Following on from the identification of the gene TBK1 as a contributory risk factor for MND in February, the plot thickens further with research published yesterday by Dr Jochen Weishaupt and colleagues.
Dr Tennore Ramesh is based at the Sheffield Institute for Translational Neuroscience (SITraN) based at the University of Sheffield. His Association funded research is investigating the early stages of MND in zebrafish, as well as screening potential drugs.
Would you ever consider that fish and humans have parallels? Interestingly it’s a “Yes”. They are vertebrates (animals with backbones) like humans and mice. They have organs that are similar to humans and have a brain and spinal cord. But wait a minute, the fish have gills not lungs and they do not have a tongue or a larynx with which to make noise or speak, which is an important symptom in MND and are generally called bulbar symptoms.
Zebrafish in the past were widely used to study early development. However, they are emerging as good models to study human diseases that occur in adulthood. Our laboratory wanted to test if zebrafish could model such complex neurological diseases. Continue reading
Natalie Rounding is a first year PhD student funded by the MND Association. Based at the Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, she gives us an insight into zebrafish and how they can be used in MND research.
A recent study by Motor Neurone Disease Association-funded researcher Dr Tennore Ramesh from the Sheffield Institute for Translational Neuroscience (SITraN) has shown that even before the symptoms of MND occur, at the earliest stages of the disease, ‘connector neurones’ known as interneurons are already becoming damaged in the zebrafish.
Zebrafish are ideal models for helping scientists understand what happens in MND. Unlike mice and fly models, zebrafish have transparent embryos which enable scientists to get a unique view of the developing neurones under a microscope! Scientists can also look at disease progression in adult zebrafish by looking at muscle strength and measuring their progress swimming against a current.
Not only are zebrafish useful for helping scientists understand what happens in MND, they are also an ideal drug screening model. Zebrafish and humans are more similar than you may think (see Kelly’s post) and potential new MND drugs can be screened quickly. Looking at how MND progresses in the zebrafish, before symptoms appear, can help us gain a better understanding of what causes the disease.
Motorways, dual carriage ways and slip roads
No, I’m not writing about travel alerts or the latest road disruptions due to flooding or snow. In fact, these road systems happen to be a perfect example of what interneurons are, how they relate to motor neurones and what goes wrong in MND.
Our body consists of two types of motor neurones, which are known as upper and lower motor neurones. The upper motor neurones are found in the motor region of our brain and connect to the spinal cord. The lower motor neurones are found between the upper motor neurones in the spinal cord and connect to the muscles (e.g. in the arms and legs). Interneurons are the vital connections between the upper and lower motor neurones.
When a signal is sent from our brain to bend an arm it starts by travelling down an upper motor neurone. The signal then travels to a lower motor neurone via an interneuron. When the signal from the lower motor neurone reaches the muscle in our arm it causes the muscle to contract and bend.
In MND these upper and lower motor neurones become damaged and they are unable to transport the nerve signal from the brain to the muscle in our arm. This means we are unable to contract and bend, even though the brain is telling it to.
In our road system scenario the upper motor neurones are the motorways (e.g. the M1), and the lower motor neurones are the dual carriageways that link the motorways to nearby towns (e.g. the A38). In order for an upper motor neurone to send a signal (e.g. a car) to a lower motor neurone it needs to go via an interneuron, which in our road system scenario is a ‘slip road’ – making these interneurons vital connections between motor neurones.
This study has given us a better understanding of what happens in MND at the early stages of the disease (before symptoms occur). The researchers found that interneurons became damaged before the motor neurones themselves. Therefore this shows that interneurons are important in the early stages of the disease and scientists can begin to look at ways of preventing interneuron damage to see whether this has an effect on MND.
Adding more evidence to the puzzle
This study showed that, in zebrafish, interneurons are involved in the early stages of MND, which adds further evidence to previous work by another MND Association-funded researcher. Dr. Martin Turner (Oxford) also found damaged interneurons at the early stages of the disease before symptoms of MND occur in humans, with other studies showing interneuron damage in SOD1 mice models.
The next step would be to look at ways of preventing these interneurons from becoming damaged, to see whether this has any effect on the progression of MND.
This research is the first article we have paid to be made available Open Access, so that it is freely accessible to all. The article was published online in the prestigious journal ANNALS of Neurology on the 31 December 2012.
