Mitochondrial quality control: the real dawn of intervertebral disc degeneration?

Abstract

Intervertebral disc degeneration is the most common disease in chronic musculoskeletal diseases and the main cause of low back pain, which seriously endangers social health level and increases people’s economic burden. Disc degeneration is characterized by NP cell apoptosis, extracellular matrix degradation and disc structure changes. It progresses with age and under the influence of mechanical overload, oxidative stress and genetics. Mitochondria are not only the energy factories of cells, but also participate in a variety of cellular functions such as calcium homeostasis, regulation of cell proliferation, and control of apoptosis. The mitochondrial quality control system involves many mechanisms such as mitochondrial gene regulation, mitochondrial protein import, mitophagy, and mitochondrial dynamics. A large number of studies have confirmed that mitochondrial dysfunction is a key factor in the pathological mechanism of aging and intervertebral disc degeneration, and balancing mitochondrial quality control is extremely important for delaying and treating intervertebral disc degeneration. In this paper, we first demonstrate the molecular mechanism of mitochondrial quality control in detail by describing mitochondrial biogenesis and mitophagy. Then, we describe the ways in which mitochondrial dysfunction leads to disc degeneration, and review in detail the current research on targeting mitochondria for the treatment of disc degeneration, hoping to draw inspiration from the current research to provide innovative perspectives for the treatment of disc degeneration.

Introduction

Low back pain is the leading cause of disability worldwide. Almost everyone will experience low back pain at some point in their life, which has a huge impact on the global social economy and quality of life [1,2,3]. A large number of studies have shown that disc degeneration causes low back pain through spinal canal stenosis, herniation, inflammation and so on, which is one of the main pathogenic factors of low back pain [4].

Intervertebral disc consists of three structures, the gelatinous nucleus pulposus as the core is surrounded by a layered fibrous ring, and the cartilaginous endplate covers the upper and lower ends of the disc and connects to the vertebral body, thereby transmitting mechanical pressure of the spine and allowing the spine to extend, bend, rotate, and bend sideways during the motion. IVDD is one of the most common age-dependent chronic diseases that begins in youth and progresses in response to genetic, mechanical stress, aging, trauma and other pathogenic factors [5,6,7]. Degradation of the nucleus pulposus matrix, decreased hydration, infiltration of inflammatory factors, spinal biomechanical changes, vascular and nerve growth are the characteristics of IVDD [8,9,10]. With the aging of nucleus pulposus, the ability to synthesize extracellular matrix decreases, and the metabolism of extracellular matrix is unbalanced and dominated by catabolism (the levels of matrix metallopeptidase (MMP), disintegrin with thrombochondroitin motif and metalloproteinases (ADAMTS) are increased), resulting in loss of agglutincan, increased proportion of type I collagen, and fibrosis of nucleus pulposus. Subsequently, the capacity of the nucleus pulposus in buffering mechanical stress declines, resulting in disc height loss, annulus fibrosus rupture, vaso-nerve growth, and inflammatory factor infiltration under sustained mechanical stress, which aggravates chronic inflammation associated with aging of the nucleus pulposus cells. A large number of infiltrating inflammatory factors, such as TNF-α and IL-1, stimulate the secretion of MMP and ADAMTS, and further aggravate the degradation of extracellular matrix to form a vicious cycle [11]. At the same time, the growth of blood vessels and nerves is also one of the causes of low back pain [12]. The current treatment for disc degeneration is mainly conservative treatment and surgery to relieve pain [13], which cannot reverse the process of degeneration. Classical intervertebral disc replacement and fusion surgery are prone to adjacent intervertebral disc lesions, while Percutaneous endoscopic lumbar discectomy has a greater risk of recurrence [13]. Elucidating the molecular mechanism is a prerequisite for the development of new and effective treatments. So, we reviewed the extensive literature to try to tease out a clear molecular network of disc degeneration and find the key points.

Mitochondria are not only the most important source of ATP, but also participating in key cellular processes such as calcium homeostasis, apoptosis, ROS-mediated signaling pathway, while plays a crucial role in aging and a variety of age-related diseases including disc degeneration [14,15,16,17]. Mitochondria are energy factories with a bilayer membrane structure. Due to invagination into cristae, the inner membrane is divided into cristae membrane and inner boundary membrane, which greatly increases the area of the inner membrane. The oxidative phosphorylation system is located above the ridge membrane, including four respiratory complexes and ATP synthase (respiratory complex V). Mitochondrial DNA is also located on the ridge membrane and is a 16.6 kb long circular DNA molecule containing 37 genes encoding 2 ribosomal Rnas (Rnas), 22 transfer Rnas (t Rnas) and 13 proteins. These 13 proteins constitute hydrophobic proteins that form the core part of the oxidative respiratory chain complex. In addition to the above 13 proteins, 1000–1500 mitochondrial proteins are encoded by nuclear genes, translated by cytoplasmic ribosomes, and transported to the mitochondria via protein transporters in the mitochondrial membrane. Mitochondrial quality control includes mitochondrial biogenesis and mitophagy. When the mitochondrial quality control system is dysfunctional, mitochondrial dysfunction will lead to a series of consequences such as cell metabolism disorder, calcium homeostasis destruction, ROS increase, and apoptosis.

To the best of our knowledge, there have been no articles systematically describing the mitochondrial quality control system and the interaction between mitochondrial dysfunction and IVDD. Therefore, in this review, we systematically introduce mitochondrial biogenesis and mitophagy, and summarize the mechanisms by which mitochondrial dysfunction causes disc degeneration. Meantime, all the references cited in this paper were searched in PUBMED, and the details were put in supplementary materials. Finally, we summarize the current intervertebral disc therapies targeting mitochondrial dysfunction in order to provide innovative perspectives for the treatment of IVDD.

