A Potential Treatment for Universal Lowering of all Polyglutamine Disease Proteins

Written by Frida Niss Edited by Dr. Hayley McLoughlin

One drug to treat them all: an approach using RNA interference to selectively lower the amount of mutant protein in all polyglutamine diseases. Work by a group in Poland shows initial success in Huntington’s Disease, DRPLA, SCA3/MJD, and SCA7 patient cells.

Can one drug treat nine heritable and fatal disorders? Polyglutamine diseases are disorders in which a gene encoding a specific protein is expanded to include a long CAG repeat. This results in the protein having a long chain of the amino acid glutamine, which disturbs the ability of the protein to fold itself and interact correctly with other proteins. This type of malfunctioning protein would normally be degraded by the cell, but in the case of polyglutamine proteins this seems unusually difficult. This causes a gradual build-up of faulty protein that disrupts several cellular pathways, eventually leading to cell death in sensitive cells. Currently there is only symptomatic treatment of these fatal diseases available, and they do not slow down the disease progression. One promising line of research is investigating the possibility of lowering the amount of these disease proteins using RNA interference.

RNA interference is the method by which a gene is silenced through a manipulation of a natural defense mechanism against viruses. When a virus attacks, it tries to inject DNA or RNA like particles to hijack the cell’s machinery for its own survival. To defend itself, the cell uses the RNA interference pathway, where the protein Dicer slices the DNA/RNA into smaller pieces and loads it into the RNA-induced silencing complex (RISC complex). The RISC complex finds all DNA/RNA particles in the cell with the same sequence and destroys them, effectively hamstringing the virus.

This machinery can be co-opted as a potential tool for treating neurodegenerative diseases caused by harmful mutant proteins. By inserting a small interfering RNA (siRNA), we can target the mRNA that codes for the harmful protein and trick the RISC complex into degrading it. In polyglutamine diseases, this has been successful when the mutant mRNA possesses a small mutation called a single nucleotide polymorphism (SNP). However, when an siRNA is delivered to a cell using a vector, which is a circular piece of DNA carrying genetic material, the Dicer protein tends to process the siRNA in unpredictable ways. This means that the treatment may not always be selective, and can end up targeting the normal protein as well. Moreover, not all patients have the same SNPs, so several drugs for every disease might be needed.

A pipette transfering liquid between small centifuge tubes
Close up picture of scientific research being conducted in a laboratory. Image courtesy of the University of Michigan SEAS.

In the paper by Kotowska-Zimmer and colleagues they have used short hairpin RNAs (shRNAs) targeting the CAG repeat tract itself instead of siRNAs targeting SNPs around the CAG repeat tract. shRNAs fold themselves like a hairpin when transcribed, and this loads them into the RISC complex through a somewhat different pathway, with less degradation along the way than conventional siRNAs. The second part that is different to other RNA interference strategies in this study is that the shRNA does not completely match the CAG repeat, but contains mismatches. This means that the RISC complex cannot actually cut and degrade the mRNA, and ends up simply sitting on the CAG repeat tract instead. The longer the repeat tract, the more RISC complexes can fit on the tract and block translation. Using this type of RNA interference Kotowska-Zimmer and colleagues have tried to lower the expression of huntingtin, atrophin-1, ataxin-3 and ataxin-7 proteins in cellular models of the corresponding polyglutamine diseases.

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Snapshot: How does CAG tract length affect ataxia symptom onset?

The instructions our bodies need to grow and function are contained in our genes. These instructions are made up of tiny structures called nucleobases. There are four types of nucleobases in DNA: adenine (A), cytosine (C), guanine (G), thymine (T). By putting these four nucleobases in different orders and patterns, this writes the instructions for our body.

artists drawing of a blue DNA molecule
A cartoon strand of DNA. Image by PublicDomainPictures from Pixabay

Some of the genes contain long sections of repeating ‘CAG” instructions, called CAG tracts. Everyone has repeating CAG tracts in these genes, but once they are over a certain length they can lead to disease. Some ataxias are caused by this type of mutation, including SCA1, SCA2, SCA3, SCA6, SCA7 and SCA17. These are often called polyglutamine expansion disorders. This is because “CAG” gives the body instructions to make the amino acid glutamine. You can read more about what is polyglutamine expansion in our past Snapshot about that subject.

For each disorder caused by a CAG expansion mutation, the number of times the CAG is repeated in a particular gene will determine whether someone will develop the disease. Repeat lengths under this number will not cause symptoms and repeat lengths over the threshold will usually lead to ataxia. When someone undergoes genetic testing for ataxia, doctors will be able to tell them the length of these CAG tracts and whether they have a CAG repeat number in one of these genes that is over the threshold. This table gives a summary of different CAG expansion mutations that can lead to ataxia and how the length of the repeat affects age of onset.

