Emerging role and therapeutic implication of mTOR signalling in intervertebral disc degeneration

Abstract Intervertebral disc degeneration (IDD), an important cause of chronic low back pain (LBP), is considered the pathological basis for various spinal degenerative diseases. A series of factors, including inflammatory response, oxidative stress, autophagy, abnormal mechanical stress, nutritional deficiency, and genetics, lead to reduced extracellular matrix (ECM) synthesis by intervertebral disc (IVD) cells and accelerate IDD progression. Mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase that plays a vital role in diverse degenerative diseases. Recent studies have shown that mTOR signalling is involved in the regulation of autophagy, oxidative stress, inflammatory responses, ECM homeostasis, cellular senescence, and apoptosis in IVD cells. Accordingly, we reviewed the mechanism of mTOR signalling in the pathogenesis of IDD to provide innovative ideas for future research and IDD treatment.

exploration of IDD-related molecular mechanisms would provide new strategies for LBP intervention.
The intervertebral disc (IVD) is a complex fibrocartilaginous tissue that is the most important functional part of the spine, located between upper and lower vertebrae, and plays an important role in carrying weight and buffering compressive loads. 6,7 Healthy IVDs are mainly composed of the central gelatinous nucleus pulposus (NP), inner and outer annulus fibrosus (AF) surrounding the NP, and cartilage endplate (CEP) above and below the NP and AF, forming a relatively closed environment. 8,9 The NP is primarily rich in type II collagen fibres, elastin fibres, and proteoglycans and is essential for spinal multiaxial flexibility and counteracting axial mechanical loads. 10,11 The AF consists of a series of concentric circular lamellar structures (type I and type II collagen fibres) divided into the inner and outer AFs. Compared with the inner AF, the outer AF contains more type I collagen fibres, which afford strong resistance to tensile load and prevent the NP from protruding outwards. 12,13 CEP is a thin hyaline cartilage that prevents the NP tissue from projecting into the vertebral body. Additionally, given that the IVD is an avascular tissue, NP and inner AF exchange nutrients and metabolic wastes by CEP diffusion, thereby maintaining normal IVD structure and function. 14 Accumulated evidence suggests that inflammatory response, oxidative stress, autophagy, abnormal mechanical stress, nutritional deficiencies, and genetics can lead to reduced ECM synthesis and secretion by IVD cells, ultimately resulting in structural and functional dysfunction of the IVD. [15][16][17] Mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase involved in the regulation of protein synthesis, cellular senescence, autophagy, apoptosis, and immunity. 18 Several studies have confirmed that the mTOR signalling pathway plays an important role in various degenerative diseases, including osteoarthritis, diabetes, atherosclerosis, and Parkinson's disease. [19][20][21][22] Liu et al. 23 used the LDH model to induce radicular pain in rats. Intraperitoneal injection of AMP-activated protein kinase (AMPK) activators can activate the AMPK signalling pathway in dorsal root ganglion neurones, inhibit mTOR signalling, and alleviate LDH-induced radicular pain, suggesting that inhibition of mTOR signalling can alleviate radicular pain caused by IVD protrusion-mediated compression. Recent studies have suggested that mTOR signalling is critical for maintaining IVD homeostasis. 24,25 Based on existing literature, we focused on the mTOR signalling pathway and its multiple biological functions in IVD cells to comprehensively clarify the role of the mTOR signalling pathway in IDD.