McGown, A. et al. Early Interneuron Dysfunction in ALS: Insights from a mutant sod1 Zebrafish Model. ANNALS of Neurology 2012 DOI: 10.1002/ana.23780 http://onlinelibrary.wiley.com/doi/10.1002/ana.23780/abstract
An international research group spanning seven countries and including 23 researchers has identified a gene that modifies survival in MND. The gene, called EPHA4 was identified through a zebrafish genetic screening project and verified in rodents and humans with MND. The study was led by Prof Wim Robberecht, who has previously been funded by the MND Association and who is the Chair of our International Symposium on ALS/MND. The findings were published in the prestigious journal Nature Medicine this week.
What did the research group find?
By screening zebrafish for genetic factors that can modify the progression of MND, the research group identified EPHA4. By stopping, or slowing down the activity of EPHA4, they identified that MND zebrafish can be rescued and rodents (mice and rats) can live longer.
They also identified that MND vulnerable motor neurones have a higher level of EPHA4 than those at a lesser risk of developing the disease. This also means that a low level of EPHA4 confers to a lower risk of MND.
The research group then looked to humans to see if anybody with MND had mistakes in the EPHA4 gene that resulted in a change in survival. The group found two people with MND with genetic differences in the EPHA4 gene. These two people lived with MND for an exceptionally long time. As these genetic differences result in a lower level of EPHA4, this suggests that EPHA4 could be a valid therapeutic target for MND.
What does EPHA4 do?
Ephrin type-A receptor 4 (EPHA4 for short), plays a vital role in the development of our nervous system, in maintaining the shape of the neurone and in preventing regeneration after injury.
As our motor neurones grow, the projecting length of the neurone (the axon) needs to be guided to grow toward the right areas to connect to its respective muscle. To do this, a complex ephrin negative signalling system is used to guide the growing neurone in the right direction.
In real life terms, this signalling pathway can be thought of as somebody blindfolded, navigating a traffic cone maze. This person (the neurone) doesn’t want to move into an area cornered off by traffic cones (the corresponding ephrin signal). As the neurone can’t see where it’s going, it feels its way around using Eph receptors like EPHA4. The neurone moves away from ephrin signals as it ‘feels’ them. This eventually leads to the neurone reaching its target destination (the muscle).
To physically make the neurone move, when an ephrin signal connects to the ephrin receptor, the inner workings of the neurone are called into action. To find out more information on this, please read our previous article about Profilin1, which was found to be a cause of MND last month.
As EPHA4 plays an integral role in stopping neurone growth, it isn’t surprising that it also plays a role in stopping regeneration of neurones after injury. For example, in mice that have spinal cord injury (this was a study unrelated to MND), by genetically stopping the EPHA4 signal, new axon growth can be seen. With mice that have spinal cord injury that have a normal level of EPHA4 signal, growth cannot be seen.
The above non-MND study, along with the current EPHA4 finding further suggests that a lower level of EPHA4 can result in a longer survival because of its inability to perform its usual function to its usual extent. This incompetance seemingly allows the protection of neurones from degenerating at it’s normal pace.
As an additional note, you may be thinking that it seems counterintuitive that humans have additional signals that stop processes like regeneration when less complex animals like frogs still have this ability. Unfortunately, it’s a bi-product of mammalian evolution that continues to baffle scientists!
What does this mean for people with MND?
This finding unfortunately does not mean that a new genetic test will become available for EPHA4.
This discovery does however, offer another target for research institutes to look into and develop therapies that could slow down disease progression by lowering the amount of EPHA4.
Although this research is likely to take years to develop toward a clinical trial* in humans, it’s promising to see yet another exciting genetic advance that could have an impact on finding a better treatment for MND in the future.
*It’s important to point out that therapeutically lowering the EPHA4 signal in humans would not necessarily mean that neurones could regenerate as seen in the zebrafish. Many different signals other than EPHA4 prevent a human motor neurone from regenerating and from finding its target muscle. However, finding a way to lower the EPHA4 signal may still slow down progression, as seen in mice in this study.
What does this mean for the future of MND research?
Further studies are needed to verify and expand on these exciting results.
This finding means that researchers can explore this pathway in more detail as it, in conjunction with the recent Profilin 1 finding, suggests that this guidance/growth system of motor neurones may play an important role in the development of MND.
