Endolysosome Dysfunction And Inter-Organellar Signalling: An \u27Iron-Ic\u27 Story

Endosomes and lysosomes (endolysosomes) are acidic organelles that are important both physiologically and pathologically. Implicated in the physiological and pathophysiological processes regulated by endolysosomes are readily releasable stores of cations including ferrous iron (Fe2+); an essential cofactor for the generation of reactive oxygen species (ROS). In determining the extent to which and mechanisms by which Fe2+ released from endolysosomes affects cellular functions it was important to determine levels of Fe2+ in endolysosomes. In some cells and by other researchers, FeRhoNox-1 was found to detect Fe2+ in acidic organelles known as Golgi. Here, using U87MG astrocytoma cells and primary cultures of rat neurons we report that FeRhoNox-1 is highly specific for Fe2+, that FeRhoNox-1 positive stores are largely localized in endolysosomes and not in Golgi, that control levels of Fe2+ were 36.3 ± 13.6 µM in endolysosomes, and that the stores of Fe2+ in endolysosomes increased to 75 ± 15.7 µM when cells were incubated with ferric ammonium citrate and decreased to 0.08 ± 0.05 µM when cells were incubated with the iron chelator deferoxamine. Furthermore, subpopulations of endolysosomes exist with extensive variability in Fe2+ content. Our findings demonstrate the utility of using FeRhoNox-1 to measure Fe2+ stores in endolysosomes and suggest that this probe will find important uses in better understanding cellular events downstream of released endolysosome Fe2+. Mitochondria are subject to iron overload under a variety of conditions and disease states, but it is not clear what are the subcellular origins of this iron nor its consequences. Endolysosomes are storage sites of ferrous iron (Fe2+), and the degree to which and the precise mechanisms by which endolysosome Fe2+ contribute to iron-dependent changes to mitochondria and cell injury remains uncertain. Here, our studies were aimed to determine the role of inter-organellar signaling of Fe2+ from iron-rich endolysosomes to mitochondria under pharmacologically-induced conditions. We demonstrated, in U87MG astrocytoma cells, mouse primary hepatocytes, and primary cultures of rat cortical neurons, that Fe2+ within endolysosomes was translocated to the mitochondria resulting in mitochondrial dysfunction and cell death. The weak-base chloroquine and the vacuolar-ATPase inhibitor bafilomycin A1 both de-acidify endolysosomes and both induced the release of Fe2+ and this resulted in increased concentrations of Fe2+ in the cytoplasm and in mitochondria. Furthermore, the endocytosed iron chelator, deferoxamine, inhibited the release of bafilomycin A1- and chloroquine-induced release of endolysosome stores of Fe2+ and prevented the induced increases of ROS in cytoplasm and mitochondria. These findings demonstrate that redox-active Fe2+ in endolysosomes plays a key upstream role in mitochondrial iron accumulation and dysfunction, and deferoxamine might be potential adjunctive therapeutic strategies in preventing neurotoxicity and enhancing therapeutic outcomes of disease. Drugs of abuse including the opioid morphine increase levels of reactive oxygen species (ROS) and predispose cells to insult-induced cell death. However, it remains uncertain as to the underlying mechanisms. Iron has long been known to be required for the generation of mitochondrial ROS and endolysosomes are major storage sites of ferrous iron (Fe2+). Yet, the degree to which and the precise mechanisms by which endolysosome iron plays a role in mitochondrial dysfunction remains uncertain. Here, our studies were aimed to determine the effects of morphine on inter-organellar signaling of Fe2+ from iron-rich endolysosomes to mitochondria. We demonstrated, in U87MG astrocytoma cells, that endolysosome Fe2+ is translocated to mitochondria and results in mitochondrial dysfunction. Morphine de-acidification of endolysosomes caused the release of Fe2+ from endolysosomes and increased levels of Fe2+ in cytosol and in. The morphine-induced effects on endolysosome Fe2+ appeared to be regulated through mu opioid receptors because naloxone blocked the de-acidification of endolysosomes by morphine and the release of endolysosome iron. Furthermore, the endocytosed iron chelator, deferoxamine, inhibited the release of redox-active Fe2+ into the cytosol and the morphine-induced increases in mitochondrial ROS. These findings demonstrate that redox-active Fe2+ in endolysosomes plays a key upstream role in mitochondrial dysfunction, and deferoxamine might be a potentially useful therapeutic strategy associated with opioid use disorders. HIV-associated neurocognitive disorder (HAND) affects 50% of people living with HIV-1 despite viral suppression achieved by antiretroviral therapies. Pathologically, brain tissue from HAND patients has shown morphological changes to intracellular organelles including endolysosomes and mitochondria. Moreover, people living with HIV-1 show elevated iron serum levels and iron chelators have been suggested as an adjuvant therapy to antiretroviral therapeutics. Mechanistically, soluble factors including the HIV-1 coat protein gp120 have been implicated in HAND pathogenesis. Here, we tested the hypothesis that HIV-1 gp120-induced de-acidification of endolysosomes leads to an efflux of iron from endolysosomes and a subsequent increase in levels of cytosolic and mitochondrial reactive oxygen species (ROS). We used U87MG glioblastoma cells and time-lapse confocal microscopy to measure gp120-induced changes in endolysosome pH, endolysosome iron, cytosolic and mitochondrial iron, and ROS levels. 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 iron chelator deferoxamine that only enters cells via endocytosis. These results suggest that cellular and subcellular effects of HIV-1 gp120 can be downstream of its ability to de-acidify endolysosomes and increase the release of iron from endolysosomes. Thus, endolysosomes might represent an early and upstream target for therapeutic strategies against HAND