Mitochondrial biogenesis

Transcription factors regulate mitochondrial biogenesis

NRF

Nuclear respiratory factor-1 (NRF-1) acts as a transcriptional activator that promotes transcription through C-terminal transcriptional activation domain (TAD) binding to promoters of multiple mitochondria-associated genes, including respiratory complex subunits [18,19,20,21]. NRF-1 is also able to promote transcription of transcription factor A and B genes by binding and activating their promoters, thereby facilitating transcription and regulation of mitochondrial DNA [22]. In addition, NRF-1 plays a positive activation role in the transcription of the mitochondrial outer membrane translocation complex TOMM gene, thereby promoting the transport of mitochondrial proteins [23, 24]. Moreover, a key role of NRF-1 in the heme synthesis pathway has also been found, which can induce the expression of its key enzymes [25]. NRF-2 is equally important for the transcription of mitochondriarelated genes. NRF-2 is a tetramer complex composed of two independent proteins (α/β) whose DNA-binding domain and transcription-activating domain exist in NRF-α and NRF-β, respectively [26]. NRF-2 was found to be a general activator of genes associated with the cytochrome c oxidase subunit [27,28,29]. In addition, NRF-2 also binds to mitochondrial transcription factor genes to promote their transcription with a weaker effect than NRF-1 [30,31,32]. Remarkably, both NRF-1 and NRF-2 have the same promoter site in many of the same mitochondria-associated genes [21, 33, 34].

ERR

Estrogen-related receptors are orphan nuclear receptors without natural ligands and play important roles in mitochondrial biogenesis and mitochondrial metabolism. Peroxisome proliferator activated Receptor-γ (PPAR-γ) coactivator factor-1 (PGC-1) is able to activate ERRs transcriptional activity. In turn, ERRs activates the expression of other transcription factors, such as GABPA, PPAR, and TBFM2, which promote mitochondrial biogenesis [35].When ERRs gene is knocked out, the role of PGC-1β in oxidative phosphorylation and fatty acid oxidation is inhibited, suggesting that ERRs may be a downstream effector of PGC-1β[36]. In addition, ERRs deficient mice are unable to regulate thermogenesis in cold environments, suggesting that ERRs is required for mitochondrial generation and oxidative phosphorylation in brown adipose tissue [37]. ERRs also plays a wide range of roles in mitochondrial metabolism by recognizing promoters of genes associated with metabolic pathways such as glycolysis, heme synthesis, lipid metabolism, and tricarboxylic acid cycle [38]. Given the harsh nutritional environment in which intervertebral discs are located, nucleus pulposus cells must undergo more precise nutrient utilization and material metabolism, as well as the important role of ERRs in cellular metabolism and mitochondrial development. The relationship between ERRs and IVDD deserves further research.

PGC-1

Peroxisome proliferator-activated receptor-γ-coactivator-1α (PGC-1α) is a major auxiliary transcription factor, which is closely related to mitochondrial biogenesis and energy metabolism, and plays an extremely important role in the physiological and pathological processes of brain, heart, skeletal muscle, fat and other high-energy metabolic tissues [39,40,41,42]. PGC-1α can stimulate the gene expression of a variety of transcriptional activators such as NRF1/2, and can amplify the role of NRF-1 in activating mitochondrial transcription factor gene transcription [43]. Numerous studies have revealed a link between locomotion and mitochondrial biogenesis that PGC-1α is a key player in this link. Exercise may promote mitochondrial biogenesis by activating PGC-1α expression in two ways. One is the activation of AMPK due to ATP depletion and increased AMP/ATP ratio, thus activating the expression of PGC-1α; The other is that a large increase in Ca activates calmodulin kinase (CaMK), which induces the expression of PGC-1α[44,45,46]. Other studies have suggested that exercise may also induce PGC-1α expression through ATF2 and MEF2 by activating the p38 mitogen-activated protein kinase (MAPK) pathway [47]. It is worth noting that the role of PGC-1α in IVDD has been paid more and more attention by researchers. Song et al. ' s research shows that nicotinamide mononucleotide (NMN) in nucleus pulposus cells restores SIRT3 function through AMPK-PGC-1α pathway to improve mitochondrial homeostasis and prevent apoptosis [48]. Interestingly, overexpression of PGC-1α attenuates apoptosis by inhibiting excessive mitophagy in annulus fibrosus cells by up-regulating SIRT2 [49]. The different modes of action of PGC-1α in different cell types of intervertebral discs deserve our further study. As a homologue of PGC-1α, PGC-1β can also act on transcription factors such as NRF and ERRs to promote mitochondrial biogenesis. Unlike PGC-1α, PGC-1β is more expressed in brown adipose tissue development, and cold stimulation has no significant effect on its content in brown adipose tissue [50]. Notably, deletion of one or the other alone did not have a significant effect on brown adipose mitochondrial biogenesis, whereas deletion of both was associated with a lack of mitochondrial biogenesis in both heart and brown adipose tissue [51, 52]. This provides evidence that PGC-1 coactivators are essential for threadgrain biogenesis, and that PGC-1α and PGC-1β have complementary effects.