 Affected Gene Normal
Repeat Size
Repeat Size
SCA6CACNA1A 4-1821-33

For SCA1, SCA2, SCA3, SCA6, and SCA7; longer CAG tracts are associated with earlier onset.

For SCA12, it is hard to predict the age of onset based on repeat length as SCA12 is so rare. Some individuals with long repeats don’t develop ataxia. One study found that longer CAG tract lengths are associated with earlier onset but that it does not affect the severity of symptoms.

For SCA17, Longer CAG tracts have separately been associated with an earlier age of onset and more severe cerebellar atrophy.

In general, people with longer repeat lengths in ataxia genes are likely to present with ataxia symptoms earlier in life. However, it is important to remember that there are many other factors involved. Other genes may have mutations that either worsen the progression of ataxia or protect against more severe symptoms. Therefore, in individual people, the length of the repeat is not always enough information to determine when that person will start showing symptoms, or how severe these symptoms will be.

If you would like more information about the genetic causes of SCAs, including information about genetic testing and what CAG repeat length might mean, take a look at these resources by the National Ataxia Foundation.

Snapshot written by Anna Cook and edited by Larissa Nitschke.

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Eyes: Windows to peek at brain function in spinocerebellar ataxias

Written by Dr. Sriram Jayabal Edited by Dr. David Bushart

Eye movement deficits occur ubiquitously in spinocerebellar ataxias, even at early disease states, highlighting their clinical importance.

Imagine the different motor movements that you make in your everyday life. Many people think of actions that we perform using our hands and legs, such as reaching for objects or walking. Let’s zoom in on a different task: catching a baseball. You need to know where the ball is going to land so you can run to that spot, then guide your arms while diving, if need be, to catch the ball. For this to work perfectly, you need to see and follow the ball. Your eyes enable you to track the ball while it is moving. How can your eyes keep the ball in focus while you are running at full speed towards the spot where you expect the ball to land? Your eyes are equipped with muscles which enable the eyes to move so as to keep the visual scene in focus. These eye movements, as demanded by the needs of the current scenario, in this case, catching a baseball, are indispensable for us to see the world without any hindrance.

Woman with hand in a "C" shape in front of her face. She's focusing in on her eye.
The eyes may provide a window into spinocerebellar ataxia, even before other symptoms show up. Photo by fotografierende on Pexels.com

Which brain region gives us the power to do this?

The cerebellum, or “little brain”, which enables one to move their arms and legs precisely, also controls the way we move our eyes. Therefore, it is logical to posit that when cerebellum goes awry, it may lead to eye movement abnormalities. Several previous studies have shown this to be true in many spinocerebellar ataxias (SCAs), where non-gait symptoms such as eye movement abnormalities have been found to accompany gait deficits in advanced stages of the disease. However, recent work from pioneers in clinical ataxia research at the Harvard Medical School have shown that eye movement abnormalities are also commonly present in SCAs even in pre-symptomatic states. This study emphasizes the critical need to better document the history of eye movement deficits and track them throughout the progression of the disease. This will help researchers to develop better rating scales for ataxia.

In this study, a population of SCA patients (134 individuals) who exhibited different types of SCA (including SCA1, SCA2, SCA3, SCA5, SCA6, SCA7, SCA8 and SCA17) were assessed for eye movement abnormalities at different stages of the disease, from pre-symptomatic (with no gait deficits) to advanced stages (those who use a wheel-chair). First, it was found that ~78% of all pre-symptomatic individuals exhibited eye movement deficits, and these deficits became even more common as the disease progressed, where every single person in advanced stages exhibited eye movement deficits. Second, when researchers examined the eye movements closely, they found that different types of ataxia might cause different kinds of eye movement deficits. However, these results are only suggestive because of the small population size of early-stage SCA individuals in this study, and the types of assessments used. Therefore, future studies will require a larger population size and a thorough quantitative analysis of specific types of eye movement deficits to help characterize eye movement abnormalities in SCAs. Finally, the Brief Ataxia Rating Scale (BARS), a recently designed simple clinical test for ataxia, was further improved in this study to account for the clinically observed eye movement deficits in SCAs. With such a nuanced metric, an improved BARS score was found to correlate with the stage, severity and duration of the disease irrespective of the type of ataxia.

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Snapshot: What is RNAi?

RNA interference, or RNAi, is a natural biological process that inhibits the expression of a specific gene. In medicine, targeted RNAi therapies can be used to silence the expression of a disease-causing gene. To understand RNAi, you first have to understand RNA.

DNA is transcribed to make mRNA, which is tranlated by the ribosome to make protein.

