New articles from Prof. Shilpa Buch, our Editorial Board Member

Our staff editors continue to share exciting, interesting, and thought-provoking reads in this recommended articles series.

This week, we would like to share several latest articles from Prof. Shilpa Buch, our Editorial Board Member.

Title: Alzheimer’s-Like Pathology at the Crossroads of HIV-Associated Neurological Disorders
Authors: Divya T. Chemparthy, Muthukumar Kannan, Lila Gordon, Shilpa Buch, Susmita Sil
Type: Review of Vaccines
Abstract:
Despite the widespread success of combined antiretroviral therapy (cART) in suppressing viremia, the prevalence of human immunodeficiency virus (HIV)-associated neurological disorders (HAND) and associated comorbidities such as Alzheimer’s disease (AD)-like symptomatology is higher among people living with HIV. The pathophysiology of observed deficits in HAND is well understood. However, it has been suggested that it is exacerbated by aging. Epidemiological studies have suggested comparable concentrations of the toxic amyloid protein, amyloid-β42 (Aβ42), in the cerebrospinal fluid (CSF) of HAND patients and in the brains of patients with dementia of the Alzheimer’s type. Apart from abnormal amyloid-β (Aβ) metabolism in AD, a better understanding of the role of similar pathophysiologic processes in HAND could be of substantial value. The pathogenesis of HAND involves either the direct effects of the virus or the effect of viral proteins, such as Tat, Gp120, or Nef, as well as the effects of antiretrovirals on amyloid metabolism and tauopathy, leading, in turn, to synaptodendritic alterations and neuroinflammatory milieu in the brain. Additionally, there is a lack of knowledge regarding the causative or bystander role of Alzheimer’s-like pathology in HAND, which is a barrier to the development of therapeutics for HAND. This review attempts to highlight the cause–effect relationship of Alzheimer’s-like pathology with HAND, attempting to dissect the role of HIV-1, HIV viral proteins, and antiretrovirals in patient samples, animal models, and cell culture model systems. Biomarkers associated with Alzheimer’s-like pathology can serve as a tool to assess the neuronal injury in the brain and the associated cognitive deficits. Understanding the factors contributing to the AD-like pathology associated with HAND could set the stage for the future development of therapeutics aimed at abrogating the disease process.
Access this article: https://doi.org/10.3390/vaccines9080930

Title: An emerging and variant viral promoter of HIV-1 subtype C exhibits low-level gene expression noise
Authors: Haider Ali, Disha Bhange, Kavita Mehta, Yuvrajsinh Gohil, Harshit Kumar Prajapati, Siddappa N. Byrareddy, Shilpa Buch, Udaykumar Ranga
Type: Research of Retrovirology
Abstract:
Background
We observe the emergence of several promoter-variant viral strains in India during recent years. The variant viral promoters contain additional copies of transcription factor binding sites present in the viral modulatory region or enhancer, including RBEIII, LEF-1, Ap-1 and/or NF-κB. These sites are crucial for governing viral gene expression and latency. Here, we infer that one variant viral promoter R2N3-LTR containing two copies of RBF-2 binding sites (an RBEIII site duplication) and three copies of NF-κB motifs may demonstrate low levels of gene expression noise as compared to the canonical RN3-LTR or a different variant R2N4-LTR (a duplication of an RBEIII site and an NF-κB motif). To demonstrate this, we constructed a panel of sub-genomic viral vectors of promoter-variant LTRs co-expressing two reporter proteins (mScarlet and Gaussia luciferase) under the dual-control of Tat and Rev. We established stable pools of CEM.NKR-CCR5 cells (CEM-CCR5RL reporter cells) and evaluated reporter gene expression under different conditions of cell activation.
Results
The R2N3-LTR established stringent latency that was highly resistant to reversal by potent cell activators such as TNF-α or PMA, or even to a cocktail of activators, compared to the canonical RN3- or the variant R2N4-LTR. The R2N3-LTR exhibited low-level basal gene expression in the absence of cell activation that enhanced marginally but significantly when activated. In the presence of Tat and Rev, trans-complemented in the form of an infectious virus, the R2N3-LTR demonstrated gene expression at levels comparable to the wild-type viral promoter. The R2N3-LTR is responsive to Tat and Rev factors derived from viral strains representing diverse genetic subtypes.
Conclusion
With extremely low-level transcriptional noise, the R2N3-LTR can serve as an excellent model to examine the establishment, maintenance, and reversal of HIV-1 latency. The R2N3-LTR would also be an ideal viral promoter to develop high-throughput screening assays to identify potent latency-reversing agents since the LTR is not affected by the usual background noise of the cell.
Access this article: https://doi.org/10.1186/s12977-021-00572-2

