Compression‐induced senescence of nucleus pulposus cells by promoting mitophagy activation via the PINK1/PARKIN pathway

Abstract The current research aimed to explore the possible relationship between PINK1/PARKIN‐mediated mitophagy and the compression‐induced senescence of nucleus pulposus cells (NPCs). Therefore, the stages of senescence in NPCs were measured under compression lasting 0, 24 and 48 hours. The mitophagy‐related markers, autophagosomes and mitochondrial membrane potential were tested to determine the levels of PINK1/PARKIN‐mediated mitophagy under compression. The PINK1 and PARKIN levels were also measured by immunohistochemistry of human and rat intervertebral disc (IVD) tissues taken at different degenerative stages. A specific mitophagy inhibitor, cyclosporine A (CSA) and a constructed PINK1‐shRNA were used to explore the relationship between mitophagy and senescence by down‐regulating the PINK1/PARKIN‐mediated mitophagy levels. Our results indicated that compression significantly enhanced the senescence of NPCs in a time‐dependent manner. Also, PINK1/PARKIN‐mediated mitophagy was found to be activated by the extended duration of compression on NPCs as well as the increased degenerative stages of IVD tissues. After inhibition of PINK1/PARKIN‐mediated mitophagy by CSA and PINK1‐shRNA, the senescence of NPCs induced by compression was strongly rescued. Hence, the excessive degradation of mitochondria in NPCs by mitophagy under continuous compression may accelerate the senescence of NPCs. Regulating PINK1/PARKIN‐mediated mitophagy might be a potential therapeutic treatment for IVD degeneration.


| INTRODUC TI ON
Intervertebral disc degeneration (IVDD), which contributes to instability, deformity and stenosis of the spinal segment, 1 has been regarded as one of the leading causes of physical disability and low back pain in recent years. 2 IVDD is an intricate process that involves age-related changes. 3 Recently, several studies have reported the accumulation of cellular senescence in human, bovine and rat degenerative intervertebral discs (IVDs). 4,5 Cellular senescence, an irreversible process induced by various stresses, arrests cellular growth and mediates various chronic diseases and ageing dysfunctions. 6 By reducing cell viability and interfering with the microenvironment, cellular senescence is closely related to the pathogenesis of IVDD. 7 Excessive or inappropriate mechanical load is considered a vital contributor to the development of IVDD. 8,9 Compression could directly influence the synthesis of extracellular matrix (ECM; collagen and proteoglycan) components in IVD cells. 10 Most studies have focused on the mechanisms of cell death in IVD cells in response to excessive compression, 11,12 and few have revealed a potential association between compression and cellular senescence in IVDs, 13 which might also participate in the development of IVDD. 14 Excessive compression was shown to promote mitochondrial dysfunction, 15 which enhanced the development of pre-ageing features of cellular senescence. 16 Hence, we have been suggested that compression-induced mitochondrial dysfunction could aggravate senescence of nucleus pulposus cells (NPCs) and further lead to IVDD. PTEN-induced putative kinase 1 (PINK1), a Ser/Thr kinase, participated in stress-related mitochondrial dysfunction and could induce mitophagy through recruiting cytosolic PARKIN to the outer surface of mitochondria. 17,18 PINK1/PARKIN-mediated mitophagy has been found to be closely related to the pathogenesis of age-associated neurodegenerative diseases, such as Parkinson's disease 19 and Huntington's disease. 20 Recent studies have also suggested that the PINK1 and PARKIN genes are involved in cardiac diseases, 21 chronic obstructive pulmonary disease, 22 diabetic kidney disease 23 and cancers. 24 However, whether excessive compression could induce Pink/PARKIN-mediated mitophagy in IVDs has not been reported. Moreover, the association between mitophagy and NPC senescence remains unclear. Thus, we conducted the current study to determine whether compression could promote Pink/PARKINmediated mitophagy and to explore the association between mitophagy and cellular senescence in IVD.

| Isolation and culture of primary rat NPCs
All animal experiments were carried out with the protocol approved by the Animal Experimentation Committee of Huazhong University of Science and Technology. The male Sprague Dawley rats (12 weeks old, weighing 250-300 g), without any musculoskeletal degenerative diseases, were chosen to use in the current study. The rats were obtained from the Experimental Animal Center of Tongji Medical College, Wuhan, China. Rat NPCs were extracted and purified as described previously by Ma et al. 11 Cells at passage two were utilized throughout the subsequent experiments.

| Application of a compression apparatus on rat NPCs
Cells were cultured in a compression apparatus, previously reported in our laboratory, to simulate the compression conditions of IVDs in vivo. 11, 25 The compression apparatus was designed to apply a static pressure of 1.0 MPa, which has been widely reported to induce degeneration of the disc. 26 The bottom of the compression apparatus was filled with sterile distilled water to maintain the humidity, which was detected by a hygrometer. Besides, the apparatus was placed in an incubator at 37°C to maintain the temperature and the concentration of CO 2 was monitored and maintained at 5% by a CO 2 indicator. Before compression administration, cells were maintained unstressed for 6 days. Then, rat NPCs were exposed to the compression apparatus at 1.0 MPa static pressure for different time periods (0, 24 and 48 hours). The compression environment was established by the compression apparatus mentioned above.

