The Brain at High Altitude: From Molecular Signaling to Cognitive Performance.

The brain requires over one-fifth of the total body oxygen demand for normal functioning. At high altitude (HA), the lower atmospheric oxygen pressure inevitably challenges the brain, affecting voluntary spatial attention, cognitive processing, and attention speed after short-term, long-term, or lifespan exposure. Molecular responses to HA are controlled mainly by hypoxia-inducible factors. This review aims to summarize the cellular, metabolic, and functional alterations in the brain at HA with a focus on the role of hypoxia-inducible factors in controlling the hypoxic ventilatory response, neuronal survival, metabolism, neurogenesis, synaptogenesis, and plasticity.

Iron- and erythropoietin-resistant anemia in a spontaneous breast cancer mouse model.

Anemia of cancer (AoC) with its multifactorial etiology and complex pathology is a poor prognostic indicator for cancer patients. One of the main causes of AoC is cancer-associated inflammation that activates mechanisms, commonly observed in anemia of inflammation, whereby functional iron deficiency and iron-restricted erythropoiesis are induced by increased hepcidin levels in response to raised levels of interleukin-6. So far only a few AoC mouse models have been described, and most of them did not fully recapitulate the interplay of anemia, increased hepcidin levels and functional iron deficiency in human patients. To test if the selection and the complexity of AoC mouse models dictates the pathology or if AoC in mice per se develops independently of iron deficiency, we characterized AoC in Trp53floxWapCre mice that spontaneously develop breast cancer. These mice developed AoC associated with high levels of interleukin-6 and iron deficiency. However, hepcidin levels were not increased and hypoferremia coincided with anemia rather than causing it. Instead, an early shift in the commitment of common myeloid progenitors from the erythroid to the myeloid lineage resulted in increased myelopoiesis and in the excessive production of neutrophils that accumulate in necrotic tumor regions. This process could not be prevented by either iron or erythropoietin treatment. Trp53floxWapCre mice are the first mouse model in which erythropoietin-resistant anemia is described and may serve as a disease model to test therapeutic approaches for a subpopulation of human cancer patients with normal or corrected iron levels who do not respond to erythropoietin.

Pro-Apoptotic and Anti-Invasive Properties Underscore the Tumor-Suppressing Impact of Myoglobin on a Subset of Human Breast Cancer Cells.

The expression of myoglobin (MB), well known as the oxygen storage and transport protein of myocytes, is a novel hallmark of the luminal subtype in breast cancer patients and correlates with better prognosis. The mechanisms by which MB impacts mammary tumorigenesis are hitherto unclear. We aimed to unravel this role by using CRISPR/Cas9 technology to generate MB-deficient clones of MCF7 and SKBR3 breast cancer cell lines and subsequently characterize them by transcriptomics plus molecular and functional analyses. As main findings, loss of MB at normoxia upregulated the expression of cell cyclins and increased cell survival, while it prevented apoptosis in MCF7 cells. Additionally, MB-deficient cells were less sensitive to doxorubicin but not ionizing radiation. Under hypoxia, the loss of MB enhanced the partial epithelial to mesenchymal transition, thus, augmenting the migratory and invasive behavior of cells. Notably, in human invasive mammary ductal carcinoma tissues, MB and apoptotic marker levels were positively correlated. In addition, MB protein expression in invasive ductal carcinomas was associated with a positive prognostic value, independent of the known tumor suppressor p53. In conclusion, we provide multiple lines of evidence that endogenous MB in cancer cells by itself exerts novel tumor-suppressive roles through which it can reduce cancer malignancy.

Novel antibodies directed against the human erythropoietin receptor: creating a basis for clinical implementation.

Recombinant human erythropoietin (rHuEPO) is an effective treatment for anaemia but concerns that it causes disease progression in cancer patients by activation of EPO receptors (EPOR) in tumour tissue have been controversial and have restricted its clinical use. Initial clinical studies were flawed because they used polyclonal antibodies, later shown to lack specificity for EPOR. Moreover, multiple isoforms of EPOR caused by differential splicing have been reported in cancer cell lines at the mRNA level but investigations of these variants and their potential impact on tumour progression, have been hampered by lack of suitable antibodies. The EpoCan consortium seeks to promote improved pathological testing of EPOR, leading to safer clinical use of rHuEPO, by producing well characterized EPOR antibodies. Using novel genetic and traditional peptide immunization protocols, we have produced mouse and rat monoclonal antibodies, and show that several of these specifically recognize EPOR by Western blot, immunoprecipitation, immunofluorescence, flow cytometry and immunohistochemistry in cell lines and clinical material. Widespread availability of these antibodies should enable the research community to gain a better understanding of the role of EPOR in cancer, and eventually to distinguish patients who can be treated safely by rHuEPO from those at increased risk from treatment.