Delegates to last weekend’s ENCALS (European Network for the Cure of ALS) meeting in Dublin were met with uncharacteristic hot and sunny weather – enjoyed by the numerous Stag and Hen parties wandering the city centre, but not by the 200 people ensconced in the impressive, new Biomedical Sciences Institute at Trinity College, from 8am to 7pm, for a packed programme of presentations and debate.
ENCALS was established to help develop the standards of clinical and biomedical MND research across Europe and create a more collaborative environment for researchers, industry, funding agencies and Patient Associations. However, the meeting had a very transatlantic flavour, thanks to the participation of several of the leading researchers from North America.
With around 40 speakers, as well as numerous poster presentations, there is too much to cover in a few hundred words, so I’ll focus on just a few of the key themes that were covered. I also apologise for the quite technical language, which may make for hard reading, but is a positive in that it reflects the increasing complexity and sophistication of MND research.
Can we block the ‘molecular funnel’?
The opening speaker, Prof Teepu Siddique, from Northwestern University in Chicago, spoke on The molecular funnel of neurodegeneration. His view of MND is that it may have a large number of different causes, but the way a motor neurone dies will probably be similar, no matter what the original cause. We’re currently finding lots of new genetic factors involved in the disease, but we don’t understand how many of these genes work in health, much less how they malfunction in disease. So, the mouth of our funnel is getting wider.
Prof Siddique’s view is that by focusing on the cellular changes that are common to all forms of the disease, it gives us possible therapeutic targets that could be relevant to all forms of MND. It’s easier to block the funnel at its narrowest point.
He discussed how the degradation of incorrectly formed or damaged proteins is a classic hallmark of all forms of MND. While the way in which the proteins are damaged may differ from one form of MND to the next, it’s the cell’s inability to correctly deal with these proteins that may be a good target. If we can normalise or improve this process, it may keep the motor neurones functioning for longer.
Prof Orla Hardiman, the meeting organiser from Dublin, discussed the need for much larger and more detailed study of large numbers of patients, to attempt to unpick the environmental influences that undoubtedly exist.
A question that many people often ask is whether MND is occuring more often in younger people that in the past. Intriguingly, Prof Hardiman’s ‘population-based’ research using the Irish MND Register suggests the opposite – the average age of symptom onset is getting older. She suggests that continued improvement in medicine and diet means that the population in general is healthier, so our ‘biological age’ is slowing. If age-related diseases such as MND are linked to ‘biological age’ rather than ‘actual age’, it would explain this surprising trend.
Good Genes/Bad Genes
While factors that cause or predispose towards MND are clearly the subject of intensive research, there is of course also interest in factors that might prevent or slow the disease. Some of these potentially ‘good’ genetic variants are being explored:
- Prof Wim Robberecht’s group (University of Leuven) is examining the function of a gene called ephA4, which appears to correlate with longer survival in humans. This work is supported by studies in zebrafish and mouse models of MND.
- Prof Kevin Talbot (University of Oxford) showed data that suggests that by increasing activity of a gene called smn1 might be beneficial to motor neurones. This is a strategy that is being followed for a predominately childhood motor neurone disease called Spinal Muscular Atrophy, so if these approaches work in this particular condition, they might be of benefit in other, adult onset motor neurone diseases.
- Prof Robert Brown (University of Massachusetts) presented early data from a study of a variant in a gene called sarn1, which appears to protect motor neurones from damage….at least in fruit flies and mice. Work is ongoing to see whether it also has relevance in humans.
In contrast, Dr Andrea Calvo (University of Torino) provided information from Italian patients confirming studies in other populations that a variation in the unc13A gene can speed up disease progression. However, the important issue about these disease-modifying genes – and it doesn’t matter whether they speed up or slow down MND – is that they all represent potential therapeutic targets.
Not just about the motor neurones!
We know that motor neurones do not die alone. Other parts of the brain and spine can be affected, but it’s the motor neurones that ‘bear the brunt’.
Dr Sharon Abrahams (University of Edinburgh) provided an excellent overview of the range of cognitive and behavioural changes that can occur in the disease, indicating damage to other part of the brain, in particular the frontal lobe. Thankfully, the ‘real world’ effects of frontal lobe changes are usually subtle, but the fact that they can be picked up by psychological tests and MRI scans will help in defining specific ‘subtypes’ of MND which may require additional approaches to managing the disease.
