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Article
The Role of SIRT2 Expressed by Oligodendrocytes in
Increasing Axonal ATP

By: Yannay Kaplan

Abstract

 

Axons require sufficient energy to be healthy, otherwise neurodegenerative diseases result. A recent study shows that oligodendrocytes (OL) send SIRT2 proteins in exosomes which increases axonal ATP levels. The delivery of Sirtuin 2 (SIRT2) was found to be critical to increase ATP axonal energy by using in vivo samples comparing wildtype OLs to SIRT2-deletions, by using neurons from knockout mice, and by preventing the expression of SIRT2 from the exosome. Only wildtype OLs exhibited strong acetylation of mitochondrial proteins ANT1 and ANT2. Additionally, SIRT2 was found to revive the mitochondrial ability of knockout mice. Using this data, a potential way to increase ATP production in neurodegenerative disease has been discovered.

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Introduction

The brain is composed of several types of cells, including neurons, oligodendrocytes, glial cells, and astrocytes. Oligodendrocytes 

(OL) are responsible for covering neurons with a myelin sheath, which allows signals to be sent quickly throughout the body. A recent study demonstrated OLs are fundamental to ensure neurons can produce ATP.1 This research provided a better understanding of a mechanism in which OLs allow axonal mitochondria to produce ATP. A lack of neuronal supply of ATP is connected to neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and MS.2,3 Previous studies have shown that OLs are vital to the function of an axon and that having the myelin sheath intact without OLs is insufficient and axons will die.4,5 These studies have demonstrated a link between the health of an axon, ATP, and OLs.6 The purpose of this paper is to explain how researchers in a study analyzed the mechanism in which OLs cause axonal mitochondria to increase the production of ATP.1 



 

OL Enhancement of ATP Production in Axons

It is first imperative to establish a significant correlation between OLs and ATP production levels in axons. Chamberlain et al extracted neurons from mice and separated the axons from their cell bodies using a microfluidic chamber. Axons were divided into two groups. A control group was created containing only the axons. In a second experimental group, OLs were added. In each group of axons, ATP levels were measured using a method called Fluorescence resonance energy transfer (FRET) which uses fluorescent markers to detect ATP binding. A significant increase of ATP (p<0.0001) in the experimental group was observed in comparison to the control group, demonstrating that OLs have an important role in ATP production in axons. The advantage to FRET is that it is highly specific, allowing for the determination of the specific organelle in which ATP generation occurs. Specificity ensures that the conclusions reached will be more precise, and that doubts regarding the accuracy of some previous studies and their corresponding conclusions are allayed.3 Additionally, using this method, results are received in real-time. Non-myelinating OLs were used for most of the experiments described, which increases the information regarding how non-myelinating OLs in gray matter support axonal ATP production.1

 

Next, they determined if the type of signal transferred between the OL and the axon which led to the eventual increase in ATP in an axon is contact dependent or secretory based. To understand the process undergone by OLs to increase ATP, the researchers left pure OLs on media/broth for 24 hours to condition the media. After 24 hours, they removed the OLs and incorporated the conditioned media with the axons. The media caused a similar increase in ATP in the axons. Due to ATP production also increasing while OLs were not present, the researchers concluded that a secretory mechanism was used by OLs to transmit energy to axons. As will be described later, SIRT2 is being expressed by OLs. Based on the above, the expression of SIRT2 by OLs is not based on axon need but is instead a continuous release of the protein.1,7

 

Possible Effect of Lactate on Axonal ATP Production

Lactate is an important energy source for axons. The molecule is broken down into pyruvate and assists mitochondria to create ATP through oxidative phosphorylation. One source of lactate for axons are OLs.8 It was hypothesized that if lactate was added to media containing only axons, axonal mitochondria would absorb the lactate and increase production of ATP. When lactate was applied to the media, no change in fluorescence was observed in the axon, indicating the addition of lactate to axons had no significant effect on axonal ATP levels.1 Thus, another mechanism responsible for increasing ATP must be found.