Imagining the highway:Anticipating infrastructural and environmental change in Belize

This article examines the social and political, as well physical, construction of infrastructure, by attending to the implications of a highway yet to be built. In southern Belize, where the development of rural road networks figures strongly in historical narratives of political and environmental change, the recent paving of a major domestic highway has had distinctive implications for livelihoods and land rights among the predominantly Maya population of rural Toledo district. At the time of research, a plan for a new paved highway to the Guatemalan border animated longstanding debates over territoriality, environment and development, even as the details remained elusive. Bringing political ecology into conversation with attention to the perception of sensory environments, and the affective power of anticipation, I argue for extending anthropological conversations about infrastructure to encompass the meanings and consequences of imagined infrastructures for the ways people encounter, experience and enact social and environmental change

Bedform migration in a mixed sand and cohesive clay intertidal environment and implications for bed material transport predictions

Many coastal and estuarine environments are dominated by mixtures of non-cohesive sand and cohesive mud. The migration rate of bedforms, such as ripples and dunes, in these environments is important in determining bed material transport rates to inform and assess numerical models of sediment transport and geomorphology. However, these models tend to ignore parameters describing the physical and biological cohesion (resulting from clay and extracellular polymeric substances, EPS) in natural mixed sediment, largely because of a scarcity of relevant laboratory and field data. To address this gap in knowledge, data were collected on intertidal flats over a spring-neap cycle to determine the bed material transport rates of bedforms in biologically-active mixed sand-mud. Bed cohesive composition changed from below 2 vol% up to 5.4 vol% cohesive clay, as the tide progressed from spring towards neap. The amount of EPS in the bed sediment was found to vary linearly with the clay content. Using multiple linear regression, the transport rate was found to depend on the Shields stress parameter and the bed cohesive clay content. The transport rates decreased with increasing cohesive clay and EPS content, when these contents were below 2.8 vol% and 0.05 wt%, respectively. Above these limits, bedform migration and bed material transport was not detectable by the instruments in the study area. These limits are consistent with recently conducted sand-clay and sand-EPS laboratory experiments on bedform development. This work has important implications for the circumstances under which existing sand-only bedform migration transport formulae may be applied in a mixed sand-clay environment, particularly as 2.8 vol% cohesive clay is well within the commonly adopted definition of “clean sand”

The dominant Anopheles vectors of human malaria in Africa, Europe and the Middle East: occurrence data, distribution maps and bionomic précis

