A new tool to search for medicines that might treat neurodegenerative diseases linked to problems with mitochondria, including amyotrophic lateral sclerosis (ALS), has been developed by a team of scientists at the Scripps Research Institute, a study reports.
This tool can screen thousands of potential medicines by directly measuring the health of the mitochondria (a cell’s energy source) in neurons. A test screening of 2,400 compounds found several able to improve mitochondrial health, and to protect these cellular powerhouses from disease-associated damage.
The study, “Neuron-based high-content assay and screen for CNS active mitotherapeutics,” was published in Science Advances.
Mitochondria are tiny, specialized structures that supply cells with most of their energy by converting oxygen and nutrients into chemical energy in the form of adenosine triphosphate (ATP).
Many of the genes associated with ALS play a role in mitochondrial function, and studies in disease models and patients implicate mitochondrial damage as a core component of ALS.
“It hasn’t yet been emphasized in the search for effective therapeutics, but mitochondrial failure is a feature of many neurodegenerative disorders and something that must be corrected if neurons are to survive, Ronald Davis, PhD, of the Florida-based Scripps Research Institute and the study’s senior author, said in a press release. “So I’m a big believer that finding mitochondria-protecting molecules is the way to go against these diseases.”
Finding new medicines to correct this damage requires testing thousands of experimental compounds — a process known as high content screening — to identify compounds with the best potential to become medicines.
Cell-based systems are typically used as a first step in the screening process, then the best candidate medicines are tested further in animal models and people. However, as cultures of neuron cells are difficult to maintain, current screening systems designed to test mitochondrial function use cells found outside the brain.
Davis and his team solved this problem by developing a system that uses cultured neurons from mouse brains grown in such a way that thousands of compounds can be tested at once.
The mitochondria within these neurons are labeled so they glow when exposed to fluorescent light. Microscopic images are collected and analyzed to record the numbers of mitochondria, their shape (which becomes small and rounded when defective), and other metabolic markers of mitochondrial health.
To validate the system, the team screened 2,400 compounds, many of which are existing medicines, and found 149 “hit” compounds that either increased the numbers of mitochondria, caused them to become more elongated, or increased their metabolic function.
The second screen narrowed down the number of confirmed compounds to 67, so-called modulators of neuronal mitostasis (MnMs). Of these, 32 increased mitochondrial elongation, 45 increased mitochondrial numbers in neurons, and 33 improved mitochondrial health. In addition, 61 of the confirmed compounds were shown to directly increase metabolic functions, including a significant increase in ATP production.
To find out if this screening method could be used in therapeutic discovery, neuron cells cultured with and without MnMs were exposed to three stresses known to harm mitochondria: toxic A-beta(1–42) oligomers, peroxide, and excessive amounts of the neurotransmitter glutamate.
In cells not treated with MnMs, mitochondria were severely damaged by all three stresses. Mitochondria in cells treated with MnMs, however, were protected from damage. Overall, seven MnMs provided complete protection against at least one of the three stresses.
One of the MnMs was dyclonine — found in throat lozenges as an anesthetic — and chosen for further analysis given its ability to increase ATP production and improve nerve cell function.
The final test was done to establish whether dyclonine, as a representative of MnMs, altered mitochondrial function in mice, as cultured cells may not reflect an in vivo (living system) environment. Mice treated for seven months with dyclonine in their water supply had enhanced mitochondrial function in their neurons.
“It remains a mystery why dyclonine and other local anesthetics have such effects on mitochondria in neurons — we certainly didn’t anticipate this,” Davis said. “But the compounds we identified give us strong hope that we’ll see beneficial effects when we test them in animal models of specific neurodegenerative diseases, as we’re now doing.”
“Future studies will include identifying molecular targets for this important set of small molecules and testing their efficacy in vivo and in animal models of disease,” the scientists concluded.
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