| Senescence-associated β-galactosidase (SA-βgal) staining
Cells were analysed using a SA-β-gal Staining Kit (Beyotime) following the manufacturer's protocol. Briefly, cells were washed with PBS (pH 7.4) and fixed in an SA-β-gal working solution (pH 6.0) at 37°C without CO 2 overnight. Then, five fields of each group were selected randomly and analysed under an optical microscope. Finally, the average percentage of total SA-β-gal-positive cells was calculated for quantitative analysis.

| Establishment of IVDD with the rat tail compression model
Fifteen male Sprague Dawley rats (12 weeks old, 300-400 g) were obtained from the Laboratory Animal Center of Huazhong University of Science and Technology. After rats were anaesthetized with 2% (w/v) chloral hydrate (40 mg/kg), IVDs in the rat tail (Co7/8 and Co8/9) were located by palpation and counting and confirmed by trial radiography. Then, a well-developed static loading apparatus 27,28 was applied with a magnitude of 1.3 MPa on the rat tail (Co7/8 and Co8/9). Rats were divided into the following three groups: the sham group (n = 5), the moderate degeneration group (n = 5) and the severe degeneration group (n = 5) as shown in Figure 2A. In the end, all the animals were euthanized, and the target discs (Co7/8 and Co8/9) were harvested for histopathological and immunohistochemical examination. All the procedures were reported in accordance with the ARRIVE guidelines. 29

| Acquisition of human NP tissues
The present study was approved by the medical ethics commit-  Table S1. Prior to sample collection, informed consent was signed by the patients.

| Histopathological and immunohistochemical examination
Human NP tissues and rat IVD tissues were fixed with 4% para-

| Mitochondrial membrane potential (MMP) analysis
The MMP was monitored using a JC-1 assay kit (Beyotime) based on the manufacturer's instructions. The JC-1 dye in mitochondria with a low or high membrane potential was present as a green or red fluorescence, respectively. The level of MMP depolarization was identi-

| Immunofluorescence
Cells were washed twice with PBS and fixed in 4% paraformaldehyde for 15 minutes at room temperature. Then, the cells were blocked for half an hour with 5% bovine serum albumin diluted

| Reactive oxygen species (ROS) flow cytometry detection
The intracellular ROS was measured using a ROS detection kit (Sigma-Aldrich) was used to detect the intracellular ROS level. The NPCs were treated with compression at 0, 24, 48 hours with or without additional CSA administration. Then, cells were digested and washed by PBS twice, and incubated with 2,7-dichlorofluorescin diacetate in the dark at 37°C for 30 minutes. Then, ROS production of the cells was measured by a flow cytometry (BD LSR II, Becton Dickinson), following the manufacturer's instructions.

| Observation by transmission electron microscopy (TEM)
Cells were fixed by glutaraldehyde and osmic acid and dehydrated by gradient acetone, after which they were immersed in embedding medium and ultrathin-sectioned using an automatic microtome (LeicaRM2235). Finally, the processed cells were stained with 1% uranylacetate. The cell sections were observed with TEM (Hitachi) to visualize the status of mitochondria and autophagic bodies in the rat NPCs.  used to quantitatively analyse the gene expression. All data were analysed using the 2 −ΔΔCT method and normalized to the β-actin.

| Statistical analysis
Each experiment was performed independently and at least three technical replicates for statistical analysis, which was performed using the IBM SPSS software package 18.0. Student's t tests were conducted to analyse the difference between the two groups.
Multiple groups of data were analysed by one-way analysis of variance (ANOVA) test, followed by Tukey's post hoc test. All data are presented as the mean ± standard deviation (SD), and statistical significance was set at P < .05.

| Compression induced the senescence of rat NPCs
To observe the senescence levels of rat NPCs exposed to compression, we used SA-gal staining to monitor cellular senescence. In addition, the proteins related to cellular senescence were detected by Western blot analyses. As the exposure to compression was prolonged, the percentage of SA-gal-positive cells strongly increased ( Figure 1A,B), and the protein levels of p21 and p53 were significantly up-regulated while a sustained low expression level of p16 was observed ( Figure 1C,D). These results suggested a positive relationship between prolonged compression and p53/p21-dependent senescence of rat NPCs.

