Dr Caroline Brennan and her research team are interested in the molecular processes controlling behaviour, in particular the genetic mechanisms underlying behaviours and diseases such as drug addiction and dementia.
Dr Brennan, who is a QMUL senior lecturer in the School of Biological and Chemical Sciences, has received funding from the NC3Rs to help her carry out her studies in a way that minimises any animal suffering.
Zebrafish, like mice, are similar to humans at a cellular and developmental level, which can give important insight into human disease processes. She uses zebrafish in preference to mice as a model system for several reasons, including:
They are easier than rodents to house in controlled, humane and non-stressful conditions, which reduces the variations due to environment between the fish, thereby increasing the consistency of results
The impact of any genetic mutation or drug treatment is easy to see because zebrafish embryos and larvae are completely transparent, enabling non-invasive imaging techniques to be used
They have larger numbers of offspring than rodents (about 200-300 per pairing) and they grow and develop very quickly
It’s easier to introduce genetic changes into zebrafish as their embryos can absorb chemicals added to the water. Zebrafish are able to withstand much higher levels of chemicals that trigger genetic changes than can be tolerated by rodents, making it possible to induce a much higher density of mutations in their genome.
The molecular processes involved in the fish are directly mirrored in humans, making it possible to translate research on possible preventions and cures.
Dr Caroline Brennan
By introducing genetic mutations into zebrafish, researchers are able to track the effect of these mutations on the development of various diseases and behaviours. This can direct research in humans by narrowing down the list of genes to investigate for their association with certain conditions.
Dr Brennan says: “Zebrafish have all the advantages of being able to apply the 3Rs – replacement, refinement, reduction – in our research, while providing us with an excellent model for human conditions, diseases and behaviours. Through our work with zebrafish, we have been able to use genetic screening techniques to identify new genes involved in addictive behaviour that make people more likely to smoke.
In addition, we have found that exposure of fish to alcohol as a developing embryo increases the propensity to addictive behaviours in adults; the molecular processes involved in the fish are directly mirrored in humans, making it possible to translate research on possible preventions and cures from zebrafish to humans.”
Described by Dr Chris Faulkes as looking like sabre-toothed sausages, naked mole-rats are the slightly improbable subject for research studies that range from understanding healthy ageing and longevity, resistance to the development of cancer and other diseases, to fertility and propensities to autism, depression and anti-social behaviour.
Dr Faulkes, who is Reader in Evolutionary Ecology, keeps one of the largest collections of naked mole-rats worldwide at QMUL. His research with these fascinating, underground-dwelling mammals follows all applicable Home Office rules but takes a non-invasive approach to studying behaviour and genetics, which doesn’t involve any procedures that are regulated by EU and UK legislation.
The discovery of their apparent resistance to cancer and the exceptional longevity for a small rodent has opened up new and important avenues of research.
Dr Chris Faulkes
Naked mole-rats have social behaviour that is akin to insects such as bees and ants. They have a queen, who suppresses the fertility of the females and most males around her, and the rest of the colony are “workers”, maintaining the burrow system, or “soldiers”, defending the colony against foreign mole-rats or predators. This, in itself, is remarkable for a mammal, but even more amazing is that they can live to over 30 years old – ten times longer than a mouse and more than five times longer than predicted for its body size – not only do they almost never get cancer, but also they resist the normal signs of ageing.
“The naked mole-rat is a unique and fascinating mammal that has excited biologists since the discovery of its ‘insect-like’ behaviour,” says Dr Faulkes. “While these animals have many unusual adaptations to their lifestyle, the discovery of their apparent resistance to cancer and the exceptional longevity for a small rodent has opened up new and important avenues of research that have implications for understanding these processes in humans and improving human health.”
He and his colleagues are studying how the social behaviour and cooperation of naked mole-rats has evolved and is maintained, and this work includes evolutionary ecology, identifying and understanding the roles played by various genes, and the interplay between genetics and the environment.
