A New Use for Old Drugs

Written by Dr. Amy Smith-Dijak Edited by Logan Morrison

Basic biology helps identify a new treatment for ataxia

Drug design doesn’t always have to start with a blank slate. Sometimes understanding how existing drugs work can help researchers to design new ones, or even to recombine old drugs in new and more effective ways. That’s what the researchers behind this paper did. They investigated the basic biology of three existing drugs: chlorzoxazone, baclofen, and SKA-31.

Two of these – chlorzoxazone and baclofen – are already FDA-approved for use as muscle relaxants, and chlorzoxazone had previously been found to have a positive effect on eye movements in spinocerebellar ataxia type 6. Looking at the results of their experiments, they realized that a combination of chlorzoxazone and baclofen would probably be an effective treatment for ataxia over a long period. They offered this drug combination to patients, who had few adverse effects and showed improvement in their diseasesymptoms. Based on these findings, the researchers recommended that larger trials of this drug combination should be conducted and that people trying to design new drugs to treat ataxia should try to interact with the same targets as chlorzoxazone.

mutliple types of drugs in pill form scattered ac
Can old drugs have potential for new types of treatment? Photo by Anna Shvets on Pexels.com.

When this paper’s authors started their research, they wanted to know more about how ataxia changes the way that brain cells communicate with each other. Brain cells do this using a code made up of pulses of electricity. They create these pulses by controlling the movement of electrically charged atoms known as ions. The main ions that brain cells use are potassium, sodium, calcium and chloride. Cells control their movement through proteins on their surface called ion channels which allow specific types of ions to travel into or out of the cell at specific times. Different types of cells use different combinations of ion channels, which causes different types of ions to move into and out of the cell more or less easily and under different conditions. This affects how these cells communicate with each other.

For example, a cell’s “excitability” is a measure of how easy it is for that cell to send out electrical pulses. Creating these pulses depends on the right ions entering and exiting the cell at the right time in order to create one of these pulses. Multiple types of spinocerebellar ataxia seem to make it difficult for Purkinje cells, which send information out of the cerebellum, to properly control the pattern of electrical signals that they send out. This would interfere with the cerebellum’s ability to communicate with the rest of the brain. The cerebellum plays an important roll in balance, posture and general motor coordination, so miscommunication between it and the rest of the brain would account for many of the symptoms of spinocerebellar ataxias.

Earlier research had found a link between this disrupted communication and a decrease in the amount of some types of ion channels that let potassium ions into Purkinje cells. Thus, this paper’s authors wanted to see if drugs that made the remaining potassium channels work better would improve Purkinje cell communication.

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Snapshot: What is an action potential?

You may have heard that nerve cells (or neurons) in the brain use electrical activity to communicate with one another. The proteins responsible for creating these electrical signals are called ion channels. How do neurons use these electrical signals to communicate with one another in a meaningful way?

A good way to think about the brain is that it is wired into circuits. These brain circuits are not unlike the electrical circuits that power our home, computers, and cell phones. This means that within these circuits, an individual neuron must be able to send and receive electrical signals to and from its neighbors. The electrical signals used to transmit information down the length of a neuron are called action potentials.

cartoon of blue neurons sening small electrical signals to eachother
An artist’s drawing of action potentials (white) being sent out by neurons (blue) to communicate with eachother. Image courtesy of Flickr.

Brain communication: the action potential

Neurons use the electricity harnessed from the opening of ion channels to generate an action potential. Normally, when a neuron is not active, it rests at a negative resting state (or, a hyperpolarized membrane potential). This means that ion channels are closed and the neuron is not actively sending any signals to other neurons. Don’t worry, these voltages aren’t large enough to zap you! Neurons operate in a range between -90 millivolts and +40 millivolts, which is thousands of times smaller than the voltages that power circuits in our homes!

A neuron can become activated with the opening of certain ion channels, particularly ones that allow sodium ions into the neuron. Each time a sodium ion flows into the neuron, the membrane potential becomes slightly more positively charged, or depolarized. Once a specific threshold of depolarization is reached, a huge number of sodium channels open all at once and the cell’s membrane potential moves up to +20 mV.

Interestingly, another major type of ion channel, called a potassium channel, becomes activated on a slight delay compared to when sodium channels open. When potassium channels open, a large amount of positively-charged potassium quickly exits the neuron. This exit of potassium causes the membrane potential returns to its negative resting state. This allows the membrane to become hyperpolarized back to where it began. Now the cycle can start all over when another signal tells the neuron it is time to act. This whole cycle, from -70 mV to +20mV and back again, is the definition of an action potential. Action potentials quickly travel down the neuron in a single direction. Once they reach the end, they help generate a different type of chemical signal that tells the next neuron to generate an action potential of its own. And thus, the information continues to travel through the circuit.

