Mitochondrially Stressed

Written by Dr. Judit M. Pérez Ortiz Edited by Dr. Brenda Toscano Márquez

Scientists describe how SCA2 oxidative stress can affect mitochondrial function, and potentially how to fix it

Mitochondrial Stress

We all have experienced stress. When cramming for an exam last minute, or getting ready for a job interview, our bodies feel stress-related energetic drive and hyperfocus. Small bursts of stress can help us get through specific demands, but too much constant stress takes a toll and makes it difficult for us to function. It turns out that the cells in our bodies experience stress too! While the stress response that we experience in our hectic lives is associated with stress hormones, the stress cells experience is from another source altogether – mitochondria. Scientists at the University of Copenhagen in Denmark identified a novel link between mitochondrial oxidative stress and spinocerebellar ataxia type 2 (SCA2).

Classically, we learn that mitochondria are the powerhouse of the cell responsible for making the bulk of the energy currency that cells need to work and survive, ATP. To do this, mitochondria rely on a cooperative group of protein complexes called the Electron Transport Chain (ETC). Albeit via a more sophisticated procedure than a hot-potato game, the complexes mediate chemical reactions (called redox reactions) by which “hot” electrons are passed from high energy molecules to lower-energy molecules, and so on. The final electron recipient (“acceptor”) is a stable oxygen molecule and their encounter is used to make water. The activity of the ETC helps harness energy that is ultimately used to make ATP in what is called oxidative phosphorylation.

Sometimes not all the electrons make it through; the hot potato “drops”. Electrons leak out and react directly with molecular oxygen (chemical formula O2), turning unstable superoxide (chemical formula O2) which in turn, can create other reactive oxygen species (ROS). The extra electron in superoxide gives it a negative charge and makes it highly reactive and toxic. Just like the small amount of stress primes your body for a challenge to come, low levels of ROS hints the cell that it needs to make some changes to optimize the system. As the superoxide levels go up, cells make more antioxidant enzymes available to keep ROS in check. Antioxidant enzymes convert the highly reactive superoxide to a less reactive hydrogen peroxide (like the one in your bathroom cabinet). This, in turn, can be converted to water and ordinary oxygen molecules. In a word, the antioxidants “detox” the cells from ROS insult.

The cell becomes “stressed out” when there’s too much ROS that can’t be compensated for. This stress caused by oxygen or “oxidative stress” can damage DNA, fats, and proteins that affect the cell and organism as a whole. For example, oxidative stress can contribute to heart disease, diabetes, cancer, and neurodegenerative diseases.

cartoon drawing of human cells that are blue
An artist’s drawing of human cells under a microscope.

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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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Zapping the brain to help ataxia

Written by Dr. Judit M. Perez Ortiz Edited by Dr. Sriram Jayabal

In a new study, scientists have found that “zapping” the brain with an electromagnetic wand may someday help patients with spinocerebellar ataxia.

In an era of ever-evolving technological advances used for personal entertainment and space travel, medical scientists are harnessing the power of electromagnetism to safely penetrate the skull and manipulate brain cells by mimicking their favorite language – electric current.

Clinicians currently have access to powerful and effective tools designed to stimulate brain cells (known as neurons) for various neurological and psychiatric conditions. Spinocerebellar ataxias (SCAs), however, are not yet in the mix. Though several techniques exist, the methods used to stimulate neurons in the brain can be broadly classified into invasive and non-invasive approaches. For instance, Vagus Nerve Stimulation is used for drug-resistant epileptic seizures, while Deep Brain Stimulation is used for Parkinson’s disease and severe depression. In both instances, a surgical procedure is required because the implanted electrodes have to come in direct contact with the target nerve or brain structure. Disadvantages associated with these surgical methods include the risk of infection, bleeding, and hardware malfunction. Non-invasive approaches to stimulate the brain include electroconvulsive (“shock”) therapy, in which electrodes are placed on the scalp surface to provoke a controlled seizure that yields a therapeutic effect. However, shock therapy requires anesthesia, and patients run the risk of memory issues as a side effect. A second non-invasive brain stimulation tool is also available, called repetitive Transcranial Magnetic Stimulation (rTMS). There are many factors that make rTMS clinically appealing: it does not require surgery, it is already FDA-approved (for severe depression), it is painless, and it has been found to be safe. Further, unlike the broad brain stimulation achieved by electroshock therapy, rTMS delivers a more precise stimulation in a defined brain region, which leaves untargeted brain regions untouched.

cartoon of neuronal brain cells and electricity flowing between them
Artist’s depiction of electrical signals in the brain. Image courtesy of flickr.

Besides its circular or figure-eight attachment, the rTMS device looks quite a bit like a magic wand. Though this is no wizard’s tool, you could say that it does cast a powerful spell: the attachments on the end of the rTMS device are electromagnetic coils, which have the power to “zap” specific brain regions. In a remarkably simple procedure, the wand is gently placed over the patient’s scalp, where it delivers electromagnetic pulses that create just enough electric current to stimulate underlying brain cells without adversely affecting them.

A new pilot study conducted at the Beth Israel Deaconess Medical Center found that using rTMS to stimulate the cerebellum of SCA patients is safe and may improve some aspects of ataxia. First, the investigators recorded the study participants’ baseline movement performance using a battery of tests designed to evaluate different features of ataxia, including balance, gait, and posture. Then, half of the study participants were randomly assigned to receive rTMS, while the other half were assigned to the control, or “sham” group.

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Snapshot: What is RAN translation?