A Comprehensive Insight and In Silico Analysis of CircRNAs in Hepatocellular Carcinoma: A Step toward ncRNA-Based Precision Medicine.

Circular RNAs (circRNAs) are cardinal players in numerous physiological and pathological processes. CircRNAs play dual roles as tumor suppressors and oncogenes in different oncological contexts, including hepatocellular carcinoma (HCC). Their roles significantly impact the disease at all stages, including initiation, development, progression, invasion, and metastasis, in addition to the response to treatment. In this review, we discuss the biogenesis and regulatory functional roles of circRNAs, as well as circRNA-protein-mRNA ternary complex formation, elucidating the intricate pathways tuned by circRNAs to modulate gene expression and cellular processes through a comprehensive literature search, in silico search, and bioinformatics analysis. With a particular focus on the interplay between circRNAs, epigenetics, and HCC pathology, the article sets the stage for further exploration of circRNAs as novel investigational theranostic agents in the dynamic realm of HCC.

Association of serum hepcidin with prostate-specific antigen levels in men from high Andean cities of Peru.

Objective: The prostate-specific antigen (PSA) is the primary biomarker to diagnose prostate cancer. Hepcidin has been reported as an alternative for this diagnosis; however, it is unclear how PSA and hepcidin function at high altitude (HA). This study aims to assess the association between hepcidin with PSA in HA residents chronically exposed to hypobaric hypoxia. Methods: We retrospectively examined data of 70 healthy males (aged 18-65-years-old) from four different altitudes cities in Peru: Lima (<150 m), Huancayo (2380 m), Puno (3800 m), and Cerro de Pasco (4320 m). Serum hepcidin, testosterone, and PSA were analyzed by chemiluminescence immunoassay. HA parameters (hemoglobin [Hb], pulse oxygen saturation [SpO2], and chronic mountain sickness [CMS] score) were also included in the study. Bivariate analyses and a multivariate linear mixed model were used to evaluate the association between hepcidin and PSA, adjusted by HA parameters, age, and body mass index (BMI). Results: Cases of excessive erythrocytosis (EE) (Hb >21 g/dL) were observed in the three highest cities. Hepcidin was positively correlated with Hb, CMS score, and BMI (P ≤ 0.05). Hepcidin was higher in Huancayo with respect to Puno, while PSA was lower in Cerro de Pasco in regard to Puno and Lima (P ≤ 0.05). Neither hepcidin nor PSA was increased by altitude in each city (P > 0.05). We did not find an association between hepcidin and PSA, even adjusted by age, BMI, Hb, and SpO2 (P ≤ 0.05). Conclusion: These findings showed no association between hepcidin and PSA levels in healthy residents at HA.

Myoglobin in Brown Adipose Tissue: A Multifaceted Player in Thermogenesis.

Brown adipose tissue (BAT) plays an important role in energy homeostasis by generating heat from chemical energy via uncoupled oxidative phosphorylation. Besides its high mitochondrial content and its exclusive expression of the uncoupling protein 1, another key feature of BAT is the high expression of myoglobin (MB), a heme-containing protein that typically binds oxygen, thereby facilitating the diffusion of the gas from cell membranes to mitochondria of muscle cells. In addition, MB also modulates nitric oxide (NO•) pools and can bind C16 and C18 fatty acids, which indicates a role in lipid metabolism. Recent studies in humans and mice implicated MB present in BAT in the regulation of lipid droplet morphology and fatty acid shuttling and composition, as well as mitochondrial oxidative metabolism. These functions suggest that MB plays an essential role in BAT energy metabolism and thermogenesis. In this review, we will discuss in detail the possible physiological roles played by MB in BAT thermogenesis along with the potential underlying molecular mechanisms and focus on the question of how BAT-MB expression is regulated and, in turn, how this globin regulates mitochondrial, lipid, and NO• metabolism. Finally, we present potential MB-mediated approaches to augment energy metabolism, which ultimately could help tackle different metabolic disorders.

