Miriam J. Schönenberger

Oxygen (O2) is an essential substrate in cellular metabolism, bioenergetics, and signaling and as such linked to the survival and normal function of all metazoans. Low O2 tension (hypoxia) is a fundamental feature of physiological processes as well as pathophysiological conditions such as cancer and ischemic diseases. Central to the molecular mechanisms underlying O2 homeostasis are the hypoxia-inducible factors-1 and -2 alpha (HIF-1α and EPAS1/HIF-2α) that function as master regulators of the adaptive response to hypoxia. HIF-induced genes promote characteristic tumor behaviors, including angiogenesis and metabolic reprogramming. The aim of this review is to critically explore current knowledge of how HIF-α signaling regulates the abundance and function of major O2-consuming organelles. Abundant evidence suggests key roles for HIF-1α in the regulation of mitochondrial homeostasis. An essential adaptation to sustained hypoxia is repression of mitochondrial respiration and induction of glycolysis. HIF-1α activates several genes that trigger mitophagy and represses regulators of mitochondrial biogenesis. Several lines of evidence point to a strong relationship between hypoxia, the accumulation of misfolded proteins in the endoplasmic reticulum, and activation of the unfolded protein response. Surprisingly, although peroxisomes depend highly on molecular O2 for their function, there has been no evidence linking HIF signaling to peroxisomes. We discuss our recent findings that establish HIF-2α as a negative regulator of peroxisome abundance and suggest a mechanism by which cells attune peroxisomal function with O2 availability. HIF-2α activation augments peroxisome turnover by pexophagy and thereby changes lipid composition reminiscent of peroxisomal disorders. We discuss potential mechanisms by which HIF-2α might trigger pexophagy and place special emphasis on the potential pathological implications of HIF-2α-mediated pexophagy for human health.

Peroxisomes play a central role in lipid metabolism, and their function depends on molecular oxygen. Low oxygen tension or von Hippel-Lindau (Vhl) tumor suppressor loss is known to stabilize hypoxia-inducible factors alpha (Hif-1α and Hif-2α) to mediate adaptive responses, but it remains unknown if peroxisome homeostasis and metabolism are interconnected with Hif-α signaling. By studying liver-specific Vhl, Vhl/Hif1α, and Vhl/Hif2α knockout mice, we demonstrate a regulatory function of Hif-2α signaling on peroxisomes. Hif-2α activation augments peroxisome turnover by selective autophagy (pexophagy) and thereby changes lipid composition reminiscent of peroxisomal disorders. The autophagy receptor Nbr1 localizes to peroxisomes and is likewise degraded by Hif-2α-mediated pexophagy. Furthermore, we demonstrate that peroxisome abundance is reduced in VHL-deficient human clear cell renal cell carcinomas with high HIF-2α levels. These results establish Hif-2α as a negative regulator of peroxisome abundance and metabolism and suggest a mechanism by which cells attune peroxisomal function with oxygen availability.

ABSTRACT. A series of new in vitro systems for the cultivation of bloodstream forms of Trypanosoma (Trypanozoon) brucei brucei, T. (T.) b. rhodesiense , and T. (T.) b. gambiense was developed. The standard system consists of a feeder layer of fibroblast‐like cells derived from embryos of New Zealand White rabbits (REF) or a mountain vole, Microtus montanus (MEF), with HEPES‐buffered Minimum Essential Medium (MEM), with Earle's salts, supplemented with 15% inactivated rabbit serum. These two and other feeder layers were cross‐checked with different sera to test for growth support of bloodstream forms of the three trypanosome subspecies studied. Cultures could be initiated with bloodstream forms from mammalian hosts or from cryopreserved stabilates. Metacyclic forms from infected Glossina m. morsitans could also be used as inoculum; they transformed within 6 h to bloodstream forms. Maintenance of cultures and growth properties are described in detail. Experiments were undertaken to confirm that the cultivated bloodstream forms still possess some of the characteristic features of pleomorphic bloodstream populations. Cultivated bloodstream forms were always infective for mice, and a surface coat could be demonstrated by electron microscopy. They could also be cyclically transmitted through tsetse flies, and the metacyclic forms from these flies could be brought back into culture. In vitro cloning with single bloodstream forms and metacyclic forms could be achieved with high cloning efficiency. The consumption of glucose and the production of pyruvate and lactate were determined.