| STRUCTURE AND FUNCTION OF MTOR SIGNALLING PATHWAY
mTOR is an atypical serine/threonine kinase belonging to the phosphatidylinositol kinase-related kinase (PIKK) family. 26 The mTOR protein is composed of five domains, each of which has a different function, including two groups of N-terminal HEAT repeat (PR65/A subunit of protein phosphatase 2A, Huntingtin, elongation factor 3) domains, potentially involved in protein interactions, membrane anchoring, and cytoplasmic trafficking. 27 The FAT (FRAP, ATM, TRRAP) domain occurs downstream of the HEAT repeat domain. In addition, the C-terminal FAT C domain, which is similar to the FAT domain, maintains the structural stability of the mTOR protein. 28 The FRB and Ser/Thr kinase domains are located between FAT and FATC domains. The FRB domain is the FKBP12-rapamycin complex-binding site of mTOR. Rapamycin binds to FKBP12 in the cytoplasm via the FRB domain, thereby inhibiting mTOR activity. 29 The Ser/Thr kinase domain is the active centre of mTOR, which achieves signal transduction or functional regulation after activation. 30 Following in-depth investigations into mTOR protein, based on their structural and functional differences, mTOR can be divided into three distinct multisubunit protein complexes: mTOR complex 1 (mTORC1), mTOR complex 2 (mTORC2), and a putative mTOR complex 3 (mTORC3). 31,32 mTORC1 is composed of the catalytic subunit mTOR, a regulatoryrelated protein of mTOR (Raptor), a proline-rich Akt substrate (PRAS40), a DEP domain-containing mTOR-interacting protein (Deptor), and mammalian lethal protein SEC13 protein 8 (mLST8). 33 In addition to the same three subunit catalytic subunits, mTOR, Deptor, and mLST8 in mTORC1, mTORC2 comprises three other subunits, including rapamycin-insensitive companion of mTOR (Rictor), mitogen-activated protein kinase-related protein 1 (mSin1), and Protor. 34 In mTORC1, Raptor acts as a bridge to PRAS40, whereas PRAS40 and mLST8 bind to mTOR as negative and positive regulatory subunits, respectively. In mTORC2, mSin1 localizes to Rictor, which, in turn, binds to mTOR and inhibits its activity. Protor is responsible for assisting in complex assembly, while mLST8 and Deptor function similarly to mTORC1. 29 However, few reports are available on mTORC3. Current studies have revealed that mTOR3 is insensitive to rapamycin and has been shown to have tumourigenic effects; it is composed of ETV7, mTOR, and other undefined components, lacking Raptor or Rictor 35 (Figure 1).

| SIGNAL TRANSDUCTION OF MTOR SIGNALLING PATHWAY
The mTOR signalling pathway plays a critical role in regulating cell growth and metabolism in eukaryotic cells by modulating transcription, translation, lipid synthesis, autophagy, and lysosomal biosynthesis. 36 Tuberous sclerosis complex (TSC1/TSC2) is a key negative regulator of mTORC1 activity. 37 On exposure of cells to hypoxia, growth factors, energy stress, stress, and amino acids, mTORC1 is activated or inhibited through different pathways and participates in various biological processes. Downstream effectors of mTORC1, including eIF-4E-binding protein (4E-BP1), sterol response element-binding protein (SREBP), hypoxia-inducible factor 1α (HIF-1α), transcription factor EB (TFEB), and activating transcription factor 4 (ATF4), can regulate mRNA translation, lipid synthesis, glucose metabolism, lysosomal biogenesis, and nucleotide metabolism. 38 The regulation of mRNA translation by mTORC1 is primarily mediated via the activation of ribosomal protein S6 kinase 1 (p70S6K) by 4E-BP1. In addition, mTORC1 plays an important role in the regulation of autophagy. The ULK1-Atg13 complex is conducive to the formation of autophagosomes, whereas mTORC1 phosphorylates ULK1 to facilitate complex formation with Atg13, thereby mediating the regulation of autophagy. 39 Amino acids induce mTORC1 activation through small GTPases of the Rag family to coordinate nutrient requirements for cell growth. 40 Growth factors then activate the PI3K-Akt axis via corresponding receptors, and the activated Akt can inhibit complex TSC1/2 formation, thereby activating mTORC1. 32 In addition, tumour necrosis factor-alpha (TNF-α) is phosphorylated by its downstream kinase IKKβ, which also leads to the activation of mTORC1 by inhibiting the formation of the TSC1/TSC2 complex. 41 AMPK is a sensor of cellular energy levels, and mTORC1 signalling can also sense intracellular energy changes, suggesting a potential regulatory relationship. Studies have found that hypoxia and stress stimulation can activate the AMPK signalling pathway and phosphorylate Raptor, which reduces mTORC1 activity through allosteric inhibition, thereby regulating cell energy metabolism and promoting cell survival. 42 However, compared with mTORC1, the molecular regulation of mTORC2 and downstream signalling was relatively reduced. mTORC2 has been shown to play important roles in regulating endocytosis, sphingolipid biosynthesis, cell survival, and actin cytoskeleton reorganization. 43 During growth factor stimulation, PI3K phosphorylates PI(4,5)P2 to generate PI (3,4,5)P3 and subsequently binds to mSIN1 to relieve mTORC2 inhibition via mSin1, further leading to mTORC2 activation, which, in turn, phosphorylates Akt, which is involved in the regulation of biological processes. 44 In response to energy stress, the AMPK signalling pathway can directly activate mTORC2 to enhance cell survival. 45,46 In addition, mTORC2 can promote cell survival through downstream genes, serum and glucocorticoid-induced protein kinase 1 (SGK1) and protein kinase C-α (PKC-α). 47 Studies have shown that mTORC1 can downregulate PI3K signalling via S6K1, resulting in mTORC2 inactivation, suggesting a potential feedback control loop between mTORC1 and mTORC2. 44 Recent studies have shown that mTORC2 is also involved in regulating autophagy, cell senescence, and induction of osteogenic differentiation. 48,49 In addition, studies have shown that mTORC3 affects the proliferation of tumour cells through 4E-BP1 35 ; however, the underlying regulatory mechanism remains elusive, warranting in-depth future investigations ( Figure 2).