Mitochondrial protein import

Human mitochondria contain about 1,300 proteins, 99% of which are encoded by nuclear genes and 1% by mitochondrial genes. In addition to the core hydrophobic subunits of the oxidative respiratory chain complex encoded by mitochondrial genes, all proteins in the outer membrane, intermembrane space, intima and matrix subregions are encoded by nuclear genes, which requires an extremely complex and well-regulated mitochondrial protein input system to support the targeted input, assembly and function of mitochondrial proteins. The outer membrane transport complex (TOM) carries the majority of mitochondrial proteins and transports them to different subregions of mitochondria through multiple pathways. The mammalian TOM complex consists of seven subunits: a central channel TOM40, three presequence protein receptors (TOM20, TOM22, TOM70), and three small TOM proteins (5, 6, 7). Most precursor proteins translated by cytoplasmic free ribosomes contain a targeted sequence called a preorder at the n-terminal, which contains a positively charged amphoteric helix. The hydrophobic and hydrophilic side chains of the presequence can be recognized by TOM20 and TOM22, respectively, and then pass through the major transport channel TOM40 [53,54,55,56,57]. Prior to transport, cytoplasmic Hsp70 and Hsp90 not only help the precursor protein maintain its unfolded state and prevent it from aggregating, but also target the precursor protein to TOM70 in the mitochondrial outer membrane [58, 59]. J protein Djp1 assists cytoplasmic Hsp70 in targeting the precursor protein to TOM70, while J protein Xdj1 interacts with TOM22 to assist in the transport of the precursor protein [60]. TOM40 forms a supercomplex with the intimal precursor transporter Tim23. The precursor protein carrying the precursor is first delivered to Tim50 subunit of the TIM23 complex after passing through the outer membrane through Tom40, and then further transported to the intima or stroma via TIM23-TIM17 channels of the TIM23 complex [61,62,63,64]. A negative membrane potential on the inner side of the intima activates the TIM23 complex and promotes the passage of positively charged presequences through the intima [65, 66]. Hsp70, the core of mitochondrial transportase associated motor protein (PAM), interacts with J-type cochaperone Pam18 and J-related protein Pam16 via Tim44, binding to the TIM23 complex in an ATP-driven manner to facilitate the transport of precursor proteins to the matrix [67, 68]. Meanwhile, the ATPase cycle of mtHsp70 is also facilitated by Pam18 and the nucleotide exchange factor Mge1. The presequence protein entering the matrix is first cleaved by mitochondrial processing peptidase, and then removed by 55 k Da intermediate cleavage peptidase (Icp55) and octapeptidase (Oct1) to remove the unstable amino acid residues, resulting in a stable amino terminal that is not easily degraded by matrix proteases [69, 70]. Chaperones such as MtHsp70 and Hsp60 help proteins fold correctly in the matrix [71, 72]. When multiple stressors cause mitochondrial damage, the unfolded protein response (UPR^mt) can dissolve accumulated unfolded proteins and restore mitochondrial protein homeostasis [73, 74]. First, by inducing an increase in mitochondrial chaperone expression such as Hsp70 and Hsp60, UPR^mt not only promotes the correct folding of newly synthesized proteins, but also folds and breaks down misfolded proteins [75, 76]. Second, UPR^mt can promote the expression of nucleo-encoded detoxification enzymes and input components of mitochondrial proteins [77, 78]. In addition, UPR^mt can also promote the expression of a large number of mitochondrial proteases, such as LONP1 and CLPP, which are important for protein maintenance and removal of damaged proteins [79]. Studies have shown that compression induces increased mitochondrial fission in nucleus pulposus cells and leads to apoptosis, while Hsp70 alleviates nucleus pulposus cell apoptosis by up-regulating SIRT3 to inhibit mitochondrial fission [80].

Some precursor proteins contain a hydrophobic transport termination signal near the N-terminal, which prevents transintimal transport of precursor proteins, and then translaterally insert them into the intimal lipid bilayer via the PAM-free TIM23 complex (consisting of TIM23 core and TIM17) in a membrane potential dependent manner [81,82,83]. In addition, the respiratory chain supercomplex composed of TIM23 complex, cytochrome bc1 complex and cytochrome c oxidase can also promote the intimal sorting of membrane potential dependent precursor proteins. Mitochondrial β-barrel proteins are all located on the outer membrane of the mitochondria and have different insertion modes. After the β precursors are transported to the intermembrane space by the TOM complex, mature barrel-like proteins are inserted into the mitochondrial outer membrane by the transport and action of the SAM complex [84]. The SAM complex consists of two SAM50 subunits and peripheral SAM35 and SAM37 subunits. During transport by the SAM complex, the last beta chain of the beta precursor pairs with the first beta chain of SAM50 by hydrogen bonding and replaces the SAM50beta signal interacting with SAM50-β1 [84, 85]. Subsequently, the β precursor extends from the carboxyl terminal until a complete barrel-like structure is formed, and the mature barrel-like protein is formed by the pairing of the first and last β chains. Remarkably, the alpha helical proteins of the outer membrane are not transported by the TOM complex, but are inserted into the mitochondrial outer membrane with the assistance of mammalian mitochondrial carrier homolog 2 (MTCH2) [86].

Although more and more attention has been paid to the study of mitochondria in IVDD, there are few studies on the pathophysiological changes of mitochondria in degenerative nucleus pulposus cells. Here we can reasonably speculate that under stress conditions, the mitochondrial potential of nucleus pulposus cells in harsh environments such as high acid and hypoxia changes, affecting the input and folding of positively charged mitochondrial proteins, resulting in mitochondrial dysfunction. In addition, different from the current mainstream strategy of promoting mitophagy to remove damaged mitochondria to alleviate IVDD, we propose to promote the formation of healthy mitochondria by interfering with all aspects of mitochondrial biogenesis to restore the mitochondrial function level of nucleus pulposus cells to prevent and reverse IVDD.

Mitophagy

Mitophagy, a form of autophagy that selectively degrades mitochondria, is a basic mechanism that regulates mitochondrial quality control and maintains mitochondrial function conserved from yeast to humans [87, 88]. Mitophagy in mammals is divided into PINK/ PrKN-dependent mitophagy and receptor-mediated mitophagy.

PINK/ PRKN-dependent mitophagy

Physiologically, with the assistance of the mitochondrial outer membrane transport enzymes TOM22 and TOM40, TOM20 recognizes the MTS sequence of PINK1 (PTEN-induced putative kinase 1) and mediates PINK1’s entry into the inner membrane through the transport pore formed by TOM40 [89,90,91]. After that, it is cleaved by the mitochondrial inner membrane protease presenilin-associated rhomboid-like protein (PARL) in the inner membrane to produce 52 kda PINK1, which is further decomposed by mitochondrial protease to maintain a low level of PINK1 [89, 92]. When the mitochondrial membrane potential decreased due to oxidative stress, mechanical overload and other external stimuli, pyruvate dehydrogenase kinase 2 phosphorylated the presenilin associated rhomboid protein to make it lose the ability to cut PINK1, thus disrupting the translocation of.

