of the lumbar spine: a preliminary analysis.

PURPOSE: The following study aimed to determine the existence of blood biomarkers in symptomatic patients with or without lumbar Modic changes (MC). METHODS: A cross-sectional sub-analyses of a prospective cohort was performed. Fasting blood samples were collected from patients with and without lumbar MC who had undergone spinal fusion or microdiscectomy. An 80-plex panel and CCL5/RANTES were used to assess preoperative plasma cytokine concentrations. Patient demographics and imaging phenotypes were also assessed. RESULTS: Thirty-one subjects were analysed (n = 18 no MC; n = 13 MC). No significant differences were found in age, sex, body mass index, smoking and alcohol history, and surgical procedure (i.e. fusion, decompression) between the two groups (p > 0.05). Several statistically significant blood biomarkers in MC patients were identified, including elevated levels of C-C Motif Chemokine Ligand 5 (CCL5, p = 0.0006), while Macrophage Migration Inhibitory Factor (MIF) was significantly lower (p = 0.009). Additionally, C-X-C Motif Chemokine Ligand 5 (CXCL5, p = 0.052), Pentraxin 3 (PTX3, p = 0.06) and Galectin-3 (Gal-3, p = 0.07) showed potential relevance. Moreover, MC patients exhibited significantly higher levels of disc degeneration (p = 0.0001) and displacement severity (p = 0.020). Based on multivariate analyses and controlling for disc degeneration/displacement, CCL5 (OR 1.02; 95% CI 1.002-1.033; p = 0.028) and MIF (OR 0.60; 95% CI 0.382-0.951; p = 0.030) were independently associated with MC patients. CONCLUSION: This "proof-of-concept" study is the first to identify specific and significantly circulating blood biomarkers associated with symptomatic patients with lumbar MC, independent of disc alterations of degeneration and/or bulges/herniations. Specifically, differences in CCL5 and MIF protein levels were significantly noted in MC patients compared to those without MC.

HIV infection of non-classical cells in the brain.

HIV-associated neurological disorders (HAND) affect up to 50% of people living with HIV (PLWH), even in the era of combination antiretroviral therapy (cART). HIV-DNA can be detected in the cerebral spinal fluid (CSF) of approximately half of aviremic ART-suppressed PLWH and its presence is associated with poorer neurocognitive performance. HIV DNA + and HIV RNA + cells have also been observed in postmortem brain tissue of individuals with sustained cART suppression. In this review, we provide an overview of how HIV invades the brain and HIV infection of resident brain glial cells (astrocytes and microglia). We also discuss the role of resident glial cells in persistent neuroinflammation and HAND in PLWH and their potential contribution to the HIV reservoir. HIV eradication strategies that target persistently infected glia cells will likely be needed to achieve HIV cure.

CD4dim CD8bright T Cells Home to the Brain and Mediate HIV Neuroinvasion.

CD4dim CD8bright T cells are a mature population of CD8+ T cells that upon activation upregulate CD4 dimly on their surface. Expression of CD4 on these cells suggests that they can be an additional source of HIV neuroinvasion and persistence in the brain. We used HIV-infected NOD/SCID/IL-2rcγ-/- (NSG) humanized mice to track CD4dim CD8bright T cell homing to the brain and define their role in HIV dissemination into the brain. We report here that CD4dim CD8bright T cells are found in the brain at a median frequency of 2.6% and in the spleen at median frequency of 7.6% of CD3+ T cells. In the brain, 10 to 20% of CD4dim CD8bright T cells contain integrated provirus, which is infectious as demonstrated by viral outgrowth assay. CD4dim CD8bright T cells in the brain exhibited significantly higher expression of the brain homing receptors CX3CR1 and CXCR3 in comparison to their single-positive CD8+ T cell counterpart. Blocking lymphocyte trafficking into the brain of humanized mice via anti-VLA4 and anti-LFA1 antibodies reduced CD4dim CD8bright T cell trafficking into the brain by 60% and diminished brain HIV proviral DNA by 72%. Collectively, our findings demonstrate that CD4dim CD8bright T cells can home to the brain and support productive HIV replication. These studies also reveal for the first time that CD4dim CD8bright T cells are capable of HIV neuroinvasion and are a reservoir for HIV. IMPORTANCE We report here a seminal finding of a novel population of T cells, termed CD4dim CD8bright T cells, that plays a role in HIV neuroinvasion and is a reservoir for HIV in the brain.