| MECHANISMS UNDERLYING MTOR SIGNALLING IN IVD CELLS
IDD is a complex degenerative disease of the musculoskeletal system mediated by multiple pathological processes. Studies have shown that autophagy, oxidative stress, inflammatory responses, imbalances in ECM synthesis and catabolism, and genetic factors can crucially contribute to IDD. 50 Numerous studies have shown that the mTOR signalling pathway plays an important role in regulating cell growth and metabolism. Recent studies have found that human IVD NP tissue exhibit the expression of molecules related to mTOR signalling; however, owing to insufficient tissue sample size, the correlation between its expression level and grade of IDD degeneration needs to be further explored. 51,52 NP cells treated with high oxygen tension showed increased levels of intracellular reactive oxygen species (ROS) and ECM degradation. 53 In addition, compared with the normal group, high oxygen tension leads to abnormal expression of various genes in NP cells, and the Kyoto Encyclopedia of Genes and Genome pathway analysis showed that the response of NP cells to high oxygen tension involves multiple pathways, including the mTOR signalling pathway. 53 In IDD, compared with mTORC2 inhibition, mTORC1 F I G U R E 1 (A) Schematic diagram of the structure of mTOR and the functions of its components. (B) Schematic structure of mTORC1, mTORC2 and mTORC3. mTOR, mammalian target of rapamycin inhibition enhanced autophagy in NP cells, thereby reducing NP cell apoptosis, senescence, and ECM degradation, suggesting that mTORC1 may play a key role in IDD progression. 51,52 Therefore, the mTOR signalling pathway participates in the regulation of autophagy, oxidative stress, inflammation, apoptosis, and ECM homeostasis in IDD ( Figure 3).

