Involvement of Endolysosomes and Aurora Kinase A in the Regulation of Amyloid ß Protein Levels in Neurons.

Aurora kinase A (AURKA) is a serine/threonine-protein kinase that regulates microtubule organization during neuron migration and neurite formation. Decreased activity of AURKA was found in Alzheimer's disease (AD) brain samples, but little is known about the role of AURKA in AD pathogenesis. Here, we demonstrate that AURKA is expressed in primary cultured rat neurons, neurons from adult mouse brains, and neurons in postmortem human AD brains. AURKA phosphorylation, which positively correlates with its activity, is reduced in human AD brains. In SH-SY5Y cells, pharmacological activation of AURKA increased AURKA phosphorylation, acidified endolysosomes, decreased the activity of amyloid beta protein (Aß) generating enzyme ß-site amyloid precursor protein cleaving enzyme (BACE-1), increased the activity of the Aß degrading enzyme cathepsin D, and decreased the intracellular and secreted levels of Aß. Conversely, pharmacological inhibition of AURKA decreased AURKA phosphorylation, de-acidified endolysosomes, decreased the activity of cathepsin D, and increased intracellular and secreted levels of Aß. Thus, reduced AURKA activity in AD may contribute to the development of intraneuronal accumulations of Aß and extracellular amyloid plaque formation.

Preparation, Characterization, and Antioxidant Activity of L-Ascorbic Acid/HP-ß-Cyclodextrin Inclusion Complex-Incorporated Electrospun Nanofibers.

L-Ascorbic acid (LAA) is a key vitamin, implicated in a variety of physiological processes in humans. Due to its free radical scavenging activity, it is extensively employed as an excipient in pharmaceutical products and food supplements. However, its application is greatly impeded by poor thermal and aqueous stability. Herein, to improve the stability and inhibit oxidative degradation, we prepared LAA-cyclodextrin inclusion complex-incorporated nanofibers (NFs). The continuous variation method (Job plot) demonstrated that LAA forms inclusions with hydroxypropyl-ß-cyclodextrin (HP-ß-CD) at a 2:1 molar stoichiometric ratio. The NFs were prepared via the single step electrospinning technique, without using any polymer matrix. The solid-state characterizations of LAA/HP-ß-CD-NF via powder x-ray diffractometry (PXRD), Fourier-transform infrared (FT-IR) analysis, differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), and nuclear magnetic resonance (1H NMR and 2D-NOESY) spectroscopy, reveal the effective encapsulation of the LAA (guest molecule) inside the HP-ß-CD (host) cavity. The SEM micrograph reveals an average fiber diameter of ~339 nm. The outcomes of the thermal investigations demonstrated that encapsulation of LAA within HP-ß-CD cavities provides improved thermal stability of LAA (by increasing the thermal degradation temperature). The radical scavenging assay demonstrated the enhanced antioxidant potential of LAA/HP-ß-CD-NF, as compared to native LAA. Overall, the study shows that cyclodextrin inclusion complex-incorporated NFs, are an effective approach for improving the limitations associated with LAA, and provide promising avenues in its therapeutic and food applications.

Mu opioid receptor-mediated release of endolysosome iron increases levels of mitochondrial iron, reactive oxygen species, and cell death.

