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.

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.

Antiretroviral Drugs Promote Amyloidogenesis by De-Acidifying Endolysosomes.

Antiretroviral therapeutics (ART) have effectively increased the long-term survival of HIV-1 infected individuals. However, the prevalence of HIV-1 associated neurocognitive disorders (HAND) has increased and so too have clinical manifestations and pathological features of Alzheimer's disease (AD) in people living with HIV-1/AIDS. Although underlying mechanisms are not clear, chronic exposure to ART drugs has been implicated in the development of AD-like symptoms and pathology. ART drugs are categorized according to their mechanism of action in controlling HIV-1 levels. All ART drugs are organic compounds that can be classified as being either weak acids or weak bases, and these physicochemical properties may be of central importance to ART drug-induced AD-like pathology because weak bases accumulate in endolysosomes, weak bases can de-acidify endolysosomes where amyloidogenesis occurs, and endolysosome de-acidification increases amyloid beta (Aß) protein production and decreases Aß degradation. Here, we investigated the effects of ART drugs on endolysosome pH and Aß levels in rat primary cultured neurons. ART drugs that de-acidified endolysosomes increased Aß levels, whereas those that acidified endolysosomes decreased Aß levels. Acidification of endolysosomes with the mucolipin transient receptor potential (TRPML) channel agonist ML-SA1 blocked ART drug-induced increases in Aß levels. Further, ART drug-induced endolysosome de-acidification increased endolysosome sizes; effects that were blocked by ML-SA1-induced endolysosome acidification. These results suggest that ART drug-induced endolysosome de-acidification plays an important role in ART drug-induced amyloidogenesis and that endolysosome acidification might attenuate AD-like pathology in HIV-1 positive people taking ART drugs that de-acidify endolysosomes. Graphical Abstract.

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.

Bioenergetic adaptations to HIV infection. Could modulation of energy substrate utilization improve brain health in people living with HIV-1?

The human brain consumes more energy than any other organ in the body and it relies on an uninterrupted supply of energy in the form of adenosine triphosphate (ATP) to maintain normal cognitive function. This constant supply of energy is made available through an interdependent system of metabolic pathways in neurons, glia and endothelial cells that each have specialized roles in the delivery and metabolism of multiple energetic substrates. Perturbations in brain energy metabolism is associated with a number of different neurodegenerative conditions including impairments in cognition associated with infection by the Human Immunodeficiency Type 1 Virus (HIV-1). Adaptive changes in brain energy metabolism are apparent early following infection, do not fully normalize with the initiation of antiretroviral therapy (ART), and often worsen with length of infection and duration of anti-retroviral therapeutic use. There is now a considerable amount of cumulative evidence that suggests mild forms of cognitive impairments in people living with HIV-1 (PLWH) may be reversible and are associated with specific modifications in brain energy metabolism. In this review we discuss brain energy metabolism with an emphasis on adaptations that occur in response to HIV-1 infection. The potential for interventions that target brain energy metabolism to preserve or restore cognition in PLWH are also discussed.

Role of endolysosomes and inter-organellar signaling in brain disease.

Endosomes and lysosomes (endolysosomes) are membrane bounded organelles that play a key role in cell survival and cell death. These acidic intracellular organelles are the principal sites for intracellular hydrolytic activity required for the maintenance of cellular homeostasis. Endolysosomes are involved in the degradation of plasma membrane components, extracellular macromolecules as well as intracellular macromolecules and cellular fragments. Understanding the physiological significance and pathological relevance of endolysosomes is now complicated by relatively recent findings of physical and functional interactions between endolysosomes with other intracellular organelles including endoplasmic reticulum, mitochondria, plasma membranes, and peroxisomes. Indeed, evidence clearly indicates that endolysosome dysfunction and inter-organellar signaling occurs in different neurodegenerative diseases including Alzheimer's disease (AD), HIV-1 associated neurocognitive disease (HAND), Parkinson's disease (PD) as well as various forms of brain cancer such as glioblastoma multiforme (GBM). These findings open new areas of cell biology research focusing on understanding the physiological actions and pathophysiological consequences of inter-organellar communication. Here, we will review findings of others and us that endolysosome de-acidification and dysfunction coupled with impaired inter-organellar signaling is involved in the pathogenesis of AD, HAND, PD, and GBM. A more comprehensive appreciation of cell biology and inter-organellar signaling could lead to the development of new drugs to prevent or cure these diseases.