Next-Generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire.

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS, a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multiparametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We applied Ig-MS to plasma from subjects with severe and mild COVID-19 and immunized subjects after two vaccine doses, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, that could use other antigens of interest to gauge immune responses to vaccination, pathogens, or autoimmune disorders.

Anti-HIV Drugs Cause Mitochondrial Dysfunction in Monocyte-Derived Macrophages.

Combination antiretroviral therapy (cART) dramatically changed the face of the HIV/AIDS pandemic, making it one of the most prominent medical breakthroughs of the past 3 decades. However, as the life span of persons living with HIV (PLWH) continues to approach that of the general population, the same cannot be said regarding their quality of life. PLWH are affected by comorbid conditions such as high blood pressure, diabetes, and neurocognitive impairment at a higher rate and increased severity than their age-matched counterparts. PLWH also have higher levels of inflammation, the drivers of which are not entirely clear. As cART treatment is lifelong, we assessed here the effects of cART, independent of HIV, on primary human monocyte-derived macrophages (MDMs). MDMs were unskewed or skewed to an alternative phenotype and treated with Atripla or Triumeq, two first-line cART treatments. We report that Triumeq skewed alternative MDMs toward an inflammatory nonsenescent phenotype. Both Atripla and Triumeq caused mitochondrial dysfunction, specifically efavirenz and abacavir. Additionally, transcriptome sequencing (RNA-seq) demonstrated that both Atripla and Triumeq caused differential regulation of genes involved in immune regulation and cell cycle and DNA repair. Collectively, our data demonstrate that cART, independent of HIV, alters the MDM phenotype. This suggests that cART may contribute to cell dysregulation in PLWH that subsequently results in increased susceptibility to comorbidities.

ß-Catenin Restricts Zika Virus Internalization by Downregulating Axl.

The latest outbreak of Zika virus (ZIKV) in the Americas was associated with significant neurologic complications, including microcephaly of newborns. We evaluated mechanisms that regulate ZIKV entry into human fetal astrocytes (HFAs). Astrocytes are key players in maintaining brain homeostasis. We show that the central mediator of canonical Wnt signaling, ß-catenin, regulates Axl, a receptor for ZIKV infection of HFAs, at the transcriptional level. In turn, ZIKV inhibited ß-catenin, potentially as a mechanism to overcome its restriction of ZIKV internalization through regulation of Axl. This was evident with three ZIKV strains tested but not with a laboratory-adapted strain which has a large deletion in its envelope gene. Finally, we show that ß-catenin-mediated Axl-dependent internalization of ZIKV may be of increased importance for brain cells, as it regulated ZIKV infection of astrocytes and human brain microvascular cells but not kidney epithelial (Vero) cells. Collectively, our studies reveal a role for ß-catenin in ZIKV infection and highlight a dynamic interplay between ZIKV and ß-catenin to modulate ZIKV entry into susceptible target cells. IMPORTANCE ZIKV is an emerging pathogen with sporadic outbreaks throughout the world. The most recent outbreak in North America was associated with small brains (microcephaly) in newborns. We studied the mechanism(s) that may regulate ZIKV entry into astrocytes. Astrocytes are a critical resident brain cell population with diverse functions that maintain brain homeostasis, including neurogenesis and neuronal survival. We show that three ZIKV strains (and not a heavily laboratory-adapted strain with a large deletion in its envelope gene) require Axl for internalization. Most importantly, we show that ß-catenin, the central mediator of canonical Wnt signaling, negatively regulates Axl at the transcriptional level to prevent ZIKV internalization into human fetal astrocytes. To overcome this restriction, ZIKV downregulates ß-catenin to facilitate Axl expression. This highlights a dynamic host-virus interaction whereby ZIKV inhibits ß-catenin to promote its internalization into human fetal astrocytes through the induction of Axl.