Endogenous myoglobin expression in mouse models of mammary carcinoma reduces hypoxia and metastasis in PyMT mice.

Myoglobin (MB) is expressed in different cancer types and may act as a tumor suppressor in breast cancer. The mechanisms by which basal MB expression level impacts murine mammary tumorigenesis are unclear. We investigated how MB expression in breast cancer influences proliferation, metastasis, tumor hypoxia, and chemotherapy treatment in vivo. We crossed PyMT and WapCreTrp53flox mammary cancer mouse models that differed in tumor grade/type and onset of mammary carcinoma with MB knockout mice. The loss of MB in WapCre;Trp53flox mice did not affect tumor development and progression. On the other hand, loss of MB decreased tumor growth and increased tissue hypoxia as well as the number of lung metastases in PyMT mice. Furthermore, Doxorubicin therapy prevented the stronger metastatic propensity of MB-deficient tumors in PyMT mice. This suggests that, although MB expression predicts improved prognosis in breast cancer patients, MB-deficient tumors may still respond well to first-line therapies. We propose that determining the expression level of MB in malignant breast cancer biopsies will improve tumor stratification, outcome prediction, and personalized therapy in cancer patients.

Voluntary exercise does not always suppress lung cancer progression.

Physical exercise can lower lung cancer incidence. However, its effect on lung cancer progression is less understood. Studies on exercising mice have shown decreased ectopic lung cancer growth through the secretion of interleukin-6 from muscles and the recruitment of natural killer (NK) cells to tumors. We asked if exercise suppresses lung cancer in an orthotopic model also. Single-housed C57Bl/6 male mice in cages with running wheels were tail vein-injected with LLC1.1 lung cancer cells, and lung tumor nodules were analyzed. Exercise did not affect lung cancer. Therefore, we also tested the effect of exercise on a subcutaneous LLC1 tumor and a tail vein-injected B16F10 melanoma model. Except for one case of excessive exercise, tumor progression was not influenced. Moderately exercising mice did not increase IL-6 or recruit NK cells to the tumor. Our data suggest that the exercise dose may dictate how efficiently the immune system is stimulated and controls tumor progression.

Erythropoietin receptor regulates tumor mitochondrial biogenesis through iNOS and pAKT.

Erythropoietin receptor (EPOR) is widely expressed in healthy and malignant tissues. In certain malignancies, EPOR stimulates tumor growth. In healthy tissues, EPOR controls processes other than erythropoiesis, including mitochondrial metabolism. We hypothesized that EPOR also controls the mitochondrial metabolism in cancer cells. To test this hypothesis, we generated EPOR-knockdown cancer cells to grow tumor xenografts in mice and analyzed tumor cellular respiration via high-resolution respirometry. Furthermore, we analyzed cellular respiratory control, mitochondrial content, and regulators of mitochondrial biogenesis in vivo and in vitro in different cancer cell lines. Our results show that EPOR controls tumor growth and mitochondrial biogenesis in tumors by controlling the levels of both, pAKT and inducible NO synthase (iNOS). Furthermore, we observed that the expression of EPOR is associated with the expression of the mitochondrial marker VDAC1 in tissue arrays of lung cancer patients, suggesting that EPOR indeed helps to regulate mitochondrial biogenesis in tumors of cancer patients. Thus, our data imply that EPOR not only stimulates tumor growth but also regulates tumor metabolism and is a target for direct intervention against progression.

In conditions of limited chromophore supply rods entrap 11-cis-retinal leading to loss of cone function and cell death.

RPE65 is a retinoid isomerase required for the production of 11-cis-retinal, the chromophore of both cone and rod visual pigments. We recently established an R91W knock-in mouse strain as homologous animal model for patients afflicted by this mutation in RPE65. These mice have impaired vision and can only synthesize minute amounts of 11-cis-retinal. Here, we investigated the consequences of this chromophore insufficiency on cone function and pathophysiology. We found that the R91W mutation caused cone opsin mislocalization and progressive geographic cone atrophy. Remnant visual function was mostly mediated by rods. Ablation of rod opsin corrected the localization of cone opsin and improved cone retinal function. Thus, our analyses indicate that under conditions of limited chromophore supply rods and cones compete for 11-cis-retinal that derives from regeneration pathway(s) which are reliant on RPE65. Due to their higher number and the instability of cone opsin, rods are privileged under this condition while cones suffer chromophore deficiency and degenerate. These findings reinforce the notion that in patients any effective gene therapy with RPE65 needs to target the cone-rich macula directly to locally restore the cones' chromophore supply outside the reach of rods.