Title: HIV TAT-mediated microglial senescence: Role of SIRT3-dependent mitochondrial oxidative stress
Authors: Annadurai Thangaraj, Ernest T. Chivero, Ashutosh Tripathi, Seema Singh, Fang Niu, Ming-Lei Guo, Prakash Pillai, Palsamy Periyasamy, Shilpa Buch
Type: Research Paper of Redox Biology
Highlights: 

• HIV TAT induces senescence-like phenotype in microglia.
• HIV TAT decreases SIRT3 with concomitant increase of mitochondrial ROS.
• Overexpression of SIRT3 attenuated HIV TAT-mediated microglial senescence.
• miR-505 negatively regulate SIRT3 expression.
• miR-505 inhibition prevents SIRT3-mediated mitochondria stress and glial senescence.

Abstract:
The advent of combined antiretroviral treatment (cART) as a treatment for HIV-1 infection has not only resulted in a dramatic decrease in the peripheral viral load but has also led to increased life expectancy of the infected individuals. Paradoxically, increased lifespan is accompanied with higher prevalence of age-related comorbidities, including HIV-associated neurocognitive disorders (HAND). Present study was aimed at exploring the role of HIV TAT protein in mediating microglial mitochondrial oxidative stress, ultimately resulting in neuroinflammation and microglial senescence. Our findings demonstrated that exposure of mouse primary microglial cells (mPMs) to HIV TAT protein resulted in a senescence-like phenotype, that was characterized by elevated expression of both p16 and p21 proteins, increased numbers of senescence-associated-β-galactosidase positive cells, augmented cell-cycle arrest, increased release of proinflammatory cytokines and decreased telomerase activity. Additionally, exposure of mPMs to HIV TAT also resulted downregulation of SIRT3 with a concomitant increase in mitochondrial oxidative stress. Dual luciferase reporter assay identified miR-505 as a novel target of SIRT3, which was upregulated in mPMs exposed to HIV TAT. Furthermore, transient transfection of mPMs with either the SIRT3 plasmid or miRNA-505 inhibitor upregulated the expression of SIRT3 and mitochondrial antioxidant enzymes, with a concomitant decrease in microglial senescence. These in vitro findings were also validated in the prefrontal cortices and striatum of HIV transgenic rats as well as cART-treated HIV-infected individuals. In summary, this study underscores a yet undiscovered novel mechanism(s) underlying HIV TAT-mediated induction of senescence phenotype in microglia, involving the miR-505-SIRT3 axis-mediated induction of mitochondrial oxidative stress.
Access this article: https://doi.org/10.1016/j.redox.2020.101843