HIV infects astrocytes in vivo and egresses from the brain to the periphery.

HIV invades the brain during acute infection. Yet, it is unknown whether long-lived infected brain cells release productive virus that can egress from the brain to re-seed peripheral organs. This understanding has significant implication for the brain as a reservoir for HIV and most importantly HIV interplay between the brain and peripheral organs. Given the sheer number of astrocytes in the human brain and their controversial role in HIV infection, we evaluated their infection in vivo and whether HIV infected astrocytes can support HIV egress to peripheral organs. We developed two novel models of chimeric human astrocyte/human peripheral blood mononuclear cells: NOD/scid-IL-2Rgc null (NSG) mice (huAstro/HuPBMCs) whereby we transplanted HIV (non-pseudotyped or VSVg-pseudotyped) infected or uninfected primary human fetal astrocytes (NHAs) or an astrocytoma cell line (U138MG) into the brain of neonate or adult NSG mice and reconstituted the animals with human peripheral blood mononuclear cells (PBMCs). We also transplanted uninfected astrocytes into the brain of NSG mice and reconstituted with infected PBMCs to mimic a biological infection course. As expected, the xenotransplanted astrocytes did not escape/migrate out of the brain and the blood brain barrier (BBB) was intact in this model. We demonstrate that astrocytes support HIV infection in vivo and egress to peripheral organs, at least in part, through trafficking of infected CD4+ T cells out of the brain. Astrocyte-derived HIV egress persists, albeit at low levels, under combination antiretroviral therapy (cART). Egressed HIV evolved with a pattern and rate typical of acute peripheral infection. Lastly, analysis of human cortical or hippocampal brain regions of donors under cART revealed that astrocytes harbor between 0.4-5.2% integrated HIV gag DNA and 2-7% are HIV gag mRNA positive. These studies establish a paradigm shift in the dynamic interaction between the brain and peripheral organs which can inform eradication of HIV reservoirs.

Bone Quality in Relation to HIV and Antiretroviral Drugs.

PURPOSE OF REVIEW: People living with HIV (PLWH) are at an increased risk for osteoporosis, a disease defined by the loss of bone mineral density (BMD) and deterioration of bone quality, both of which independently contribute to an increased risk of skeletal fractures. While there is an emerging body of literature focusing on the factors that contribute to BMD loss in PLWH, the contribution of these factors to bone quality changes are less understood. The current review summarizes and critically reviews the data describing the effects of HIV, HIV disease-related factors, and antiretroviral drugs (ARVs) on bone quality. RECENT FINDINGS: The increased availability of high-resolution peripheral quantitative computed tomography has confirmed that both HIV infection and ARVs negatively affect bone architecture. There is considerably less data on their effects on bone remodeling or the composition of bone matrix. Whether changes in bone quality independently predict fracture risk, as seen in HIV-uninfected populations, is largely unknown. The available data suggests that bone quality deterioration occurs in PLWH. Future studies are needed to define which factors, viral or ARVs, contribute to loss of bone quality and which bone quality factors are most associated with increased fracture risk.

Canonical Wnts Mediate CD8+ T Cell Noncytolytic Anti-HIV-1 Activity and Correlate with HIV-1 Clinical Status.

CD8+ T cells do not rely solely on cytotoxic functions for significant HIV control. Moreover, the noncytotoxic CD8+ T cell antiviral response is a primary mediator of natural HIV control such as that seen in HIV elite controllers and long-term nonprogressors that does not require combined antiretroviral therapy. In this study, we investigated the biological factors contributing to the noncytotoxic control of HIV replication mediated by primary human CD8+ T cells. We report that canonical Wnt signaling inhibits HIV transcription in an MHC-independent, noncytotoxic manner and that mediators of this pathway correlate with HIV controller clinical status. We show that CD8+ T cells express all 19 Wnts and CD8+ T cell-conditioned medium (CM) induced canonical Wnt signaling in infected recipient cells while simultaneously inhibiting HIV transcription. Antagonizing canonical Wnt activity in CD8+ T cell CM resulted in increased HIV transcription in infected cells. Further, Wnt2b expression was upregulated in HIV controllers versus viremic patients, and in vitro depletion of Wnt2b and/or Wnt9b from CD8+ CM reversed HIV inhibitory activity. Finally, plasma concentration of Dkk-1, an antagonist of canonical Wnt signaling, was higher in viremic patients with lower CD4 counts. This study demonstrates that canonical Wnt signaling inhibits HIV and significantly correlates with HIV controller status.

