HOW IS THE STUDY OF NON-MODEL ORGANISMS SUCH AS MOLE RATS ADVANCING MEDICAL RESEARCH?
A DISSERTATION BY CHRISTOPHER CLARK
Table of Contents
Chapter 1: Introduction to Non-model Organisms 1
1.1. Model Organisms VS Non-Model Organisms 1
1.2. Why Non-Model Organism are Important in Medical Research 2
Chapter 2: Mole-Rats and Medical Research 3
2.1. Introduction to Mole Rats – The Naked Mole-Rat 3
2.2. Features of the Naked Mole Rat Including Those That Make Them Suitable for Medical Research 4
2.3. How Naked Mole-Rats Are Advancing Medical Research 5
2.3.1. Human Aging Research: 5
2.3.2. Cancer Research: 7
2.3.3. Nociception/Pain Research 10
2.3.4. Development of Drugs against Hypoxia Mediated Neuronal Damage 12
Chapter 1: Introduction to Non-model Organisms
1.1. Model Organisms VS Non-Model Organisms
Model organisms such as Escherichia coli, Fruitfly Drosophila melanogaster and mice have been crucial in the analysis of cellular and molecular processes that underpin life. However, model organisms have proven to be insufficient in unlocking mechanisms by which biological functions cope with complexity of environmental conditions in which life develops. Thus, to understand the potential mechanisms of adaptation of biological organisms to changing environmental conditions, it is important to increase the diversity of organisms used in medical research (1, 2).
To define a non-model organism, it is important to identify the characteristics that constitute a model organism. A model organism is an organism with a representational scope, characterized by easy cultivation as a stable isolated species in a controlled laboratory condition; by its small size and short life span; by a description of its development and traits, and by its ability to easily be genetically manipulated (1, 3). Thus, to define non-model organisms, they are organisms that are not well characterized and cultivated as a stable species in controlled conditions. They require more effort in protocol adaptation and method development. As a result, make difficult experimental organisms (1, 3).
1.2. Why Non-Model Organism are Important in Medical Research
Non-model organisms are important organisms used in medical research, because not only do they differ from current models, their study constitutes the heart of comparative biology, exposing mechanisms that underlie biological diversity. That is, non-model organisms provide an understanding of certain processes and physiological changes that occur in life, such as aging. Non-model organisms therefore, serve as surrogates for human studies in environmental science because they are capable of surviving and coping with environmental alterations. Studying the molecular mechanisms of adaptation of non-model organisms is important in developing drugs against certain conditions and diseases such as cancer (1).
Chapter 2: Mole-Rats and Medical Research
2.1. Introduction to Mole Rats – The Naked Mole-Rat
There are over 30 different mole rats. However, the most common one is the Naked Mole-Rat (NMR) (4). The NMR, also known as Heterocephalus glaber is a subterranean mammal of the family, Bathyergidae. They are endemic to semi-arid regions of East Africa, mainly Kenya, Ethiopia and Somalia (5, 6). Mole rats including NMRs spend their entire life living underground, in burrows comprising mazes of tunnels with numerous chambers (5, 6). NMRs are therefore, poikilothermic and like social insects, such as ants and bees, they have a castle system with up to 20 to 300 individuals produced by a single breeding queen (7). The queen supresses the fertility of females, and most male members of the colony, except for one to three male members (6). As seen with insects, the colony exhibits division of labour, that is, some members of the colony are workers whose responsibility is to dig out and maintain the burrow system; the other members are soldiers whose role is to defend and protect the colony from predation and attack mainly by snakes (6).
2.2. Features of the Naked Mole Rat Including Those That Make Them Suitable for Medical Research
NMRs are unique and fascinating mammals. Since the discovery of their insect-like behaviours, NMRs has gained interest from biologist across the world: and because the molecular mechanisms that enable their unusual biology are now understood (5, 6). NMRs are typically, about 7cm long and weigh between 30 to 80 grams. They have no hair on their skin, hence their name, and like all rodents, two of their front protruding teeth continue to grow throughout their lives due to the continuous wear and tear during the process of digging and making tunnels. Thus, their teeth are often regenerated (7).