<p>Abstract</p> <p>Background</p> <p>This is the second in a series of three articles documenting the geographical distribution of 41 dominant vector species (DVS) of human malaria. The first paper addressed the DVS of the Americas and the third will consider those of the Asian Pacific Region. Here, the DVS of Africa, Europe and the Middle East are discussed. The continent of Africa experiences the bulk of the global malaria burden due in part to the presence of the <it>An. gambiae </it>complex. <it>Anopheles gambiae </it>is one of four DVS within the <it>An. gambiae </it>complex, the others being <it>An. arabiensis </it>and the coastal <it>An. merus </it>and <it>An. melas</it>. There are a further three, highly anthropophilic DVS in Africa, <it>An. funestus</it>, <it>An. moucheti </it>and <it>An. nili</it>. Conversely, across Europe and the Middle East, malaria transmission is low and frequently absent, despite the presence of six DVS. To help control malaria in Africa and the Middle East, or to identify the risk of its re-emergence in Europe, the contemporary distribution and bionomics of the relevant DVS are needed.</p> <p>Results</p> <p>A contemporary database of occurrence data, compiled from the formal literature and other relevant resources, resulted in the collation of information for seven DVS from 44 countries in Africa containing 4234 geo-referenced, independent sites. In Europe and the Middle East, six DVS were identified from 2784 geo-referenced sites across 49 countries. These occurrence data were combined with expert opinion ranges and a suite of environmental and climatic variables of relevance to anopheline ecology to produce predictive distribution maps using the Boosted Regression Tree (BRT) method.</p> <p>Conclusions</p> <p>The predicted geographic extent for the following DVS (or species/suspected species complex*) is provided for Africa: <it>Anopheles </it>(<it>Cellia</it>) <it>arabiensis</it>, <it>An. </it>(<it>Cel.</it>) <it>funestus*</it>, <it>An. </it>(<it>Cel.</it>) <it>gambiae</it>, <it>An. </it>(<it>Cel.</it>) <it>melas</it>, <it>An. </it>(<it>Cel.</it>) <it>merus</it>, <it>An. </it>(<it>Cel.</it>) <it>moucheti </it>and <it>An. </it>(<it>Cel.</it>) <it>nili*</it>, and in the European and Middle Eastern Region: <it>An. </it>(<it>Anopheles</it>) <it>atroparvus</it>, <it>An. </it>(<it>Ano.</it>) <it>labranchiae</it>, <it>An. </it>(<it>Ano.</it>) <it>messeae</it>, <it>An. </it>(<it>Ano.</it>) <it>sacharovi</it>, <it>An. </it>(<it>Cel.</it>) <it>sergentii </it>and <it>An. </it>(<it>Cel.</it>) <it>superpictus*</it>. These maps are presented alongside a bionomics summary for each species relevant to its control.</p

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

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

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

Lanthanoid and alkaline earth complexes involving new substituted pyrazolates

From the pyrazoles 3,5-di-(2′-furanyl)pyrazole (fu2pzH), 3-phenyl-5-(2′-thienyl)pyrazole (PhtpzH) and 3-(2′-furanyl)-5-(2′′-naphthyl)pyrazole (funappzH), a range of alkaline earth and lanthanoid pyrazolate complexes has been prepared by redox transmetallation/protolysis reactions between free metals, Hg(C6F5)2 and the pyrazoles, by reaction of I2-activated metals with the pyrazoles, and in one case by a similar reaction of unactivated metal, in the donor solvents tetrahydrofuran (thf) and 1,2-dimethoxyethane (dme). Thus the divalent [Ca(Phtpz)2(thf)4], [Ba(Phtpz)2(thf)4] and [Ca(funappz)2(thf)4]·(thf) complexes, the heteroleptic [Yb(Phtpz)I(thf)4] and the trivalent [La(fu2pz)3(thf)3]·2thf complex have been prepared and structurally characterized, as well as the dme complexes [Yb(Phtpz)2(dme)2] and [Eu(Phtpz)3(dme)2]. Highlights include the first trans-[LnII(pz)I(thf)4] complex, a rare transoid [Ln(pz)2(dme)2] complex and a complex with both chelating and unidentate dme. In all cases, the Phtpz complexes exhibit pronounced positional disorder of the 2-thienyl and phenyl groups in the solid state, as do the two polymorphs of the parent pyrazole