Negative regulation of IL-8 in human astrocytes depends on ß-catenin while positive regulation is mediated by TCFs/LEF/ATF2 interaction.

Expression of cytokines/chemokines is tightly regulated at the transcription level. This is crucial in the central nervous system to maintain neuroimmune homeostasis. IL-8 a chemoattractant, which recruits neutrophils, T cells, and basophils into the brain in response to inflammation and/or injury is secreted predominantly by neurons, microglia, and astrocytes. Here, we investigated the mechanism by which astrocytes regulate IL-8 expression. We demonstrate that while ß-catenin negatively regulated IL-8 transcription, its canonical transcriptional partners, members of the TCF/LEF transcription factors (TCF1, TCF3, TCF4 and LEF1) and Activating transcription factor 2 (ATF2) positively regulated IL-8 transcription. We further identified a putative TCF/LEF binding site at -175nt close to the minimal transcription region on the IL-8 promoter, mutation of which caused a significant reduction in IL-8 promoter activity. Chromatin immunoprecipitation demonstrated binding of TCF1, TCF4, LEF1 and ATF2 on the IL-8 promoter suggesting that TCFs/LEF partner with ATF2 to induce IL-8 transcription. These findings demonstrate a novel role for ß-catenin in suppression of IL-8 expression and for TCFs/LEF/ATF2 in inducing IL-8. These findings reveal a unique mechanism by which astrocytes tightly regulate IL-8 expression.

ß-Catenin and TCFs/LEF signaling discordantly regulate IL-6 expression in astrocytes.

BACKGROUND: The Wnt/ß-catenin signaling pathway is a prolific regulator of cell-to-cell communication and gene expression. Canonical Wnt/ß-catenin signaling involves partnering of ß-catenin with members of the TCF/LEF family of transcription factors (TCF1, TCF3, TCF4, LEF1) to regulate gene expression. IL-6 is a key cytokine involved in inflammation and is particularly a hallmark of inflammation in the brain. Astrocytes, specialized glial cells in the brain, secrete IL-6. How astrocytes regulate IL-6 expression is not entirely clear, although in other cells NFκB and C/EBP pathways play a role. We evaluated here the interface between ß-catenin, TCFs/LEF and C/EBP and NF-κB in relation to IL-6 gene regulation in astrocytes. METHODS: We performed molecular loss and/or gain of function studies of ß-catenin, TCF/LEF, NFκB, and C/EBP to assess IL-6 regulation in human astrocytes. Specifically, siRNA mediated target gene knockdown, cDNA over expression of target gene, and pharmacological agents for regulation of target proteins were used. IL-6 levels was evaluated by real time quantitative PCR and ELISA. We also cloned the IL-6 promoter under a firefly luciferase reporter and used bioinformatics, site directed mutagenesis, and chromatin immunoprecipitation to probe the interaction between ß-catenin/TCFs/LEFs and IL-6 promoter activity. RESULTS: ß-catenin binds to TCF/LEF to inhibits IL-6 while TCFs/LEF induce IL-6 transcription through interaction with ATF-2/SMADs. ß-catenin independent of TCFs/LEF positively regulates C/EBP and NF-κB, which in turn activate IL-6 expression. The IL-6 promoter has two putative regions for TCFs/LEF binding, a proximal site located at -91 nt and a distal site at -948 nt from the transcription start site, both required for TCF/LEF induction of IL-6 independent of ß-catenin. CONCLUSION: IL-6 regulation in human astrocytes engages a discordant interaction between ß-catenin and TCF/LEF. These findings are intriguing given that no role for ß-catenin nor TCFs/LEF to date is associated with IL-6 regulation and suggest that ß-catenin expression in astrocytes is a critical regulator of anti-inflammatory responses and its disruption can potentially mediate persistent neuroinflammation. Video Abstract.