Objectives: Opioids including morphine and DAMGO activate mu-opioid receptors (MOR), increase intracellular reactive oxygen species (ROS) levels, and induce cell death. Ferrous iron (Fe2+) through Fenton-like chemistry increases ROS levels and endolysosomes are "master regulators of iron metabolism" and contain readily-releasable Fe2+ stores. However, mechanisms underlying opioid-induced changes in endolysosome iron homeostasis and downstream-signaling events remain unclear. Methods: We used SH-SY5Y neuroblastoma cells, flow cytometry, and confocal microscopy to measure Fe2+ and ROS levels and cell death. Results: Morphine and DAMGO de-acidified endolysosomes, decreased endolysosome Fe2+ levels, increased cytosol and mitochondria Fe2+ and ROS levels, depolarized mitochondrial membrane potential, and induced cell death; effects blocked by the nonselective MOR antagonist naloxone and the selective MOR antagonist ß-funaltrexamine (ß-FNA). Deferoxamine, an endolysosome-iron chelator, inhibited opioid agonist-induced increases in cytosolic and mitochondrial Fe2+ and ROS. Opioid-induced efflux of endolysosome Fe2+ and subsequent Fe2+ accumulation in mitochondria were blocked by the endolysosome-resident two-pore channel inhibitor NED-19 and the mitochondrial permeability transition pore inhibitor TRO. Conclusions: Opioid agonist-induced increases in cytosolic and mitochondrial Fe2+ and ROS as well as cell death appear downstream of endolysosome de-acidification and Fe2+ efflux from the endolysosome iron pool that is sufficient to affect other organelles.

Recent progress in covalent organic frameworks for cancer therapy.

Covalent organic frameworks (COFs) have gained tremendous interest in cancer therapy owing to their multifunctional properties, such as biocompatibility, tunable cavities, excellent crystallinity, ease of modification/functionalization, and high flexibility. These unique properties offer multiple benefits, such as high loading capacity, prevention from premature leakage, targeted delivery to the tumor microenvironment (TME), and release of therapeutic agents in a controlled manner, which makes them effective and excellent nanoplatforms for cancer therapeutics. In this review, we outline recent advances in using COFs as delivery system for chemotherapeutic agents, photodynamic therapy (PDT), photothermal therapy (PTT), sonodynamic therapy (SDT), cancer diagnostics, and combinatorial therapy for cancer therapeutics. We also summarize current challenges and future directions of this unique research field.

Recent advances in dual-ligand targeted nanocarriers for cancer therapy.

Nanocarriers (NCs) containing targeting ligands have received significant attention in recent years because of their ability to enhance cancer cell recognition, which in turn improves both their accuracy and the therapeutic efficacy of their payloads. A promising approach in this area is the use of dual ligands, in which NCs are functionalized with two different targeting ligands, enabling them to specifically recognize and interact with two different biomarkers present on cancer cells for more efficient targeting compared with single-ligand targeted nanocarriers. Herein, we highlight recent advances in dual-ligand targeted NCs with particular emphasis on their potential for improving therapeutic outcomes for cancer therapy.

Heterogeneity of ferrous iron-containing endolysosomes and effects of endolysosome iron on endolysosome numbers, sizes, and localization patterns.

Endolysosomes are key regulators of iron metabolism and are central to iron trafficking and redox signaling. Iron homeostasis is linked to endolysosome acidity and inhibition of endolysosome acidity triggers iron dysregulation. Because of the physiological importance and pathological relevance of ferrous iron (Fe2+ ), we determined levels of Fe2+ specifically and quantitatively in endolysosomes as well as the effects of Fe2+ on endolysosome morphology, distribution patterns, and function. The fluorescence dye FeRhoNox-1 was specific for Fe2+ and localized to endolysosomes in U87MG astrocytoma cells and primary rat cortical neurons; in U87MG cells the endolysosome concentration of Fe2+ ([Fe2+ ]el ) was 50.4 µM in control cells, 73.6 µM in ferric ammonium citrate (FAC) treated cells, and 12.4 µM in cells treated with the iron chelator deferoxamine (DFO). Under control conditions, in primary rat cortical neurons, [Fe2+ ]el was 32.7 µM. Endolysosomes containing the highest levels of Fe2+ were located perinuclearly. Treatment of cells with FAC resulted in endolysosomes that were less acidic, increased in numbers and sizes, and located further from the nucleus; opposite effects were observed for treatments with DFO. Thus, FeRhoNox-1 is a useful probe for the study of endolysosome Fe2+ , and much more work is needed to understand better the physiological significance and pathological relevance of endolysosomes classified according to their heterogeneous iron content Cover Image for this issue: https://doi.org/10.1111/jnc.15396.

