| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Julius H. Comroe Memorial Lecture |
1 Departments of Pediatrics and Neuroscience, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0735, USA
Abstract
There have been extensive studies and experiments on cells, tissues and animals that are susceptible to low O2, and many pathways have been discovered that can lead to injury in mammalian tissues. But other pathways that can help in the survival of low O2 have also been discovered in these same tissues. It should be noted, however, that the mechanisms that can lead to better survival in susceptible mammalian tissues have quantitatively a ‘narrow range’ for recovery, since these tissues are inherently at risk. Another strategy for understanding the susceptibility of organisms is to learn about pathways used by anoxia-resistant animals. Approximately a decade ago, I and my co-workers discovered that one such animal, Drosophila melanogaster, is very tolerant of low O2. Here, I detail some of the studies that we performed and the strategies that we developed to understand the mechanisms that underlie the fascinating resistance of Drosophila to measured partial pressure of O2 of zero. We employed three ideas to try to address our questions: (1) mutagenesis screens to identify loss-of-function mutants; (2) microarrays on adapted versus naïve flies; and (3) studying cell biology and physiology of genes that seem important in flies and mammals. The hope is to learn from these studies about the fundamental basis of tolerance to the lack of O2, and with this knowledge be able to develop better therapies for the future.
(Received 9 December 2005;
accepted after revision 16 January 2006; first published online 23 January 2006)
Corresponding author G. G. Haddad: Department of Pediatrics, School of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0735, USA. Email: ghaddad{at}ucsd.edu
‘Man was created of the Earth, and lives by virtue of the air; for there is in the air a secret food for life ... whose invisible congealed spirit is better than the whole Earth.’Michael Sendivogius, 1604
Although Lavoisier has been credited with the discovery of oxygen in the 18th century, indeed he was standing on the shoulders of a number of insightful individuals at that time. Even in the 15th century, about 200 years before the Frenchman's work on combustion, Leonardo DaVinci had realized that air is composed of at least two components, and that the part that does not support fire does not support life either. In the 17th century, Boyle showed that in a partial vacuum, animals die and flames go out. Furthermore, at the beginning of that century, Sendivogius was able to produce O2 by heating nitrates such as KNO3. Lavoisier, however, gave O2 its name, finally proving that O2 was the active component in air.
What motivates a large number of clinicians and basic scientists to be interested in oxygen and oxygenation at present? It is mostly questions that are related to clinical conditions for which answers to improve human health are lacking. In addition, there are still many questions of biological and physiological significance that are of importance and fascinating to address. Such clinical and fundamental questions have motivated many investigators. Consider, for example, the following questions.
Although there have been extensive investigations studying these questions, they are still largely unsolved despite major advances in our knowledge of the effects of hypoxia on cells and tissues. While all of these questions are of interest to researchers in our laboratory, this paper will be limited to the recent work we have performed addressing questions related to the genetics of susceptibility or tolerance using an invertebrate animal model that we have used over the past decade (Haddad, 1996, 1999, 2000; Haddad & Ma, 2001).
From a clinical point of view, answers to such questions would be very welcome, since many conditions are still poorly treated. Consider, for example, children with sickle cell anaemia and the lesions in the CNS that result from the disease. Patients spanning the whole age spectrum who are admitted to intensive care units generally experience episodes of hypoxia and often suffer as a result. Intra-uterine insufficiency can result in fetal demize, and early postnatal patients with obstructive sleep apnoea/hypoventilation often succumb to episodes of hypoxia and asphyxia or succumb to the sudden infant death syndrome, and no effective treatment is available.
Responses to hypoxia
Systemic and inherent responses in vertebrates. Responses to low O2 can either be a result of a feedback system involving multiple organs or be dependent on the inherent cellular properties and their ability to tolerate low O2 concentrations. The response of pinnipeds (e.g. seals) is an example of the first type of response (Hochachka, 2000; Hurford et al. 1996). Seals drop their heart rate and cardiac output, and redistribute blood flow so that the brain does not have a diminished blood flow despite the major drop in cardiac output. Furthermore, seals tremendously increase their haematocrit by displacing red blood cells (‘squeezing’) from their spleens into the bloodstream just as the dive starts to take place (Fig. 1). Therefore, they increase their O2-carrying capacity as they reduce heart rate, cardiac output and respiration with diving.