PINK1 and promoting the accumulation of PINK1 in the outer membrane of mitochondria [93, 94]. Subsequently, the accumulated PINK1 directly or indirectly activates the ubiquitin ligase activity of PRKN through phosphorylation of the Ser65 site of the ubiquitin (UBL) domain of PRKN or phosphorylation of the Ser65 site of ubiquitin (Ub) and polyubiquitin chains, and promotes the recruitment and activation of PRKN [95,96,97,98]. In addition, activated PRKN mediates ubiquitination of a variety of mitochondrial outer membrane proteins, such as mitochondrial fusion protein 1/2 (MFN1/2), mitochondrial Rho GTPase protein (Miro), voltage-dependent anion channel protein (VDAC), BAK protein, etc [99,100,101,102]. Mitochondrial fusion proteins are degraded in a proteasome-dependent and P97 (an AAA + ATPase) manner after PRKN ubiquitination in the depolarized mitochondrial outer membrane, thereby preventing the fusion of damaged mitochondria and promoting mitophagy [99]. Miro, an atypical GTase, mediates mitochondrial cytoskeleton transport by anchoring driver proteins to the mitochondrial surface and also controls Ca2 + mitochondrial homeostasis at the endoplasmic reticulum mitochondrial junction (ERMCS) by regulating polo kinase-mediated phosphorylation of miro [102,103,104,105]. Phosphorylation of Miro by PINK accumulated in the mitochondrial outer membrane promotes Miro interaction with PRKN, accelerates Miro ubiquitination and degradation, and thus promotes mitophagy [106]. These ubiquitination actions of recruited and activated PRKN can in turn promote the ubiquitination phosphorylation of PINK to form a positive feedback loop [95, 107]. Interestingly, PRKN can ubiquitinate the foreign protein green fluorescent protein (GFP) and myelin basic protein (MBP) targeted by artificial mitochondria, suggesting that PRKN does not require a consistent substrate recognition sequence [108]. In contrast, PRKN has spatial selectivity for depolarized mitochondria rather than substrate specificity similar to general ubiquitin ligase, which may allow PRKN to ubiquitinate damaged mitochondria efficiently and rapidly. Subsequently, PINK1/ PRKN-dependent autophagy junction proteins (p62/SQSTM1, NBR1, NDP52/ CALCOCO2, TAX1BP1, and OPTN) are recruited to the mitochondria [109,110,111,112,113,114]. These autophagy adaptors contain a ubiquitin binding domain that selectively identifies ubiquitin chains in damaged mitochondrial membranes, and an LC3 interaction region (LIR) that is used to recruit LC3-wrapped autophagosomal vesicles to phagocyse damaged mitochondria [115]. At present, some studies have confirmed that PRKN is a potential target for the treatment of IVDD. Knocking down the expression of leucine-rich repeat kinase 2 (LRRK2) in nucleus pulposus cells can enhance mitophagy by promoting recruitment of PRKN, thereby inhibiting oxidative stress-induced apoptosis of nucleus pulposus cells [116]. In addition, overexpression of Tank-binding kinase 1 (TBK1) also promotes mitophagy in nucleus pulposus cells by relying on PRKN to alleviate puncture-induced nucleus pulposus degeneration [117]. Moreover, up-regulation of PRKN and Nrf-2 expression protected endplate chondrocytes from H2O2-induced mitochondrial dysfunction, oxidative stress, and apoptosis [118]. Many studies have highlighted the key role of targeting PRKN-dependent mitophagy in the treatment of IVDD, but the exact molecular mechanism of PRKN downstream has not been verified in IVDD.It is of great significance to find the specific autophagy adaptor protein expressed in the downstream nucleus pulposus for the study of mitophagy in IVDD.

Receptor-mediated mitophagy

Mitophagy, which is not dependent on PINK/PRKN, is mediated mainly by binding the LIR motif of the autophagy receptor in the mitochondrial outer membrane directly to LC3 on the surface of the autophagy body. BNIP3 and NIX, members of the BH3 only Bcl-2 protein family, induce initiation of mitophagy by direct binding to LC3-GABARAP in a nonubiquitinating manner. BNIP3 inhibits optic atrophy protein (Opa1) -mediated mitochondrial fusion and induces Drp1 translocation to the mitochondria, thereby promoting mitochondrial fragmentation and separation of damaged mitochondria [119, 120]. Notably, the inactivation of BNIP3 leads to increased hydrolysis of PINK1, while hypoxia-induced BNIP3 expression can induce increased PINK1 expression and its accumulation in the mitochondrial outer membrane, thereby promoting recruitment of PRKN to amplify PINK/ PRKN-dependent mitophagy [121]. The role of NIX-mediated mitophagy in cell differentiation and maturation has been demonstrated. In reticulocytes, NIX-mediated mitophagy promotes the clearance of mitochondria, thus helping reticulocytes complete the transition to mature red blood cells [122, 123]. Lack of NIX results in compensatory expansion of erythroid progenitor cells, while overexpression of BNIP3 restores mitochondrial clearance in NIX-/- reticulocytes. In addition, NIX-mediated mitophagy is a key factor in mitochondrial network formation decline during cardiac cell differentiation [124]. Both BNIP3 and NIX are transcriptionally regulated by HIF-1 A, the alpha subunit of hypoxia-inducing factor-1. Under hypoxia conditions, HIF1A can up-regulate the expression of BNIP3 and NIX to promote mitophagy [125, 126]. Since intervertebral disc is a naturally hypoxic tissue, the role of HIF1A-BNIP3/NIX pathway in the occurrence and progression of IVDD has attracted the attention of many researchers. Vedavathi et al. found an increased autophagy throughput in BNIP3 knocked out mice, which confirmed that the absence of a single autophagy receptor does not cause a decrease in autophagy, and showed early signs of disc mutation such as reduced disc height, loss of chondrocyte hypertrophy associated protein expression, and phenotype drift [127].