An action potential is an all-or-none response. This means that if the threshold voltage is not reached, the neuron will remain silent and no action potential will be fired. In most neurons, a signal from a neighboring neuron causes the opening of sodium channels to help this signal initiate. However, in certain cell types (such as Purkinje neurons), action potentials can happen spontaneously, and all the time – sometimes even hundreds of times per second!

Approximate plot of a typical action potential shows its various phases as the action potential passes a point on a cell membrane. The membrane potential starts out at approximately −70 mV at time zero. A stimulus is applied at time = 1 ms, which raises the membrane potential above −55 mV (the threshold potential). After the stimulus is applied, the membrane potential rapidly rises to a peak potential of +40 mV at time = 2 ms. Just as quickly, the potential then drops and overshoots to −90 mV at time = 3 ms, and finally the resting potential of −70 mV is reestablished at time = 5 ms.
A diagram of how researchers plot a typical action potential. Image courtesy of WikiMedia.

Why do neuronal action potentials matter for cerebellar ataxia?

Researchers who study mouse models of ataxia have noticed that Purkinje neuron action potentials can undergo big changes during disease. Depending on the type of cerebellar ataxia, Purkinje neuron action potentials may take on a different shape or even disappear completely. Either of these situations could make it difficult, or even impossible, for a neuron to send proper signals to its neighbors. Some researchers suggest that improving action potential firing might be one way to improve ataxia symptoms. For this reason, identifying drugs that improve action potential firing is a major area of therapeutic research in ataxia.

If you would like to learn more about action potential, take a look at these resources by Khan Academy and Wikipedia.

Key Definitions

Membrane potential: the electrical voltage of a cell’s outer membrane. Changes in membrane potential are controlled by the opening and closing of many different types of ion channels.

Hyperpolarized: a negatively-charged membrane potential. A neuron usually rests at -70 mV when it is silent. It returns to that voltage after an action potential is completed.

Depolarized: a positively-charged membrane potential. This usually occurs when sodium channels open during the early part of an action potential. This cuases the cell quickly jumps up to +20 mV.

Snapshot written by David Bushart and edited by Celeste Suart.

Snapshot: What is poly-ADP-ribose (PAR)?

DNA repair is an important topic when talking about of neurodegenerative disorders. The amount of biochemical stress the brain experiences increases naturally as we age. Some connections have been made between the amount of stresses on the brain and the age people develop neurodegenerative disorders.

Many of these natural stresses can damage DNA. For this reason, many researchers are trying to find ways of helping or fixing DNA repair. Chemicals that effect DNA repair could be used as new drugs. Here, we will focus on just one part of the DNA damage response that has been a great success in cancer drug discovery.

PAR is like a net that pulls in proteins that repair DNA

Poly-ADP-ribose, also called PAR, are long molecules in the cell. They are made of of the same building blocks cells use to store enegry. PARylation is when these long chains of PAR are made and attached to different parts of the cell. This happens in response to many different types of stress. For example, a stress could be if a cell’s DNA is damaged or it is infected with a virus.

When DNA damage happens, PAR molecules are attached on the surface of proteins and can act as a basket to trap other proteins. PAR is made and woven together by PAR polymerase proteins (called PARPs). PARPs add PAR chains all around a site of damage to let other parts of the cell know that damage has happened. This attracts DNA repair proteins to DNA damage by binding to PAR and performing their role to fix the damage.

a lage black fishing net on a white background, it is worn in some placed.
PAR can act like a fishing net that “catches” and pulls in proteins to help fix DNA damage. Image of a fishing net by Nikodem Nijaki on Wikimedia.
To much PAR causes cells to run out of energy

Even though PAR does a good job of signalling that DNA damage has happened, it takes a lot of energy to make. If the damage can not be fixed, the cell will keep trying to make PAR until runs out of energy. This can lead to PAR molecules causing cell death. This effect of too much PAR can be seen in multiple types of neurodegenerative diseases.

A type of cerebellar ataxia called AOA-XRCC1 is known for having higher levels of PAR due to DNA damage. When researchers reduced the amount of PAR in a mouse model of AOA-XRCC1, the mouse had fewer ataxia symptoms and lost fewer neurons. This type of ataxia is caused by a mutation in a protein called XRCC1, which normally helps fix DNA and binds to PAR chains. But in the disease, the XRCC1 gets stuck at DNA along with the long chains of PAR.

These findings may be applicable to other types of ataxia and neurodegenerative disorders because of their link to higher levels of DNA damage. A lot more work to be done on PARylation and its role in neurodegeneration. But a lot of research has been done on PAR in cancer. Many drugs have been FDA approved for cancer patients as safe and effective. Cancer and ataxia are very different diseases. But all the work that has previously been done has laid the groundwork for new research in neurodegeneration.

If you would like to learn more about poly-ADP-ribose , take a look at these resources by the National Cancer Institute and Cancer Research UK.

Snapshot written by Carlos Barba-Bazan and edited by Dr. Ray Truant

Continue reading “Snapshot: What is poly-ADP-ribose (PAR)?”

