Non-invasive imaging of neurodegeneration in live animals

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.

A dozen zebrafish swim in deep blue water. Zebra fish are narrow and long. They have two to three black stripes running down their side.
A school of Zebrafish (Photo by Lynn Ketchum, courtesy of Oregon State University)

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.

Continue reading “Non-invasive imaging of neurodegeneration in live animals”

Brain-derived neurotrophic factor: A new (old) hope for the treatment of SCA1

Written by Eviatar Fields Edited by Dr. Vitaliy Bondar

Scientists use Brain Derived Neurotrophic Factor to delay motor symptom onset and cell death in a mouse model of Spinocerebellar Ataxia Type 1

Spinocerebellar ataxia type 1 (SCA1) is a rare neurodegenerative disease that affects about 2 out of 100,000 individuals. Patients with SCA1 present with motor symptoms such as disordered walking, poor motor coordination and balance problems by their mid-thirties and will progressively get worse symptoms over the next two decades. No treatments for SCA1 exists. These motor symptoms cause a significant decrease in patient independence and quality of life. Scientists use mouse models that recreate many SCA1 symptoms to understand the cause of this disease and test new treatments.

In this paper, Mellesmoen and colleagues use a mouse model of SCA1 which presents with severe motor symptoms by adulthood. In order to measure the severity of the motor problems in the SCA1 mouse model, the researchers use a test called a rotarod. The rotarod test is similar to a rolling log balance: mice are placed on a rotating drum that slowly accelerates. Mice that can stay on the drum for longer durations have better motor coordination than mice who fall off the drum earlier. Mellesmoen was trying to find a way to get the mice to stay on the drum for longer.

artistic cartoon of male doctor sin from of a microscope and large DNA model
Cartoon of a medical researcher holding a clipboard. 

Purkinje cells, the main cells of the cerebellum, eventually die in SCA1 mouse models and in patients later in life. However, it remains unclear how and why these brain cells, which are responsible for the fine-tuning of movement and motor coordination, die. This is an important question as its answer might lead to new treatments that prevent brain cells from dying which might improve SCA1 symptoms. One possibility is that some changes in gene expression (that is, how “active” or “inactive” a gene is) causes the cells to die in SCA1 mice. To test this hypothesis, the authors used a technique called RNA-seq to examine how gene expression is altered in SCA1 mice compared to healthy mice.

Continue reading “Brain-derived neurotrophic factor: A new (old) hope for the treatment of SCA1”

Snapshot: What is RNA?

RNA is an important molecule that helps with regulating the function of cells. To fully understand how RNA fits in here, we must first look at the bigger picture: genetics. The central dogma of molecular biology, depicted below, states that DNA is copied (transcribed) into RNA, which is later decoded (translated) into proteins, which perform many vital functions in the cell. So, when the cell needs a specific protein, it locates the stretch of DNA that contains the code for this protein and starts to write a copy of that stretch of DNA. This copy is made using RNA, or ribonucleic acid, as a backbone. RNA is very similar to DNA, but contains one extra oxygen atom in the basic building block. Only one strand of the DNA is copied, so RNA ends up looking like half a DNA molecule. The RNA molecule can be seen as the messenger between the archive of your genes (DNA) and the protein production site. However, RNA is very versatile and is also involved in protein regulation, transport of molecules and as a structural component of large complexes in the cell.

The "central dogma" of molecular biology: DNA makes RNA, then RNA makes protein.
The “central dogma” of molecular biology: DNA makes RNA, then RNA makes protein. Adapted from Wikimedia.

The shifting stream of RNA

Apart from small random mutations during the course of a lifetime, the DNA contained in every cell remains the same from birth to death. However, since different cells need different proteins at different stages of growth, there needs to be a selection of which genes are copied and translated into proteins. This means that the process of making RNA has to be very flexible. This flexibility is achieved through a large network of signals that tell the cell which regions of DNA should be transcribed into RNA, and at what rate. To keep up with the demands of the cell, there are millions of RNAs being made at all times, to send out instructions to makes proteins.

How can RNA cause disease?

In some spinocerebellar ataxias, such as e.g. SCA8, the messenger RNA molecules contain long repetitive sequences that become sticky to other copies of the same RNA or to proteins, forming both small and large clumps in the cell. There is still controversy surrounding which steps in the process that ultimately causes cell death in large brain areas, but it seems that unsolicited binding of these sticky RNAs to proteins and other RNAs causes disruption to several functions in the cell simultaneously. Therefore, many researchers are hopeful that reducing the amount of these RNAs in the cell using Antisense Oligonucleotides or RNA interference can help treat spinocerebellar ataxias and other similar diseases.

If you would like to learn more about RNA, take a look at these resources by the Encyclopedia Britannica and Khan Academy.

Snapshot written by Frida Niss and edited by Dr. Hayley McLoughlin.

