Dev Cell︱ Lactic acid promotes peripheral nerve injury and repair B side: Long-term increase in lactic acid metabolism of axons can lead to oxidative stress and axon degeneration

Written by Deng Chen, edited by Ke, Wang Sizhen The nervous system is mainly composed of neurons and glial cells to form neural functional units
.

Neuroglia (neuroglia) play a role in supporting and promoting impulse conduction of neurons in the nervous system-on the one hand, they can wrap axons to form an insulating myelin sheath to prevent nerve impulses from interfering with each other; on the other hand, they can also Provides metabolic substrates such as lactate/pyruvate for neighboring energy-intensive neurons to help maintain the normal function of neurons [1]
.

 The myelinated glial cells in the peripheral nervous system are called Schwann cells.
They appear as two main structural units in the peripheral nervous system: around large-diameter axons, Schwann cells will "one Encapsulate a segment of neuron axons to form a sandwich cylinder structure; and around small diameter axons, Schwann cells will "engulf" many small diameter axons, forming a "one-to-many" "Remak" Fibers" structure [2]
.

 Lactic acid (lactate) is one of the metabolites of the human body.
The pyruvate produced by glycolysis is produced by the reduction action of lactate dehydrogenase (LDH).
In the case of sufficient oxygen, lactic acid will be Under the reverse action of lactate dehydrogenase, pyruvate is regenerated and enters the aerobic metabolic pathway for metabolism, so lactic acid can also be used as a nutrient to provide energy for cells [3]
.

 Rheb protein is a type of small G protein with GTPase activity.
It is an important protein in the mTORC1 pathway and regulates cell growth, proliferation and differentiation
.

In this article, it is also introduced that Rheb protein can independently regulate the activity of PDH, thereby controlling the respiratory metabolism of cells [4]
.

 Previous studies have found that: in the case of peripheral axon injury, Schwann cells will change their metabolism to provide more lactic acid to neurons, thereby supporting the survival of axons [5]
.

From this point of view, the lactic acid provided by glial cells seems to be better for neurons to perform normal functions and resist external stress, but is this really the case? On October 6, 2021, the research team led by Professor Xiao Bo from the School of Life Sciences of Southern University of Science and Technology published an article "Rheb-regulated mitochondrial pyruvate metabolism of Schwann cells linked to axon stability" in the Journal of Developmental Cell.
The lactic acid has conducted in-depth research on maintaining the stability of peripheral axons, and proposed that the lactic acid provided by glial cells may be a "double-edged sword" for neurons-increased lactic acid in a short period of time can promote damaged axons However, excessive lactic acid metabolism over a long period of time will cause oxidative damage to neurons and cause axon degeneration [6]
.

 Previous studies by the research team found that in oligodendrocyte precursor cells (OPCs), the deletion of the small and medium G protein Rheb gene can lead to severe hypomyelination of brain and spinal cord nerves mediated by mTORC1
.

Based on this fact, the team is interested in whether the loss of Rheb in peripheral glial cells will have the same effect on its corresponding peripheral nerves
.

To this end, the team constructed Schwann cell-specific Rheb gene knockout (Rheb cKO) mice
.

Transmission electron microscopy results showed that the thickness of the myelin sheath of the sciatic nerve of this strain of mice was reduced to a certain extent, accompanied by obvious axon degeneration
.

Consistent with this, the results of immunofluorescence and immunoblotting of nerve axons showed that the content of nerve damage marker SMI32 was significantly up-regulated
.

These results together show that the peripheral nerves of Rheb cKO mice have lesions (Figure 1)
.

Figure 1 Rheb cKO mouse sciatic nerve axon degeneration and damage (picture quoted from: Jia et al.
Dev Cell.
2021) Next, the author contacted the function of peripheral nerve axons and predicted that degenerated nerve axons would cause mice to behave Changes in learning
.

The gait analysis results of Rheb cKO mice suggest that the coordination ability of the mice is impaired, and it also reflects the pathological changes of peripheral motor neurons in this strain of mice
.

 How did this phenotype arise? The author has learned in previous articles that the lactic acid provided by glial cells can be used as a metabolic substrate of axons, and when axons are damaged, the lactic acid provided by glial cells to axons will increase [6], which is likely to increase The load of mitochondria, and the author found in previous studies that axon neurons are extremely vulnerable to oxidative damage due to their high unsaturated fatty acid content, high oxygen consumption, and relatively weak antioxidant defense mechanism
.

Based on this information, the author predicts that the axon damage is due to the excessive lactic acid provided by Schwann cells lacking Rheb protein, which causes the axon mitochondria to continuously metabolize lactic acid and produce more reactive oxygen species (ROS).
It causes oxidative stress on the axons, destroys the proteins and lipids in the axons, resulting in damage to the axons
.

 Based on such a hypothesis, the author first detected the oxidative stress of the sciatic nerve of Rheb cKO mice, with the content of reduced glutathione/oxidized glutathione (GSH/GSSH), compound dismutase 1 (SOD1), etc.
As a comparative indicator of oxidative stress, the results showed that the GSH/GSSH in the sciatic nerve was significantly reduced, and the SOD1 content and DHE fluorescence intensity were significantly increased, indicating that nerve cells are being subjected to a higher degree of oxidative stress
.