Snapshot: What is Non-Homologous End Joining?

What is DNA damage?

As we go through life, our DNA undergoes a lot of stress, which can ultimately lead to DNA damage. The different stressors that can cause DNA damage are environmental factors such as UV light, radiation, and certain toxins. Additionally, DNA damage can be caused by metabolic processes that occur naturally in our body. DNA damage can manifest in multiple forms. One form of DNA damage are DNA breaks, which arise when either one or both of the DNA strands break, creating a physical separation between the DNA bases. Every day, each cell will experience about 50,000 single-stranded breaks, and every cell division will lead to about ten double-stranded breaks.

A blue DNA molecule that is broken in half, like it has been cut with a pair of scissors
An artist’s drawing of a double-stranded DNA break. Image part of the public domain.

How is the DNA repaired?

Thankfully, although DNA damage is a common occurrence in our cells, there are many ways our body can repair the damage. The type of repair is thereby determined by the type of DNA damage and the growth stage of the cell. A commonly used repair pathway to fix double-strand breaks is non-homologous end joining (NHEJ).

How NHEJ can repair DNA.  DNA damage cuses either a single stranded or double stranded break. If it is a double stranded break, then NHEJ can "correct" the damage, but dome genetic information will be lost.
How NHEJ can repair DNA. Damage to DNA can cause Single Strand (SS) or Double Strand (DS) breaks. Non-Homologous End Joining can repair double stranded break, but also causes small deletions of DNA. Image by Eder Xhako, Created with BioRender

NHEJ occurs mainly when the cell is actively growing. Double-stranded breaks are very harmful to the cell. If left unrepaired, the break can make the whole DNA chromosome unstable, and result in death of the cell. In order to minimize the damage caused by the breaks, NHEJ is a very fast, although often imprecise, method of DNA repair. When DNA undergoes a double-stranded break, NHEJ will act like glue and stick the two broken DNA ends together. Depending on where the break occurred and how much damage happened, the repair can then either lead to a complete repair or the loss of some DNA base pairs at the junctions resulting in permanent deletions. It is more common for NHEJ to lead to small deletions rather than a precise repair. Depending on the location of the deletion, it can have either no consequence or be potentially damaging to a gene

How do scientists use NHEJ to their advantage?

Outside of the context of natural DNA damage, scientists have learned how to utilize the naturally occurring NHEJ repair pathway to inactivate genes of interest. As such, scientists have developed methods, such as the CRISPR-Cas9 system, to artificially introduce double-stranded breaks in essential and functional parts of the gene. The double-strand break then causes the cell to repair the double-stranded break using NHEJ. As NHEJ often leads to small deletions, which will damage the essential part of the gene, the gene will often be rendered non-functional. In this way, scientists can study what happens in the absence of the gene.

If you would like to learn more about Non-Homologous End Joining, take a look at this video by Oxford University Press.

Snapshot written by Eder Xhako and edited by Larissa Nitschke

Finding New Off-Balance Protein Networks in SCA7

Written by Frida Niss Edited by Dr. Siddharth Nath

Can neurodegeneration in SCA7 in part be due to faulty calcium homeostasis in the cerebellum?

Polyglutamine diseases are caused by an increase in the length of CAG repeats within a specific gene. The mutation for spinocerebellar ataxia type 7 (SCA7) was discovered more than two decades ago, but many of the details surrounding how the mutation actually causes disease remain fuzzy. We know that the increased repeat length in the gene makes it difficult for the resulting protein to arrange or fold itself properly. We also know that the mutated protein binds to itself and to other proteins in an unusual way. It building up large deposits of seemingly useless debris in the cell, called ‘aggregates’. However, the exact pathways this leads to cell death, and subsequently neurodegeneration, is not completely clear.

There is currently research underway to directly target and inhibit the repeat proteins themselves. However, finding other pathways in the cell that are easier to target with medication is also a priority. In this research, Stoyas and her colleagues wanted to find out more about which cellular pathways are disturbed in the polyglutamine disease SCA7.

A pair of hands in plastics gloves writes down scientific findings on a chart. Beside the hands are racks of tubes with lables of different samples and dates collected.
A laboratory scientist documents research findings. Image courtesy of the National Institutes of Health on Flickr.
SCA7 mice have disordered productions of proteins that help balance ions concentrations

In SCA7, the protein that carries the mutation is Ataxin-7. Ataxin-7 participates in transcription through complexes of proteins that together can change some signalling particles on the DNA. Depending on what signalling particles are attached to a certain gene, the gene is either transcribed and made into a protein, or “silenced” and skipped over. In the case of Ataxin-7 and its complex, they work together to cause transcription of genes. One of the main theories of how a polyglutamine mutation can be toxic in Ataxin-7 is that the mutation disturbs Ataxin-7’s normal function within this transcription activating complex. Instead of being regular and orderly, ataxin-7 starts acting unpredictably. Some things that should be transcribed are not, some that shouldn’t be transcribed are.

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