HIV-1 Tat endocytosis and retention in endolysosomes affects HIV-1 Tat-induced LTR transactivation in astrocytes.

The presence of latent HIV-1 reservoirs in the periphery and brain represents a major obstacle to curing HIV-1 infection. As an essential protein for HIV-1 viral replication, HIV-1 Tat, mostly intracellular, has been implicated in latent HIV-1 infection. From HIV-1 infected cells, HIV-1 Tat is actively secreted and bystander cells uptake the released Tat whereupon it is endocytosed and internalized into endolysosomes. However, to activate the HIV-1 LTR promoter and increase HIV-1 replication, HIV-1 Tat must first escape from the endolysosomes and then enter the nucleus. Here, we tested the hypothesis that HIV-1 Tat can accumulate in endolysosomes and contribute to the activation of latent HIV-1 in astrocytes. Using U87MG astrocytoma cells expressing HIV-1 LTR-driven luciferase and primary human astrocytes we found that exogenous HIV-1 Tat enters endolysosomes, resides in endolysosomes for extended periods of time, and induces endolysosome de-acidification as well as enlargement. The weak base chloroquine promoted the release of HIV-1 Tat from endolysosomes and induced HIV-1 LTR transactivation. Similar results were observed by activating endolysosome Toll-like receptor 3 (TLR3) and TLR7/8. Conversely, pharmacological block of TLRs and knocking down expression levels of TLR3 and TLR7, but not TLR8, prevented endolysosome leakage and attenuated HIV-1 Tat-mediated HIV-1 LTR transactivation. Our findings suggest that HIV-1 Tat accumulation in endolysosomes may play an important role in controlling HIV-1 transactivation.

HIV-1 gp120-Induced Endolysosome de-Acidification Leads to Efflux of Endolysosome Iron, and Increases in Mitochondrial Iron and Reactive Oxygen Species.

The HIV-1 coat protein gp120 continues to be implicated in the pathogenesis of HIV-1 associated neurocognitive disorder (HAND); a condition known to affect ~50% of people living with HIV-1 (PLWH). Autopsy brain tissues of HAND individuals display morphological changes to mitochondria and endolysosomes, and HIV-1 gp120 causes mitochondrial dysfunction including increased levels of reactive oxygen species (ROS) and de-acidification of endolysosomes. Ferrous iron is linked directly to ROS production, ferrous iron is contained in and released from endolysosomes, and PLWH have elevated iron and ROS levels. Based on those findings, we tested the hypothesis that HIV-1 gp120-induced endolysosome de-acidification and subsequent iron efflux from endolysosomes is responsible for increased levels of ROS. In U87MG glioblastoma cells, HIV-1 gp120 de-acidified endolysosomes, reduced endolysosome iron levels, increased levels of cytosolic and mitochondrial iron, and increased levels of cytosolic and mitochondrial ROS. These effects were all attenuated significantly by the endolysosome-specific iron chelator deferoxamine, by inhibitors of endolysosome-resident two-pore channels and divalent metal transporter-1 (DMT-1), and by inhibitors of mitochondria-resident DMT-1 and mitochondrial permeability transition pores. These results suggest that oxidative stress commonly observed with HIV-1 gp120 is downstream of its ability to de-acidify endolysosomes, to increase the release of iron from endolysosomes, and to increase the uptake of iron into mitochondria. Thus, endolysosomes might represent early and upstream targets for therapeutic strategies against HAND.

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

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

Aptamer-based sensing of breast cancer biomarkers: a comprehensive review of analytical figures of merit.