FUNDC1, another widely expressed mitophagy receptor, regulates mitophagy through phosphorylation and dephosphorylation at different serine sites of its LIR motif [128,129,130]. In times of energy deficiency and stress, AMPK (AMP-activated protein kinase) can induce the recruitment of the autophagy initiating molecule ULK1 to mitochondria to promote mitophagy [131, 132]. ULK1 recruited to mitochondria phosphorylates Ser17 of the FUNDC1 LIR motif to promote its binding to LC3¹³⁰. At the same time, the side chain of LC3B undergoes structural rearrangement to accommodate FUNDC1 phosphorylation. However, Src kinase phosphorylates the Tyr18 site of FUNDC1 and prolongs its side chain, thereby interfering with the hydrophobic pocket of LC3B and disrupting the interaction between them [133]. Tyrosine protease 2 (CK2) phosphorylates Ser13 of FUNDC1 and also interferes with the FUNDC1-LC3B interaction, whereas PGAM5 phosphatase in hypoxia dephosphorylates Ser13 to promote mitophagy [129, 134]. Cellular hypoxia induces FUNDC1 phosphorylation at Ser17 and dephosphorylation at Ser13 and Tyr18 to enhance the interaction with LC3B and promote mitophagy. The phosphorylated state of FUNDC1 is also able to regulate mitochondrial division and fusion. Dephosphorylated FUNDC1 may lead to dissociation of the Fundc1-OPA1 complex, promote the formation of the FUDNC1-Drp complex, mediate mitochondrial division, and thus promote mitophagy [135]. Under hypoxia, USP19, a mitochondria-associated endoplasmic reticulum (MEM) protein, deubiquitinized FUNDC1 at the mitochondria-endoplasmic reticulum contact site, promoting Drp1 oligomerization and Drp1 GTPase activity, thereby promoting mitochondrial division [136]. Meanwhile, the interaction of FUNDC1 with calponin (ER protein) and Drp1 at the mitochondrial ER contact site can also promote mitochondrial cleavage and mitophagy [137]. More importantly, the intervertebral disc is in a hypoxic environment for a long time. Therefore, the important role of HIF-1 / FUNDC1 / mitophagy in maintaining the homeostasis of intervertebral disc cells is self-evident. When disc degeneration occurs, changes in the expression of HIF-1 and FUNDC1 during loss of hypoxic environment deserve our attention. In addition, autophagy receptors such as cardiolipin, BCL2L13, SAMM50 and AMBRA1 have important effects on the regulation of mitophagy [138,139,140,141].

Many E3 ubiquitin ligases are involved in this pathway. ARIH1 is an E3 ubiquitin ligase that belongs to the RBR family (Ring-in-in-ring) with PRKN and has a completely different substrate. PINK can activate ARIH1, and the activated ARIH1 ubiquitination mitochondrial outer membrane protein regulates mitophagy [142]. SIAH1, a ring-type E3 ubiquitin ligase, is involved in the mitophagy pathway via the PINK1-synphilin-1 (alpha-synuclein gene interaction protein) -SIAH-1 complex. Synphilin-1, recruited by PINK to the mitochondria, induces SIAH1 ubiquitination of mitochondrial outer membrane proteins, thereby recruiting LC3 and Lamp1 (lysosomal marker) to the mitochondria to initiate autophagy [143]. MUL1 is a unique E3 ubiquitin ligase embedded in the mitochondrial outer membrane, which can ubiquitinate the mitochondrial outer membrane proteins such as Drp1 and MFN, and stimulate mitochondrial division by ubiquitinating Drp1 and MFN2, thereby promoting mitophagy [144, 145]. HUWE1, an E3 ubiquitin protein ligase 1 containing the HECT, UBA and WWE domains, can exert different effects on mitophagy by ubiquitinating different substrates. On the one hand, HUWE1 assembles the K6-linked ubiquitin chains, which are particularly important for the recognition of mitophagy, and the ubiquitination MFN initiates autophagy [146]. On the other hand, HUWE1 inhibits mitophagy by promoting its degradation by ubiquitinating ATG101 [147].

Mitochondrial dysfunction and IVDD

A large number of studies have revealed that many factors leading to disc degeneration are related [80, 148, 149]. Mitochondrial injury is considered to be the pathologic basis for the occurrence and progression of IVDD. Mitochondrial dysfunction refers to reduced mitochondrial bioproduction, altered membrane potential, decreased number of mitochondria, and altered oxidized protein activity due to accumulation of ROS in cells and tissues. Dysfunctional mitochondria cause a lack of energy in cells and also negatively affect cell function by releasing various harmful molecules. In addition, mitochondrial dysfunction leads to overproduction of ROS, calcium disturbances, and leakage of factors such as cytochrome C and apoptosis-inducing factors, eventually leading to apoptosis [48, 150,151,152](Fig. 1).