In addition to these features, NMRs have a long lifespan, living over 30 years with no signs of aging. They have evolved numerous physiological mechanisms of adaptations that enable them to live and survive within extreme environmental conditions of low oxygen and high carbon dioxide levels. NMRs also tend not to develop cancer. Thus, a wide spectrum of social organizations such as eusociality and poikilothermy, coupled with their adaptations to extreme living conditions, make NMRs ideal and suitable animal subjects for medical studies that embrace broad principles in physiology. This in turn, opens up important avenues of research that have implications for understanding these processes in humans and improving human life (5, 6).
2.3. How Naked Mole-Rats Are Advancing Medical Research
2.3.1. Human Aging Research:
Human aging remains one of the most poorly understood biological phenomena, mainly because it is an integrative and complex process. However, aging can be defined as a gradual, pervasive, and irresistible process that causes a decrease in function, Including physiological processes and reproductive capabilities, such that overtime, fit individuals become weak, and turn into older individuals with an increase in age-associated damage such as neurodegeneration (8, 9 13).
Aging is therefore, a major factor that affects late life and healthy living. Thus, understanding why and how we age, and the physiological changes that occur during aging is a major medical research challenge in science (8 – 10, 13). Most of the Information we know about the mechanism involved in human aging has been mainly gleaned using a variety of model organisms (described in chapter one), such as laboratory mice (11, 13). These organisms only provide limited knowledge due to their short life span and therefore, cause us to miss important factors that contribute to aging. Therefore, species with longer extraordinary lifespans are more suitable for the studies of human aging research (10). One of such species is the NMR (13).
NMRs have a life span of up to 32 years, they are the longest-lived rodents known. This is because they properly maintain the quality of their proteins and gene expression levels for most of their lives. That is, the level of activity and metabolic rates, as well as sustained muscle mass, bone density, cardiac health, and neuron numbers remain unchanged throughout their lives (26). NMRs eventually age, showing signs of muscle loss, and cardiac dysfunction, however, they delay the onset of aging and compress the period of decline into a small fraction of their overall lifespan (26).
These findings of sustained good health are surprising given that the NMR is an exception to many current theories of aging which attempt to elucidate the mechanisms involved in human aging (13). For example, “The Telomerase Length Theory”, a theory that states that telomeres, the repetitive DNA that caps the ends of chromosomes, undergo senescence and become shorter with consecutive cell divisions, is a biomarker of aging (12, 26). This theory is invalid in NMRs because they possess similar lengths and size of telomeres when compared to humans. Another theory is the widely accepted “Oxidative Damage/Stress Theory of Aging”, which gives an insight of aging at the molecular level (11). The oxidative theory postulates that, aging results from accumulated damage of reactive oxygen species (ROS), a by-product of aerobic respiration (26). This theory is particularly appealing because ROS, is constantly produced which, in turn causes cell membrane, protein and DNA damage, which in turn leads to malfunction of physiological systems (26). However, this is not the case for NMRs, even at a young age, NMRs have increased levels of accumulated DNA oxidative damage, yet cellular functions are not impaired. Thus, NMRs possess mechanisms that enable them to reduce and resist oxidative damage as they grow which in turn contributes and increases their life span. Thus, the study of NMRs does not only provide a better insight into the timing, mechanisms and processes involved in how humans age but also, help shine light on theories postulated to cause human aging (11).
NMRs also satisfy the principle of negligible senescence, which is characterised by the lack of age related changes in reproductive and physiological functions. NMRs do not develop symptoms associated with aging such as cancer, which would be discussed later (6).
Therefore, due to their extraordinary life span coupled with their lack of age-related changes in reproductive and physiological function, NMRs possess outstanding anti-aging defences. Thus, studying NMRs are advancing aging research in the sense that, despite subtle differences in physiological function, the mechanisms that enhance them to live long and impede senescence may be of great prevalence and useful for human aging studies, providing an insight into the biology of mammalian aging (11, 12, 13).
2.3.2. Cancer Research:
According to Cancer Research UK, there were more than 356,000 new cases of cancer and over 164,000 reported deaths in 2014 in the UK (14). These statistics point to the fact that we need to improve our knowledge and understanding of the biological mechanisms that cause cancer in order to identify new therapeutic targets (6).
NMRs do not develop cancer and furthermore, subjecting NMRs to ionizing radiation does not induce much DNA damage, as seen in other animals, nor does it result in the development of tumor, even 5 years later (26). This was shown in a study by Liang et al. were upon injection of an oncogene (a retrovirus encoding SV40 large T antigen and oncogene Ras (G12V)) in NMRs cells, tumorigenesis failed to elicit, cells rather entered a state of crisis, a tumor suppressor mechanism in response to oncogene–expression in NMR cells (26, 27).
