Papers by Hanane Hadj-Moussa
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology
Journal of Experimental Zoology Part A: Ecological and Integrative Physiology
The freshwater red-eared slider turtle, Trachemys scripta elegans, experiences weeks to months of... more The freshwater red-eared slider turtle, Trachemys scripta elegans, experiences weeks to months of anoxia at the bottom of ice-locked bodies of water in the winter. While this introduces anoxia-reoxygenation cycles similar to the ischemia-reperfusion events that mammals experience, T. s. elegans does not suffer any apparent tissue damage. To survive prolonged anoxia and prevent cellular damage associated with reactive oxygen species, these turtles have developed numerous adaptions, including highly effective antioxidant defenses. Herein, we examined the subcellular localization and protein expression of nuclear factor erythroid-2-related factor 2 (Nrf2), a central transcription factor responsible for modulating cellular antioxidant responses, that was found to be upregulated and localized to the nucleus in anoxic turtles. Additionally, we examined protein levels of glutathione S-transferases (GSTs) and manganese superoxide dismutase (MnSOD) antioxidant enzymes in anoxic liver, kidney, heart, and skeletal muscle tissues. MnSOD levels were significantly higher in heart and muscle during anoxia, and the four GST isozymes (GSTK1, GSTT1, GSTP1, and GSTM3) were elevated in a tissue-specific manner during anoxia and/or aerobic recovery. Together, these results indicate that Nrf2 is likely involved in activating downstream antioxidant genes in response to anoxic stress. These results provide a possible Nrf2-mediated transcriptional mechanism that supports existing findings of enhanced antioxidant defenses that allow T. s. elegans to cope with anoxia-reoxygenation cycles, and subsequent oxidative stress.
Physiological and Biochemical Zoology, 2021
The limitations that hypoxia imparts on mitochondrial oxygen supply are circumvented by the activ... more The limitations that hypoxia imparts on mitochondrial oxygen supply are circumvented by the activation of anaerobic metabolism and prosurvival mechanisms in hypoxia-tolerant animals. To deal with the hypoxia that jumbo squid (Dosidicus gigas) experience in the ocean’s depth, they depress their metabolic rate by up to 52% relative to normoxic conditions. This is coupled with molecular reorganization to facilitate their daily descents into the ocean’s oxygen minimum zone, where they face not only low oxygen levels but also higher pressures and colder frigid waters. Our current study explores the tissue-specific hypoxia responses of three central processes: (1) antioxidant enzymes responsible for defending against oxidative stress, (2) early apoptotic machinery that signals the activation of cell death, and (3) mitogen-activated protein kinases (MAPKs) that act as central regulators of numerous cellular processes. Luminex xMAP technology was used to assess protein levels and phosphorylation states under normoxic and hypoxic conditions in brains, branchial hearts, and mantle muscles. Hypoxic brains were found to activate apoptosis via upregulation of phospho-p38, phospho-p53, activated caspase 8, and activated caspase 9, whereas branchial hearts were the only tissue to show an increase in antioxidant enzyme levels. Hypoxic muscles seemed the least affected by hypoxia. Our results suggest that hypoxic squid do not undergo large dynamic changes in the phosphorylation states of key apoptotic and central MAPK factors, except for brains, suggesting that these mechanisms are involved in squid hypometabolic responses.