INTRODUCTION: Accurate determination of the aberrantly expressed biomarkers such as human epidermal growth factor receptor 2 (HER2), carcinoembryonic antigen (CEA), platelet-derived growth factor (PDGF), mucin 1 (MUC1), and vascular endothelial growth factor VEGF165 have played an essential role in the clinical management of the breast cancer. Assessment of these cancer-specific biomarkers has conventionally relied on time-taking methods like the enzyme-linked immunosorbent assay and immunohistochemistry. However, recent development in the aptamer-based diagnostics has allowed developing tools that may substitute the conventional means of biomarker assessment in breast cancer. Adopting the aptamer-based diagnostic tools (aptasensors) to clinical practices will depend on their analytical performance on clinical samples. AREAS COVERED: In this review, we provide an overview of the analytical merits of HER2, CEA, PDGF, MUC1, and VEGF165 aptasensors. Scopus and Pubmed databases were searched for studies reporting aptasensor development for the listed breast cancer biomarkers in the past one decade. Linearity, detection limit, and response time are emphasized. EXPERT OPINION: In our opinion, aptasensors have proven to be on a par with the antibody-based methods for detection of various breast cancer biomarkers. Though robust validation of the aptasensors on significant sample size is required, their ability to detect pathophysiological range of biomarkers suggest the possibility of future clinical adoption.

Lysosomal Stress Response (LSR): Physiological Importance and Pathological Relevance.

Extensive work has characterized endoplasmic reticulum (ER) and mitochondrial stress responses. In contrast, very little has been published about stress responses in lysosomes; subcellular acidic organelles that are physiologically important and are of pathological relevance. The greater lysosomal system is dynamic and is comprised of endosomes, lysosomes, multivesicular bodies, autophagosomes, and autophagolysosomes. They are important regulators of cellular physiology, they represent about 5% of the total cellular volume, they are heterogeneous in their sizes and distribution patterns, they are electron dense, and their subcellular positioning within cells varies in response to stimuli, insults and pH. These organelles are also integral to the pathogenesis of lysosomal storage diseases and it is increasingly recognized that lysosomes play important roles in the pathogenesis of such diverse conditions as neurodegenerative disorders and cancer. The purpose of this review is to focus attention on lysosomal stress responses (LSR), compare LSR with better characterized stress responses in ER and mitochondria, and form a framework for future characterizations of LSR. We synthesized data into the concept of LSR and present it here such that the definition of LSR can be modified as new knowledge is added and specific therapeutics are developed.

Trehalose limits opportunistic mycobacterial survival during HIV co-infection by reversing HIV-mediated autophagy block.

Opportunistic bacterial infections amongst HIV-infected individuals contribute significantly to HIV-associated mortality. The role of HIV-mediated modulation of innate mechanisms like autophagy in promoting opportunistic infections, however, remains obscure. Here we show, HIV reactivation in or infection of macrophages inhibits autophagy and helps the survival of pathogenic Mycobacterium tuberculosis (Mtb) and nonpathogenic non-tuberculous mycobacterial strains (NTMs). The HIV-mediated impairment of xenophagy flux facilitated bacterial survival. Activation of autophagy by trehalose could induce xenophagy flux and kill intracellular Mtb or NTMs either during single or co-infections. Trehalose, we delineate, activates PIKFYVE leading to TFEB nuclear translocation in MCOLN1-dependent manner to induce autophagy. Remarkably, trehalose significantly reduced HIV-p24 levels in ex-vivo-infected PBMCs or PBMCs from treatment-naive HIV patients and also controlled mycobacterial survival within Mtb-infected animals. To conclude, we report leveraging of HIV-mediated perturbed host innate-immunity by opportunistic bacterial pathogens and show an attractive therapeutic strategy for HIV and associated co-morbidities.Abbreviations: AIDS: acquired immune deficiency syndrome; AMPK: AMP-activated protein kinase; ATG5: autophagy related 5; BafA1: bafilomycin A1; CFU: colony forming unit; CTSD: cathepsin D; CD63: CD63 molecule; EGFP: enhanced green fluorescent protein; FRET: Förster resonance energy transfer; GABARAP: gamma-aminobutyric acid receptor-associated protein; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GLUT: glucose transporter; HIV: human immunodeficiency virus; hMDMs: human monocyte derived macrophages; IL2: interleukin 2; LAMP1: lysosomal-associated membrane protein 1; LC3B-II: lipidated microtubule-associated proteins 1A/1B light chain 3B; Mtb: Mycobacterium tuberculosis; MTOR: mechanistic target of rapamycin; mRFP: monomeric red fluorescent protein; M6PR: mannose-6-phosphate receptor; NAC: N- acetyl- L -cysteine; NTM's: non-tuberculous mycobacteria; PBMC: Peripheral Blood Mononuclear cells; PIKFYVE: phosphoinositide kinase; FYVE-Type Zinc Finger; PHA: phytohemagglutinin; PMA: phorbol 12-myristate 13-acetate; PtdIns(3,5)P2: Phosphatidylinositol 3,5-bisphosphate; ptfLC3: pEGFP-mRFP-LC3; ROS: reactive oxygen species; SQSTM1: sequestosome1; TFEB: transcription factor EB; MCOLN1/TRPML1: mucolipin 1; PIP4P1/TMEM55B: Human trans-membrane Protein 55B; UVRAG: UV Radiation Resistance Associate; VPS35: vacuolar protein sorting associated protein 35; WDR45: WD repeat domain 45; YCAM: Yellow Chameleon.