| mTOR signalling and autophagy in NP
Autophagy is the catabolic mechanism employed by eukaryotic cells to maintain nutrient homeostasis, which captures and degrades misfolded proteins and damaged or aged organelles in lysosomes, while recycling intracellular components to maintain intracellular homeostasis. 54,55 Recent studies have suggested that autophagy disorders in IVD cells may be an important factor in IDD. 56,57 Compared with normal IVD, the IVD of IDD exhibited fewer autophagosomes, Beclin-1, and a lower ratio of LC3-II to LC3-I. 58 Interestingly, Gruberde et al. found increased expression of autophagy-related genes (Beclin-1, ATG8, and ATG12) in the IVD of IDD. 59 Furthermore, studies have found that appropriate autophagy activity is beneficial to the survival of NP cells during serum deprivation, while excessive autophagy leads to NP cell death. 60 Therefore, body-induced autophagy increases in the early stages of in vitro degeneration, which may provide a protective mechanism for in vitro cells. Under the continuous action of various unfavourable factors, it can lead to excessive activation of autophagy, engulfing normal organelles or degrading normal proteins, thereby increasing the potential for cell death and accelerating the IDD process.
The mTOR signalling pathway and autophagy have attracted increasing attention from researchers. 61 Studies have shown that the mTOR signalling pathway can regulate autophagy in chondrocytes and has a beneficial effect on rat osteoarthritis (OA). 62 Tu et al. 63 found that the expression of sestrins was lower in NP tissues of patients with IDD. Overexpression of sestrins could inhibit mTOR activity in NP cells, resulting in an increased LC3-II/I ratio and decreased p62 expression, reducing endoplasmic stressinduced apoptosis and ECM degeneration. However, decorin, increased in NP tissues of patients with IDD, reportedly suppresses mTOR phosphorylation by inhibiting the PI3K/AKT signalling pathway, thereby promoting autophagy and reducing apoptosis in rat NP cells. 64 We previously reported that bromodomain-containing protein 4 (BRD4) expression was elevated in NP tissues of patients with IDD. Silencing of BRD4 can activate the AMPK pathway in human NP cells, inhibit mTOR activity, and promote the phosphorylation of ULK1, increasing the LC3-II/LC3-I ratio and Beclin-1 levels and decreasing the level of P62. 65 Immunofluorescence (LC3) and transmission electron microscopy (autophagosome formation) further confirmed that silencing BRD4 promoted autophagy and reduced apoptosis and senescence. 65 In short, the above-listed studies have shown that inhibition of the mTOR signalling pathway promotes autophagy, which is beneficial for the survival of NP cells and the maintenance of physiological functions. In addition, various natural compounds can inhibit mTOR activity, which provides a new approach for the development of drugs targeting mTOR to treat IDD (Table 1).
The mTOR-mediated signalling pathway. Growth factors, hypoxia and stress, amino acids, energy stress, and TNF-α activate mTORC1 by stimulating different signalling axes involved in regulating protein translation, lipid and sugar metabolism, lysosomal biogenesis, nucleotide metabolism, and autophagy. The activation of mTORC2 is involved in autophagy, cell survival, osteogenic differentiation, and senescence. The mTORC3 participates in cell proliferation. mTOR, mammalian target of rapamycin; TNF-α, tumour necrosis factor-alpha