“We are planning to further our research on how the behaviour of genes can be affected by non-genetic influences such as the environment, known as epigenetics, with colleagues both at QMUL and the Marie Curie Institute in Paris. This encompasses understanding the dynamic and fine scale control of reproduction and fertility, sociality and healthy ageing. Other ongoing studies take a comparative approach, looking at the evolution of genes that are implicated in human disease, and how these vary across other mammals.”
There are about 100,000 people living with multiple sclerosis (MS) in the UK and it is a disease for which there is currently no cure, although there are treatments that can help to alleviate some of the symptoms.
MS is an autoimmune condition, in which the immune system attacks the myelin sheath – the layer that surrounds the nerves in the brain and spinal cord. It can cause serious disability with symptoms ranging from problems with vision, movement of arms and legs, numbness, stiffness, muscle spasms, sensation, balance and problems with thinking, learning and planning. People with MS have an average life expectancy five to ten years shorter than the general population.
At QMUL we have researchers who are investigating new drugs to target some of the symptoms and slow down the progression of the disease.
Mice provide a good model for the condition in humans and enable researchers to investigate how the disease progresses and what treatments may be effective. However, to do this, genetically modified mice are bred that will develop MS. QMUL researchers are working on ways to reduce the numbers of mice that are needed and to refine the types of procedures that are carried out so as to minimise suffering.
Researchers at the Blizard Institute led by David Baker, Professor of Neuroimmunology, found that a way to measure nerve loss in MS was to track what was happening in the visual systems of mice that had been genetically engineered to develop inflammation of the optic nerve. The mice could be followed over time and any nerve damage measured non-invasively via various scanning methods and tests of visual sensitivity. In this way the effect of certain drugs that inhibited or slowed the damage to the nerve cells could also be measured. This not only reduced the numbers of mice that needed to be used, but also meant the animals were not required to develop the more extreme symptoms of MS such as paralysis.
“These new approaches are relevant to the future treatment of neurodegeneration of MS in humans, which has so far evaded treatment,” says Professor Baker.
Further research in mice discovered that there was a key window of opportunity when a drug would act most effectively after disease onset. This time window between the development of signs and the beginning of treatment was set at two weeks in subsequent clinical trials of the drug in humans based on this knowledge. After this time too much damage may have occurred that would be difficult to treat.
It may prove possible to apply our findings to other conditions such as spinal cord injury, autism, glaucoma and epilepsy.
Professor David Baker
The class of drugs that the QMUL researchers are investigating are known as sodium channel inhibitors. Animal studies have shown that accumulation of too much sodium in nerve cells can trigger a chain of events that lead to the nerve damage seen in MS. Building on their work in mice, the researchers tested a sodium channel inhibitor (phenytoin) in people at the onset of MS in whom the disease was causing inflammation of their optic nerves and consequent deterioration in their sight. Results from 81 patients in a phase II clinical trial at two hospitals in the UK (London and Sheffield) found that patients who were given the drug for three months had less damage to their eyesight after six months compared to patients given a placebo drug. The researchers measured the thickness of the retinal nerve fibre layer (RNFL) and found that there was a 30 per cent reduction in the extent of RNFL loss in patients receiving phenytoin compared to those receiving the placebo.
The findings from all of this work have enabled QMUL researchers to start investigating whether another sodium-channel inhibitor called oxcarbazepine, which they believe is more potent than phenytoin, given in later stages of MS in addition to anti-inflammatory drugs, could limit loss of nerve function in a clinical trial called PROXIMUS*. The phase IIa trial is being conducted at the clinical research centre at The Royal London Hospital.
In addition, Professor Baker says: “From our work initially in mice we have also found a new mechanism, which could be targeted with new drugs to protect their nervous systems from damage. As part of this research, we have developed new ways of working with mice that reduces the numbers we have to use and the severity of the conditions that they are bred to develop. We have synthesised new drugs to target this mechanism and have moved the idea from invention through animal studies and have shown that the drug is very well tolerated in humans. We are currently undertaking additional trials in people with MS. We aim to recruit 160 people with MS within the UK, including people at Barts Health. It may prove possible to apply our findings to other conditions such as spinal cord injury, autism, glaucoma and epilepsy. We are also continuing to develop our work on reduction, refinement and replacement of animals.”