Journal of Cellular Physiology, 2020
Naked mole–rats are among the mammalian champions of hypoxia tolerance. They evolved adaptations ... more Naked mole–rats are among the mammalian champions of hypoxia tolerance. They evolved adaptations centered around reducing metabolic rate to overcome the challenges experienced in their underground burrows. In this study, we used next‐generation sequencing to investigate one of the factors likely supporting hypoxia tolerance in naked mole–rat brains, posttranscriptional microRNAs (miRNAs). Of the 212 conserved miRNAs identified using small RNA sequencing, 18 displayed significant differential expression during hypoxia. Bioinformatic enrichment revealed that hypoxia‐mediated miRNAs were suppressing energy expensive processes including de novo protein translation and cellular proliferation. This suppression occurred alongside the activation of neuroprotective and neuroinflammatory pathways, and the induction of central signal transduction pathways including HIF‐1α and NFκB via miR‐335, miR‐101, and miR‐155. MiRNAs also coordinated anaerobic glycolytic fuel sources, where hypoxia‐upregulated miR‐365 likely suppressed protein levels of ketohexokinase, the enzyme responsible for catalyzing the first committed step of fructose catabolism. This was further supported by a hypoxia‐mediated reduction in glucose transporter 5 proteins that import fructose into the cell. Yet, messenger RNA and protein levels of lactate dehydrogenase, which converts pyruvate to lactate in the absence of oxygen, were elevated during hypoxia. Together, this demonstrated the induction of anaerobic glycolysis despite a lack of reliance on fructose as the primary fuel source, suggesting that hypoxic brains are metabolically different than anoxic naked mole–rat brains that were previously found to shift to fructose‐based glycolysis. Our findings contribute to the growing body of oxygen‐responsive miRNAs “OxymiRs“ that facilitate natural miRNA‐mediated mechanisms for successful hypoxic exposures.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2020
Hibernators have evolved effective mechanisms to overcome the challenges of torpor-arousal cyclin... more Hibernators have evolved effective mechanisms to overcome the challenges of torpor-arousal cycling. This study focuses on the antioxidant and inflammatory defenses under the control of the redox-sensitive and inflammatory-centered NFκB transcription factor in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus), a well-established model of mammalian hibernation. While hibernators significantly depress oxygen consumption and overall metabolic rate during torpor, arousal brings with it a rapid increase in respiration that is associated with an influx of reactive oxygen species. As such, hibernators employ a variety of antioxidant defenses to combat oxidative damage. Herein, we used Luminex multiplex technology to examine the expression of key proteins in the NFκB transcriptional network, including NFκB, super-repressor IκBα, upstream activators TNFR1 and FADD, and downstream target c-Myc. Transcription factor DNA-binding ELISAs were also used to measure the relative degree of NFκB binding to DNA during hibernation. Analyses were performed across eight different tissues, cerebral cortex, brainstem, white and brown adipose tissue, heart, liver, kidney, and spleen, during euthermic control and late torpor to highlight tissue-specific NFκB mediated cytoprotective responses against oxidative stress experienced during torpor-arousal. Our findings demonstrated brain-specific NFκB activation during torpor, with elevated levels of upstream activators, inactive-phosphorylated IκBα, active-phosphorylated NFκB, and enhanced NFκB-DNA binding. Protein levels of downstream protein, c-Myc, also increased in the brain and adipose tissues during late torpor. The results show that NFκB regulation might serve a critical neuroprotective and cytoprotective role in hibernating brains and selective peripheral tissue.
Methods in molecular biology, 2021
The critical role microRNAs play in modulating global functions is emerging, both in the maintena... more The critical role microRNAs play in modulating global functions is emerging, both in the maintenance of homeostatic mechanisms and in the adaptation to diverse environmental stresses. When stressed, cells must divert metabolic requirements toward immediate survival and eventual recovery and the unique features of miRNAs, such as their relatively ATP-inexpensive biogenesis costs, and the quick and reversible nature of their action, renders them excellent "master controllers" for rapid responses. Many animal survival strategies for dealing with extreme environmental pressures involve prolonged retreats into states of suspended animation to extend the time that they can survive on their limited internal fuel reserves until conditions improve. The ability to retreat into such hypometabolic states is only possible by coupling the global suppression of nonessential energy-expensive functions with an activation of prosurvival networks, a process in which miRNAs are now known to p...
Proteins: Structure, 2020
The African clawed frog (Xenopus laevis) withstands prolonged periods of extreme whole‐body dehyd... more The African clawed frog (Xenopus laevis) withstands prolonged periods of extreme whole‐body dehydration that lead to impaired blood flow, global hypoxia, and ischemic stress. During dehydration, these frogs shift from oxidative metabolism to a reliance on anaerobic glycolysis. In this study, we purified the central glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) to electrophoretic homogeneity and investigated structural, kinetic, subcellular localization, and post‐translational modification properties between control and 30% dehydrated X. laevis liver. GAPDH from dehydrated liver displayed a 25.4% reduction in maximal velocity and a 55.7% increase in its affinity for GAP, as compared to enzyme from hydrated frogs. Under dehydration mimicking conditions (150 mM urea and 1% PEG), GAP affinity was reduced with a Km value 53.8% higher than controls. Frog dehydration also induced a significant increase in serine phosphorylation, methylation, acetylation, beta‐N‐acetylg...