 

Possible Effect of OL Exosomes on Axonal ATP Production 

OLs release exosomes responsible for the development of neurons and their maintenance. Further research was conducted to determine whether exosomes possess an influence on the production of ATP in neurons. Media was conditioned with wildtype OLs and incorporated into a plate containing axons. A second plate of axons also received conditioned media from OLs as well as a neutral sphingomyelinase inhibitor to inhibit the expulsion of exosomes from the multivesicular membrane. FRET was used to measure ATP binding. A significant difference in ATP production was observed when OLs were added to the respective plates. This difference demonstrated that exosomes are an important variable in an axon’s energy production. Additionally, exosomes were stained and detected in axons. Researchers determined that exosomes from mature but not necessarily myelinating OLs increase axons’ capability to produce more ATP.1

Enrichment of SIRT2 in OLs and Secretion in Exosomes

Once researchers determined that a secretory mechanism is used by exosomes, they investigated the protein SIRT2 as a possible protein being secreted. SIRT2 is a deacetylase commonly found in OL exosomes (it is found 40x more in OLs than neurons) and has been recently found to travel to the mitochondria and deacetylate mitochondrial proteins.1 Acetylation of mitochondria is known to control ATP production.9 Neuron cultures taken from mice containing both OLs and axons were stained to assess the expression of SIRT2 in OLs. The finding was that only mature OLs expressed SIRT2.1,7

Scientists found a directly proportional connection between myelination from mature OLs and SIRT2 levels. They observed SIRT2 present in both the brain and spinal cord of mice. A later experiment examines this further. Through this experiment, researchers concluded that OLs secrete exosomes containing SIRT2, which increases ATP production in neurons.1

 

Levels of ATP Increased by Presence of SIRT2 

The next experiment conducted consisted of three parts, all suggesting that SIRT2 is the vital component which causes an increase in ATP production in neurons. One part examined the effect on axonal energy when OLs containing exosomes without SIRT2 were grown with axons, compared to when OLs with SIRT2 expressing exosomes were used. The former batch of OLs were taken from general SIRT2 knockout mice. No increase in axonal energy was found when OLs without SIRT2 were used, while a significant increase of axonal ATP was measured when OLs expressing exosomes containing SIRT2 were used.1

In the second stage, a trial was conducted in which SIRT2 was placed in a viral vector and added to axons. No OLs were used in this stage. If no SIRT2 was added to the vector, the level of ATP in an axon did not change; however, when SIRT2 was added to the vector, axons increased their production of ATP. Thus, the researchers concluded that the vital component in the process of increasing production of ATP in an axon is due to SIRT2 in an OL’s exosomes (Figure 1).1

Screen Shot 2022-06-10 at 3.14.39 AM.png

 

The third stage used a small molecule of RNA which targets SIRT2 mRNA and causes degradation. The exosome of the OL loses a majority of its SIRT2 protein. As a result of the significant decrease of SIRT2 expression, cultures which contained SIRT2-deficient OLs and axons caused similar ATP production levels as individually cultured axons. This was in contrast to axons which were cultured with OLs which expressed SIRT2, which showed higher ATP production levels. All three experiments described show a directly proportional correlation between SIRT2 expression and ATP production.1

 

Neuron Mitochondrial Deacetylation Caused by SIRT2 

After establishing a correlation between OL exosome secretion of SIRT2 and an increase in axonal ATP, further experiments were done to determine the mechanism in which the presence of SIRT2 in a neuron’s axon causes ATP production to increase. Two groups of neurons were produced, one with wild type OLs in media and the other with SIRT2 knockout OLs present, which previously showed no increase in ATP compared to axons alone. Subsequently, mitochondrial proteins were isolated from neurons through centrifugation and mitochondrial fractionation. These mitochondrial proteins were placed in two batches of OLs – one which expressed SIRT2 and one which did not – and the quantity of deacetylated proteins was measured. As aforementioned, SIRT2 is known to be a deacetylase, so the wild-type batch was hypothesized to contain more deacetylated proteins. In addition, research has shown that deacetylation of mitochondrial proteins increases their activity, which increases ATP production. As predicted, the researchers found that the wildtype axons produced more ATP, signifying that SIRT2 deacetylated proteins.1

