The year was 1850, and Augustus Volney Waller was beginning to observe the degeneration of the hypoglossal and glossopharyngeal cranial nerves in frogs, which in simple terms control the movement of the throat and tongue. What he reported was that following a damaging stimulus, the damaged nerve would begin to die, and that this degeneration occurred from “distal to proximal of the neuron body,” meaning the degeneration began far from the center of the neuron, but the damage progressed until it reached the center and eventually caused the death of the entire cell.
Today we know that the Peripheral nervous systemthat is, those nerves and pathways that are not contained and protected by meninges and bones (such as the skull and spine), can regenerate in mammals, animals that correspond to a group of common origin in evolution to which mice, rats, cats, dogs, chimpanzees and humans belong.
In this group, after extensive injuries, where the ability to partially perceive the extremities is lost (such as when someone burns their hand and loses sensitivity), it is possible to regain perception of those areas. However, the Central Nervous Systemwhich corresponds to the spinal cord and the brain, lack this capacity; which means that any damage that occurs in these, such as a Cerebrovascular Accident (CVA), an Aneurysm or Whiplashis very difficult to counteract and almost impossible to reverse or remedy. This challenge is magnified in the case of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease.
This is why understanding the factors that determine axonal degeneration and the regeneration of these fibers is an important problem for current neuroscience, which is why a group of researchers from Interdisciplinary Center for Neuroscience of Valparaiso (CINV) Led by doctors Andrea Calixto and Chiayu Chiu, they used the animal model Caenorhabditis elegansto find factors common to the determinants of neuronal regeneration and protection previously described, such as gene expression, diet, calcium and mitochondrial function, by observing one of the 300 neurons in this animal. The results of this study were published in 2023 in the journal eNeuro.
At the beginning, the article shows us the results of a transcriptomics experiment, which consists of recognizing all the messenger RNAs, which will serve as a template for protein synthesis (something like reviewing all the ‘recipes’ that cells are using at a certain time to build their proteins), and comparing it between different stages of development and diets. Based on a previous publication by the same group in 2020, the important time points for neuron protection were known, so samples were taken from animals at 12, 24, and 48 hours postnatally, which had not yet reached the adult state (approximately 3 days), to sequence and recognize the sequences (recipes) present at these different stages.
Of the approximately 20,000 protein-coding genes found in most animals, 40 of them were reported to be overexpressed (i.e., produced more in a pro-regenerative condition) in animals consuming a protective diet and another 53 had lower than normal expression. This result would suggest that the neuronal protection provided by this diet requires a specific pattern of genes for neuronal protection to occur. A gene interference experiment was then performed to determine the gene expression of the protective diet. ARNwhich consists of a complementary nucleic acid that binds to the protein ‘recipe’ and prevents it from being able to exist, blocking one at a time, that is, a special molecule is used to attach itself to the protein ‘recipes’ and prevent them from being made, blocking one ‘recipe’ at a time. They used this technique to check the participation of the over-expressed genes found, and noticed that not all of the genes that were in greater number were actually necessary, only 15 of them were required at a systemic, or whole-body, level, and 7 of them were also essential at the neuron level.
In general, many processes depend on calcium, such as cell death, so the results described below were quite surprising. This is why they sought to find out what would happen if this calcium was absent in both the degeneration and regeneration of neurons. Therefore, they raised animals in two conditions: one with and one without calcium, which decreased the speed of neuron degeneration. However, this had already been reported previously. What was interesting and surprising was that the absence of calcium affected neuronal protection under two different protective conditions, such as a good diet and diapause (a process in which some animals enter a kind of ‘suspended animation’ of their vital functions in order to survive in adverse environmental conditions). These results would suggest that calcium, although it is needed to signal cell death, would also be necessary to slow down death by eating a good diet and also regenerate damaged neurons, especially during diapause.
With this result, the researchers used a complementary nucleic acid again (in order to block specific ‘recipes’) to study the role of different transport proteins, which allow the presence of calcium to be regulated inside the cell (cytoplasm), since they can sequester it in compartments, and thus, they could accelerate or slow down neuronal damage. This experiment is innovative, since during ‘suspended animation’ (diapause) it is not possible to introduce the complementary nucleic acid. Therefore, the mothers of the animals were treated and their effect on the offspring was observed. Because it is known that nucleic acids are inherited from the mother (such as RNA) and, in some cases, from the father. The results show that during diapause, fragility was greater, as it was necessary to block one transporter to intervene in regeneration, while outside of ‘suspended animation’ it was necessary to block at least two of them. This suggests that calcium control in the neuron is redundant, and that it is crucial during diapause.
With this in mind, the researchers analyzed the morphology of one of the organelles, or intracellular functional components, in order to understand the role of cellular functions in this process. For the sake of simplicity, they observed, photographed, quantified and measured the size of mitochondria, organelles responsible for energy production in the cell, during the degeneration, protection and regeneration of the neuron’s axon. The morphology of mitochondria has previously been described as an important factor in approximating the physiological state of cells. Through these observations, they concluded that, during regeneration, mitochondria exhibit a more elongated morphology, which would indicate a better metabolic state. In contrast, during degeneration, more fragmented mitochondria, that is, more damaged, were observed in the neurons of these animals. With this idea, the genes associated with mitochondrial elongation or fragmentation were silenced, finding that only mitochondrial elongation was decisive in sustaining the effect.
Finally, to relate the function of this organelle to neuronal regeneration, they analyzed the effect of inhibiting or silencing the expression of mitochondrial genes using a complementary nucleic acid, similarly to the experiments shown above. Using this approach, they found that these genes are necessary for regeneration, which could suggest that the best-known mitochondrial function, the production of energy in the cell, is also necessary for regeneration.
This article is an example of how a phenomenon such as the inhibition of neuron degeneration can be analyzed, both from the gene expression profile and from the function of these genes themselves, which underlie axonal protection and regeneration. It is expected that similar effects can be found in brains with greater complexity. C. eleganswhich, with 300 neurons, shows us phenomena common to all animals even when the number of neurons is billions, as is the case in humans.
As participants in this work, we find it interesting how mitochondria can determine not only energy production, but can also interfere in different cellular processes. By searching the literature, we can find that studies of human muscle physiology have reported the role that low-impact, but long-duration exercise plays in the elongation of mitochondria; if we let ourselves be carried away by the idea that all our mitochondria are affected in the same way, this could be one more reason to get up and lift weights or jog to improve the function of our mitochondria and protect our cells.
Original article:
*This article arises from the agreement with the Interdisciplinary Center for Neuroscience of the University of Valparaíso.