In addition to entering a state of crisis, NMRs have other mechanisms that researchers might be able to leverage for the treatment of cancer (6). For example, in humans, one common mechanism used to prevent cancer is, replicative senescence, defined as a mechanism of limiting the number of times a cell can divide, accomplished by telomerase suppression in somatic cells during adult life (6).
However, in rodents, such as NMRs, this is not the case because they retain their telomerase activity in somatic cells, suggesting other mechanisms help them resist developing cancer (6).
The mechanism that confers cancer resistance in NMR is a phenomenon termed “Early Contact Inhibition (ECI)”. ECI is a mechanism that causes cell cycle arrest when cells encounter each other or the extracellular matrix (16). The effect of ECI is mediated via the retinoblastoma (Rb) pathway which contains regulators of cell control, and is backed up by an apoptotic response via the p53 pathway, associated with the induction of p16 (6). NMRs also rely on the upregulation of p27 for contact inhibition, which causes cells to become dense and ultimately undergo apoptosis. Thus, as ECI is an important mechanism that enables NMRs to resist developing cancer, understanding what activates ECI, could be a stepping-stone for inhibiting cancer in humans (6).
Researches have pinpointed at a natural substance that causes the activation of ECI (6). ECI is therefore, thought to be triggered by the secretion of a high molecular mass hyaluronic acid (HMM-HA) (6, 15). HMM-HA is an unbranched disaccharide glucuronic acid/N-acetylglucosamine polymer of the extracellular matrix, that accumulate abundantly in the tissues owing to a decreased activity of HA-degrading enzymes and the unique sequence of hyaluronan synthase 2 (HAS2) (16). The HMM-HA produced in NMRs is five times larger than the HA produced in humans, and as with humans, HMM-HA acts via CD44 receptor which also interacts with NF2 encoding merlin (a tumour suppressor) to activate the induction of p16 which coincides with ECI. Thus, ECI is mediated by the interaction of HMM HA with CD44–NF2 as shown in the diagram below (5, 15, 16). Furthermore, NMRs also produce a tumour suppressor protein, which is an isoform of INK4 in response to HMM-HA stimulation, which leads to the induction of contact inhibition, and thus, cancer resistance (17).
Cancer is therefore, a disease that generally affects most mammalian species, believed to be an unavoidable accompaniment of aging. Therefore, understanding the basis for cancer resistance in the NMR could be important in defining causes of cancer susceptibility and resistance in humans (17).
2.3.3. Nociception/Pain Research
The ability to detect pain is crucial to an organism’s well-being. The neurobiology of pain is therefore, of fundamental interest to medical researchers (18, 19). Pain is a complex experience triggered by real or perceived tissue damage, which, in turn, causes manifestations of certain autonomic, psychological and behavioural reactions (20). Pain can be acute or chronic. Chronic pain is more severe and serves no survival benefit. As much as 19% of the adult human population are expected to experience chronic pain at some point of their life (6, 19).
Recent studies using NMRs in nociception/pain research, has helped identify some molecular mechanisms that drive pain (6). In humans and most mammals, the sensory nerve cells, which detect and respond to pain, are nociceptors, which are either myelinated (Aδ) fibres or unmyelinated C-fibres (18). The unmyelinated C-fibres express substance P (SP) and calcitonin gene-related peptide (CGRP). SP and CGRP are signalling neuropeptides, found in the brain and spinal cord responsible for mediating the effect of pain by binding to neurokinin 1 receptors and G-protein coupled receptors respectively (6). However, in NMRs although, they possess nerves that contain unmyelinated C-fibres, their C-fibres completely lack SP and CGRP, which function to stimulate pain (6). Thus, NMRs are insensitive to pain or exhibit avoidance behaviours to noxious stimuli. This effect, demonstrated in a study by laVinka PC et al. and Smith ES et al. where they reveal that NMRs are insensitive to histamine-induced itch, capsaicin, and ammonia fumes, which activate C-fibers to mediate nociception in other organisms such as mice (21, 22). In others studies by Park TJ et al., laVinka PC et al., and Smith ES et al. they demonstrated that neurons in NMRs are also, insensitive to acidic-induced pain (23, 24). They concluded this to be due to the actions of their voltage-gated sodium channels (NaV1.7), which undergoes mutations to cause them to shut down under acidic conditions thereby, strongly inhibiting and avoiding pain signals which are normally transduced along nerve fibres (19, 23, 24).