Subsequently, the experimenters chose specific mitochondrial proteins which they hypothesized SIRT2 would bind to that may cause an increase in axonal ATP. The proteins chosen included ANT1, ANT2, PHB2, ATP5A, and NDUFA5. They found that SIRT2 deacetylates ANT1 and ANT2 but not PHB2, ATP5A, and NDUFA5 (Figure 2). The reasoning behind the inconsistency of protein deacetylation is left open to future experiments. The study researchers suggest that perhaps other sirtuin proteins are responsible for deacetylating the proteins left untouched.1

Screen Shot 2022-06-10 at 3.14.26 AM.png

 

Revival of Mitochondrial Function via SIRT2

In a final experiment, the researchers assessed SIRT2 expression in myelinated axons using knockout mice which do not have OLs that express SIRT2. Although these mice have a similar number of mitochondria in their axons as compared to wildtype mice, their mitochondrial function is compromised. Mitochondrial membrane potential – which is correlated with the health and ATP production of an axon – was measured using a dye which was inserted into the spinal cord. Researchers introduced exosomes from wild type mice into the spinal cords of the knockout mice. Using the dye that shows mitochondrial health, they observed that the injected SIRT2 exosomes in knockout mice increased mitochondrial ATP production. This increase was not seen with a separate group of control mice.1

 

Conclusion

OLs have been found to increase axonal ATP production by secreting SIRT2 via exosomes. In axons, SIRT2 deacetylates certain mitochondrial proteins which cause ATP production to increase. Further research is still required to discover whether the SIRT2 protein interacts with the mitochondria via a signal cascade or by entering the cell. Furthermore, SIRT1 has been implicated in changing histone acetylation.10 Additional research could be conducted to determine if SIRT2 enters the nucleus and changes histone acetylation as-well.7 Additional experimentation with SIRT2 expression between white and gray matter oligodendrocytes will be helpful to understand the applications of this knowledge. Clarity with regards to how axonal energy is generated and increased will be helpful in developing methods to combat neurodegenerative diseases.

References

1. Chamberlain, Kelly A et al. “Oligodendrocytes enhance axonal energy metabolism by deacetylation of mitochondrial proteins through transcellular delivery of SIRT2.” Neuron vol. 109,21 (2021): 3456-3472.e8. doi:10.1016/j.neuron.2021.08.011

2. Camandola, Simonetta, and Mark P Mattson. “Brain metabolism in health, aging, and neurodegeneration.” The EMBO journal vol. 36,11 (2017): 1474-1492. doi:10.15252/embj.201695810

3. Pathak, Divya et al. “Energy failure: does it contribute to neurodegeneration?.” Annals of neurology vol. 74,4 (2013): 506-16. doi:10.1002/ana.24014

4. Oluich, Laura-Jane et al. “Targeted ablation of oligodendrocytes induces axonal pathology independent of overt demyelination.” The Journal of neuroscience : the official journal of the Society for Neuroscience vol. 32,24 (2012): 8317-30. doi:10.1523/JNEUROSCI.1053-12.2012

5. Edgar, Julia M et al. “Oligodendroglial modulation of fast axonal transport in a mouse model of hereditary spastic paraplegia.” The Journal of cell biology vol. 166,1 (2004): 121-31. doi:10.1083/jcb.200312012

6. Chamberlain, Kelly Anne, and Zu-Hang Sheng. “Mechanisms for the maintenance and regulation of axonal energy supply.” Journal of neuroscience research vol. 97,8 (2019): 897-913. doi:10.1002/jnr.24411

7. TWiN 28: Oligodendrocyte performance enhancing exosomes.” YouTube, uploaded by Vincent Racaniello. 4 April 2022, https://www.youtube.com/watch?v=pCsuxLR1Osc

8. Fünfschilling, Ursula et al. “Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity.” Nature vol. 485,7399 517-21. 29 Apr. 2012, doi:10.1038/nature11007

9. Liu, Guoxiang et al. “Loss of NAD-Dependent Protein Deacetylase Sirtuin-2 Alters Mitochondrial Protein Acetylation and Dysregulates Mitophagy.” Antioxidants & redox signaling vol. 26,15 (2017): 849-863. doi:10.1089/ars.2016.6662

10. Rifaï, Khaldoun et al. “SIRT1-dependent epigenetic regulation of H3 and H4 histone acetylation in human breast cancer.” Oncotarget vol. 9,55 30661-30678. 17 Jul. 2018, doi:10.18632/oncotarget.25771

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