Erythropoietin promotes hippocampal mitochondrial function and enhances cognition in mice.

Erythropoietin (EPO) improves neuronal mitochondrial function and cognition in adults after brain injury and in those afflicted by psychiatric disorders. However, the influence of EPO on mitochondria and cognition during development remains unexplored. We previously observed that EPO stimulates hippocampal-specific neuronal maturation and synaptogenesis early in postnatal development in mice. Here we show that EPO promotes mitochondrial respiration in developing postnatal hippocampus by increasing mitochondrial content and enhancing cellular respiratory potential. Ultrastructurally, mitochondria profiles and total vesicle content were greater in presynaptic axon terminals, suggesting that EPO enhances oxidative metabolism and synaptic transmission capabilities. Behavioural tests of hippocampus-dependent memory at early adulthood, showed that EPO improves spatial and short-term memory. Collectively, we identify a role for EPO in the murine postnatal hippocampus by promoting mitochondrial function throughout early postnatal development, which corresponds to enhanced cognition by early adulthood.

Myoglobin, expressed in brown adipose tissue of mice, regulates the content and activity of mitochondria and lipid droplets.

The identification of novel physiological regulators that stimulate energy expenditure through brown adipose tissue (BAT) activity in substrate catalysis is of utmost importance to understand and treat metabolic diseases. Myoglobin (MB), known to store or transport oxygen in heart and skeletal muscles, has recently been found to bind fatty acids with physiological constants in its oxygenated form (i.e., MBO2). Here, we investigated the in vivo effect of MB expression on BAT activity. In particular, we studied mitochondrial function and lipid metabolism as essential determinants of energy expenditure in this tissue. We show in a MB-null (MBko) mouse model that MB expression in BAT impacts on the activity of brown adipocytes in a twofold manner: i) by elevating mitochondrial density plus maximal respiration capacity, and through that, by stimulating BAT oxidative metabolism along with the organelles` uncoupled respiration; and ii) by influencing the free fatty acids pool towards a palmitate-enriched composition and shifting the lipid droplet (LD) equilibrium towards higher counts of smaller droplets. These metabolic changes were accompanied by the up-regulated expression of thermogenesis markers UCP1, CIDEA, CIDEC, PGC1-α and PPAR-α in the BAT of MB wildtype (MBwt) mice. Along with the emergence of the "browning" BAT morphology, MBwt mice exhibited a leaner phenotype when compared to MBko littermates at 20 weeks of age. Our data shed novel insights into MB's role in linking oxygen and lipid-based thermogenic metabolism. The findings suggest potential new strategies of targeting the MB pathway to treat metabolic disorders related to diminishing energy expenditure.

LIF-dependent JAK3 activation is not essential for retinal degeneration.

Retinal degeneration causes the induction of a leukemia inhibitory factor (LIF)-controlled survival pathway which includes Janus kinase/signal transducer and activator of transcription signaling. Lack of LIF prevents activation of this signaling cascade and accelerates disease progression leading to a fast loss of photoreceptor cells. In this study, we show that expression of Janus kinase 3 (Jak3), but not of the other members of the family of Janus kinases, is induced in four different models of retinal degeneration and that LIF is essential and sufficient to activate Jak3 gene expression. We also show that the induction of Jak3 and Lif may not depend directly on cell death but rather on the retinal stress during photoreceptor degeneration. However, despite its dependence on LIF, JAK3 is not essential for LIF-mediated photoreceptor protection or gene expression. Also, absence of JAK3 in knockout mice did not affect immune-related responses in the degenerating retina. JAK3 may therefore play a different, yet unknown, role in the retinal response to photoreceptor injury.

R91W mutation in Rpe65 leads to milder early-onset retinal dystrophy due to the generation of low levels of 11-cis-retinal.

