Written by Dr. Marija Cvetanovic   Edited by Larissa Nitschke

Purkinje cells (a type of neuron in the cerebellum) are the most vulnerable cells in many Spinocerebellar Ataxias (SCAs). While animal models of SCA have been very fruitful in understanding the mechanisms of Purkinje cell neurodegeneration, none of these models have allowed for visualization of neurodegenerative processes in live animals as the disease progresses – until now. In the laboratory of Dr. Reinhard Köster, researchers have developed a zebrafish model of SCA that allows for the expression of SCA-causing mutant protein in Purkinje cells and proteins that can be used to monitor Purkinje cell changes. As zebrafish larvae are almost transparent, researchers can now study pathogenic changes in neurons in a live animal during disease progression.

Since the 1993 discovery of the mutation that causes Spinocerebellar Ataxia Type 1 (SCA1), we have significantly increased our understanding of disease pathogenesis using animal models. While there are advantages and disadvantages of using any model, most researchers would agree that the similarity between humans and the animal used, plus the cost of creating and caring for the animals, are critical determinants of which model to choose. Mouse models, for instance, are useful to study pathogenesis at the molecular, cellular, tissue and behavioral level, but are costly to house and maintain. Fruit fly models, on the other hand, allow high-throughput studies (that is, studies that can produce a lot of relevant data quickly) of disease modifying properties but are much farther from human beings evolutionarily. Unfortunately, neither of these animal models allow us to follow up changes in neurons in the same animal throughout disease progression – to study the neurons, the animal must be euthanized and the brain must be dissected. Understanding how neurons are affected during disease progression, however, is very important. Observing the same neurons over time could increase our understanding of disease processes and inform us about the optimal timing for therapies. For example, if we were to identify changes in neurons that occur just prior to the onset of motor symptoms, this might mean that these changes are a contributing factor to behavioral pathology. This could also tell us the stage at which neurons start dying and disease thus becomes irreversible.

In an effort to examine how cells behave over time, many researchers use zebrafish. The fact that zebrafish embryos (larvae) are mostly transparent means that we can follow changes in neurons throughout disease progression. Moreover, in most SCAs, Purkinje cells in the cerebellum are the neurons that are most affected by the disease-causing mutant protein, and the zebrafish cerebellum has an anatomy and function that is quite similar to the human cerebellum. Zebrafish are also inexpensive and produce hundreds of offspring weekly, providing researchers with a large number of animals to study.

Using state-of-the-art genetic approaches, Dr. Reinhard Köster’s laboratory at the Technical University of Braunschweig in Germany created a zebrafish model of SCA that expresses two types of protein in their Purkinje cells: a disease-causing SCA mutant protein, and a fluorescent reporter protein to monitor degenerative changes and cell death.

The authors started by identifying the specific DNA sequence that would allow for the selective expression of proteins only in Purkinje cells. They then modified this sequence, adding in the genes for the expression of both the disease-causing protein and the disease-monitoring protein. After taking the fertilized eggs from these genetically-modified zebrafish, the researchers then used very precise microscopy to follow up on changes in the  Purkinje cells: specifically, they assessed the morphology (size and shape) of the Purkinje cells in live, behaving animals over the period of several days. Because different SCAs require different quantities of mutant proteins to cause disease, authors also included the option to have different amounts of mutant protein expression in the zebrafish. By allowing the DNA sequence to be adjustable between low, middle, and high quantities of protein, many types of SCAs could be modeled with this system.

As a proof of concept, the authors then used these tools to model SCA13. SCA13 is an inherited SCA caused by a mutation in the gene KCNC3, which encodes the ion channel Kv3.3. SCA13 patients with this mutation, known as KCNC3^R420H, have progressive cerebellar atrophy and motor symptoms.

The authors created zebrafish that express either normal or mutant kcnc3 in  Purkinje cells, plus a red fluorescent reporter gene that marked the surface of the neurons and a green fluorescent reporter gene that marked the nucleus of the neurons. This allowed researchers to monitor changes in neuronal morphology in red and also quantify the loss of neurons by counting the number of green nuclei. They imaged zebrafish larvae at four different time points in disease progression (4, 7, 11 and 14 days after fertilization) and were able to see shrinkage of  Purkinje cells and a gradual reduction in the number of Purkinje cells in the SCA13 zebrafish larvae when compared to normal zebrafish larvae. In addition, SCA13 larvae demonstrated changes in behavior, such as impaired eye movement, which provided the team with a relevant behavioral test (impaired eye movement is one of the symptoms exhibited by some SCA patients).

In summary, Dr. Köster and his team have developed a versatile tool for SCA research: using zebrafish as a model system, researchers can now track progressive neurodegenerative changes in live animals and connect these to the appearance and progression of motor symptoms. In addition, since zebrafish are relatively cheap and can produce a large number of offspring quickly, this model system will allow researchers to perform reproducible, high-throughput studies. For these reasons, we think these zebrafish models would also be quite useful for future studies aimed at testing the effectiveness of new therapeutic approaches.

Key Terms

Cerebellum: A primary area of pathology in the spinocerebellar ataxias. This brain region sits toward the back of the skull and, though small in stature, contains the majority of the nerve cells (neurons) in the central nervous system. Contains the circuits that fine-tune our movements, giving us the ability to move with precision.

Purkinje Cells: A type of neuron in the cerebellum. They are some of the largest cells in the brain. They help regulate fine movement.  Purkinje cell loss/pathology is a common feature in cerebellar ataxia.

Zebrafish: A small freshwater fish of the minnow family whose larvae are almost transparent, which allows researchers to examine changes in the living animal

Conflict of Interest Statement

The authors and editor declare no conflict of interest.

Citation of Article Reviewed

Namikawa, K., Dorigo, A., Zagrebelsky, M., Russo, G., Kirmann, T., Fahr, W., … & Köster, R. W. (2019). Modeling Neurodegenerative Spinocerebellar Ataxia Type 13 in Zebrafish Using a Purkinje Neuron Specific Tunable Coexpression System. Journal of Neuroscience, 39(20), 3948-3969.