Next, the author detected lipid and protein peroxidation markers 4HNE and DNP and found that lipid and protein oxidative damage occurred in the peripheral axons of Rheb cKO mice.
At the same time, further transmission electron microscopy results showed that mitochondria appeared in the axons.
Swelling and other damages, which strongly proves that the sciatic nerve of Rheb cKO mice has a high level of oxidative stress, which leads to the damage of mitochondria and the oxidative damage of proteins and lipids in cells (Figure 2)
.

Figure 2 The high level of oxidative stress in the sciatic nerve of Rheb cKO mice caused the oxidative damage of intracellular proteins and lipids (picture quoted from: Jia et al.
Dev Cell.
2021) Next, based on the previous assumptions, the author needs To verify whether it is the excess lactic acid produced by Schwann cells lacking Rheb protein that enters the axon, which forces the axon mitochondria to carry out continuous lactate metabolism.
For this reason, the author first measured the oxygen consumption rate (OCR) in Schwann cells cultured in vitro.
And extracellular acidification degree (ECAR), it was found that Schwann cell OCR was significantly reduced, while ECAR was significantly increased at the same time
.

This implies that the respiratory metabolism of Schwann cells is transforming from aerobic respiration to anaerobic respiration that produces lactic acid
.

Based on the understanding of the signaling pathways involved in Rheb protein, these changes are largely due to the decrease in PDH activity regulated by Rheb protein
.

Western blotting experiments on phosphorylated PDH (p-PDH) confirmed this result, and this metabolic change is likely to cause axon damage (Figure 3)
.

Figure 3 Metabolic changes of Rheb cKO Schwann cells cultured in vitro lead to increased production and release of lactic acid (picture quoted from: Jia et al.
Dev Cell.
2021) Next, the use of 2DG to inhibit the production of lactic acid effectively inhibits peripheral nerves.
Oxidative damage and mitochondrial swelling further confirm that excessive lactic acid is the key to inducing oxidative damage and axon degeneration (Figure 4)
.

Figure 4 The oxidative damage of the sciatic nerve of Rheb cKO mice is reduced after 2DG treatment (picture quoted from: Jia et al.
Dev Cell.
2021) The increased lactic acid output of Schwann cells does not directly confirm that it flows to the axon of the peripheral nerve, for confirmation In this regard, the author learned from previous studies that the transport of metabolites such as lactate and pyruvate between axons and neighboring Schwann cells is done by monocarboxylic acid transporter (MCT).
In order to confirm the above hypothesis, the author Using real-time quantitative reverse transcription PCR (qRT-PCR) and western blotting, the mRNA and protein levels of MCT1 in Rheb cKO mice were detected, and it was found that both were significantly up-regulated, indicating a significant increase in MCT transporters and increased lactate The flux of transport therefore shows that Schwann cells will transfer the large amount of lactic acid produced into the axons
.

 Furthermore, the authors examined the content of various metabolites in the sciatic nerve of Rheb cKO mice and found that although the metabolic activity of the mitochondria of Schwann cells knocked out by Rheb was reduced, the metabolites involved in mitochondrial metabolism in the entire sciatic nerve tissue were significantly increased, and lactic acid and pyruvate were also significantly increased.
To increase
.

Considering that the sciatic nerve is mainly composed of axons and Schwann cells, it is speculated that the mitochondria in the peripheral axons of Rheb cKO mice are undergoing high-load metabolism of lactic acid
.

In theory, overloaded mitochondria will inevitably produce more reactive oxygen species
.

For this reason, the author then used a reactive oxygen indicator to detect the reactive oxygen content in the dorsal root neurons (DRG) of Rheb cKO mice, and supplemented the detection of the reactive oxygen content of the DRG neurons in the neurons cultured in vitro after additional lactic acid was added.
The significant increase in the number of patients proved that the peripheral neurons of Rheb cKO mice produced more ROS due to excessive lactic acid metabolism (Figure 5)
.

Figure 5 The massive input of lactic acid into the Schwann cells of Rheb cKO mice caused the mitochondria in the sciatic nerve to overload and produce high doses of ROS (picture quoted from: Jia et al.
Dev Cell.
2021).
Growth, the input of a large amount of lactic acid leads to impaired mitochondrial function, lactic acid begins to accumulate due to incomplete metabolism, mitochondrial production of ATP is reduced, in the early stage, nerve cells can also increase the intensity of glycolysis to ensure energy supply, but in the later stage, In old mice, with the increase of oxidative damage, the damage of energy metabolism cannot be restored by enhanced glycolysis, which reduces the synthesis of ATP, and the lack of energy aggravates the degeneration of peripheral axons in mice, suggesting late axon mitochondrial oxidation Insufficient cell energy supply caused by injury and self-oxidative damage together lead to axon degeneration (Figure 6)
.