Methods in molecular biology, 2021
Mature microRNAs (miRNAs) are short RNA sequences about 18-24 nucleotide long, which provide the ... more Mature microRNAs (miRNAs) are short RNA sequences about 18-24 nucleotide long, which provide the recognition key within RISC for the posttranscriptional regulation of target RNAs. Considering the canonical pathway, mature miRNAs are produced via a multistep process. Their transcription (pri-miRNAs) and first processing step via the microprocessor complex (pre-miRNAs) occur in the nucleus. Then they are exported into the cytosol, processed again by Dicer (dsRNA) and finally a single strand (mature miRNA) is incorporated into RISC (miRISC). The sequence of the incorporated miRNA provides the function of RNA target recognition via hybridization. Following binding of the target, the mRNA is either degraded or translation is inhibited, which ultimately leads to less protein production. Conversely, it has been shown that binding within the 5' UTR of the mRNA can lead to an increase in protein product. Regulation of homeostasis is very important for a cell; therefore, all steps in the ...
Journal of Insect Physiology, 2021
The goldenrod gall moth (Epiblema scudderiana) is a cold hardy insect that survives subzero tempe... more The goldenrod gall moth (Epiblema scudderiana) is a cold hardy insect that survives subzero temperatures during the winter by supercooling bodily fluids to approximately -40 °C, allowing the insect to remain unfrozen despite the freezing temperatures. This is characterized by a drastic increase of cryoprotectant glycerol along with widespread downregulation of non-essential genes and processes to conserve cellular energy. This study examined the role of epigenetic enzymes in regulating this freeze-avoidant process across a range of freezing temperatures experienced in nature. Cold and subzero temperature exposure in E. scudderiana resulted in upregulation of select DNA methyltransferase (DNMT) enzymes with concurrent decreases in DNMT activity and no change in activity of the Ten-Eleven Translocation (TET) demethylation enzyme activities. Levels of histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity decreased during cold exposures. The increase in DNMT expression and concurrent decrease in HAT activity suggests a role for DNA methylation to assist with transcriptional suppression. These findings propose that epigenetic regulation of genes and histones underpin the winter survival strategies of this insect.
Cryobiology, 2021
Hypothermia is a valuable clinical tool in mitigating against the consequences of ischemia in sur... more Hypothermia is a valuable clinical tool in mitigating against the consequences of ischemia in surgery, stroke, cardiac arrest and organ preservation. Protection is afforded principally by a reduction of metabolism, manifesting as reduced rates of oxygen uptake, preservation of ATP levels, and a curtailing of ischemic calcium overload. The effects of non-ischemic hypothermic stress are relatively unknown. We sought to investigate the effects of clinically mild-to-severe hypothermia on mitochondrial morphology, oxygen consumption and protein expression in normoxic hearts and cardiac cells. Normoxic perfusion of rat hearts at 28-32°C was associated with inhibition of mitochondrial fission, evidenced by a reduced abundance of the active phosphorylated form of the fission receptor Drp1 (pDrp1S616). Abundance of the same residue was reduced in H9c2 cells subjected to hypothermic culture (25-32°C), in addition to a reduced abundance of the Drp1 receptor MFF. Hypothermia-treated H9c2 cardiomyocytes exhibited elongated mitochondria and depressed rates of mitochondrial-associated oxygen consumption, which persisted upon rewarming. Hypothermia also promoted a reduction in mRNA expression of the capsaicin receptor TRPV1 in H9c2 cells. When normothermic H9c2 cells were transfected with TRPV1 siRNA we observed reduced pDrp1S616 and MFF abundance, elongated mitochondria, and reduced rates of mitochondrial-associated oxygen consumption, mimicking the effects of hypothermic culture. In conclusion hypothermia promoted elongation of cardiac mitochondria via reduced pDrp1S616 abundance which was also associated with suppression of cellular oxygen consumption. Silencing of TRPV1 in H9c2 cardiomyocytes reproduced the morphological and respirometric phenotype of hypothermia. This report demonstrates a novel mechanism of cold-induced inhibition of mitochondrial fission.
The Journal of Experimental Biology, 2020
From squid at the bottom of the ocean to humans at the top of mountains, animals have adapted to ... more From squid at the bottom of the ocean to humans at the top of mountains, animals have adapted to diverse oxygen-limited environments. Surviving these challenging conditions requires global metabolic reorganization that is orchestrated, in part, by microRNAs that can rapidly and reversibly target all biological functions. Herein, we review the involvement of microRNAs in natural models of anoxia and hypoxia tolerance, with a focus on the involvement of oxygen-responsive microRNAs (OxymiRs) in coordinating the metabolic rate depression that allows animals to tolerate reduced oxygen levels. We begin by discussing animals that experience acute or chronic periods of oxygen deprivation at the ocean's oxygen minimum zone and go on to consider more elevated environments, up to mountain plateaus over 3500 m above sea level. We highlight the commonalities and differences between OxymiR responses of over 20 diverse animal species, including invertebrates and vertebrates. This is followed b...