Possible Role of Adenosine in COVID-19 Pathogenesis and Therapeutic Opportunities.

The outbreak of the novel coronavirus disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) requires urgent clinical interventions. Crucial clinical needs are: 1) prevention of infection and spread of the virus within lung epithelia and between people, 2) attenuation of excessive lung injury in Advanced Respiratory Distress Syndrome, which develops during the end stage of the disease, and 3) prevention of thrombosis associated with SARS-CoV-2 infection. Adenosine and the key adenosine regulators adenosine deaminase (ADA), adenosine kinase (ADK), and equilibrative nucleoside transporter 1 may play a role in COVID-19 pathogenesis. Here, we highlight 1) the non-enzymatic role of ADA by which it might out-compete the virus (SARS-CoV-2) for binding to the CD26 receptor, 2) the enzymatic roles of ADK and ADA to increase adenosine levels and ameliorate Advanced Respiratory Distress Syndrome, and 3) inhibition of adenosine transporters to reduce platelet activation, thrombosis and improve COVID-19 outcomes. Depending on the stage of exposure to and infection by SARS-CoV-2, enhancing adenosine levels by targeting key adenosine regulators such as ADA, ADK and equilibrative nucleoside transporter 1 might find therapeutic use against COVID-19 and warrants further investigation.

Two-pore channels regulate Tat endolysosome escape and Tat-mediated HIV-1 LTR transactivation.

HIV-1 Tat is essential for HIV-1 replication and appears to play an important role in the pathogenesis of HIV-associated neurological complications. Secreted from infected or transfected cells, Tat has the extraordinary ability to cross the plasma membrane. In the brain, Tat can be taken up by CNS cells via receptor-mediated endocytosis. Following endocytosis and its internalization into endolysosomes, Tat must be released in order for it to activate the HIV-1 LTR promoter and facilitate HIV-1 viral replication in the nucleus. However, the underlying mechanisms whereby Tat escapes endolysosomes remain unclear. Because Tat disrupts intracellular calcium homeostasis, we investigated the involvement of calcium in Tat endolysosome escape and subsequent LTR transactivation. We demonstrated that chelating endolysosome calcium with high-affinity rhodamine-dextran or chelating cytosolic calcium with BAPTA-AM attenuated Tat endolysosome escape and LTR transactivation. Significantly, we demonstrated that pharmacologically blocking and knocking down the endolysosome-resident two-pore channels (TPCs) attenuated Tat endolysosome escape and LTR transactivation. This calcium-mediated effect appears to be selective for TPCs because knocking down TRPML1 calcium channels was without effect. Our findings suggest that calcium released from TPCs is involved in Tat endolysosome escape and subsequent LTR transactivation. TPCs might represent a novel therapeutic target against HIV-1 infection and HIV-associated neurological complications.