Methamphetamine decreases K+ channel function in human fetal astrocytes by activating the trace amine-associated receptor type-1.

Methamphetamine (Meth) is a potent and commonly abused psychostimulant. Meth alters neuron and astrocyte activity; yet the underlying mechanism(s) is not fully understood. Here we assessed the impact of acute Meth on human fetal astrocytes (HFAs) using whole-cell patch-clamping. We found that HFAs displayed a large voltage-gated K+ efflux (IKv ) through Kv /Kv -like channels during membrane depolarization, and a smaller K+ influx (Ikir ) via inward-rectifying Kir /Kir -like channels during membrane hyperpolarization. Meth at a 'recreational' (20 µM) or toxic/fatal (100 µM) concentration depolarized resting membrane potential (RMP) and suppressed IKv/Kv-like . These changes were associated with a decreased time constant (Ƭ), and mimicked by blocking the two-pore domain K+ (K2P )/K2P -like and Kv /Kv -like channels, respectively. Meth also diminished IKir/Kir-like , but only at toxic/fatal levels. Given that Meth is a potent agonist for the trace amine-associated receptor type-1 (TAAR1), and TAAR1-coupled cAMP/cAMP-activated protein kinase (PKA) cascade, we further evaluated whether the Meth impact on K+ efflux was mediated by this pathway. We found that antagonizing TAAR1 with N-(3-Ethoxyphenyl)-4-(1-pyrrolidinyl)-3-(trifluoromethyl)benzamide (EPPTB) reversed Meth-induced suppression of IKv/Kv-like ; and inhibiting PKA activity by H89 abolished Meth effects on suppressing IKv/Kv-like . Antagonizing TAAR1 might also attenuate Meth-induced RMP depolarization. Voltage-gated Ca2+ currents were not detected in HFAs. These novel findings demonstrate that Meth suppresses IKv/Kv-like by facilitating the TAAR1/Gs /cAMP/PKA cascade and altering the kinetics of Kv /Kv -like channel gating, but reduces K2P /K2P -like channel activity through other pathway(s), in HFAs. Given that Meth-induced decrease in astrocytic K+ efflux through K2P /K2P -like and Kv /Kv -like channels reduces extracellular K+ levels, such reduction could consequently contribute to a decreased excitability of surrounding neurons. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Correction to: Astrocytes as an HIV CNS reservoir: highlights and reflections of an NIMH-sponsored symposium.

In the original article the name of author Lena Al-Harthi was misspelled. It is correct here.

Wnt7a induces a unique phenotype of monocyte-derived macrophages with lower phagocytic capacity and differential expression of pro- and anti-inflammatory cytokines.

The variation of macrophage functions suggests the involvement of multiple signalling pathways in fine tuning their differentiation. Macrophages that originate from monocytes in the blood migrate to tissue in response to homeostatic or 'danger' signals and undergo substantial morphological and functional modifications to meet the needs of the dominant signals in the microenvironment. Wnts are secreted glycoproteins that play a significant role in organ and cell differentiation, yet their impact on monocyte differentiation is not clear. In this study, we assessed the role of Wnt1 and Wnt7a on the differentiation of monocytes and the subsequent phenotype and function of monocyte-derived macrophages (MDMs). We show that Wnt7a decreased the expression of CD14, CD11b, CD163 and CD206, whereas Wnt1 had no effect. The Wnt7a effect on CD11b was also observed in the brain and spleen of Wnt7a-/- adult brain mouse tissue and in embryonic Wnt7a-/- tissue. Wnt7a reduced the phagocytic capacity of M-MDMs, decreased interleukin-10 (IL-10) and IL-12 secretion and increased IL-6 secretion. Collectively, these findings demonstrate that Wnt7a generates an MDM phenotype with both pro-inflammatory and alternative MDM cytokine profiles and reduced phagocytic capacity. As such, Wnt7a can have a significant impact on macrophage responses in health and disease.

Aging alters voltage-gated calcium channels in prefrontal cortex pyramidal neurons in the HIV brain.