Genomics, 2020
The intertidal marine periwinkle, Littorina littorea, have developed various strategies to deal w... more The intertidal marine periwinkle, Littorina littorea, have developed various strategies to deal with cyclic exposures to anoxic and/or freezing stresses when out of water at low tide. With promising translational research potential, evolutionarily conserved microRNAs (miRNAs) have recently become a focus of animal stress response studies. Using RNA-seq, the current study explores the conserved hepatopancreas miRNAs in facilitating snail stress survival. Overall, stress-specific miRNA responses were overserved. Anoxia led to substantial differential miRNA expression patterns, whereas freezing stress showed a relatively high degree of individual variance in miRNA expression. Pathway analysis identified miRNA-related stress survival adaptations, such as cell proliferation. Additionally, machine learning-based gene selection identified seven hepatopancreas miRNAs critical to distinguish between snails under either stress conditions. Our study demonstrated that conserved miRNAs reflect survival adaptations by marine periwinkles under anoxic or frozen conditions, and thus further establishes these snails as an optimal stress model suited for translational research.
Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cryobiology, 2019
chemoembolization (TACE) combined with cryoablation (TACE-cryoablation) in large (main tumor 5 cm... more chemoembolization (TACE) combined with cryoablation (TACE-cryoablation) in large (main tumor 5 cm in diameter) hepatocellular carcinomas (HCCs). Methods: From January 2010 to December 2015, 56 lesions in 56 patients were treated with combination therapy via a single TACE session followed by one to three percutaneous cryoablation sessions twice a week (TACEcryoablation group). A total of 54 lesions in 54 patients were treated with TACE alone for two to six sessions once a month (TACE group). The decision between TACE and TACE cryoablation was based on patient choice. Outcomes of patients in two groups were compared according to the largest tumor diameter (subgroup): Group A (5 cm tumor <10 cm), Group B (10 cm tumor <15 cm), and Group C (tumor 15 cm). Results: The mean number of cryoablation sessions per patient was 2.3 (range: 1e6). Within Group B, TACE-cryoablation significantly improved survival compared with TACE alone (11.0 vs 6.0 months; p ¼ .008). This was also seen in Group C (8.0 vs 5.0 months; p ¼.001). However, no significant difference was noted in Group A (17.0 vs 13.0 months; p ¼ .674). The complications related to TACE were comparable between the two groups. Two adverse events of grade 3-4 related to cryoablation occurred in two patients (3.6%).
Journal of Experimental Zoology Part A: Ecological and Integrative Physiology, 2019
Naked mole rats are a long-lived animal model that age much like humans, but that can also withst... more Naked mole rats are a long-lived animal model that age much like humans, but that can also withstand oxidative damage, cancer, neurodegenerative diseases, and severe hypoxic conditions, which is of particular interest to this study. The conditions of their underground burrows result in competition for oxygen consumption, yet despite this oxygen deprivation they emerge unscathed. To understand the mechanisms in place to facilitate neuronal preservation during hypoxia, we investigated the protein levels of well-known cell-stress factors. We found that under hypoxic conditions, nearly half of the proteins measured increased expression in brain, while only a few decreased. Under hypoxic conditions there appeared to be a HIF1α-centered response, where HIF1α and its interactors carbonic anhydrase 9, CITED2, p21/CIP1, and NFκB1, among others, were upregulated. Concurrently, a hypoxia-induced decrease of cytochrome c was consistent with decreased mitochondrial function and protection from apoptosis. The picture that emerges is one of neuroprotection, cell-cycle arrest, and the promotion of antiapoptotic functions, all of which are consistent with conserving energy and maintaining neural integrity under low oxygen levels. These results suggest how this species may be poised to face hypoxia and contribute to its remarkable ability to deal with myriad of other damaging factors and sets the stage for future work on the neuroprotective facilitators we identified.