Figure 6 The mitochondrial function of the neurons in the aged Rheb cKO mice decreased, and the energy metabolism was disturbed (picture quoted from: Jia et al.
Dev Cell.
2021) Fig.
7 work summary picture (picture quoted from: Jia et al.
Dev Cell.
2021) ) Conclusion and discussion of the article, enlighten and prospect the structure and interaction of neuron-Schwann cells in the peripheral nervous system is a necessary condition for the peripheral nerve to maintain its normal function
.

Signal transmission and metabolic coupling between neurons and neighboring glial cells play an important role in maintaining the stability of the nervous system, and glial cell pathology is the culprit of many neurodegenerative diseases, so the difference between the two The interaction is being paid attention to by more and more researchers
.

 The author of this article found that knocking out Rheb in Schwann cells would reduce PDH activity, leading to more pyruvate metabolism into lactic acid; and the long-term increase in lactic acid metabolism of axons would lead to oxidative stress and axon degeneration
.

Past research work has proposed that the lactic acid provided by Schwann cells supports and protects peripheral nerves [5], while this study proposes the other side of lactic acid on the maintenance of axon integrity, that is, excessive lactic acid intake over a long period of time has an effect on axons.
The toxic effect
.

At the same time, in many disease states such as diabetes and brain aging, there will be an abnormal increase in lactic acid [6], so this study may provide new research ideas for elucidating the pathogenesis of aging and neurodegenerative diseases
.

 Of course, there are still some unsolved problems in this article.
As mentioned in the article, this article did not study the effect of cell acidification that may be caused by increased lactic acid on axon stability; at the same time, follow-up studies need to verify whether antioxidants are effective The axon degeneration caused by lactic acid can relieve or protect
.

com/developmental-cell/fulltext/S1534-5807(21)00731-0 Selected articles from previous issues [1] Nat Commun︱ selective inhibition of microglia activation is expected to alleviate pathological α-syn transmission [2] Science︱ serum Does vegetarian help overcome cocaine addiction? [3] Mol Psychiatry︱ Gao Tianming’s research group reveals the different roles of astrocytes and neurons in synaptic plasticity and memory [4] Sci Transl Med︱ Xiang Xianyuan and others reveal that brain immune cells madly eat sugar and help nerves Early diagnosis of degenerative diseases [5] A new mechanism of Mol Cell︱ Alzheimer's disease: Tau protein oligomerization induces nuclear cell transport of RNA binding protein HNRNPA2B1 and mediates enhancement of m6A-RNA modification [6] Cereb Cortex | Li Tao project The group reported the abnormality of the cortical myelin covariation network with the deep characteristics of the cerebral cortex in schizophrenia [7] Cell︱ hold hands, advance and retreat together! The formation of a cellular network between microglia to work together to degrade pathological α-syn [8] lipids and Alzheimer's disease! The lack of sulfatide in the myelin sheath of the central nervous system in adulthood can lead to Alzheimer’s disease-like neuroinflammation and cognitive impairment [9] Brain︱ new method! Plasma soluble TREM2 can be used as a potential detection marker for white matter damage in cerebral small vessel diseases [10] EMBO J︱ neuronal Miro1 protein deletion destroys mitochondrial autophagy and overactivates the integrated stress response [11] Science Frontier Review Interpretation︱ Nicotinic Acetylcholine The regulatory mechanism of receptor accessory molecules and the application prospects of disease treatment and transformation [12] Neurosci Bull︱ synapse-associated protein Dlg1 improves depression-like behavior in mice by inhibiting microglia activation [13] Brain | For the first time! PAX6 may be a key factor in the pathogenesis of Alzheimer’s disease and a new therapeutic target [14] Nat Biomed Eng︱Ye Yuru’s team developed a new strategy for whole-brain gene editing-mediated treatment of Alzheimer’s disease [15] Luo Liqun Science Center Interpretation of the system ︱Nerve loop structure-a high-quality scientific research training course recommendation for the system that makes the brain "computer" run at high speed [1] A guide to data graphs! How good is it to learn these software? 【2】JAMA Neurol︱ Attention! Young people are more likely to suffer from "Alzheimer's disease"? [3] Patch Clamp and Optogenetics and Calcium Imaging Technology Seminar (October 30-31) References (slide up and down to view) [1] Dale Purves · George, JADFWCHA-SLRDMMLP NEUROSCIENCE.
6th Edition.
edn, (Oxford University Press 2017-12-10, 2017).
【2】Harty, BL & Monk, KR Unwrapping the unappreciated: recent progress in Remak Schwann cell biology.
Curr Opin Neurobiol 47, 131-137, doi:10.
1016/j.
conb.
2017.
10.
003 (2017).
[3] Mathews, CK Biochemistry.
4th ed.
.
edn, (Toronto: Pearson, c2013, 2013).
[4] Saxton, RA & Sabatini, DM mTOR Signaling in Growth, Metabolism, and Disease .
Cell 168, 960-976, doi:10.
1016/j.
cell.
2017.
02.
004 (2017).
【5】Babetto, E.
, Wong, KM & Beirowski, B.
A glycolytic shift in Schwann cells supports injured axons.
Nat Neurosci 23, 1215-1228, doi:10.
1038/s41593-020-0689-4 (2020).
[6] Jia, L.
et al.