Circulating levels of ATP is a biomarker of HIV cognitive impairment.

BACKGROUND: In developed countries, Human Immunodeficiency Virus type-1 (HIV-1) infection has become a chronic disease despite the positive effects of anti-retroviral therapies (ART), but still at least half of the HIV infected population shown signs of cognitive impairment. Therefore, biomarkers of HIV cognitive decline are urgently needed. METHODS: We analyze the opening of one of the larger channels expressed by humans, pannexin-1 (Panx-1) channels, in the uninfected and HIV infected population (n = 175). We determined channel opening and secretion of intracellular second messengers released through the channel such as PGE2 and ATP. Also, we correlated the opening of Panx-1 channels with the circulating levels of PGE2 and ATP as well as cogntive status of the individuals analyzed. FINDINGS: Here, we demonstrate that Panx-1 channels on fresh PBMCs obtained from uninfected individuals are closed and no significant amounts of PGE2 and ATP are detected in the circulation. In contrast, in all HIV-infected individuals analyzed, even the ones under effective ART, a spontaneous opening of Panx-1 channels and increased circulating levels of PGE2 and ATP were detected. Circulating levels of ATP were correlated with cognitive decline in the HIV-infected population supporting that ATP is a biomarker of cognitive disease in the HIV-infected population. INTERPRETATION: We propose that circulating levels of ATP could predict CNS compromise and lead to the breakthroughs necessary to detect and prevent brain compromise in the HIV-infected population.

Importance of measuring endolysosome, cytosolic, and extracellular pH in understanding the pathogenesis of and possible treatments for glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM) is a very aggressive form of brain cancer that carries with it a tragically poor prognosis. As with many other forms of cancer, the extracellular environment near GBM tumors is acidified and is relevant to the pathogenesis of GBM because decreased pH promotes tumor cell invasion, increases angiogenesis, decreases immune surveillance, and increases resistance to possible treatments. Recently, vacuolar ATPase (v-ATPase), a proton pump that helps maintain the acidic environment in endosomes and lysosomes (hereafter referred to endolysosomes) as well as proton gradients across the plasma membrane, was identified as a novel therapeutic target for GBM. However, information is lacking about cancer cell and tissue pH of endolysosomes, cytosol and extracellular fluid. AIM: Here, we measured endolysosome, cytosolic, and extracellular pH in U87MG cells in the absence and presence of the v-ATPase inhibitor bafilomycin A1. METHODS: In vitro measurements of U87MG cells were conducted using LysoSensor dye and a Lysosome-RFP dye for lysosome pH, BCECF-AM for cytosolic pH, and a pH-sensitive microprobe for extracellular pH. RESULTS: Bafilomycin A1 increased endolysosome pH from 5.28 to 5.57, decreased cytosolic pH from 7.01 to 6.46, and increased extracellular pH from 7.18 to 7.40. CONCLUSIONS: Here, we report the ability to make pH measurements in U87MG glioblastoma cells and discuss these results in the context of GBM pathogenesis and possible treatment. This might be of some importance in understanding the pathogenesis of GBM because the highly regulated stores of hydrogen (H+) ions in endolysosomes can influence cytosolic and extracellular pH as well as the distribution, numbers, and sizes of endolysosomes.

Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation.