ROS is a byproduct of oxidative phosphorylation of mitochondria, and mitochondria are the source of the vast majority of ROS in mammals [153]. Normally, ROS production and antioxidant enzyme levels are in balance to prevent excess ROS damage. However, the intervertebral disc is in an unfavorable microenvironment of hypoxia and hyperosmosis. Under stress such as aging, mechanical overload, oxidative stress, inflammation, and metabolic disorder, the mitochondrial permeability transition pore will abnormally open, resulting in the reduction of mitochondrial membrane potential, mitochondrial swelling, mitochondrial endometrium ridge loss, oxidative respiratory chain damage, and finally mitochondrial dysfunction [154,155,156,157]. More electrons overflow from the damaged respiratory chain, thus generating more ROS; At the same time, excess ROS can exacerbate mitochondrial dysfunction in a vicious cycle. Excessive ROS causes cellular oxidative stress, which in turn damages cellular proteins, lipids, DNA, and extracellular matrix through metabolic control and inflammation [7]. For example, through the NF-kB pathway, ROS causes the activation of NLRP3 in NPCS and promotes the release of intracellular inflammatory factors, thus promoting aging and death of myeloid cells [158]. In addition, ROS also causes disc degeneration by activating MAPK-ERK, DDR, and small RNA signaling pathways.

MtDNA mutations accumulate continuously in mitochondria with aging and cell senescence, and colocalization with oxidative respiratory chain also makes mtDNA vulnerable to ROS damage and accelerates mutation [159, 160]. Because mtDNA encodes the core subunit of the respiratory chain complex, mtDNA mutations can cause structural damage of the respiratory chain, decrease the efficiency of ATP production, and increase the production of ROS, thus leading to aging and various age-related diseases (Fig. 1). Genetically engineered mice with mtDNA mutations show signs of premature aging, such as anemia, shortened lifespan, graying hair, osteoporosis and hair loss [161,162,163]. During mitochondrial stress, the mitochondrial outer membrane becomes permeable and mtDNA is released into the cytoplasm. mtDNA in the cytoplasm can promote the aging phenotype SASP by activating the cGAS-STING pathway [164]. Although there are few studies on mtDNA in IVDD, given the close relationship between IVDD, mitochondria and aging, it is very promising to explore the influence of mtDNA mutation on intervertebral disc mutation.

Mitochondria are a dynamic network that constantly fuses and divides in response to damage signals and metabolic changes [165,166,167]. The major proteins involved in the mitochondrial fusion process that are balanced by mitochondrial division and fusion and contribute to many diseases include mitochondrial fusion protein 1 (MFN1), mitochondrial fusion protein 2 (MFN2), and optic atrophy type 1 (OPA1). Mitochondrial fission proteins include motor protein-associated protein 1 (DRP1), mitochondrial fission factor (MFF), mitochondrial fission protein 1 (Fis1), mitochondrial motor proteins MiD49 and MiD51 [168, 169]. The imbalance of mitochondrial dynamics is a key factor in the pathogenesis of aging, cancer, cardiovascular disease and other diseases [170, 171]. Changes in mitochondrial dynamics in IVDD have been found. For example, the accumulation of prosenin in NP cells can shift mitochondrial dynamics toward fission events by decreasing levels of Opa1 and Mfn1/2 and increasing levels of Drp1. However, Taxus chinensis can significantly reduce presenilininduced mitochondrial dysfunction and NP cell senescence [152]. In addition, under mechanical overload, the protein levels of Drp1, Mff and Fis1 are significantly up-regulated, while the protein levels of Opa1 and Mfn1/2 are down-regulated, which leads to impaired mitochondrial function and increased apoptosis of NP cells (Fig. 1). Meanwhile, mitoQ can ameliorate such damage [172]. Similar results suggest that mitochondrial dynamic balance is important for maintaining mitochondrial function and preventing damage in NP cells.

Fig. 1

Mitochondrial quality control and IVDD. Under the effects of mechanical load, aging, hypoxia, inflammation and other stress, mitochondria of NP cells undergoes a series of changes. First, mitochondrial membrane potential changes, which impinge on voltage-dependent mitochondrial protein input, thus affecting mitochondrial biogenesis. At the same time, the change of membrane potential will hinder the transport of PRKN and make it stay in the outer membrane of mitochondria, thus promoting mitochondrial autophagy. Second, the vicious cycle between oxidative respiratory chain, ROS, and mtDNA is an important cause of mitochondrial dysfunction. The damaged oxidative respiratory chain produces an excess of ROS, which in turn causes it to be further damaged and causes mutations in mtDNA (the gene encoding the respiratory complex). Excessive ROS not only activates NF-κB/MAPK, NLPR3/Caspases, Ca + overload, but also promotes the expression of SASP, which leads to mitochondrial dysfunction and apoptosis. However, the complex composed of Nrf2 and MAF can act on the antioxidant reaction element ARE in the promoter region of the target gene, thus promoting the expression of antioxidant genes. In addition, Sirt3 plays an antioxidant role by acting on FOXO3 to promote the expression of SOD2. In addition, the expression of mitochondrial mitogen increased under stress, which damaged mitochondrial integrity and caused mitochondrial dysfunction

Therapeutic strategies targeting mitochondrial dysfunction of IVDD

Sirtuins

Sirtuins are enzymes that depend on NAD+, and a total of 7 sirtuins are present in the human body. Among them, sirt3-sirt5 contains an n-terminal mitochondrial signaling sequence that is localized in the mitochondria. They are involved in the process of cellular aging by regulating multiple mitochondrial signaling pathways. Sirtuins catalyze a variety of NAD + dependent reactions, such as deacetylation, deacylation, and ADP ribosylation. Dependence on NDA + as a coenzyme also makes Sirtuins a key sensor of cellular metabolism and REDOX status [173, 174]. Reduced mitochondrial NAD+/NADPH ratios result in the loss of Sirtuins activity to cope with acute mitochondrial stress, which leads to severe impairment of cell homeostasis [175]. In addition, many studies have found that restoring NAD + levels during stress or aging can directly activate mitochondrial Sirtuins to restore cell homeostasis [176, 177]. The proteomic characterization of the mitochondrial Sirtuins protein interaction network by Wen Yang et al. revealed multiple functional plate interactions between Sirtuins and mitochondria [178]. Among them, sirt3 was found to be most correlated with mitochondrial proteins, which not only interacts with proteins in amino acid metabolism, fatty acid oxidation, tricarboxylic acid cycle and oxidative respiratory chain, but also related to mtDNA replication, transcription and translation. The interaction network demonstrated the dynamic regulatory capacity of Sirtuins in mitochondrial homeostasis and stress. Sirt3 binds to ATP5O, a subunit of ATP synthase, under homeostasis conditions, and dissociates and deacetylates substrate with ATP synthase during mitochondrial membrane depolarization and mitochondrial matrix PH reduction, which promots oxidative metabolism to restore proton gradient [178].