Cellular and Molecular Life Sciences, 2018
When temperatures plummet below 0 °C, wood frogs (Rana sylvatica) can endure the freezing of up t... more When temperatures plummet below 0 °C, wood frogs (Rana sylvatica) can endure the freezing of up to ~ 65% of their body water in extracellular ice masses, displaying no measurable brain activity, no breathing, no movement, and a flat-lined heart. To aid survival, frogs retreat into a state of suspended animation characterized by global suppression of metabolic functions and reprioritization of energy usage to essential survival processes that is elicited, in part, by the regulatory controls of microRNAs. The present study is the first to investigate miRNA biogenesis and regulation in the brain of a freeze tolerant vertebrate. Indeed, proper brain function and adaptations to environmental stimuli play a crucial role in coordinating stress responses. Immunoblotting of miRNA biogenesis factors illustrated an overall reduction in the majority of these processing proteins suggesting a potential suppression of miRNA maturation over the freeze-thaw cycle. This was coupled with a large-scale RT-qPCR analysis of relative expression levels of 113 microRNA species in the brains of control, 24 h frozen, and 8 h thawed R. sylvatica. Of the 41 microRNAs differentially regulated during freezing and thawing, only two were significantly upregulated. Bioinformatic target enrichment of the downregulated miRNAs, performed at the low temperatures experienced during freezing and thawing, predicted their involvement in the potential activation of various neuroprotective processes such as synaptic signaling, intracellular signal transduction, and anoxia/ischemia injury protection. The predominantly downregulated microRNA fingerprint identified herein suggests a microRNA-mediated cryoprotective mechanism responsible for maintaining neuronal functions and facilitating successful whole brain freezing and thawing.
PeerJ, 2017
The wood frog (Rana sylvatica) can endure freezing of up to 65% of total body water during winter... more The wood frog (Rana sylvatica) can endure freezing of up to 65% of total body water during winter. When frozen, wood frogs enter a dormant state characterized by a cessation of vital functions (i.e., no heartbeat, blood circulation, breathing, brain activity, or movement). Wood frogs utilize various behavioural and biochemical adaptations to survive extreme freezing and component anoxia and dehydration stresses, including a global suppression of metabolic functions and gene expression. The stress-responsive myocyte enhancer factor-2 (MEF2) transcription factor family regulates the selective expression of genes involved in glucose transport, protein quality control, and phosphagen homeostasis. This study examined the role of MEF2A and MEF2C proteins as well as select downstream targets (glucose transporter-4, calreticulin, and muscle and brain creatine kinase isozymes) in 40% dehydration and 24 h anoxia exposure at the transcriptional, translational, and post-translational levels usi...
FEBS Letters, 2019
A degree of regulation continues into death according to post-mortem transcriptome studies, which... more A degree of regulation continues into death according to post-mortem transcriptome studies, which have identified 'zombie genes' that come alive hours and days after organismal death. We hypothesized that hibernation, representing the closest natural mammalian phenomenon to death, would display similar gene expression profiles. Exploring zombie genes using qPCR and available transcriptomic resources from multiple torpid tissues in 13-lined ground squirrels showed little in common with gene profiles observed following death. Hibernators repress transcription, surviving only on the transcripts required during profound slowdowns of metabolic rate and of most physiological functions, therefore not requiring zombie gene expression that could be the cell's last resort during stress. This is the first study to explore zombie gene responses to a near-death situation in a living system.
Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 2018
† Indicates equal input into this study.
Journal of Experimental Biology, 2018
Some animals must endure prolonged periods of oxygen deprivation to survive. One such extreme mod... more Some animals must endure prolonged periods of oxygen deprivation to survive. One such extreme model is the Northern Crayfish (Orconectes virilis), that regularly survives year-round hypoxic and anoxic stresses in its warm stagnant summer waters and in its cold, ice-locked winter waters. To elucidate the molecular underpinnings of anoxia-resistance in this natural model, we surveyed the expression profiles of 76 highly-conserved microRNAs in crayfish hepatopancreas and tail muscle from normoxic, acute 2hr anoxia, and chronic 20hr anoxia exposures. MicroRNAs are known to regulate a diverse array of cellular functions required for environmental stress adaptations, and here we explore their role in anoxia tolerance. The tissue-specific anoxia responses observed herein, with 22 anoxia-responsive microRNAs in hepatopancreas and only 4 changing microRNAs in muscle, suggest that microRNAs facilitate a reprioritization of resources to preserve crucial organ functions. Bioinformatic microRNA ...
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Papers by Hanane Hadj-Moussa