RPE65 is a retinal pigment epithelial protein essential for the regeneration of 11-cis-retinal, the chromophore of cone and rod visual pigments. Mutations in RPE65 lead to a spectrum of retinal dystrophies ranging from Leber's congenital amaurosis to autosomal recessive retinitis pigmentosa. One of the most frequent missense mutations is an amino acid substitution at position 91 (R91W). Affected patients have useful cone vision in the first decade of life, but progressively lose sight during adolescence. We generated R91W knock-in mice to understand the mechanism of retinal degeneration caused by this aberrant Rpe65 variant. We found that in contrast to Rpe65 null mice, low but substantial levels of both RPE65 and 11-cis-retinal were present. Whereas rod function was impaired already in young animals, cone function was less affected. Rhodopsin metabolism and photoreceptor morphology were disturbed, leading to a progressive loss of photoreceptor cells and retinal function. Thus, the consequences of the R91W mutation are clearly distinguishable from an Rpe65 null mutation as evidenced by the production of 11-cis-retinal and rhodopsin as well as by less severe morphological and functional disturbances at early age. Taken together, the pathology in R91W knock-in mice mimics many aspects of the corresponding human blinding disease. Therefore, this mouse mutant provides a valuable animal model to test therapeutic concepts for patients affected by RPE65 missense mutations.

Iron Regulation in Elderly Asian Elephants (Elephas maximus) Chronically Infected With Mycobacterium tuberculosis.

Restriction of nutrients to pathogens (nutritional immunity) is a critical innate immune response mechanism that operates when pathogens such as Mycobacterium tuberculosis have the potential to evade humoral immunity. Tuberculosis is of growing concern for zoological collections worldwide and is well-illustrated by infections of Asian and African elephants, where tuberculosis is difficult to diagnose. Here, we investigated hematological parameters and iron deposition in liver, lung, and spleen of three Asian elephants (Elephas maximus) infected with Mycobacterium tuberculosis. For reference purposes, we analyzed tissue samples from control M. tuberculosis-negative elephants with and without evidence of inflammation and/or chronic disease. Molecular analyses of bacterial lesions of post mortally collected tissues confirmed M. tuberculosis infection in three elephants. DNA sequencing of the bacterial cultures demonstrated a single source of infection, most likely of human origin. In these elephants, we observed moderate microcytic anemia as well as liver (mild), lung (moderate) and spleen (severe) iron accumulation, the latter mainly occurring in macrophages. Macrophage iron sequestration in response to infection and inflammation is caused by inhibition of iron export via hepcidin-dependent and independent mechanisms. The hepatic mRNA levels of the iron-regulating hormone hepcidin were increased in only one control elephant suffering from chronic inflammation without mycobacterial infection. By contrast, all three tuberculosis-infected elephants showed low hepcidin mRNA levels in the liver and low serum hepcidin concentrations. In addition, hepatic ferroportin mRNA expression was high. This suggests that the hepcidin/ferroportin regulatory system aims to counteract iron restriction in splenic macrophages in M. tuberculosis infected elephants to provide iron for erythropoiesis and to limit iron availability for a pathogen that predominantly proliferates in macrophages. Tuberculosis infections appear to have lingered for more than 30 years in the three infected elephants, and decreased iron availability for mycobacterial proliferation may have forced the bacteria into a persistent, non-proliferative state. As a result, therapeutic iron substitution may not have been beneficial in these elephants, as this therapy may have enhanced progression of the infection.

A Single 60.000 IU Dose of Erythropoietin Does Not Improve Short-Term Aerobic Exercise Performance in Healthy Subjects: A Randomized, Double-Blind, Placebo-Controlled Crossover Trial.