|
|
Drosophila melanogasterand invertebrate models. Drosophila melanogaster has been used as an animal model for more than a century, but its power as a genetic model has only been harnessed in the past two decades to solve biological questions of interest to mammalian and human health (Metzger & Krasnow, 1999; St John et al. 1999; Haddad & Ma, 2001; Potter et al. 2000; Driscoll & Gerstbrein, 2003; Farahani & Haddad, 2003). To name a few examples, fruit flies have been used to understand the basis for organ development, ageing, memory and learning, circadian rhythm, alcohol intoxication and cancer. We have, in addition, investigated the possibility of using flies to look at the notion of tolerance versus susceptibility in the Drosophila cells and tissues to O2 deprivation. It should be recognized that some of the major discoveries in physiology and medicine have only been made in model systems in the past 50 years. Examples abound. Consider for instance, the giant squid axon and the discovery of the basis of action potential generation by Hodgkin and Huxley; Aplysia and the discovery of the basis for long-term potentiation and memory by Kandel and colleagues; fruit flies and the genes that control embryonic development by both Nusslein-Volhard and Wieschaus; Caenorhabditis elegans and the genes that control programmed cell death by Horwitz and his group; and finally the discovery of the K+ channel structure in bacteria by Rod MacKinnon and colleagues.
Drosophila melanogaster and tolerance to low O2.
Why use Drosophila to address hypoxia tolerance or susceptibility? In the early 1990s we discovered that fruit flies can sustain anoxia (0 mmHg) environments for hours (
5 h) without morphological abnormalities on light or ultrastructural electron microscopic levels (G. Haddad, E. Ma, unpublished observations from our laboratory). Furthermore, after such an exposure, flies can perform complex behaviours such as mating, flying and seeing (G. G.Haddad, unpublished observations). The major question for us about a decade ago was related to what approaches we should use in order to unfold the secrets of how the fly resists the extreme atmosphere of anoxia so well.
The advantages of using Drosophila were imminently appreciated as being that: (1) genetic markers and mutant lines were readily available; (2) the generation time is relatively short (10 days) at 25°C; (3) each generation has a large size which yields itself to the study of recombinations and genetic analysis; (4) molecular biological and physiological tools are also available, and the Drosophila genome has been available in the past several years; and (5) most importantly, the conservation of biochemical and genetic pathways are striking. Indeed, about 75% of human disease genes have a fly orthologue.
Questions and approaches in Drosophila. The three major questions that we asked, therefore, are as follows.
These questions can be translated into three approaches that we have used. These approaches allowed us to use specific techniques and test specific hypotheses. Indeed, we have used: (1) classic or forward genetics, in which a phenotype is dissected genetically and a gene or genes are identified that could be responsible for the phenotype of tolerance to O2 deprivation; (2) reverse genetics, in which gene expression is studied, and differential gene expression can lead to the understanding of the phenotype of interest; and (3) the study of specific genes in flies in order to understand the cell biology and physiology that is not currently understood in mammals; flies are used as a model.
Using the first strategy, or forward genetics, one focuses on defining the phenotype and then isolating the gene(s) responsible for the phenotype after using a mutagenesis screen, which includes a behavioural and/or physiological assay that is essential to uncover those mutants that have either lost or gained function. After such mutants are isolated, the mutations are mapped and the genes are cloned. To prove that such genes are responsible for the phenotype, wild-type cDNA of the gene of interest can be injected into a mutant embryo, for example, to rescue the phenotype. This is precisely what we have done, and we obtained very interesting results pertaining to the gene hypnos-2P or ADAR, which is a pre-mRNA adenosine deaminase of mRNA (Fig. 3; Ma et al. 2001). We have further characterized the phenotype and studied how this mutant behaves under various conditions. For example, this mutant, although it is anoxia sensitive, is very resistant to oxidant injury. In addition, we have looked at the phenotype not only from a behavioural viewpoint but also from a physiological angle. These mutant flies recover their evoked potentials after anoxia at much later times than wild-type flies. Such results are very similar to the behavioural results described above. We have also done rescue experiments to ascertain that hypnos-2P or ADAR is the gene that induces the sensitivity to anoxia (Fig. 4).