We assessed firing and voltage-gated Ca2+ influx in medial prefrontal cortex (mPFC) pyramidal neurons from older (12 months old) HIV-1 transgenic (Tg) rats. We found that neurons from older Tg rats showed increased firing compared to non-Tg rats, but Ca2+ spikes were unchanged. However, stronger excitatory stimulation was needed to evoke Ca2+ spikes, which was associated with reduced mPFC Cav1.2 L-type Ca2+ channel (L-channel) protein. In contrast, L-channel protein was unaltered in younger (6-7 weeks old) Tg rats, which we previously found had enhanced neuronal Ca2+ influx. These studies demonstrate that aging alters HIV-induced Ca2+ channel dysfunction that affects mPFC activity.

Migration of CD8+ T Cells into the Central Nervous System Gives Rise to Highly Potent Anti-HIV CD4dimCD8bright T Cells in a Wnt Signaling-Dependent Manner.

The role of CD8(+) T cells in HIV control in the brain and the consequences of such control are unclear. Approximately 3% of peripheral CD8(+) T cells dimly express CD4 on their surface. This population is known as CD4(dim)CD8(bright) T cells. We evaluated the role of CD4(dim)CD8(bright) and CD8 single positive T cells in HIV-infected brain using NOD/SCID/IL-2rcγ(-/-) mice reconstituted with human PBMCs (NSG-huPBMC). All three T cell populations (CD4 single positive, CD8 single positive, and CD4(dim)CD8(bright)) were found in NSG-huPBMC mouse brain within 2 wk of infection. Wnts secreted from astrocytes induced CD4(dim)CD8(bright) T cells by 2-fold in vitro. Injection of highly purified CD8 single positive T cells into mouse brain induced CD4(dim)CD8(bright) T cells by 10-fold, which were proliferative and exhibited a terminally differentiated effector memory phenotype. Brain CD4(dim)CD8(bright) T cells from HIV-infected mice exhibited anti-HIV-specific responses, as demonstrated by induction of CD107ab post exposure to HIV peptide-loaded targets. Further, higher frequency of CD4(dim)CD8(bright) T cells (R = -0.62; p ≤ 0.001), but not CD8 single positive T cells (R = -0.24; p ≤ 0.27), negatively correlated with HIV gag mRNA transcripts in HIV-infected NSG-huPBMC brain. Together, these studies indicate that single positive CD8(+) T cells entering the CNS during HIV infection can give rise to CD4(dim)CD8(bright) T cells, likely through a Wnt signaling-dependent manner, and that these cells are associated with potent anti-HIV control in the CNS. Thus, CD4(dim)CD8(bright) T cells are capable of HIV control in the CNS and may offer protection against HIV-associated neurocognitive disorders.

Combined chronic blockade of hyper-active L-type calcium channels and NMDA receptors ameliorates HIV-1 associated hyper-excitability of mPFC pyramidal neurons.

Human Immunodeficiency Virus type 1 (HIV-1) infection induces neurological and neuropsychological deficits, which are associated with dysregulation of the medial prefrontal cortex (mPFC) and other vulnerable brain regions. We evaluated the impact of HIV infection in the mPFC and the therapeutic potential of targeting over-active voltage-gated L-type Ca(2+) channels (L-channel) and NMDA receptors (NMDAR), as modeled in HIV-1 transgenic (Tg) rats. Whole-cell patch-clamp recording was used to assess the membrane properties and voltage-sensitive Ca(2+) potentials (Ca(2+) influx) in mPFC pyramidal neurons. Neurons from HIV-1 Tg rats displayed reduced rheobase, spike amplitude and inwardly-rectifying K(+) influx, increased numbers of action potentials, and a trend of aberrant firing compared to those from non-Tg control rats. Neuronal hyper-excitation was associated with abnormally-enhanced Ca(2+) influx (independent of NMDAR), which was eliminated by acute L-channel blockade. Combined chronic blockade of over-active L-channels and NMDARs with open-channel blockers abolished HIV effects on spiking, aberrant firing and Ca(2+) potential half-amplitude duration, though not the reduced inward rectification. In contrast, individual chronic blockade of over-active L-channels or NMDARs did not alleviate HIV-induced mPFC hyper-excitability. These studies demonstrate that HIV alters mPFC neuronal activity by dysregulating membrane excitability and Ca(2+) influx through the L-channels. This renders these neurons more susceptible and vulnerable to excitatory stimuli, and could contribute to HIV-associated neuropathogenesis. Combined targeting of over-active L-channels/NMDARs alleviates HIV-induced dysfunction of mPFC pyramidal neurons, emphasizing a potential novel therapeutic strategy that may effectively decrease HIV-induced Ca(2+) dysregulation in the mPFC.

