Interview

Redoxcope by Maria Célia Wider

For decades, caloric restriction has been known to enhance longevity and the prevent age-related diseases. However, the processes responsible for these effects are not yet fully understood. Now, Redoxoma Network researchers have found the mechanism in which caloric restriction facilitates mitochondrial calcium retention capacity in the brain, resulting in protection against excitotoxic damage, which is related to neuronal loss in diseases such as stroke, Parkinson's and Alzheimer’s. “Because we determined caloric restriction´s mechanism of action, we may be able to develop drugs to increase mitochondrial calcium uptake in mitochondria in a manner that is not dependent on the diet. Finding how to protect the brain against excitotoxicity means we can act against many diseases associated with this process”, said Ignacio Amigo, the main author of the study, published in Aging Cell and conducted in Professor Alicia Kowaltowski´s laboratory in the Chemistry Institute of the University of São Paulo.

Using both in vivo and in vitro experiments, the researchers found that caloric restriction is highly effective in preventing excitotoxic cell death because it increases mitochondrial electron transport activity, enhances antioxidant defenses and promotes brain mitochondrial calcium uptake.

Mechanism

Excitotoxicity is the ability of excitatory neurotransmitters such as glutamate to mediate neuronal cell death due to excessive glutamate receptor activation. In certain neurological and neurodegenerative diseases, high amounts of glutamate are released into the extracellular space, promoting calcium entry into the cell, and resulting in neuronal death. Because mitochondria can buffer this calcium, they play a central role in excitotoxicity. Cell death occurs when the mitochondrial buffering capacity is surpassed.

To investigate the neuroprotective effects of caloric restriction against excitotoxicity, the researchers initially worked with Swiss mice that were separated in two groups: one kept on a calorically restricted diet for 14 weeks and another fed ad libitum (without any restrictions). The animals then received kainic acid injections, a molecule that works similarly to glutamate, which promotes seizures, neuronal damage and death. Kainic acid acts by over-activating glutamate receptors in the hypothalamus and is an often-used model to study excitotoxicity. Mice maintained on a calorically-restricted diet did not have seizures, demonstrating the effects of the diet in vivo.

Next, the authors isolated mitochondria from the brains of Sprague Dawley rats, also kept on caloric restriction or ad libitum diets. In mitochondria from calorically-restricted animals, the authors saw an increase in the levels of some antioxidant enzymes, such as glutathione peroxidase and glutathione reductase, as well as an increase in superoxide dismutase activity. These results indicate that caloric restriction increases brain redox capacity, protecting the organ against oxidative damage, such as occurs in excitotoxicity. The levels of some mitochondrial proteins and activity of electron transport chain components was also found to be enhanced by caloric restriction.

Amigo, however, believes the experiments measuring calcium uptake yielded the most interesting results.

To evaluate calcium uptake, the researchers added more and more calcium to the isolated rat mitochondria, comparing the ability to maintain this calcium in both groups. “We found that calcium uptake is larger in mitochondria from calorically-restricted animals. This is a new result, that had never been seen before”, says Amigo.

Further investigating their finding, the authors used cyclosporin, a compound that increases mitochondrial calcium uptake by inhibiting a protein called cyclophillin D. They found that cyclosporin only affected calcium uptake in ad libitum mitochondria, suggesting that cyclophillin D was inhibited in caloric restriction. However, the levels of cyclophillin D were the same in both groups.

“Many post-translational modifications change cyclophillin D activity. One of these modifications is acetylation. When the protein is deacetylated, it is inhibited”, describes the researcher.

This is where a family of proteins called sirtuins acts. One of these proteins, SIRT3, is mitochondrial, and is more highly expressed in caloric restriction. This is the sirtuin that deacetylates cyclophillin D.

Knowing this, the researchers looked at the levels of SIRT3 in the animal brains, and found it to be elevated. They also found that cyclophillin D was less acetylated in caloric restriction.

“When the sirtuin is enhanced, it deacetylates cyclophillin D, which is then inhibited, increasing mitochondrial calcium uptake. This is the mechanism we propose”, concludes the researcher.

The article “Caloric Restriction Increases Brain Mitochondrial Calcium Retention Capacity and Protects Against Excitotoxicity”, by Ignacio Amigo, Sergio Luiz Menezes-Filho, Luis Alberto Luévano-Martínez, Bruno Chausse and Alicia J. Kowaltowski, can be accessed here.