SIRT3 is essential for the regulation of mitochondrial ROS. First, sirt3 directly deacetylates superoxide dismutase (SOD2) to promote ROS clearance [179, 180]. Second, sirt3 deacetylates all oxidative respiratory complex proteins to improve electron transport efficiency, reduce ROS production, and increase ATP production [181,182,183]. Finally, SIRT3 reduces ROS levels by activating FOXO3A to induce transcriptional antioxidant programs, as well as inducing transcription of nuclear and mitochondrial genes involved in antioxidant programs and ECT function [184, 185](Fig. 1). The regulation of ROS by the Sirtuin protein family influences many pathological processes of aging and IVDD. sirt3 prevents apoptosis of nucleus pulposus cells induced by advanced glycation products by maintaining mitochondrial REDOX balance [48]. Sirt3 can also promote anti-oxidative stress and delay IVDD by up-regulating SIRT3/FOXO3/SOD2 signaling pathway [186]. Lin et al. ‘s study showed that overexpression of SIRT3 in vivo can alleviate oxidative stress in nucleus pulpocytes, delay aging and matrix degradation, and thus delay disc degeneration [187]. Treatment with magnolia magnolia can activate AMPK-PGC-1α-SIRT3 signaling pathway to maintain mitochondrial antioxidant capacity, promote mitochondrial dynamics, activate mitophagy and inhibit the aging and apoptosis of nucleus pulpocytes induced by TBHP [188]. In conclusion, sirtuin protein family plays an important role in mitochondrial reoxidation, mitochondrial metabolism, signal transduction and cell homeostasis, making it one of the research focuses on anti-aging and delay of IVDD.

Nrf2

Nuclear factor E2 related factor 2 (Nrf2), also known as nuclear factor erythrocyte 2-like 2 (NFE2L2), is a major antioxidant transcription factor encoded by the human NFE2L2 gene, capable of coordinating stress activation and basal activation of a large number of protective genes [189]. Nrf2 protein contains seven highly conserved domains, Neh1 through Neh7, each with a different function [190, 191]. Keap1, an aptamer of Cul3’s E3 ubiquitin ligase, interacts negatively with the Neh2 domain of Nrf2 to regulate Nrf2 activity and promote continuous ubiquitination and degradation of Nrf2 [192,193,194]. When ROS levels in cells are increased, the cysteine residue of Keap1 is covalently modified, resulting in Keap1 inactivation and the inhibition of Nrf2 ubiquitination and the accumulation of new synthetic Nrf2 [195]. Subsequently, Nrf2 is transported to the nucleus, where it forms a heterodimer with the small muscle aponeurotic fibrosarcoma (Maf) protein [196, 197]. Next, Nrf2-MAF dimerization binds to antioxidant response elements (ARE) in the promoter region of the Nrf2 target gene, promoting the transcription of downstream antioxidant genes, Including superoxide dismutase (SOD), heme oxygenase-1 (HO-1), catalase (CAT), glutathione (GSH), and NADPH quinone dehydrogenase 1 (NQO1) [191, 198](Fig. 1). Since oxidative stress is the main pathological mechanism leading to IVDD, the Nrf2 antioxidant system plays an extremely important role in delaying the progression of IVDD. In compression-induced NP cells, MitoQ increases Nrf2 activity by inhibiting the expression of Keap1, thereby maintaining REDOX balance and restoring mitochondrial dynamic balance, and reversing the stress-induced apoptosis of NP cells [172]. Hua et al. confirmed that icariin promoted mitochondrial biogenesis by activating the Nrf-2 signaling pathway to up-regulate the expression of nuclear respiratory factori-1 (NRF-1) and mitochondrial transcription factor (TFAM) proteins, thereby alleviating hydrogen peroxidation-induced apoptosis of nucleus pulpocytes [199]. Studies have shown that many antioxidants, such as dimethyl fumarate, ocyanin-3-glucoside, ullistatin and sulforaphane, can enhance the activity of NRF2-mediated HO-1 pathway to relieve oxidative stress of NP cells and delay disc degeneration [200,201,202,203]. Levulinic acid not only alleviates TBHP-induced oxidative stress by upregulating the production of antioxidant proteins such as HO-1、 NQO1 and SOD induced by Nrf2, but also alleviates TBHP-induced inflammatory response and extracellular matrix degradation, thereby delaying the progression of IVDD [204]. Rapamycin induced autophagy enhances Nrf2/Keap1 signal transduction and promotes the expression of antioxidant proteins, thereby eliminating ROS, alleviating cell senescence, reducing osteogenic differentiation of CESCs, and ultimately protecting CEPs from chronic inflammatory induced degeneration [205]. In addition, hydrogen peroxide leads to mitochondrial dysfunction and apoptosis by inducing oxidative stress on the cartilage endplate, whereas polydatin can up-regulate the expression of Parkin and Nrf-2 to counteract this damaging effect of H2O2 [118]. Interestingly, Tang et al. demonstrated that Nrf2 can drive the Keap1-Nrf2-p62 feedback loop to activate autophagy in response to excessive oxidative stress during disc degeneration [206]. In summary, Nrf2, as the most important role of cellular antioxidant defense system, plays an important role in the protection of intervertebral disc cells under ROS, inflammation and other stimuli, and is a potential therapeutic target.