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.

HIV-1 gp120 Promotes Lysosomal Exocytosis in Human Schwann Cells.

Human immunodeficiency virus type 1 (HIV-1) associated neuropathy is the most common neurological complication of HIV-1, with debilitating pain affecting the quality of life. HIV-1 gp120 plays an important role in the pathogenesis of HIV neuropathy via direct neurotoxic effects or indirect pro-inflammatory responses. Studies have shown that gp120-induced release of mediators from Schwann cells induce CCR5-dependent DRG neurotoxicity, however, CCR5 antagonists failed to improve pain in HIV- infected individuals. Thus, there is an urgent need for a better understanding of neuropathic pain pathogenesis and developing effective therapeutic strategies. Because lysosomal exocytosis in Schwann cells is an indispensable process for regulating myelination and demyelination, we determined the extent to which gp120 affected lysosomal exocytosis in human Schwann cells. We demonstrated that gp120 promoted the movement of lysosomes toward plasma membranes, induced lysosomal exocytosis, and increased the release of ATP into the extracellular media. Mechanistically, we demonstrated lysosome de-acidification, and activation of P2X4 and VNUT to underlie gp120-induced lysosome exocytosis. Functionally, we demonstrated that gp120-induced lysosome exocytosis and release of ATP from Schwann cells leads to increases in intracellular calcium and generation of cytosolic reactive oxygen species in DRG neurons. Our results suggest that gp120-induced lysosome exocytosis and release of ATP from Schwann cells and DRG neurons contribute to the pathogenesis of HIV-1 associated neuropathy.

Acidifying Endolysosomes Prevented Low-Density Lipoprotein-Induced Amyloidogenesis.

Cholesterol dyshomeostasis has been linked to the pathogenesis of sporadic Alzheimer's disease (AD). In furthering the understanding of mechanisms by which increased levels of circulating cholesterol augments the risk of developing sporadic AD, others and we have reported that low-density lipoprotein (LDL) enters brain parenchyma by disrupting the blood-brain barrier and that endolysosome de-acidification plays a role in LDL-induced amyloidogenesis in neurons. Here, we tested the hypothesis that endolysosome de-acidification was central to amyloid-ß (Aß) generation and that acidifying endolysosomes protects against LDL-induced increases in Aß levels in neurons. We demonstrated that LDL, but not HDL, de-acidified endolysosomes and increased intraneuronal and secreted levels of Aß. ML-SA1, an agonist of endolysosome-resident TRPML1 channels, acidified endolysosomes, and TRPML1 knockdown attenuated ML-SA1-induced endolysosome acidification. ML-SA1 blocked LDL-induced increases in intraneuronal and secreted levels of Aß as well as Aß accumulation in endolysosomes, prevented BACE1 accumulation in endolysosomes, and decreased BACE1 activity levels. LDL downregulated TRPML1 protein levels, and TRPML1 knockdown worsens LDL-induced increases in Aß. Our findings suggest that endolysosome acidification by activating TRPML1 may represent a protective strategy against sporadic AD.

Ten years of PCV2 vaccines and vaccination: Is eradication a possibility?

More than two decades after its emergence, porcine circovirus type 2 (PCV2) remains an economically important swine pathogen. Commercial vaccines which were first introduced to the U.S in 2006, have been highly effective in reducing clinical signs and improving production. Recent studies have indicated a declining level of PCV2 prevalence and viremia in the field. However, reports on the emergence of new viral variants have also continued to increase. This article reviews topics of current interest in the field of PCV2 vaccines; including the comparative efficacy of the available commercial products, efficacy of current vaccines against new and emerging strains, findings on the differences between immunity in natural infection versus vaccination, limitations of current experimental models for PCV2 vaccine studies, and new developments in novel experimental vaccines. The discussion is framed in the context of attempts for the possible eradication of PCV2 in the future.