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.
The sod1 zebrafish
In approximately 5-10% of cases MND is inherited and caused by a genetic mutation. The sod1 gene mutation was the first to be identified by researchers in 1993. We created the world’s first adult onset neurodegeneration in zebrafish by developing the mutant sod1 zebrafish. The mutant sod1 transgenic zebrafish developed normally and even over a period of a year when we observe them in a fish tank they appear normal. They are able to swim, reach food and so it initially appeared that they did not show any MND symptoms. However, one must remember that unlike mice, zebrafish live in almost zero gravity as they have an air bladder. This allows them to regulate floating and sinking, meaning they do not require a lot of muscles to move within the fish tank where the water is almost static. These fish in the lab, unlike those in the wild, are not challenged by strong water currents that we see in rivers and streams.
Therefore we replicated these natural settings of the fish by putting them in swim tunnels where we could increase the strength of the water current. In these real life conditions the mutant zebrafish were weak and unable to swim under strong water currents as compared to the healthy ‘normal’ zebrafish. Additionally, their muscle showed atrophy, changes in muscle neurone connectivity and had fewer motor neurones in the spinal cord. Thus, the sod1 zebrafish showed symptoms that was almost identical to those seen in humans.
Early changes stop the neurones from applying the brakes!
This zebrafish can also be used to identify the earliest changes that occur in MND. Mutant sod1 is made by all the cells in the body right from early development throughout life in people living with MND. However, the disease is observed only in adults and primarily affects the nervous system. This was an interesting problem for us to solve. To study how mutant sod1 affected cells, we added a fluorescent gene that glowed red when cells exhibit cellular stress. Thus, whenever any cell is stressed they should glow red. We observed that mutant sod1 zebrafish showed stress at the earliest stages of their development, in embryonic neurones, which then continued to grow into adult neurones. Most interestingly, the neurones that showed stress in the embryos were the neurones that ‘apply the brakes’ on the motor neurones!
Now, one can imagine driving a car without brakes. The car may eventually crash. However, the car would still be functional. Over time, this inability of ‘breaking’ and lack or regulation takes its toll on the motor neurones. As expected, we observed that when the fish get older (in 6 months), their motor neurones start showing signs of stress. The stressed motor neurones also fail to form normal connectivity with the muscle. Thus, we were able to see how early changes in MND may have affected the motor neurones later in life and may explain why its takes decades to see the symptoms of MND in human patients. Recent data suggests that such changes prior to the onset of symptoms are also seen in human MND patients, suggesting that both humans and zebrafish with the disease share a lot of similarities. Read more about this research here.
We tested whether Riluzole, the only drug that has been approved to treat MND, can reduce stress in neurones. As expected we observed a reduction in the stress of the neurones in the zebrafish. Since we observed these findings, we have used ‘neuronal stress’ as a biomarker in the zebrafish to help us identify potential new drugs. To date we have screened a library of 2,000 compounds, many of which are already approved for treatment in humans. Through this screening process, we have identified one compound that has shown promise, as it had similar effects to riluzole. We are now in the process of testing this drug, in combination with riluzole, and our initial studies indicate that a combination of low doses of riluzole and the novel compound have a good ability to reduce the levels of neuronal stress in the zebrafish.
Zebrafish – a model of bulbar MND?
In addition to testing drugs we are also exploring if the zebrafish show bulbar MND, as seen in humans. Difficulty in speech and swallowing is an important aspect of bulbar symptoms in MND. A third of people with MND will present with bulbar symptoms at diagnosis (Green et al 2013) and nearly all people living with MND (85-90%) will develop these symptoms as the disease develops (Beukelman et al 2011). Degeneration of specific types of motor neurones, known as the ‘cranial motor neurones’, leads to bulbar symptoms. The nerve innervating the tongue (known as the hypoglossal) is also found to be the most damaged in human MND patients. However, other muscles including the muscles found in the throat (the pharyngeal muscles) are required for swallowing, and can also be affected in MND. These muscles are supplied by other cranial motor neurones and modelling these clinical symptoms of MND in an animal model is important to understand the disease process and in testing potential new drugs.
So how can one test bulbar dysfunction in zebrafish? While the zebrafish cannot speak (they lack a voice box in their throat), they do have a pharynx, which is important in swallowing.
Additionally, the nerves in the brain that connect to the throat are conserved from human to zebrafish. In order to determine if zebrafish show bulbar MND, we are investigating whether zebrafish show any swallowing deficits. The way in which we are studying this is by feeding fluorescent beads (which have been added to the food) and to see how much of the beads they can eat. Although preliminary, we are seeing that mutant zebrafish may have a slightly reduced ability to eat food as larvae (young fish). Interestingly we have observed that the pharyngeal muscles of the mutant zebrafish show stress, suggesting that these muscles may have some defects starting at a very early stage.
We are now extremely keen to further understand ‘when’ and ‘what’ changes occur in the brain and swallowing muscles of the zebrafish. Additionally, we are also interested in understanding what leads these muscles in the mutant zebrafish to become stressed, when other skeletal muscles of the body do not show stress at such an early stage. We will keep you posted on any interesting developments on this in the future!
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