HERV-K (HML-2) Envelope Protein Induces Mitochondrial Depolarization and Neurotoxicity via Endolysosome Iron Dyshomeostasis.

Human endogenous retroviruses (HERVs) are associated with the pathogenesis of amyotrophic lateral sclerosis (ALS); a disease characterized by motor neuron degeneration and cell death. The HERV-K subtype HML-2 envelope protein (HERV-K Env) is expressed in the brain, spinal cord, and cerebrospinal fluid of people living with ALS and through CD98 receptor-linked interactions causes neurodegeneration. HERV-K Env-induced increases in oxidative stress are implicated in the pathogenesis of ALS, and ferrous iron (Fe2+) generates reactive oxygen species (ROS). Endolysosome stores of Fe2+ are central to iron trafficking and endolysosome deacidification releases Fe2+ into the cytoplasm. Because HERV-K Env is an arginine-rich protein that is likely endocytosed and arginine is a pH-elevating amino acid, it is important to determine HERV-K Env effects on endolysosome pH and whether HERV-K Env-induced neurotoxicity is downstream of Fe2+ released from endolysosomes. Here, we showed using SH-SY5Y human neuroblastoma cells and primary cultures of human cortical neurons (HCNs, information on age and sex was not available) that HERV-K Env (1) is endocytosed via CD98 receptors, (2) concentration dependently deacidified endolysosomes, (3) decreased endolysosome Fe2+ concentrations, (4) increased cytosolic and mitochondrial Fe2+ and ROS levels, (5) depolarized mitochondrial membrane potential, and (6) induced cell death, effects blocked by an antibody against the CD98 receptor and by the endolysosome iron chelator deferoxamine. Thus, HERV-K Env-induced increases in cytosolic and mitochondrial Fe2+ and ROS as well as cell death appear to be mechanistically caused by HERV-K Env endocytosis, endolysosome deacidification, and endolysosome Fe2+ efflux into the cytoplasm.

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.

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.

Role of endolysosome function in iron metabolism and brain carcinogenesis.

Iron, the most abundant metal in human brain, is an essential microelement that regulates numerous cellular mechanisms. Some key physiological roles of iron include oxidative phosphorylation and ATP production, embryonic neuronal development, formation of iron-sulfur clusters, and the regulation of enzymes involved in DNA synthesis and repair. Because of its physiological and pathological importance, iron homeostasis must be tightly regulated by balancing its uptake, transport, and storage. Endosomes and lysosomes (endolysosomes) are acidic organelles known to contain readily releasable stores of various cations including iron and other metals. Increased levels of ferrous (Fe2+) iron can generate reactive oxygen species (ROS) via Fenton chemistry reactions and these increases can damage mitochondria and genomic DNA as well as promote carcinogenesis. Accumulation of iron in the brain has been linked with aging, diet, disease, and cerebral hemorrhage. Further, deregulation of brain iron metabolism has been implicated in carcinogenesis and may be a contributing factor to the increased incidence of brain tumors around the world. Here, we provide insight into mechanisms by which iron accumulation in endolysosomes is altered by pH and lysosome membrane permeabilization. Such events generate excess ROS resulting in mitochondrial DNA damage, fission, and dysfunction, as well as DNA oxidative damage in the nucleus; all of which promote carcinogenesis. A better understanding of the roles that endolysosome iron plays in carcinogenesis may help better inform the development of strategic therapeutic options for cancer treatment and prevention.

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.

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.

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.

Readily Releasable Stores of Calcium in Neuronal Endolysosomes: Physiological and Pathophysiological Relevance.

Neurons are long-lived post-mitotic cells that possess an elaborate system of endosomes and lysosomes (endolysosomes) for protein quality control. Relatively recently, endolysosomes were recognized to contain high concentrations (400-600 µM) of readily releasable calcium. The release of calcium from this acidic organelle store contributes to calcium-dependent processes of fundamental physiological importance to neurons including neurotransmitter release, membrane excitability, neurite outgrowth, synaptic remodeling, and cell viability. Pathologically, disturbances of endolysosome structure and/or function have been noted in a variety of neurodegenerative disorders including Alzheimer's disease (AD) and HIV-1 associated neurocognitive disorder (HAND). And, dysregulation of intracellular calcium has been implicated in the neuropathogenesis of these same neurological disorders. Thus, it is important to better understand mechanisms by which calcium is released from endolysosomes as well as the consequences of such release to inter-organellar signaling, physiological functions of neurons, and possible pathological consequences. In doing so, a path forward towards new therapeutic modalities might be facilitated.