Long thought to be "junk DNA", in recent years it has become clear that a substantial fraction of intergenic genomic DNA is actually transcribed, forming long noncoding RNA (lncRNA). Like mRNA, lncRNA can also be spliced, capped, and polyadenylated, affecting a multitude of biological processes. While the molecular mechanisms underlying the function of lncRNAs have just begun to be elucidated, the conditional regulation of lncRNAs remains largely unexplored. In genome-wide studies our group and others recently found hypoxic transcriptional induction of a subset of lncRNAs, whereof nuclear-enriched abundant/autosomal transcript 1 (NEAT1) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) appear to be the lncRNAs most ubiquitously and most strongly induced by hypoxia in cultured cells. Hypoxia-inducible factor (HIF)-2 rather than HIF-1 seems to be the preferred transcriptional activator of these lncRNAs. For the first time, we also found strong induction primarily of MALAT1 in organs of mice exposed to inspiratory hypoxia. Most abundant hypoxic levels of MALAT1 lncRNA were found in kidney and testis. In situ hybridization revealed that the hypoxic induction in the kidney was confined to proximal rather than distal tubular epithelial cells. Direct oxygen-dependent regulation of MALAT1 lncRNA was confirmed using isolated primary kidney epithelial cells. In summary, high expression levels and acute, profound hypoxic induction of MALAT1 suggest a hitherto unrecognized role of this lncRNA in renal proximal tubular function.

Oxygen (O2) is an essential substrate in cellular metabolism and signaling and as such is linked to the survival and normal function of metazoans. Central to the molecular mechanisms underlying O2 homeostasis are hypoxia-inducible factors (HIFs), heterodimeric transcription factors composed of O2-regulated α subunits (HIF1A/HIF-1α or EPAS1/HIF-2α), and a constitutively expressed ARNT/HIF-1β subunit, that serve as master regulators of the adaptive response to hypoxia. HIF1A and EPAS1 have both unique and overlapping functions in the regulation of diverse cellular processes, but so far there has been no evidence linking HIF signaling to peroxisomes. In a recent study we identified a unique function of EPAS1 as promoter of pexophagy in hepatocytes. Here we summarize our findings and discuss potential mechanisms by which EPAS1 might trigger pexophagy.

Ketohexokinase (KHK) is the first and rate-limiting enzyme of fructose metabolism. Expression of the two alternatively spliced KHK isoforms, KHK-A and KHK-C, is tissue-specific and KHK-C is predominantly expressed in liver, kidney and intestine and responsible for the fructose-catabolizing function. While KHK isoform choice has been linked to the development of disorders such as obesity, diabetes, cardiovascular disease and cancer, little is known about the regulation of total KHK expression. In the present study, we investigated how hypoxic signaling influences fructose metabolism in the liver. Hypoxia or von Hippel-Lindau (VHL) tumor suppressor loss leads to the stabilization of hypoxia-inducible factors alpha (HIF-1α and HIF-2α) and the activation of their signaling to mediate adaptive responses. By studying liver-specific Vhl, Vhl/Hif1a, and Vhl/Epas1 knockout mice, we found that KHK expression is suppressed by HIF-2α (encoded by Epas1) but not by HIF-1α signaling on mRNA and protein levels. Reduced KHK levels were accompanied by downregulation of aldolase B (ALDOB) in the livers of Vhl and Vhl/Hif1a knockout mice, further indicating inhibited fructose metabolism. HIF-1α and HIF-2α have both overlapping and distinct target genes but are differentially regulated depending on the cell type and physiologic or pathologic conditions. HIF-2α activation augments peroxisome degradation in mammalian cells by pexophagy and thereby changes lipid composition reminiscent of peroxisomal disorders. We further demonstrated that fructose metabolism is negatively regulated by peroxisome-deficiency in a Pex2 knockout Zellweger mouse model, which lacks functional peroxisomes and is characterized by widespread metabolic dysfunction. Repression of fructolytic genes in Pex2 knockout mice appeared to be independent of PPARα signaling and nutritional status. Interestingly, our results demonstrate that both HIF-2α and peroxisome-deficiency result in downregulation of Khk independent of splicing as both isoforms, Khka as well as Khkc, are significantly downregulated. Hence, our study offers new and unexpected insights into the general regulation of KHK, and therefore fructolysis. We revealed a novel regulatory function of HIF-2α, suggesting that HIF-1α and HIF-2α have tissue-specific opposing roles in the regulation of Khk expression, isoform choice and fructolysis. In addition, we discovered a previously unknown function of peroxisomes in the regulation of fructose metabolism.