Title: NLRP3 Inflammasome Is Involved in Cocaine-Mediated Potentiation on Behavioral Changes in CX3CR1-Deficient Mice
Authors: Ming-Lei Guo, Ernest T. Chivero, Shannon E. Callen, Shilpa Buch
Type: Article of Journal of Personalized Medicine
Abstract:
Microglia, the primary immunocompetent cells of the brain, are suggested to play a role in the development of drug addiction. Previous studies have identified the microglia-derived pro-inflammatory factor IL1β can promote the progression of cocaine addiction. Additionally, the activation status of microglia and “two-hit hypothesis” have been proposed in the field of drug addiction to explain how early life stress (ELS) could significantly increase the incidence of drug addiction in later life. However, the mechanisms underlying microglia prime and full activation and their roles in drug addiction remain greatly unexplored. Here, we employed CX3CR1-GFP mice (CX3CR1 functional deficiency, CX3CR1−/−) to explore whether primed microglia could potentiate cocaine-mediated behavioral changes and the possible underlying mechanisms. CX3CR1−/− mice revealed higher hyperlocomotion activity and conditional place preference than wild-type (WT) mice did under cocaine administration. In parallel, CX3CR1−/− mice showed higher activity of NLR family pyrin domain-containing 3 (NLRP3) inflammasome than WT mice. Interestingly, CX3CR1 deficiency itself could prime NLRP3 signaling by increasing the expression of NLPR3 and affect lysosome biogenesis under basal conditions. Taken together, our findings demonstrated that the functional status of microglia could have an impact on cocaine-mediated reward effects, and NLRP3 inflammasome activity was associated with this phenomenon. This study was consistent with the two-hit hypothesis and provided solid evidence to support the involvement of microglia in drug addiction. Targeting the NLRP3 inflammasome may represent a novel therapeutic approach for ameliorating or blocking the development of drug addiction.
Access this article: https://doi.org/10.3390/jpm11100963

Title: Astrocytes & Astrocyte derived Extracellular Vesicles in Morphine Induced Amyloidopathy: Implications for Cognitive Deficits in Opiate Abusers
Authors: Susmita Sil, Seema Singh, Divya T Chemparathy, Ernest T Chivero, Lila Gordon, Shilpa Buch
Type: Original Article of Aging and disease
Abstract:
While opiates like morphine play a major role in the pharmacotherapy for the control of pain associated with various diseases, paradoxically, their long-term use is associated with cognitive impairments. Furthermore, morphine administration has been shown to result in neuronal synaptodendritic injury in rodent brains, leading to neurodegeneration. We recently reported the role of astrocytes as contributors of amyloidosis associated with HIV-associated neurological disorders. Herein we hypothesize that morphine could induce astrocytic amyloidosis, which, in turn, could be disseminated to various regions in the brain by astrocyte-derived EVs (ADEVs). In this study we demonstrate brain region-specific up-regulation of astrocytic amyloids in morphine dependendent rhesus macaques. In addition, herein we also demonstrate increased expression of β-site cleaving enzyme (BACE1), APP, and Aβ in human primary astrocytes (HPAs) exposed to morphine. Mechanisms involved in this process included the up-regulation of hypoxia-inducible factor (HIF-1α), its translocation and binding to the promoter of BACE1, leading to increased BACE1 activity and, generation of Aβ 1-42. Gene silencing approaches confirmed the regulatory role of HIF-1α in BACE1 mediated amyloidosis leading to astrocyte activation and neuroinflammation. We next sought to assess whether morphine-stimulated ADEVs could carry amyloid cargoes. Results showed that morphine exposure induced the release of morphine-ADEVs, carrying amyloids. Interestingly, silencing HIF-1α in astrocytes not only reduced the numbers of ADEV released, but also the packaging of amyloid cargos in the ADEVs. These findings were further validated in brain derived EVs (BEVs) isolated from macaques, wherein it was shown that BEVs from morphine-dependent macaques, carried varieties of amyloid cargoes including the cytokine IL-1β. This is the first report implicating the role of HIF-1α-BACE1 axis in morphine-mediated induction of astrocytic amyloidosis, leading, in turn, to neuroinflammation and release of the amyloid cargoes via ADEVs. These findings set the groundwork for the future development of therapeutic strategies for targeting cognitive deficits in chronic opiate users.
Access this article: https://doi.org/10.14336/AD.2021.0406