BACKGROUND: Apolipoprotein E (ApoE) is the major carrier protein that mediates the transport and delivery of cholesterol and other lipids in the brain. Three isoforms of ApoE (ApoE2, ApoE3, ApoE4) exist in humans, and their relative expression levels impact HIV-1 infection, HIV-1/AIDS disease progression, and cognitive decline associated with HIV-1-associated neurocognitive disorder. Because HIV-1 Tat, a viral protein essential for HIV-1 replication, can bind to low-density lipoprotein receptor-related protein 1 (LRP1) that controls ApoE uptake in the brain, we determined the extent to which different isoforms of ApoE affected Tat-mediated HIV-1 LTR transactivation. METHODS: Using U87MG glioblastoma cells expressing LTR-driven luciferase, we determined the extent to which LRP1 as well as ApoE2, ApoE3, and ApoE4 affected Tat-mediated HIV-1 LTR transactivation. RESULTS: A specific LRP1 antagonist and siRNA knockdown of LRP1 both restricted significantly Tat-mediated LTR transactivation. Of the three ApoEs, ApoE4 was the least potent and effective at preventing HIV-1 Tat internalization and at decreasing Tat-mediated HIV-1 LTR transactivation. Further, Tat-mediated LTR transactivation was attenuated by an ApoE mimetic peptide, and ApoE4-induced restriction of Tat-mediated LTR transactivation was potentiated by an ApoE4 structure modulator that changes ApoE4 into an ApoE3-like phenotype. CONCLUSIONS: These findings help explain observed differential effects of ApoEs on HIV-1 infectivity and the prevalence of HAND in people living with HIV-1 infection and suggest that ApoE mimetic peptides and ApoE4 structure modulator might be used as a therapeutic strategy against HIV-1 infection and associated neurocognitive disorders.

Mycobacterium tuberculosis and Human Immunodeficiency Virus Type 1 Cooperatively Modulate Macrophage Apoptosis via Toll Like Receptor 2 and Calcium Homeostasis.

The emergence of drug resistant strains of Mycobacterium tuberculosis (M. tuberculosis) together with reports of co-infections with the human immunodeficiency virus (HIV) has renewed interest to better understand the intricate mechanisms prevalent during co-infections. In this study we report a synergistic effect of M. tuberculosis and HIV-1, and their antigens Rv3416 and Nef, respectively, in inhibiting apoptosis of macrophages. This inhibition involves the TLR2 pathway and second messengers that play complementing and contrasting roles in regulating apoptosis. Interestingly, the route of calcium influx into cells differentially regulates apoptosis during antigenic co-stimulation. While calcium released from intracellular stores was anti-apoptotic, calcium influx from the external milieu was pro-apoptotic. Further, molecular sensors of intracellular calcium release aid in antigen mediated inhibition of apoptosis. A cross-regulation between oxidative burst and differential routing of calcium influx governed apoptosis. Interestingly, the HIV-1 Nef supported anti-apoptotic responses in macrophages whereas Vpu had no significant effect. These results point to a synergistic liaison between M. tuberculosis and HIV-1 in regulating macrophage apoptosis.

The SARS Coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor.

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is reported to cause apoptosis of infected cells and several of its proteins including the 3a accessory protein, are pro-apoptotic. Since the 3a protein localizes to the endoplasmic reticulum (ER)-Golgi compartment, its role in causing ER stress was investigated in transiently transfected cells. Cells expressing the 3a proteins showed ER stress based on activation of genes for the ER chaperones GRP78 and GRP94. Since ER stress can cause differential modulation of the unfolded protein response (UPR), which includes the inositol-requiring enzyme 1 (IRE-1), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) pathways, these were individually tested in 3a-expressing cells. Only the PERK pathway was found to be activated in 3a-expressing cells based on (1) increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2alpha) and inhibitory effects of a dominant-negative form of eIF2alpha on GRP78 promoter activity, (2) increased translation of activating transcription factor 4 (ATF4) mRNA, and (3) ATF4-dependent activation of the C/EBP homologous protein (CHOP) gene promoter. Activation of PERK affects innate immunity by suppression of type 1 interferon (IFN) signaling. The 3a protein was found to induce serine phosphorylation within the IFN alpha-receptor subunit 1 (IFNAR1) degradation motif and to increase IFNAR1 ubiquitination. Confocal microscopic analysis showed increased translocation of IFNAR1 into the lysosomal compartment and flow cytometry showed reduced levels of IFNAR1 in 3a-expressing cells. These results provide further mechanistic details of the pro-apoptotic effects of the SARS-CoV 3a protein, and suggest a potential role for it in attenuating interferon responses and innate immunity.