| Compression induced the activation of PINK1/ PARKIN-mediated mitophagy in vitro and in vivo
To observe the mitophagy levels of rat NPCs under compression,

| Down-regulation of compression-induced mitophagy by CSA
To regulate the compression-induced mitophagy, we employed CSA, a mitophagy inhibitor, and the mitophagy level and PINK1/ PARKIN pathway were detected. The ROS accumulation was

| PINK1 molecule is indispensable in compression-induced mitophagy
To further explore the role of PINK1 in compression-induced mitophagy, we transfected PINK1-shRNA into rat NPCs. After transfection for 12 hours, the rat NPCs were observed by a fluorescence microscope, and RT-qPCR was applied to examine the transfection efficiency. The key molecules of mitophagy (PINK1, PARKIN, p62 and LC3B) were detected by Western blot analysis. LC3B expression was also observed with immunofluorescence using a fluorescence microscope. The MMP was monitored using a JC-1 assay kit by flow cytometry. The photographs verified the high efficiency of transfection [the much more intense green fluorescence of the PINK1-shRNA group (shPINK1) and mock-transfected group (shC-TRL) than the blank control group] ( Figure 5A). In addition, the shPINK1 transfection significantly down-regulated mRNA level of PINK1 ( Figure S3). As a result, PINK1, PARKIN and LC3II/β-actin levels were down-regulated, while p62 levels were up-regulated in the PINK1-shRNA group compared with the mock-transfected group ( Figure 5B,C). Additionally, immunofluorescence showed that LC3B decreased sharply ( Figure 5D,E) and that MMP was substantially rescued ( Figure 5F,G) after PINK1-shRNA transcription. Therefore, we concluded that PINK1 was an indispensable molecule in compression-induced mitophagy.

| PINK1/PARKIN-mediated mitophagy was involved in the senescence of rat NPCs under compression
To demonstrate the relationship between mitophagy and rat NPC senescence, we used CSA to inhibit mitophagy. Moreover, PINK1-shRNA was constructed to silence the endogenous PINK1/PARKIN pathway of rat NPCs. After treatment with CSA or silencing of PINK1 by lentivirus, the percentage of SA-gal-positive cells dropped significantly ( Figure 6A

| D ISCUSS I ON
Abnormal spinal loading (increased compressive axial and tensile radial strains) is one of the major causes of IVDD. 30 Excessive compression has been shown to induce cellular senescence in IVD tissues. 14, 31 The accumulation of these senescent disc cells was shown to accelerate the process of IVDD through their aberrant paracrine functions by which senescent cells induce the senescence of neighbouring cells and up-regulate matrix catabolism and inflammation in IVD tissues. 32,33 Although this is a common phenomenon, the mechanisms and signalling pathways linked to compression-induced cellular senescence and IVDD are still obscure.
Mitophagy, the selective autophagic clearance of malfunctional mitochondria, plays a vital role in the cellular response to various stressors, such as bioenergetics stress, oxidative stress and proteotoxic stress. 34 The PINK1/PARKIN pathway, a critical pathway mediating mitophagy, has been shown to be related to mitochondrial dysfunction. 35 Compression, which is one of the major endogenous stresses exerted on disc cells, has been reported to be associated with mitochondrial dysfunction of disc cells. 25 To decrease the level of mitophagy, we added a specific mitophagy inhibitor, CSA, or knocked down PINK1 by lentivirus transfections, during compression exposure conditions. Malfunction of mitochondria could affect oxidative phosphorylation and cause overgeneration of ROS. 41 Compression has been reported to increase the intracellular ROS accumulation, 42,43 and inhibition of ROS significantly relieved morphological denaturation of mitochondria. 42,44 Therefore, ROS accumulation may be considered to be an initial promoter of mito-dysfunction induced by compression. CSA has been shown to decrease the ROS level induced by compression. 36 Also, it has been reported that CSA could exert a strong effect on mitochondria via preventing the MTP from opening. 45 [49][50][51] Generally, mitophagy plays a Janus-type role in the process of ageing. Baseline mitophagy benefits the cells, while abnormal mitophagy could promote cellular injury. 39,53,54 In summary, the current study identified a positive relationship between PINK1 expression in IVD tissues and the severity of IVDD.
F I G U R E 5 Down-regulation of PINK1 relieved compression-induced mitophagy of rat NPCs. Compression with 1.0 MPa for 0, 24 and 48 h was employed on the rat NPCs with PINK1-shRNA/mock-shRNA transfection. A, The rat NPCs were observed by a fluorescence microscope after PINK1-shRNA transfection for 24. Scale bar, 50 μm. B, The levels of p62, LC3-I, LC3-II, PINK1 and PARKIN were detected by Western blotting. C, A quantitative analysis of Western blotting shown by the p62/β-actin, LC3-II/β-actin, PINK1/β-actin and PARKIN/βactin ratios. D, LC3B was observed by a fluorescence microscope through immunofluorescence. Scale bar, 20 μm. E, Quantification of LC3B positive dots per cell from immunofluorescence staining as depicted in D; quantification was performed using ImageJ software. F, The MMP was monitored using a JC-1 assay kit by flow cytometry. G, Quantitative and statistical analysis of MMP in rat NPCs; the data are expressed as the ratio of red to green fluorescence intensity, as assessed by flow cytometry. Data are presented as the mean ± SD (n = 3). *Indicates a significant difference (P < .05) and **indicates a significant difference (P < .01): between shCTRL and shPINK1 groups at the same treatment durations in (C) and (G); between the two groups in (E). shCTRL, mock-transfected group; shPINK1, PINK1-shRNA group

ACK N OWLED G EM ENTS
We would like to thank all the people who helped us in the current study.

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.