| mTOR signalling and oxidative stress in NP
Oxidative stress is defined as an imbalance between ROS production in the body and endogenous antioxidant defence mechanisms, resulting in disrupted redox signalling and related molecular damage. 66 Mitochondria are major sites of ROS production. Under the stimulation by unfavourable factors, excessive ROS production by cells can cause protein and lipid peroxidation and DNA damage, leading to cellular dysfunction or irreversible cell damage and death. 67 Chen et al. 68 reported that t-butyl hydroperoxide (TBHP)induced oxidative stress promoted senescence and apoptosis in NP cells in vitro. In addition, ROS can lead to increased CEP apoptosis and promote CEP calcification via the MAPK/NF-κB pathway. 69 Therefore, oxidative stress may be an important contributing factor to the acceleration of IDD.
The mTOR signalling pathway has been confirmed to be closely related to the regulation of oxidative stress. The neurological function and oxidative stress state of patients with acute stroke were improved through the mTOR signalling pathway. 70 Dai et al. 71 reported that mTOR-mediated autophagy could alleviate oxidative stress damage in chondrocytes and inhibit the progression. According to Wu et al.,72 compared with the normoxia group, pramlintide showed superior inhibition of ROS production and reduced apoptosis and ECM degeneration in NP cells under hypoxic conditions, which activated the AKT-AMPK/mTOR signalling pathway. Similarly, Chen et al. 60 Challenges of mTOR-based treatment of IDD. Autophagy, oxidative stress, inflammation, ECM homeostasis, senescence, and apoptosis mediated by mTOR signalling can markedly influence IVD cell fate and IDD pathophysiology. Inhibiting or activating mTOR signalling can affect the above-mentioned pathophysiological processes and is beneficial to delay the progression of IDD. Therefore, the function of targeting mTOR signalling in IDD requires further clarification. IDD, intervertebral disc degeneration; IVD, intervertebral disc; mTOR, mammalian target of rapamycin by balancing ATP demand and ATP supply pathways, of which activation of the mTOR signalling pathway may be a potential regulatory mechanism. Interestingly, upon the continuous action of unfavourable factors, NP cells may increase autophagic flux to remove intracellular ROS and reduce cell damage by inhibiting mTOR activity. This suggests that autophagy, mediated by the mTOR signalling pathway, plays a crucial role in regulating oxidative stress under unfavourable conditions, but the underlying mechanism remains unclear. Therefore, it is necessary to comprehensively explore the specific mechanism of mTOR signalling in the regulation of oxidative stress (Table 1).

| mTOR signalling and inflammation in NP
Inflammation is an important driver accelerating the pathogenesis of IDD. IVD regression is often accompanied by the infiltration of mast cells, macrophages, and neutrophils, which secrete various inflammatory mediators, including TNF-α, interleukin (IL)-1α/β, IL-6, IL-8, IL-17, and prostaglandin E2 (PGE2). 74 As TNF-α and IL-1β exhibit potent pro-inflammatory activities, they have been extensively studied in IDD. It has been reported that mTOR signalling is also involved in the regulation of inflammatory responses. 77 Xue et al. 78 reported that the mTOR signalling pathway is involved in the regulation of autophagy and inflammatory responses in chondrocytes. TNF-α-inducible protein 3 (TNFAIP3) is a ubiquitin-modifying enzyme primarily involved in the regulation of mTOR signalling pathway. 79 Chen et al. 24 found that TNFAIP3 inhibited the mTOR signalling pathway to promote autophagy, reduce the expression of TNF-α and IL-1β, and inhibit ECM T A B L E 1 Drugs or genes that positively or negatively regulate mTOR activation in IDD degeneration in human NP cells. While pretreatment with mTOR inhibitor (Torin1), TNFAIP3-induced autophagy was attenuated, and inflammation was exacerbated, suggesting that activation of autophagy reduced inflammation. 24 Moracin inhibited the expression of TNF-α, IL-6, and IL-1β in LPS-induced rat NP cells and promoted autophagy by suppressing the PI3K/AKT/mTOR signalling pathway. 80 In addition, Yi et al. 81 reported that p65-siRNA transfection inhibited the NF-κB pathway, significantly reduced p-AKT and p-mTOR expression, promoted autophagy, and reduced LPS-induction inflammation in human NP cells. Autophagy mediated by the mTOR signalling pathway may play a key role in regulating NP cell inflammation. Thus, further research will provide novel insights into the inflammatory regulation of mTOR in NP cells (Table 1).   16 In addition, SASP can cause changes in the cellular microenvironment in an autocrine or paracrine manner, further accelerating the senescence of self or neighbouring cells. 95  for "spare macromolecular parts" to provide raw materials for proliferating cells. In summary, the mTOR signalling pathway is crucial for regulating cellular senescence (Table 1).