In many diseases caused by repeat expansion mutations in the DNA, harmful proteins containing repetitive stretches are found to build up in the brain. The repeat expansion mutation, when translated into a protein, results in an abnormally expanded repeat tract that can affect the function of the protein and have harmful consequences for the cells. Following a study published in 2011, we know that repeat expansion mutations can make additional harmful repeat-containing proteins by a process called Repeat Associated Non-AUG translation or RAN translation.

How are proteins made?

To get from DNA to protein, there are two main steps. The first step involves the conversion of a gene in the DNA into an instructional file called messenger RNA (mRNA). The second step is translation, this is where the cellular machinery responsible for making proteins uses mRNA as a template to make the protein encoded by the gene.

During translation mRNA is “read” in sets of three bases. Each set of three bases is called a codon and each codon codes for one amino acid. There is a specific codon that signals where to start making the protein, this codon is AUG. From the point where the cellular machinery “reads” the start codon, the mRNA is “read” one codon at a time and the matching amino acid is added onto the growing protein.

What happens when there is a repeat expansion mutation?

As the name suggests, Repeat Associated Non-AUG (RAN) translation is a protein translation mechanism that happens without a start codon. RAN translation occurs when the mRNA contains a repeat expansion that causes the mRNA to fold into RAN-promoting secondary structures. Because RAN translation starts without an AUG start codon, the mRNA can be “read” in different ways.

Let’s consider a CAG repeat expansion to illustrate this process. In the CAG “reading frame” a polyglutamine containing protein would be made because the codon CAG leads to incorporation of the amino acid glutamine. But a CAG repeat expansion could also be “read” as an AGC or a GCA repeat expansion if you don’t know where in the sequence to start “reading”. When “read” as AGC, the cellular machinery would incorporate the amino acid serine, making a polyserine repeat protein. In the GCA frame a polyalanine repeat protein would be made. This has been shown to happen in Huntington’s disease (HD). In HD, RAN-translated polyserine and polyalanine proteins accumulate in HD patients’ brains, along with the AUG-initiated mutant huntingtin protein containing a polyglutamine expansion.

Diagram show how different DNA sequences can be "read" and translated as different proteins
Overview of repeat proteins that can be produced by RAN-translation from a CAG expansion transcript. Designed by Mónica Bañez-Coronel.

To complicate matters more, RAN translation can happen from different repeat expansions, including those in regions of the DNA that aren’t normally made into proteins at all. Through the process of RAN translation, repeat expansion mutations in the DNA can give rise to multiple different proteins that aren’t made in healthy individuals. RAN proteins have now been identified in several neurodegenerative diseases where they have been shown to be toxic to cells, including in HD, spinocerebellar ataxia type 8, myotonic dystrophy type 1 and 2, and C9orf72 amyotrophic lateral sclerosis (ALS).

To learn more about the implications of RAN proteins for repeat expansion diseases see this article by Stanford Medicine News Center.

To learn more about the process of translation see this article by Nature.

https://www.nature.com/scitable/topicpage/translation-dna-to-mrna-to-protein-393/

For the original article describing RAN translation see this article by PNAS, and this article by Neuron about RAN translated proteins in Huntington’s disease.

Snapshot written by Dr. Hannah Shorrock and edited by Dr. Mónica Bañez-Coronel.

Recovering Purkinje cell health could improve quality of life in SCA3

Written by Jorge Diogo Da Silva Edited by Dr. David Bushart

Normalizing neuronal dysfunction in SCA3/MJD by activating a receptor inside cells

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an inherited neurodegenerative disease that typically begins in mid-adulthood. This disease causes loss of coordination and balance (a group of symptoms known as ataxia), abnormal eye movements, and other motor symptoms, all of which limit a patient’s daily life activities. Treating SCA3 patients is currently very challenging, since there are no drugs or other treatments that slow or stop the progression of this disease. While several therapeutic options have been tested in clinical trials, none have shown considerable and consistent effects in improving disease symptoms. Therefore, it is imperative that other treatments are investigated and tested in the clinical setting, in the hopes that we might find a way to improve the lives of SCA3 patients.

The cause of this disease is very well-characterized: patients with SCA3 have an abnormal form of a protein called ataxin-3. All proteins are made up of a sequence of several smaller building blocks known as amino acids. In ataxin-3’s sequence, there is a region where one type of amino acid, glutamine, is repeated consecutively. SCA3 arises when the number of these repeated amino acids is very high (an abnormality known as a polyglutamine expansion), which is toxic for cells.

One of the regions of the brain that is most responsible for regulating balance and movement coordination is the cerebellum, which is located just behind the brainstem (the region connecting the spinal cord to the rest of the brain). As expected, the cerebellum is one of the most affected brain regions in SCA3, since it helps control gait and coordination. Purkinje cells, which are some of the largest neurons in the brain, make up a substantial portion of the cerebellum. These cells receive information from other neurons that detect our surroundings, then emit a signal to the brain regions that control muscles and regulate our movement. This allows us to make movements that are coherent and fluid.

cross section of the cerebellum with purkinje cells stained blue
Cerebellum Cross Section with Purkinje Cells. Image courtesy of Berkshire Community College Bioscience Image Library

Since Purkinje cells are dysfunctional in SCA3, it is reasonable to think that improving the well-being of these cells could also reduce symptoms. In a recent publication, Watanave and colleagues described how they explored a strategy to improve Purkinje cell function using drugs in a mouse model of SCA3, with findings that could be relevant for future studies in patients.

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