An overview of  RNA is the messager between the DNA (the instructions) and the protein (the product). RNA is transcribed from the DNA. The ribosome translates the mRNA into protein. Graphic designed by Colleen Stoyas and illustrated by Celeste Suart.

Genes encode the instruction manual of our biology, but this material cannot leave the nucleus of your cells. Think of genes as a lecturer that provides instruction for your homework, which you must copy and take home to use later. The equivalent of copying this message in the cell is RNA, which transcribes the gene instructions and leaves the nucleus to be read and translated into protein. This protein then performs functions within the cell (see above image).

How can RNAi be used in ataxia?

In specific forms of ataxia, a gene mutation may provide the instructions for a protein that acts improperly and leads to disease. RNAi is a method of silencing RNA that interferes with the reading of this message, keeping a protein from being made. It works by generating a small interfering RNA in the laboratory that matches the gene of interest. When this small interfering RNA enters the cell, it binds the matching messenger RNA copied from a gene. When these two RNAs bind, the cell is triggered to cut up the message and destroy it. This means the disease-causing protein is never made. (see below image)

RNAi works by binding the mRNA, preventing it from being transcribed by the ribosome. This stops protein from being made.
How does RNAi work? It binds matching messenger RNA. This stops it from being translated by the ribosome into protein. Graphic designed by Colleen Stoyas and illustrated by Celeste Suart.

While RNAi is straightforward in the lab, getting it to work in humans can be tricky. The small interfering RNA cannot be taken in a pill, because it will not survive digestion. Additionally, the small interfering RNA is degraded along with the target messenger RNA, and so it must be continually administered. Using a viral payload, or encapsulating the interfering RNA in the coat proteins of a virus, has successfully delivered RNAi therapies in mouse models of SCA1, SCA3, and SCA7. In this method the virus integrates into your cells, which can then continue to produce the small interfering RNA. This means a single dose could potentially be all that is needed. Viral delivery to the brain is complicated, but not impossible. More work remains to be done clinically in order to determine if RNAi therapy is viable in a viral payload to treat multiple forms of spinocerebellar ataxia.

If you would like to learn more about RNAi, take a look at this video by TED-ED or entry in the Encyclopedia Britannica.

Snapshot written by Dr. Colleen Stoyas and edited by Frida Niss.

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Mitochondrial impairments identified in SCA7 mouse model and patient cells

Written by Dr. Colleen A. Stoyas Edited by Dr. Monica Banez 

Duke University researchers have found that altered cellular metabolism and mitochondrial dysfunction play a central role in spinocerebellar ataxia type 7 (SCA7), a result that has therapeutic implications for this disease.

Spinocerebellar ataxia type 7 (SCA7) is a dominantly-inherited ataxia characterized by retinal degeneration and cerebellar atrophy. As retinal degeneration advances, patients experience progressive central vision loss. Atrophy (i.e., cell loss) in the cerebellum causes a progressive loss of balance, as the cerebellum is the region of the brain that controls coordinated movement and motor learning. SCA7 patients also experience difficulty speaking and swallowing in later stages of the disease. Symptoms can manifest at any age, though the disease is particularly aggressive when found in infants and children. SCA7 is caused by an expansion mutation in the Ataxin-7 (ATXN7) gene, which produces a protein containing extra repeats of the amino acid glutamine. These additional glutamines make the protein fold in an incorrect shape. Much like an umbrella turned inside-out, this protein, once it loses its shape, does not work in the way it’s meant to. Dr. Albert La Spada has previously shown that the ataxin-7 protein is necessary for the expression of genes that are central to the normal function of the eye – particularly, the retina. Now, his group has provided evidence that abnormal cellular metabolism underlies the brain changes observed in SCA7.

Mice whose brains carry the SCA7 mutation model the juvenile forms of this disease. Using this mouse model, the La Spada group observed changes in the network and physical size of the brain’s mitochondria. Mitochondria are the cell’s “power plants,” and are responsible for the chemical reactions (known as cellular metabolism) that generate the energy our cells need to function. Cellular metabolism is assessed by measuring metabolites, which are the products of these chemical reactions. The La Spada group’s researchers identified dysfunction in the mitochondria in SCA7 due to an underlying decrease in one specific metabolite: NAD+.

microscope and sample slide
Photo by Pixabay on Pexels.com

Short for nicotinamide adenine dinucleotide, NAD+ is necessary for proper mitochondrial function. A general reduction of NAD+ occurs as humans age, as well as in a host of other neurodegenerative disorders (many of which exhibit mitochondrial dysfunction). This recent recent by Dr. La Spada and his team has shown that NAD+ is also reduced in mitochondria in SCA7.

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