| mTOR signalling and apoptosis in NP
Apoptosis is programmed cell death regulated by multiple signal transduction pathways, characterized by chromosome condensation, cell shrinkage, DNA degradation, and apoptotic body formation. 98 A high apoptosis rate was observed in IVD-degenerated tissue specimens. 99 Increased apoptosis leads to a decreased number of cells within the IVD, which, in turn, disrupts tissue homeostasis and plays an important role in IDD pathogenesis. 100 Therefore, inhibition of apoptosis could be a potentially attractive therapeutic strategy for IDD. mTOR signalling also plays an important role in the regulation of apoptosis.
In IDD, compression can activate the JNK signalling pathway and inhibit the Akt/mTOR signalling pathway to promote autophagy in NP cells and reduce apoptosis. 101 Xie et al. 96  ished their apoptosis-inhibiting effect. 104,105 Collectively, the mechanism underlying mTOR signalling in the regulation of apoptosis remains unclear, but the activation of autophagy may inhibit excessive apoptosis. Therefore, it is necessary to further investigate the potential regulatory relationship between mTOR, autophagy, and apoptosis to provide a basis for developing biological therapies for IDD (Table 1).

| mTOR signalling in AF and CEP
AF plays a critical role in IVD homeostasis. AF consists of a series of concentric circular lamellae with fibres in adjacent lamellae, approximately ±60 from the orientation of the spinal axis, which helps AF tissue support multidirectional loading during normal activity. 106 When the spine is subjected to axial compression, the tightly packed annulus fibrosus absorbs pressure from the NP to the AF wall. 107 Therefore, AF degeneration accelerates the progression of IDD.
Recent studies have shown that the mTOR signalling pathway partici- research on its potential mechanism is needed, which would be of considerable significance for IDD therapy (Table 1).

| MTOR SIGNALLING AND NONCODING RNAS (NCRNAS) IN IDD
Previous studies have shown that approximately 70% of patients with IDD exhibit genetic variants, suggesting that genetics may be a key factor in the pathogenesis of IDD. 113  and miR-654-5p has been reported in IVD tissues. Wang et al. 116 found that the expression of miRNA-21 was increased in the IVD tis-  Table 2).
lncRNAs are a group of RNA transcripts longer than 200 nucleotides that do not encode proteins and play important functional roles in regulating the transcription and translation of metabolism-related genes. 122 Recent studies have shown that lncRNAs are closely associated with the occurrence of IDD. 123 Zhan et al. 120 60,120 ; therefore, regulating mTOR to maintain an appropriate level of autophagy and exert a protective effect on cells is urgently needed in IDD. Additionally, a few studies have shown that activation of the mTOR signalling pathway can also reduce cell apoptosis and inflammation-induced damage. However, these studies have rarely investigated whether autophagy is involved in regulating apoptosis and inflammation, which may be the main reason for the divergent results. Therefore, there is an urgent need to further explore the specific mechanism of mTOR signalling in IDD, which brings both opportunities and challenges to its treatment. are recruited and react with NP to produce autoantibodies, thereby triggering an immune response and releasing cytokines to amplify inflammation, accelerating IDD progression. 127,128 However, mTOR signalling is yet to be implicated in the regulation of immune function in IDD, which might be a potential target for developing new therapies. Moreover, the mTOR signalling pathway is known to be involved in glycolysis, pyroptosis, and ferroptosis in various diseases. [129][130][131] In IDD, glycolysis is the main pathway of energy metabolism in NP cells, and inhibition of glycolysis in NP cells can significantly impact their normal physiological functions. 132 Inhibition of pyroptosis or ferroptosis was found to delay IDD progression. 133,134 Furthermore, the functions of the mTOR signalling pathway in glycolysis, pyroptosis, and ferroptosis are yet to be explored in IVDs, which might provide new ideas for assessing the mTOR pathway in IDD. In conclusion, a comprehensive understanding of the relationship between mTOR and IDD will provide a clear theoretical basis for the development of safe and effective mTOR pathway-targeted drugs for IDD.

AUTHOR CONTRIBUTIONS
Hai-Wei Chen, Jian-Wei Zhou, and Guang-Zhi.Zhang contributed equally to this work and is listed as a co-first author. All authors contributed to the revision and approved the submitted version.