Therefore, further investigations of the biology of pain and/or how the processes that confer alterations of pain in NMRs, may provide a significant insight into the components necessary for transduction of painful stimuli, this in turn, could provide vital implications for the treatment of chronic pain in humans such as inflammatory pain, joint pain and muscle pain etc. (26).
2.3.4. Development of Drugs against Hypoxia Mediated Neuronal Damage
Most mammalian brains, including those of humans, suffer serious damage after just 3 to 4 minutes of oxygen deprivation (26). This is because brain tissues do not store much energy, and a steady supply of oxygen is required to generate more (26). In other words, cells in the brain primarily require oxygen during aerobic metabolism to produce adenosine triphosphate (ATP), which, in turn is, used to maintain the concentration gradient and membrane potential of cells during synaptic transmission (6). Therefore, a decrease in ATP because of oxygen deprivation leads to disruption of ion and neurotransmitter homeostasis, ultimately causing neuronal damage and cellular death (6).
Hypoxia is the term given to the lack of oxygen supply to cells in the body and as a result, is involved in many pathological conditions including neurodegenerative disorders such as Huntington’s disease. Hypoxia is also a major concern for victims of heart attack and strokes, where, the blood supply to the brain is interrupted (26).
The use of hypoxic sensitive models organisms such as mice has, to an extent advanced our understanding of some mechanisms that contribute to hypoxia induced neuronal damage, and has enabled us develop some therapeutic targets. However, studying organisms that have evolved effective physiological, molecular, and cellular strategies to survive hypoxic insults would enable us to have a better understanding and thus, aid in discovering new targets for preventing and treating hypoxia-associated damages. Organisms with subterranean lifestyles have, yet again received great attention for their ability to cope with hypoxia (6).
In NMRs, the brain tissues remain functional, even with low or no oxygen supply. In addition, when oxygen levels are restored, the brain tissues recovers fully, even after several minutes of inactivity (26). Thus, NMRs possess special metabolic and circulatory functions that enable them to cope with hypoxic conditions (25).
The ability of NMRs to tolerate hypoxic insults is due to their low basal metabolic rate, further reduced, to cause them to; slowly consume oxygen, so that the supply matches the demand when pools of ATP are decreased during hypoxic conditions. NMRs also possess haemoglobin with a high affinity for oxygen (6, 25). That is, their blood is better at capturing what little oxygen there is in there environment (26).
In addition to the physiological adaptations explained above, NMRs are also tolerant to hypoxia at the neuronal level; where their brain tissues possess adaptive characteristics that enables them survive chronic hypoxic conditions (25, 26). NMRs have mechanism at the neuronal level that mirrors that used by the brains of infant in the womb (26). Infant babies somehow, cope with and tolerate low levels of oxygen where calcium is a key factor. Starving nerve cells of oxygen as mentioned earlier, results in lack of energy formed to regulate the amount of calcium entering the cells, in turn causing an influx of too much calcium, which poisons the cell and ultimately causes neuronal death. In infants, calcium channels close during lack of oxygen, which in turn protects brain cells from calcium overdose in the womb (26). However, when and after the baby is born, these channels are replaced by channels that open in response to oxygen deprivation, thus, can cause neuronal damage and cellular death (26).
In a recent study by Peterson et al. on NMRs, NMRs retain an infant style of calcium channels into adulthood (28). NMRs therefore, avoid hypoxic insults by reducing signals that function to transport calcium into the cell thus, avoiding the resulting neurotoxicity. However, they do this by modulating the GluN2D subunit of their neuronal N-Methyl-D-aspartate receptor (NMDAR). This makes NMRs one of a few organisms known to modulate their glutamatergic activity to effectively deal with hypoxic insults (6, 25).
Therefore, investigating and understanding the strategies by which NMRs cope with and survive hypoxic insults will help us improve our knowledge of the mechanisms involved in reducing neurotoxicity during hypoxia, which in turn, would help in identifying new therapeutic targets for preventing and effectively tackling hypoxic associated injuries such as heart attacks, strokes, and epilepsy etc.
ay in here…