Weak base drug-induced endolysosome iron dyshomeostasis controls the generation of reactive oxygen species, mitochondrial depolarization, and cytotoxicity.

Objectives: Approximately 75 % of marketed drugs have the physicochemical property of being weak bases. Weak-base drugs with relatively high pKa values enter acidic organelles including endosomes and lysosomes (endolysosomes), reside in and de-acidify endolysosomes, and induce cytotoxicity. Divalent cations within endolysosomes, including iron, are released upon endolysosome de-acidification. Endolysosomes are "master regulators of iron homeostasis", and neurodegeneration is linked to ferrous iron (Fe2+)-induced reactive oxygen species (ROS) generation via Fenton chemistry. Because endolysosome de-acidification-induced lysosome-stress responses release endolysosome Fe2+, it was crucial to determine the mechanisms by which a functionally and structurally diverse group of weak base drugs including atropine, azithromycin, fluoxetine, metoprolol, and tamoxifen influence endolysosomes and cause cell death. Methods: Using U87MG astrocytoma and SH-SY5Y neuroblastoma cells, we conducted concentration-response relationships for 5 weak-base drugs to determine EC50 values. From these curves, we chose pharmacologically and therapeutically relevant concentrations to determine if weak-base drugs induced lysosome-stress responses by de-acidifying endolysosomes, releasing endolysosome Fe2+ in sufficient levels to increase cytosolic and mitochondria Fe2+ and ROS levels and cell death. Results: Atropine (anticholinergic), azithromycin (antibiotic), fluoxetine (antidepressant), metoprolol (beta-adrenergic), and tamoxifen (anti-estrogen) at pharmacologically and therapeutically relevant concentrations (1) de-acidified endolysosomes, (2) decreased Fe2+ levels in endolysosomes, (3) increased Fe2+ and ROS levels in cytosol and mitochondria, (4) induced mitochondrial membrane potential depolarization, and (5) increased cell death; effects prevented by the endocytosed iron-chelator deferoxamine. Conclusions: Weak-base pharmaceuticals induce lysosome-stress responses that may affect their safety profiles; a better understanding of weak-base drugs on Fe2+ interorganellar signaling may improve pharmacotherapeutics.

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.

Endolysosome Iron Chelation Inhibits HIV-1 Protein-Induced Endolysosome De-Acidification-Induced Increases in Mitochondrial Fragmentation, Mitophagy, and Cell Death.

People with human immunodeficiency virus-1 (PLWH) experience high rates of HIV-1-associated neurocognitive disorders (HANDs); clinical symptoms range from being asymptomatic to experiencing HIV-associated dementia. Antiretroviral therapies have effectively prolonged the life expectancy related to PLWH; however, the prevalence of HANDs has increased. Implicated in the pathogenesis of HANDs are two HIV-1 proteins, transactivator of transcription (Tat) and gp120; both are neurotoxic and damage mitochondria. The thread-like morphological features of functional mitochondria become fragmented when levels of reactive oxygen species (ROS) increase, and ROS can be generated via Fenton-like chemistry in the presence of ferrous iron (Fe2+). Endolysosomes are central to iron trafficking in cells and contain readily releasable Fe2+ stores. However, it is unclear whether the endolysosome store is sufficient to account for insult-induced increases in levels of ROS, mitochondrial fragmentation, autophagy, and cell death. Using U87MG astrocytoma and SH-SY5Y neuroblastoma cells, we determined that chloroquine (CQ), Tat, and gp120 all (1) de-acidified endolysosomes, (2) decreased endolysosome numbers and increased endolysosome sizes, (3) increased mitochondrial numbers (fragmentation), (4) increased autophagosome numbers, (5) increased autolysosome numbers, (6) increased mitochondrial fragments within endolysosomes, and (7) increased cell death. These effects were all blocked by the endolysosome-specific iron chelator deferoxamine (DFO). Thus, the endolysosome de-acidification-induced release of endolysosome Fe2+ is sufficient to account for inter-organellar signaling events and cell biology consequences of HIV-1 proteins, including mitochondrial fragmentation, autophagy, and cell death.

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.

Overcoming Chemoresistance: Altering pH of Cellular Compartments by Chloroquine and Hydroxychloroquine.