Title: HIV Tat-Mediated Induction of Monocyte Transmigration Across the Blood–Brain Barrier: Role of Chemokine Receptor CXCR3
Authors: Fang Niu, Ke Liao, Guoku Hu, Shamsudheen Moidunny, Sabita Roy, Shilpa Buch
Type: Article of Frontiers in Cell and Developmental Biology
Abstract:
HIV trans-activator of transcription (Tat), one of the cytotoxic proteins secreted from HIV-infected cells, is also known to facilitate chemokine-mediated transmigration of monocytes into the brain leading, in turn, to neuroinflammation and thereby contributing to the development of HIV-associated neurocognitive disorders (HAND). The mechanism(s) underlying HIV Tat-mediated enhancement of monocyte transmigration, however, remain largely unknown. CXC chemokine receptor 3 (CXCR3) that is expressed by the peripheral monocytes is known to play a role in the monocyte influx and accumulation. In the present study, we demonstrate for the first time that exposure of human monocytes to HIV Tat protein resulted in upregulated expression of CXCR3 leading, in turn, to increased monocyte transmigration across the blood–brain barrier (BBB) both in the in vitro and in vivo model systems. This process involved activation of toll-like receptor 4 (TLR4), with downstream phosphorylation and activation of TANK-binding kinase 1 (TBK1), and subsequent phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3), ultimately leading to enhanced expression of CXCR3 in human monocytes. These findings imply a novel molecular mechanism underlying HIV Tat-mediated increase of monocyte transmigration across the BBB, while also implicating a novel role of CXCR3-dependent monocyte transmigration in HIV Tat-mediated neuroinflammation.
Access this article: https://doi.org/10.3389/fcell.2021.724970

Title: Chronic Morphine Administration Differentially Modulates Viral Reservoirs in a Simian Immunodeficiency Virus SIVmac251-Infected Rhesus Macaque Model
Authors: Arpan Acharya, Omalla A. Olwenyi, Michellie Thurman, Kabita Pandey, Brenda M. Morsey, Benjamin Lamberty, Natasha Ferguson, Shannon Callen, Qiu Fang, Shilpa J. Buch, Howard S. Fox, Siddappa N. Byrareddy
Type: Research Article from Journal of Virology
Abstract:
Human immunodeficiency virus (HIV) persists in cellular reservoirs despite effective combined antiretroviral therapy (cART), and there is viremia flare-up upon therapy interruption. Opioids modulate the immune system and suppress antiviral gene responses, which significantly impact people living with HIV (PLWH). However, the effect of opioids on viral reservoir dynamics remains elusive. Here, we developed a morphine-dependent simian immunodeficiency virus SIVmac251-infected rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs. RMs on a morphine (or saline control) regimen were infected with SIVmac251. cART was initiated in approximately half the animals 5 weeks postinfection, and morphine/saline administration continued until the end of the study. Among the untreated RMs, we did not find any difference in plasma/cerebrospinal fluid (CSF) viral loads or in cell-associated DNA/RNA viral loads in anatomical tissues. On the other hand, within the cART-suppressed macaques, there was a reduction in cell-associated DNA load, intact proviral DNA levels, and in inducible SIV reservoirs in lymph nodes (LNs) of morphine-administered RMs. In distinction to those in LNs, in the central nervous system (CNS), the size of latent SIV reservoirs was larger in the CD11b+ microglia/macrophages in morphine-dependent RMs. These results suggest that in the proposed model, morphine plays a differential role in SIV reservoirs by reducing the CD4+ T-cell reservoir in lymphoid tissues while increasing the microglia/macrophage reservoir size in CNS tissue. The findings from this preclinical model will serve as a tool for screening therapeutic strategies to reduce/eliminate HIV reservoirs in opioid-dependent PLWH.
Access this article: https://doi.org/10.1128/JVI.01657-20