Dr Martin Turner (University of Oxford) outlined evidence from a number of clinical research studies, including his own that nerve cells, called interneurones, might be involved early in the disease. These particular neurones usually play a role in calming down motor neurones, so if they are damaged or lost, the motor neurones themselves become over-excited and stressed, which leads ultimately to their degeneration.
Dr Turner’s evidence comes mainly from clinical imaging and electrophysiology studies in MND patients, but his theory was supported by a presentation from Dr Tennore Ramesh (University of Sheffield) who works with zebrafish models of MND. He showed results using zebrafish that carry a human SOD1 gene known to cause MND. The fish develop a form of MND in adulthood, but the very earliest signs of nerve damage actually occurs in specific types of interneurones that connect with the motor neurones, with the motor neurone damage occurring much later, closer to the onset of symptoms.
Presentations also covered the role of non-neuronal support cells, such as microglia and astrocytes, both of which have been the subject of extensive research in recent years, as they appear to play a role in the speed of progression of the disease. Prof Jeff Rothstein (Johns Hopkins University) introduced a new cellular player to the MND field, called the oligodendrocyte. These specialised cells have been known for many years to play a role in helping neurones to carry electrical signals, as well as helping them to maintain energy levels. Although they are known to be involved in multiple sclerosis, they hadn’t attracted much attention in MND.
Prof Rothstein showed that in human post mortem MND brain tissue, there is evidence that the brain has been making oligodenrocytes. This is certainly very clear in SOD1 mice, where a massive production of new oligodendrocytes occurs. However the total number of these cells was not increased in the mice, suggesting that older oligodendrocytes were being killed and getting replaced.
He suggested that the new ‘immature’ oligodendrocytes are not nearly as efficient in their supporting role, especially when it comes to supporting motor neurones in maintaining their energy balance. This provides two possible treatment approaches – either try to keep the existing oligodendrocytes healthier or find a way of making sure that their replacements reach their full functional maturity.
I’ve no doubt we’ll be hearing a lot more about these cells in the future.
A study on zebrafish has increased our understanding of how motor neurones work and has provided potential clues for the development of future treatments for MND. The study, led by Dr Catherina Becker from University of Edinburgh, showed that a unique motor neurone repair system found in zebrafish can be enhanced if a particular signal called Notch1 is stopped. The results were published in the 29 February edition of Journal of Neuroscience.
What did they find?
Unlike us, zebrafish have the ability to regenerate motor neurones when they’re damaged. This means that zebrafish can provide scientists with an excellent tool to find out about motor neurone repair. A better understanding how zebrafish achieve neurone regeneration could provide clues to develop new treatment strategies for MND.
From their studies, Dr Becker and colleagues found that a chemical signaling pathway called Notch1 is increased when the neurones are damaged in zebrafish.
By over activating the Notch1 signal, they found that new motor neurones would not grow well, and would not grow in the same prolific numbers as normal. This means that an over active Notch1 signal can stop the regeneration of motor neurones that occurs in zebrafish.
To test whether the Notch1 signal could be counteracted, the research group treated zebrafish with an anti-Notch1 chemical to stop the Notch1 signal. This increased the generation of motor neurones.
As well as having a deeper understanding of the basic biology of how zebrafish regenerate their motor neurones, this study may help to provide new clues for the development of future treatments for MND.
This type of research is vital to lay the foundations for future studies. With a solid foundation of understanding of how motor neurones work, we can work toward identifying new and better treatments for MND.
What does this mean for people with MND?
This work is still at a very early stage of development. It unfortunately doesn’t mean that a treatment coming from this research will be available soon.
Researchers still need to identify whether developing a drug that can stop, or slow down the Notch1 signaling pathway would be a beneficial treatment strategy. To do this, researchers will need to carry out tests in a cellular, and animal models of MND. This is an important step, as it determines whether treatments are safe and effective before testing the treatment in people.
Zebrafish are increasingly becoming the organism of choice to study both early development and disease. But why are zebrafish important to MND research and can we really learn anything from a fish?
Shall I compare thee to a zebrafish?
Amazingly, we share many of our genes with our finned-friend the zebrafish which means that we really can compare what happens in zebrafish with what happens in humans.
With transparent embryos, zebrafish offer a unique view into the developing fish which means that researchers can study their neurones under a microscope – a feat that is not possible in humans or other mammals. We can also learn about how the disease progresses in fish by measuring their muscle strength by the amount they move, and by measuring their progress swimming against a current in a tube.