Discovery in mice sheds light on how the brain learns to adjust how we walk (video)

Written by Dr. Ambika Tewari Edited by Dr. Sriram Jayabal

New research identifies the cell type in the cerebellum that is vital for a specific form of motor learning

Locomotion – the process of moving oneself from one place to another – is highly adaptive. Depending on our current needs, we can alter the way we walk (known as our gait) without much trouble. For instance, we might increase our speed to get to a meeting in time or, if we have time for a more relaxing stroll, reduce our speed. This locomotor adaptation may seem effortless, but it actually involves a high level of coordination. It is quite apparent when witnessing a toddler trying to walk that figuring out our adaptive mechanisms plays an important role in fine-tuning movements. Because of this, determining how locomotor adaptation works has become a focus of research in the field of rehabilitative therapy, especially with patient populations that experiences gait deficits.

Young toddler boy learning how to walk and balance
Photo of a toddler how to walk. How does of brain learn how to fine tune our movements to help us balance? Photo by Aleksandr Balandin on Pexels.com

In an effort to better understand the intricate details of locomotor adaptation, researchers at the University of Portugal recently performed a study using adult mice. In this study, mice performed a task on a specialized piece of equipment called a split-belt treadmill, which consists of two separate belts running parallel to each other. The speed of these belts can be independently controlled, allowing researchers to impose different demands to the limbs on the right side of the body versus the left. Though split-belt treadmills are used in rehabilitative therapy for patients with post-stroke hemiparesis (where one side of the body is weakened after stroke), this was the first study that adapted the use of this treadmill for mice.

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Snapshot: What is Gene Therapy?

Gene therapy is using nucleic acids to treat a genetic disorder.  These nucleic acids can be designed in a variety of ways to achieve the same therapeutic outcome. Gene therapy tools can be used to correct a mutant gene by one of three ways:

  1. Expressing a healthy copy of a gene
  2. Silencing or inactivating the mutant gene transcript
  3. Using genome editing tools to repair or turn-off the mutated gene.
computer desk laptop stethoscope
Photo of a stethoscope by Negative Space on Pexels.com

How is gene therapy used?

Monogenic disorders, like some spinocerebellar ataxias (SCAs), are excellent targets for gene therapy approaches. Gene therapies are currently being used throughout ataxia research for studying disease mechanisms and for preclinical therapeutic application.

Overview of how gene therapy works. First, Package the healthy gene, RNAi, or gene editing tools into the AAV (can also deliver as naked DNA or in a nanoparticle). Second, Inject the packaged AAV into the tissue of interest. Third, AAV will enter the cell and release the genetic material. The cell will become healthy by either 1) expressing the normal gene, 2) repressing the mutant RNA, or by 3) correcting the mutant gene.
Overview of gene therapy, designed by Stephanie Coffin using Biorender.

One gene therapy approach for rescuing SCA1 phenotypes involves overexpressing a healthy gene, ataxin-1-like, which competes with the mutant ATXN1 protein for complex formation. This work, conducted by Keiser and colleagues in 2016, showed phenotypic rescue in a mouse model of SCA1.

There are two common technologies for silencing or inactivating disease genes: RNA interference (RNAi) or antisense oligonucleotides (ASOs). RNAi strategies utilize small RNA molecules to knock down the expression of target mutant RNA transcripts, while ASOs are DNA molecules used to knock down or correct mutant RNA transcripts. Both therapeutic approaches are being pursued in SCAs. For example, Carmo and colleagues in 2013 showed that using RNAi against the SCA3 disease gene, ATXN3, could longitudinally decrease mutant ATXN3 levels. See the SCAsource snapshot on ASOs for further information about their use in SCAs.

The most common genome editing tool is the CRISPR/Cas9 system, which uses an RNA guide to direct the Cas9 nuclease to the region of the genome to be edited. One can then knockout that gene or correct the mutant gene. It is early days for this technology as a potential therapeutic option due to the challenges of delivery and the risk of off-target editing.

How is gene therapy delivered?

One of the most difficult aspects of gene therapy is how to deliver these various molecules to the cells of interest. One of the most common delivery methods is through viral delivery.  The “drug” nucleic acid is transferred into the disease cells by a vector, which is a virus that has been modified to remove viral components. The most common viral vectors for gene therapies currently are adeno-associated viruses (AAVs). Other delivery methods include non-viral vectors such as naked DNA and nanoparticles.

How long-lasting is gene therapy?

Viral delivery of gene therapy products provides a longitudinal expression of the nucleic acid, while naked DNA and nanoparticles express the nucleic acid drug transiently, thus typically requiring ongoing treatment.

If you would like to learn more about gene therapy, take a look at these resources by the National Institutes of Health and KidsHealth.

Snapshot written by Stephanie Coffin and edited by Dr.Hayley McLoughlin.