Erythropoietin (EPO) boosts exercise performance through increase in oxygen transport capacity following regular administration of EPO but preclinical study results suggest that single high dose of EPO also may improve exercise capacity. Twenty-nine healthy subjects (14 males/15 females; age: 25 ± 3 years) were included in a randomized, double-blind, placebo-controlled crossover study to assess peak work load and cardiopulmonary variables during submaximal and maximal cycling tests following a single dose of 60.000 IU of recombinant erythropoietin (EPO) or placebo (PLA). Submaximal exercise at 40%/60% of peak work load revealed no main effect of EPO on oxygen uptake (27.9 ± 8.7 ml min-1⋅kg-1/ 37.1 ± 13.2 ml min-1⋅kg-1) versus PLA (25.2 ± 3.7 ml min-1⋅kg-1/ 33.1 ± 5.3 ml min-1⋅kg-1) condition (p = 0.447/p = 0.756). During maximal exercise peak work load (PLA: 3.5 ± 0.6 W⋅kg-1 vs. EPO: 3.5 ± 0.6 W kg-1, p = 0.892) and peak oxygen uptake (PLA: 45.1 ± 10.4 ml⋅min-1 kg-1 vs. EPO: 46.1 ± 14.2 ml⋅min-1 kg-1, p = 0.344) reached comparable values in the two treatment conditions. Other cardiopulmonary variables (ventilation, cardiac output, heart rate) also reached similar levels in the two treatment conditions. An interaction effect was found between treatment condition and sex resulting in higher peak oxygen consumption (p = 0.048) and ventilation (p = 0.044) in EPO-treated males. In conclusion, in a carefully conducted study using placebo-controlled design the present data failed to support the hypothesis that a single high dose of EPO has a measurable impact on work capacity in healthy subjects.

Leukemia inhibitory factor extends the lifespan of injured photoreceptors in vivo.

Survival and death of photoreceptors in degenerative diseases of the retina is controlled by a multitude of genes and endogenous factors. Some genes may be involved in the degenerative process itself whereas others may be part of an endogenous defense system. We show in two models of retinal degeneration that photoreceptor death strongly induces expression of leukemia inhibitory factor (LIF) in a subset of Muller glia cells in the inner nuclear layer of the retina. LIF expression is essential to induce an extensive intraretinal signaling system which includes Muller cells and photoreceptors and is characterized by an upregulation of Edn2, STAT3, FGF2 and GFAP. In the absence of LIF, Muller cells remain quiescent, the signaling system is not activated and retinal degeneration is strongly accelerated. Intravitreal application of recombinant LIF induces the full molecular pathway including the activation of Muller cells in wild-type and Lif(-/-) mice. Interruption of the signaling cascade by an Edn2 receptor antagonist increases whereas activation of the receptor decreases photoreceptor cell death. Thus, LIF is essential and sufficient to activate an extensive molecular defense response to photoreceptor injury. Our data establish LIF as a Muller cell derived neuronal survival factor which controls an intrinsic protective mechanism that includes Edn2 signaling to support photoreceptor cell survival and to preserve vision in the injured retina.

Retinal neuroprotection by hypoxic preconditioning is independent of hypoxia-inducible factor-1 alpha expression in photoreceptors.

Hypoxic preconditioning stabilizes hypoxia-inducible factor (HIF) 1 alpha in the retina and protects photoreceptors against light-induced cell death. HIF-1 alpha is one of the major transcription factors responding to low oxygen tension and can differentially regulate a large number of target genes. To analyse whether photoreceptor-specific expression of HIF-1 alpha is essential to protect photoreceptors by hypoxic preconditioning, we knocked down expression of HIF-1 alpha specifically in photoreceptor cells, using the cyclization recombinase (Cre)-lox system. The Cre-mediated knockdown caused a 20-fold reduced expression of Hif-1 alpha in the photoreceptor cell layer. In the total retina, RNA expression was reduced by 65%, and hypoxic preconditioning led to only a small increase in HIF-1 alpha protein levels. Accordingly, HIF-1 target gene expression after hypoxia was significantly diminished. Retinas of Hif-1 alpha knockdown animals did not show any pathological alterations, and tolerated hypoxic exposure in a comparable way to wild-type retinas. Importantly, the strong neuroprotective effect of hypoxic preconditioning against light-induced photoreceptor degeneration persisted in knockdown mice, suggesting that hypoxia-mediated survival of light exposure does not depend on an autocrine action of HIF-1 alpha in photoreceptor cells. Hypoxia-mediated stabilization of HIF-2 alpha and phosphorylation of signal transducer and activator of transcription 3 (STAT 3) were not affected in the retinas of Hif-1 alpha knockdown mice. Thus, these factors are candidates for regulating the resistance of photoreceptors to light damage after hypoxic preconditioning, along with several potentially neuroprotective genes that were similarly induced in hypoxic knockdown and control mice.