Unlike us, zebrafish are also able to regenerate motor neurones if they become damaged. Interestingly – it is not that we do not have this capacity; we have extra signals that tell our motor neurones not to regenerate.
Zebrafish can therefore be used in MND research to gain a greater understanding of the processes that govern both the degeneration and regeneration of motor neurones to develop new and better treatments.
In the past 30 years, the number of scientific articles published about zebrafish has increased 465 fold. Not only does this show the increased use of this model, but also represents our collective increase in understanding more about human diseases and human development.
We’re fishing our way to a world free of MND
One of our newest projects, set to begin later this year, will be using a new zebrafish model of MND to screen over 2,000 potential new drugs to test for their effectiveness. This work will be carried out at the University of Sheffield by Dr Tennore Ramesh and Prof Pam Shaw.
This project will join the ranks of many other MND Association funded projects that are developing new models of MND to learn more about the causes of MND so that we can be in a better position to develop treatments.
We have also recently supported the development of new guidelines for the use of models in MND research in order to improve our confidence in pre-clinical (laboratory) studies and hopefully the success rate of MND clinical trials.
Zebrafish will not be able to provide us with all of the answers as to what causes the disease, or how we can treat it. But, when used in combination with a number of other exciting disease models, including chick embryos, flies and mice, we can push MND research to a new and exciting level.
Our recent blog articles describe lots of fascinating science and the progress in the care and treatment of MND/ALS that was presented at the symposium. Personally, another really positive aspect was the opportunity to meet some of the researchers face-to-face. This included several senior scientists and clinicians whose work we support, some of whom gave lectures or chaired sessions. The symposium also gave presentation opportunities to PhD students and post-doctoral scientists, some of whom were attending their first International Symposium. We invited several from the UK to an ‘ice-breaker’ social on the Friday evening before the main lecture sessions began. Not all were able to attend, but a good group gathered with us, getting to know each other, and we met others later as they presented their posters.
Sheffield University was well represented, including current grantees Emily Goodall and Clare Wood-Allum who both presented posters. Newer to ALS research was Guiseppe (‘Bepe’) Battaglia part of a cross-disciplinary group of collaborators who call themselves ViNCeNS for ‘Virus-like Nanoparticles for targeting the Central Nervous System’ with a website at www.vincens.group.shef.ac.uk/index.htm.
‘Fishing’ for new animal models of MND
Two others at our ‘ice-breaker’ gave lectures during the Saturday session on ‘Emerging Disease Models’. Marc Da Costa (also from Sheffield) described some outcomes of his PhD project developing a new zebrafish with a mutated SOD1 gene. Zebrafish are popular models for neurological conditions (there was a second presentation from an US-based group), as the fish embryos are transparent, so their neurones can be studied easily under the microscope. Their muscular strength can be judged by the amount they move (studied by automated analysis of video) or by their progress swimming against a ‘current’ in a tube. Marc studies the differences between fish with normal or mutant SOD1. The latter have more difficulty swimming, and are more vulnerable to stress (for example, added toxic chemicals). Marc can test the effects of potential drugs on the stressed fish, and has already seen some promising results.
Looking in a ‘library’ for MND mice
Another new animal model was presented by Peter Joyce (MND Association funded, based at the Medical Research Council Laboratories near Oxford). His mouse strain carries a mutation (mistake) in its native (mouse) SOD1 gene, matching one recently reported from a human family with ALS. This is different from the more established SOD1 mouse model, in which the MND-like symptoms develop as a result of multiple copies of an added human mutant gene. Peter is studying the timescale in which muscle problems develop, relating these to changes in the neurones. The MRC has a huge ‘library’ of mouse mutations available, so Peter will be investigating if others match mutations reported in different ALS families, possibly looking for collaborators to work on these.
Presenting the impact of healthcare decisions
A completely different type of research was presented as posters by two researchers working with Carolyn Young at the University of Liverpool. Hikari Ando has been studying the reasons why some people with MND decide not to accept the offer of non-invasive ventilation (NIV), and the extent to it is actually used by at home. Chris Gibbons’ work covered the assessment of quality of life for people with MND, particularly the role of fatigue and depression.
It was particularly inspiring to meet so many UK-based researchers, filling all of us from the MND Association with more incurable optimism. Of course we also met many other people too – the attendees, many of whom collaborate internationally, came from more than 30 countries.