Resistance to the anti-cancer effects of chemotherapeutic agents (chemoresistance) is a major issue for people living with cancer and their providers. A diverse set of cellular and inter-organellar signaling changes have been implicated in chemoresistance, but it is still unclear what processes lead to chemoresistance and effective strategies to overcome chemoresistance are lacking. The anti-malaria drugs, chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) are being used for the treatment of various cancers and CQ and HCQ are used in combination with chemotherapeutic drugs to enhance their anti-cancer effects. The widely accepted anti-cancer effect of CQ and HCQ is their ability to inhibit autophagic flux. As diprotic weak bases, CQ and HCQ preferentially accumulate in acidic organelles and neutralize their luminal pH. In addition, CQ and HCQ acidify the cytosolic and extracellular environments; processes implicated in tumorigenesis and cancer. Thus, the anti-cancer effects of CQ and HCQ extend beyond autophagy inhibition. The present review summarizes effects of CQ, HCQ and proton pump inhibitors on pH of various cellular compartments and discuss potential mechanisms underlying their pH-dependent anti-cancer effects. The mechanisms considered here include their ability to de-acidify lysosomes and inhibit autophagosome lysosome fusion, to de-acidify Golgi apparatus and secretory vesicles thus affecting secretion, and to acidify cytoplasm thus disturbing aerobic metabolism. Further, we review the ability of these agents to prevent chemotherapeutic drugs from accumulating in acidic organelles and altering their cytosolic concentrations.

SARS-CoV-2 S1 Protein Induces Endolysosome Dysfunction and Neuritic Dystrophy.

SARS-CoV-2 is the viral cause of the COVID-19 pandemic. Increasingly, significant neurological disorders have been associated with COVID-19. However, the pathogenesis of these neurological disorders remains unclear especially because only low or undetectable levels of SARS-CoV-2 have been reported in human brain specimens. Because SARS-CoV-2 S1 protein can be released from viral membranes, can cross the blood-brain barrier, and is present in brain cells including neurons, we tested the hypothesis that SARS-CoV-2 S1 protein can directly induce neuronal injury. Incubation of primary human cortical neurons with SARS-CoV-2 S1 protein resulted in accumulation of the S1 protein in endolysosomes as well as endolysosome de-acidification. Further, SARS-CoV-2 S1 protein induced aberrant endolysosome morphology and neuritic varicosities. Our findings suggest that SARS-CoV-2 S1 protein directly induces neuritic dystrophy, which could contribute to the high incidence of neurological disorders associated with COVID-19.

BK channels regulate extracellular Tat-mediated HIV-1 LTR transactivation.

HIV-1 Tat is essential for HIV-1 replication and plays an important role in latent HIV-1 infection, HIV-1 associated neurological complication, and other HIV-1 comorbidities. Secreted from HIV-1 infected or transfected cells, Tat can be up-taken into cells by receptor-mediated endocytosis and internalized into endolysosomes. To reach nucleus where it can facilitate HIV-1 viral replication, exogenous Tat has to escape the degradation by endolysosomes. Because of findings that endolysosome de-acidification with, for example, the weak-base anti-malarial drug chloroquine prevents exogenous Tat degradation and enhances the amount of Tat available to activate HIV-1 LTR, we hypothesize that acidifying endolysosomes may enhance Tat degradation in endolysosomes and restrict LTR transactivation. Here, we determined the involvement of endolysosome-resident transient receptor potential mucolipin 1 channel (TRPML1) and the big conductance Ca2+-activated potassium (BK) channel in regulating endolysosome pH, as well as Tat-mediated HIV-1 LTR transactivation in U87MG cells stably integrated with HIV-1 LTR luciferase reporter. Activating TRPML1 channels with ML-SA1 acidified endolysosomes and restricted Tat-mediated HIV-1 LTR transactivation. These effects of ML-SA1 appeared to be mediated through activation of BK channels, because the effects of ML-SA1 on Tat-mediated HIV-1 LTR transactivation were blocked using pharmacological inhibitors or shRNA knock-down of BK channels. On the other hand, activating TRPML1 and BK channels enhanced cellular degradation of exogenous Tat. These results suggest that acidifying endolysosomes by activating TRPML1 or BK channels may provide therapeutic benefit against latent HIV-1 infection, HIV-1 associated neurocognitive disorders, and other HIV-1 comorbidities.