|
|
Using the second strategy, we asked whether we can determine which genes are important during hypoxia. We had two ideas. Firstly, we exposed flies to various relatively short durations of hypoxia (minutes to hours) and determined gene expression; subsequently we had to prove that the alterations in gene expression played a role in hypoxia. Secondly, starting from a relatively large pool of alleles, we exposed flies to hypoxia over generations and determined whether the offspring selected under hypoxic pressure had differential gene expression; we had to prove, here again, that these alterations are important. For this latter experiment we started by including a number of isogenic lines which provided diversity. After pilot experiments, we decided to set triplicate populations of flies in three separate chambers and expose them to 8% O2 at the outset and to lower this level every three to five generations. We obtained, after more than 35 generations, flies that lived perpetually at very low levels of O2, levels at which naïve flies could not survive. Since we have saved embryos, larvae and adult flies from every generation, we will be able to find out what gene families are important in inducing this tolerant phenotype.
Why does it matter if flies are resistant to low O2?
That flies can become more tolerant or are naturally more tolerant to low O2 than mammals may not be surprising or, to some investigators, not relevant to human health. However, we believe that this can be a narrow view and that work in Drosophila can enhance our ability to understand biological normative processes and most probably human diseases. I will illustrate below with an example as a proof of concept.
One of the important questions that we asked fairly recently was whether trehalose, which is a glucose dimer, can play an important role in protecting flies against anoxic stress. We had discovered in nuclear magnetic resonance (NMR) experiments that trehalose was present in abundance in Drosophila heads. We first cloned the gene for trehalose-6-phosphate synthase (tps1), which synthesizes trehalose, and examined the effect of tps1 overexpression or mutation on the resistance of Drosophila to anoxia (Chen et al. 2002; Chen & Haddad 2004). Upon induction of tps1, trehalose increased, and this was associated with increased tolerance to anoxia. A transposable genetic element (p-element) inserted into an intron of the tps1 gene resulted in an embryonic lethal fly (Chen et al. 2003). To determine whether trehalose could protect against anoxic injury in mammalian cells, we transfected the Drosophilatps1 gene (dtps1) into human embryonic kidney cells (Chen et al. 2004). Glucose starvation in culture showed that HEK 293 cells transfected with pcDNA3.1 (–) dtps1 (HEK-dtps1) did not metabolize intracellular trehalose and, interestingly, these cells accumulated intracellular trehalose during hypoxic exposure.
In contrast to HEK 293 cells transfected with pcDNA3.1 (–) (HEK-v), cells with trehalose were more resistant to low oxygen stress (1% O2; Fig. 5). Insoluble proteins were three times more abundant in HEK-v than in HEK-dtps1 after 3 days of exposure to low O2. The amount of Na+–K+ ATPase present in the insoluble proteins dramatically increased in HEK-v cells after 2 and 3 days of exposure, whereas there was no significant change in HEK-dtps1 cells. Ubiquitinated proteins increased dramatically in HEK-v cells but not in HEK-dtps1 cells over the same period. Our results indicate that increased trehalose in mammalian cells following transfection by the Drosophila tps1 gene protects cells from hypoxic injury.
|
Footnotes
Experimental Biology, San Diego, CA, USA, April 2005.
References
Chen Q, Behar KL, Xu T, Fan C & Haddad GG (2003). Expression of Drosophila trehalose-phosphate synthase in HEK-293 cells increases hypoxia tolerance. J Biol Chem 278, 49113–49118.
Chen Q & Haddad GG (2004). Role of trehalose phosphate synthase and trehalose during hypoxia: from flies to mammals. J Exp Biol 207, 3125–3129.
Chen Q, Ma E, Behar KL, Xu T & Haddad GG (2002). Role of trehalose phosphate synthase in anoxia tolerance and development in Drosophila melanogaster. J Biol Chem 277, 3274–3279.