Others

A variety of drugs can restore mitophagy flux to restore mitochondrial function, thereby delaying disc degeneration. Curcumin inhibits oxidative stress and mitochondrial dysfunction induced by tert-butylhydrogen peroxide (TBHP-), thereby inhibiting apoptosis, senescence and ECM degradation of human NP cells; Autophagy was induced and enhanced in AMPK/mTOR/ ULK1-dependent manner, increasing phosphorylation of AMPK/ULK1 and decreasing phosphorylation of mTOR. CUR mitigated TBHP-induced disruption of autophagosome - lysosome fusion and impaired lysosome function, thereby contributing to the restoration of blocked autophagy clearance [207]. Urolithin A activates AMPK signaling pathway to induce mitophagy to alleviate TBHP-induced NP cell apoptosis [208]。SIRT1 protects NPC from apoptosis of nucleus pulposus cells under various stress conditions by upregulating mitophagy [209, 210].Zn²⁺ transport is a key regulator of mitochondrial dynamics and mitophagy. Song et al. ‘s study revealed that NLRX1 agonist NX-13 can enhance the interaction between NLRX1 and the Zn²⁺ transporter SLC39A7 to regulate mitochondrial Zn²⁺ transport, thereby reducing mitochondrial damage, delaying NPC aging and alleviating disc degeneration [211]. However, upregulating the HIF-1 α/NDUFA4L2 pathway alleviates nucleus pulposus apoptosis by inhibiting excessive mitophagy [212]. In addition, it has been demonstrated that collopyrite nanomases inhibit oxidative stressinduced aging by inhibiting p53-p21 signaling pathway, thereby delaying disc degeneration [213].

It is worth noting that the powerful protective effect of melatonin on disc degeneration has been confirmed by a large number of studies. Secreted by the pineal gland, melatonin is a well-known hormone regulating biological rhythm, and because of its amphipathic properties, it can easily cross the plasma membrane to reach the mitochondria, exert its ability to improve the activity of respiratory complex I and IV, thereby restoring mitochondrial δPSI, increasing ATP production, and reducing cytochromic c leakage and apoptosis to treat IVDD [214,215,216]. The protective effect of melatonin on mitochondria is formed by upregulation of Bcl-2 and downregulation of the expression of Bax, cytochrome c and caspase 3, and activation of Parkin-dependent mitophagy [217]. In addition, melatonin exerts anti-inflammatory effects through the positive feedback loop of IL-1β/NFκB-NLRP3 inflammasome and reduces the production of ROS, and can promote the expression of lectin and collagen II [218]. To sum up, the significant anti-inflammatory, antiapoptosis, anti-aging and mitochondrial function of melatonin in intervertebral disc cells make it a future star in the treatment of IVDD, worthy of further study.

Conclusion

In this review, we systematically elucidate the molecular network of mitochondrial quality control. More importantly, based on the inseparable relationship between mitochondrial dysfunction, aging, and IVDD, we reveal the specific molecular mechanism of mitochondrial dysfunction in IVDD, and review the current research results of IVDD therapy targeting mitochondria in detail. Although the treatment of disc degeneration by targeting Sirtuins, Nrf2, AMPK and other signaling pathways has been extensively studied, to our knowledge, the effect of mtDNA mutation accumulation in senescence cells on the function of disc cells has not been studied. mtDNA encodes the core subunits of various respiratory complexes, as well as ribosomal RNA and transfer RNA, which are important for the transcription and translation of mitochondrial proteins and the integrity of oxidative respiratory chain function. Therefore, the study of the interaction between mtDNA mutation and aging intervertebral disc cells, as well as the molecular mechanism that may induce IVDD, is of great significance to expand the treatment of IVDD. Although mitophagy has become a hot topic in current research, many studies show that mitochondrial biogenesis also plays an irreplaceable role in the prevention and treatment of diseases. A study in ovulated rats found that genistein improves aging of OVX-BMMSCs by inducing mitochondrial biogenesis and mitochondrial autophagy through ERRα [219]. Meanwhile, mitochondria-targeting biomimetic nanoparticles improve motor disorders and anxiety behavior in Parkinson’s disease mice by promoting mitochondrial biogenesis and restoring mitochondrial function [220]. Moreover, disruption of Nrf1-mediated mitochondrial biogenesis leads to mitochondrial damage and slow, progressive degeneration of rod photoreceptor cells [221]. More importantly, the special environment of intervertebral disc determines the high sensitivity of NP cells to changes in the microenvironment, which is easy to cause mitochondrial homeostasis imbalance under stress. At present, lots of biotechnologies targeting mitochondria have been developed. Chen et al. reported that mitochondria-targeted metallic phenolic nanoparticles mitigated disc degeneration by alleviating oxidative stress and promoting mitochondrial fusion [222]. In addition, a multi-functional and microenvironment-responsive metal-phenolic network release platform targeting mitochondria alleviates disc degeneration by clearing ROS, reducing ECM degradation, and inhibiting pyroptosis. He et al. reported that a selenium nanoparticle could not only restore the hydration of the nucleus pulposus but also maintain mitochondrial homeostasis and improve mitochondrial function by activating glutathione peroxidase 1 [223]. However, these mitochondria-targeting therapies are mainly applied to animal and cellular models, and there are not enough clinical trials to verify the safety, biocompatibility, and sustainability of various biomimetic nanoparticles. At present, there are relatively few studies on mitochondrial quality control targets for IVDD. Therefore, we innovatively propose that intervertebral disc injection of exosomes carrying NRF, PGC-1 and other transcription factors or delivery of nanoparticles loaded with songorine, astaxanthin and other mitochondrial biogenic drugs can effectively promote mitochondrial regeneration in degraded nucleus pulposus cells, and alleviate intervertebral disc degeneration. We sincerely hope that this review can inspire readers and contribute to the progress of the treatment of IVDD.