Bloodstream forms of Trypanosoma brucei brucei, T. b. rhodesiense and T. b. gambiense can be grown continuously in vitro (Hirumi et al., 1977, Science 196: 992-994; Brun et al., 1981, J. Protozool. 28: 470-479). The culture system consists of a mammalian feeder layer of fibroblast-like cells in tissue culture medium containing 20% serum. The standard system, which supports continuous growth of all three Trypanozoon subspecies, is Microtus montanus whole embryo fibroblasts (MEF) in HEPES-buffered Minimum Essential Medium with 20% heat-inactivated rabbit, goat, horse or human serum (the latter only for human serum resistant stocks). The aim of the present study was to compare the ability of different cell lines to support growth of bloodstream forms of T. b. brucei and T. b. rhodesiense. The T. b. brucei stock STIB 247 was isolated in 1971 in the Serengeti National Park from a hartebeest. The T. b. rhodesiense stock STIB 704 was isolated in 1982 in Tanzania from the blood of a male patient. Cell lines were isolated from embryonic tissue of wild animals using the technique described earlier (Brun et al., 1981, J. Protozool. 28: loc. cit.). Lung, kidney and heart muscle were the main organs which were processed. Embryos of eland (Taurotragus oryx), impala (Aepyceros melampus) and Grant's gazelle (Gazella granti), and also an embryo of an East African goat were obtained from farms in Laikipia district, 140200 km north of Nairobi. Most of the isolated cell lines were of the fibroblast cell type, a few were epithelial or a mixture of fibroblast and epithelial cells. The cell line isolated from the impala embryo kidney contained fibroblasts and muscle cells (Table I). The experiments were carried out in 24-well tissue culture plates containing a confluent feeder layer 2 days after subpassage. The medium was Minimum Essential Medium with Earle's salts, 25 mM HEPES buffer, 1 mM L-glutamine, 1% MEM nonessential amino acids (100 x) and 1 g/ liter additional glucose. The medium was supplemented with 15% heat-inactivated serum and 10 ,tg/ml gentamycin. Each well received 1.0 ml medium and 2 x 105 trypanosomes isolated from infected ICR mice. The bloodstream forms were separated from blood cells by differential centrifugation. The cultures were incubated in a humidified incubator at 37 C in 4% CO2 in air. Every 24 hr the trypanosome density was estimated and the medium changed by gently removing about 90% from the top of the well and replacing it by fresh medium. Each feeder layer/ serum combination was run in duplicate and followed for at least 10 days. The results are summarized in Table II. Many feeder layer/serum combinations supported continuous growth of T. b. brucei and T. b. rhodesiense. With all feeder layers, inactivated fetal bovine serum (iFBS) turned out to be unsuitable for the cultivation of bloodstream forms though it was possible to maintain the culture for a few days. Inactivated rabbit serum, human serum and the homologous sera as medium supplements resulted in good or even excellent growth. An exception was the serum from Grant's gazelle which could not support growth of T. b. brucei. For T. b. rhodesiense this serum was even cy-

In the central nervous system (CNS) peroxisomes are present in all cell types, namely neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells. Brain peroxisomes are smaller in size compared to peroxisomes from other tissues and are therefore referred to as microperoxisomes. We have established a purification procedure to isolate highly purified peroxisomes from the central nervous system that are well separated from the endoplasmic reticulum and mitochondria and are free of myelin contamination. The major difficulty in purification of brain peroxisomes compared to peroxisomes from liver or kidney is the presence of large amounts of myelin in the CNS, which results in contamination of the subcellular fractions. Hence, the crucial step of the isolation procedure is the elimination of myelin by the use of a sucrose gradient, since without the elimination of myelin no significant enrichment of purified peroxisomes can be achieved. Another difficulty is that in brain tissue the abundance of peroxisomes decreases significantly during postnatal development. We provide a detailed protocol for the isolation of peroxisomes from mouse central nervous system as well as a protocol for the isolation of peroxisomes from the liver and kidney using a continuous Nycodenz gradient.

Journal Article Cultivation of bloodstream forms of pleomorphic Trypanosoma brucei stocks Get access R. Brun, R. Brun Swiss Tropical Institute, Socinstrasse 57, Basel, Switzerland Search for other works by this author on: Oxford Academic PubMed Google Scholar L. Jenni, L. Jenni Swiss Tropical Institute, Socinstrasse 57, Basel, Switzerland Search for other works by this author on: Oxford Academic PubMed Google Scholar M. Tanner, M. Tanner Swiss Tropical Institute, Socinstrasse 57, Basel, Switzerland Search for other works by this author on: Oxford Academic PubMed Google Scholar M. Schonenberger, M. Schonenberger Swiss Tropical Institute, Socinstrasse 57, Basel, Switzerland Search for other works by this author on: Oxford Academic PubMed Google Scholar K-F. Schell K-F. Schell Swiss Tropical Institute, Socinstrasse 57, Basel, Switzerland Search for other works by this author on: Oxford Academic PubMed Google Scholar Transactions of The Royal Society of Tropical Medicine and Hygiene, Volume 75, Issue 1, 1981, Page 182, https://doi.org/10.1016/0035-9203(81)90064-X Published: 01 January 1981