Chen L, Rio DC, Haddad GG & Ma E (2004). Regulatory role of dADAR in ROS metabolism in Drosophila CNS. Brain Res Mol Brain Res 131, 93–100.[Medline]
Driscoll M & Gerstbrein B (2003). Dying for a cause: invertebrate genetics takes on human neurodegeneration. Nat Rev Genet 4, 181–194.[Medline]
Farahani R & Haddad GG (2003). Understanding the molecular responses to hypoxia using Drosophila as a genetic model. Respir Physiol Neurobiol 135, 221–229.[Medline]
Haddad GG (1996). O2 deprivation in tolerant & non-tolerant organisms: phylogeny and ontogeny. In Tissue Oxygen. Developmental, Molecular and Integrated Function, ed. Haddad GG & Lister G, pp. 573–594. Marcel Dekker, New York.
Haddad GG (1999). Use of genetic models in respiratory neurobiology and sleep. Curr Opin Pulm Med 5, 349–354.[Medline]
Haddad GG (2000). Enhancing our understanding of the molecular responses to hypoxia in mammals using Drosophila melanogaster. J Appl Physiol 88, 1481–1487.
Haddad GG & Ma E (2001). Neuronal tolerance to O2 deprivation in drosophila: novel approaches using genetic models. Neuroscientist 7, 538–550.[Abstract]
Haddad GG, Sun Y, Wyman RJ & Xu T (1997a). Genetic basis of tolerance to O2 deprivation in Drosophila melanogaster. Proc Natl Acad Sci U S A 94, 10809–10812.
Haddad GG, Wyman RJ, Mohsenin A, Sun Y & Krishnan SN (1997b). Behavioral and electrophysiologic responses of Drosophila melanogaster to prolonged periods of anoxia. J Insect Physiol 43, 203–210.[CrossRef][Medline]
Hochachka PW (2000). Pinniped diving response mechanism and evolution: a window on the paradigm of comparative biochemistry and physiology. Comp Biochem Physiol A Mol Integr Physiol 126, 435–458.[CrossRef][Medline]
Hurford WE, Hochachka PW, Schneider RC, Guyton GP, Stanek KS, Zapol DG, Liggins GC & Zapol WM (1996). Splenic contraction, catecholamine release, and blood volume redistribution during diving in the Weddell seal. J Appl Physiol 80, 298.
Ma E, Gu XQ, Wu X, Xu T & Haddad GG (2001). Mutation in pre-mRNA adenosine deaminase markedly attenuates neuronal tolerance to O2 deprivation in Drosophila melanogaster. J Clin Invest 107, 685–693. Erratum: J Clin Invest107, 1203.[Medline]
Ma E, Tucker MC, Chen Q & Haddad GG (2002). Developmental expression and enzymatic activity of pre-mRNA deaminase in Drosophila melanogaster. Brain Res Mol Brain Res 102, 100–104.[Medline]
Ma E, Xu T & Haddad GG (1999). Gene regulation by O2 deprivation: an anoxia-regulated novel gene in Drosophila melanogaster. Brain Res Mol Brain Res 63, 217–224.[Medline]
Metzger RJ & Krasnow MA (1999). Genetic control of branching morphogenesis. Science 284, 1635–1639.
Potter CJ, Turenchalk GS & Xu T (2000). Drosophila in cancer research. An expanding role. Trends Genet 16, 33–39.[CrossRef][Medline]
St John MA, Tao W, Fei X, Fukumoto R, Carcangiu ML, Brownstein DG, Parlow AF, McGrath J & Xu T (1999). Mice deficient of Lats1 develop soft-tissue sarcomas, ovarian tumours and pituitary dysfunction. Nature Genet 21, 182–186.[CrossRef][Medline]
Xia S, Yang J, Su Y, Qian J, Ma E & Haddad GG (2005). Identification of new targets of Drosophila pre-mRNA adenosine deaminase. Physiol Genomics 20, 195–202.
This article has been cited by other articles:
|
|
 |
|
  K. B. Storey and J. M. Storey Tribute to P. L. Lutz: putting life on `pause' - molecular regulation of hypometabolism J. Exp. Biol., May 15, 2007; 210(10): 1700 - 1714. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Huang and G. G. Haddad Drosophila dMRP4 regulates responsiveness to O2 deprivation and development under hypoxia Physiol Genomics, May 11, 2007; 29(3): 260 - 266. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. D. Higgins, E. Bancalari, M. Willinger, and T. N.K. Raju Executive Summary of the Workshop on Oxygen in Neonatal Therapies: Controversies and Opportunities for Research Pediatrics, April 1, 2007; 119(4): 790 - 796. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