ß-Catenin signaling positively regulates glutamate uptake and metabolism in astrocytes.

BACKGROUND: Neurological disorders have been linked to abnormal excitatory neurotransmission. Perturbations in glutamate cycling can have profound impacts on normal activity, lead to excitotoxicity and neuroinflammation, and induce and/or exacerbate impairments in these diseases. Astrocytes play a key role in excitatory signaling as they both clear glutamate from the synaptic cleft and house enzymes responsible for glutamate conversion to glutamine. However, mechanisms responsible for the regulation of glutamate cycling, including the main astrocytic glutamate transporter excitatory amino acid transporter 2 (EAAT2 or GLT-1 in rodents) and glutamine synthetase (GS) which catalyzes the ATP-dependent reaction of glutamate and ammonia into glutamine, remain largely undefined. METHODS: Gain and loss of function for ß-catenin in human progenitor-derived astrocyte (PDAs) was used to assess EAAT2 and GS levels by PCR, western blot, luciferase reporter assays, and chromatin immunoprecipitation (ChIP). Further, morpholinos were stereotaxically injected into C57BL/6 mice and western blots measured the protein levels of ß-catenin, GLT-1, and GS. RESULTS: ß-Catenin, a transcriptional co-activator and the central mediator of Wnt/ß-catenin signaling pathway, positively regulates EAAT2 and GS at the transcriptional level in PDAs by partnering with T cell factor 1 (TCF-1) and TCF-3, respectively. This pathway is conserved in vivo as the knockdown of ß-catenin in the prefrontal cortex results in reduced GLT-1 and GS expression. CONCLUSIONS: These studies confirm that ß-catenin regulates key proteins responsible for excitatory glutamate neurotransmission in vitro and in vivo and reveal the therapeutic potential of ß-catenin modulation in treating diseases with abnormal glutamatergic neurotransmission and excitotoxicity.

Dynamic interaction between astrocytes and infiltrating PBMCs in context of neuroAIDS.

HIV-mediated neuropathogenesis is a multifaceted process involving several players, including resident brain cells (neurons, astrocytes, and microglia) and infiltrating cells [peripheral blood mononuclear cells (PBMCs)]. We evaluated the dynamic interaction between astrocytes and infiltrating PBMCs as it impacts HIV in the CNS. We demonstrate that human primary-derived astrocytes (PDAs) predominantly secrete Wnt 1, 2b, 3, 5b, and 10b. Wnts are small secreted glycoproteins that initiate either ß-catenin-dependent or independent signal transduction. The Wnt pathway plays a vital role in the regulation of CNS activities including neurogenesis, neurotransmitter release, synaptic plasticity, and memory consolidation. We show that HIV infection of PDAs altered astrocyte Wnt profile by elevating Wnts 2b and 10b. Astrocyte conditioned media (ACM) inhibited HIV replication in PBMCs by 50%. Removal of Wnts from ACM abrogated its ability to suppress HIV replication in PBMCs. Inversely, PBMCs supernatant activated PDAs, as demonstrated by a 10-fold increase in HLA-DR and a 5-fold increase in IFNγ expression, and enhanced astrocyte susceptibility to HIV by 2-fold, which was mediated by IFNγ in a Stat-3-dependent manner. Collectively, these data demonstrate a dynamic interaction between astrocytes and PBMCs, whereby astrocyte-secreted Wnts exert an anti-HIV effect on infected PBMCs and PBMCs, in turn, secrete IFNγ that enhance astrocyte susceptibility to productive HIV infection and mediate their activation.