The song of the zebra finch is helping researchers at QMUL to understand the processes involved in learning and memory – not just in birds but humans too.
Zebra finches communicate via “learned vocalisation” – a process of acquiring new sounds via imitation and the ability to modify and produce vocalisations (sounds generated in the bird’s syrinx, the equivalent of the human larynx). Vocal learning is rare among animals, but zebra finches share this trait with humans.
David Clayton, QMUL’s Professor of Neuroscience, says: “Vocal learning ability is a prerequisite for the development of language, and so understanding the biochemical, molecular and genetic changes that occur in zebra finches while they are learning their songs, and when they stop learning, helps us to understand what is happening in humans.”
Human babies are born receptive to the sounds of all languages, but start to lose this receptivity after a year; by adulthood they are losing their ability to learn new languages and to speak them without an accent. Why does this happen and, in addition, what is happening as humans become more forgetful as they age - a process exacerbated by conditions such as dementia and Alzheimer’s disease? These are some of the questions that the study of zebra finches is helping to answer.
“Only the male zebra finch learns to sing,” explains Professor Clayton. “When he’s an adolescent, from about one month old, he copies his song from an older tutor. It is during the first two months of life that he is sensitive to interactions with his tutor, and also with other male birds. In the third month he practices and polishes his song and after that he reaches adulthood; this is the point at which the song is set for the rest of his life and he can’t learn any new songs. This is similar to human speech learning.
If we can understand how the ability to learn and memorise is initiated or stopped [...] it could lead to new treatments for diseases of old age such as Alzheimer’s and dementia.
Professor David Clayton
“Now, we’re trying to understand the molecular processes involved when a zebra finch is learning a song and when it stops learning. We know that there are numerous changes in gene expression that can occur within 24 hours or over a longer period of time during this learning process. In fact, there are variations in gene expression occurring all the time, influenced, for example, by the environment and social interactions with other birds. For instance when a bird hears another bird singing, it will react and you can see a change in gene expression; but once it has got used to the other bird’s song, then it tunes it out and there are no further changes in gene expression.”
Gene expression is the process by which genetic instructions in a gene are used to make gene products such as proteins, which go on to perform essential functions in cells, including nerve cells. Gene expression can be altered by a process known as DNA methylation, which is an essential part of normal development. DNA methylation is an example of an epigenetic process – heritable changes in gene expression that do not involve changes to the underlying genetic make-up of a cell and that can be influenced by several factors including age and the environment.
“The neural circuitry controlling songbird vocal behavior is well worked out, and bears similarities to human circuits for speech and language – but the mechanisms that promote or limit learning are unknown. However, there is evidence pointing to a likely role for epigenetic mechanisms – in particular, DNA methylation. So we are looking at whether shifts in DNA methylation in specific brain regions underlie the progression and termination of the critical period for song learning in the zebra finch,” says Professor Clayton.
He and his colleagues will be using the latest techniques in genome-wide sequencing – the ability to identify the complete genetic make-up of an animal or other organism - to compare DNA methylation profiles in vocal communication circuits from birds that are in different stages of the learning process. Drugs that disrupt normal methylation will be tested for their effects on the learning period and the stability of song memory. Comparative data from other songbird species (and humans, where available) will be used to see whether the mechanisms identified in the zebra finch can be generalised to other species.
“If we can understand how the ability to learn and memorise is initiated or stopped, then it might be possible for us to find ways to manipulate this process with drugs, so that learning ability can be re-started. This could lead to new treatments for diseases of old age such as Alzheimer’s and dementia,” says Professor Clayton.
Most of QMUL’s work with zebra finches is regulated by EU and UK legislation on the use of animals in research; some of it involves observing the birds in their social environments, but research on the molecular, genetic and other processes underlying learning and memory does involve killing the birds humanely at specific points in their life cycle so that the tissue samples can be taken for analysis.