Mfn2 is involved in intervertebral disc degeneration through autophagy modulation

Journal Pre-proof Mfn2 is involved in intervertebral disc degeneration through autophagy modulation Yu Chen, Jialiang Lin, Jiaoxiang Chen, Chongan Huang, Zengjie Zhang, Jianle Wang, Ke Wang, Xiangyang Wang PII:

S1063-4584(20)30004-2

DOI:

https://doi.org/10.1016/j.joca.2019.12.009

Reference:

YJOCA 4575

To appear in:

Osteoarthritis and Cartilage

Received Date: 30 July 2019 Revised Date:

15 December 2019

Accepted Date: 31 December 2019

Please cite this article as: Chen Y, Lin J, Chen J, Huang C, Zhang Z, Wang J, Wang K, Wang X, Mfn2 is involved in intervertebral disc degeneration through autophagy modulation, Osteoarthritis and Cartilage, https://doi.org/10.1016/j.joca.2019.12.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.

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Mfn2 is involved in intervertebral disc degeneration through

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autophagy modulation

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Yu Chen1,2,#, Jialiang Lin1,2,#, Jiaoxiang Chen1,2, Chongan Huang1,2, Zengjie Zhang1,2,

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Jianle Wang1,2, Ke Wang1, Xiangyang Wang1,2,*

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Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China

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Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s

Zhejiang Provincial Key Laboratory of Orthopaedics, Wenzhou 325000, Zhejiang

Province, China

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*Correspondence to:

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Xiangyang Wang ([email protected]), Department of Orthopaedics, The

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Second Affiliated Hospital of Wenzhou Medical University, Xueyuan Xi Road,

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Wenzhou 325000, Zhejiang Province, China

Yu Chen and Jialiang Lin contributed equally to this work

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Abstract Objective: To explore whether Mitofusin 2 (Mfn2) is implicated in the pathogenesis of intervertebral disc degeneration (IVDD). Methods: We detected the protein content of Mfn2 in degenerated human nucleus pulposus (NP) tissues and investigated the effects of Mfn2 knockdown and Mfn2 overexpression on rat nucleus pulposus cells (NPCs) under oxidative stress by using a range of biological techniques. Afterwards, we confirmed the effects of Mfn2 overexpression on NPCs in vivo and further evaluated the therapeutic action of adenovirus (AV)-Mfn2 injection in a rodent IVDD model. Results: Mfn2 expression was decreased in human NP tissues during IVDD. Mfn2 knockdown aggravated the impairment of autophagic flux, mitochondrial dysfunction and cellular apoptosis in rat NPCs after Tert-Butyl hydroperoxide (TBHP) treatment, while Mfn2 overexpression significantly reversed these alterations. Besides, Mfn2 overexpression promoted an ROS (reactive oxygen species)-dependent mitophagy via PINK1 (PTEN-induced putative kinase protein 1)/Parkin pathway in TBHP-treated NPCs. Inhibition of autophagy with chloroquine (CQ) disordered the protective effects of Mfn2 overexpression on NPCs. Furthermore, Mfn2 overexpression in discs by AV-Mfn2 injection ameliorated the development of IVDD in rats. Conclusion: Mfn2 repression is deeply involved in the pathogenesis of IVDD with its impairment on autophagy, leading to the aggravation of mitochondrial dysfunction and apoptotic cell death, which ought to be a promising therapeutic target for IVDD. Key words: Mfn2, IVDD, autophagic flux, mitophagy, apoptosis Introduction Low back pain induced by intervertebral disc degeneration (IVDD) is the main cause of disability worldwide and imposes a massive clinical and socioeconomic burden on society[1]. Although the aetiological factors of IVDD includes excessive biomechanical loading, poor nutrient supply, genetic predisposition and ageing[2-4], they are all associated with a common disease phenotype: loss of nucleus pulposus cells (NPCs). As the primary cells resident in nucleus pulposus (NP) tissue, NPCs produce extracellular matrix proteins such as collagen II and proteoglycan, which are the main components of the gelatinous tissue and enable the disc to turgor against compressive loads[5, 6]. Excessive apoptosis of NPCs has long been proved to be a vital contributor for IVDD[7, 8]. Reactive oxygen species (ROS) serve as essential messengers in NPCs at a physiological level[9]. However, abnormal generation of ROS is a vital mediator in the pathological mechanism of IVDD[10]. ROS overproduction induced by virous initial factors causes mitochondrion dysfunction. Meanwhile, the impairment of mitochondria reinforces ROS generation in NPCs with a positive feedback regulation[11, 12]. This vicious circle can trigger a range of proapoptotic proteins in NPCs through the mitochondrial apoptosis pathway[13, 14]. Autophagy is an intracellular degradation of dysfunctional organelles and cytosolic macromolecules, which is essential for cellular survival and homeostasis under stress 2

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conditions[15]. This homeostatic process includes the sequestration of cytoplasmic components in double-membraned autophagosomes, the fusion of autophagosomes and lysosomes, and the digestion of cargoes in lysosomes, which is termed autophagic flux[16]. Mitophagy is a selective degradation of damaged mitochondria through autophagy pathway, which keeps the homoeostasis of mitochondrial dynamics and mitigates mitochondrial dysfunction during oxidative stress[17]. Dysfunction of autophagy, including mitophagy, has been involved in numerous degenerative diseases, such as ageing, neurodegeneration, osteoarthritis, as well as IVDD[18-21]. According to a recent report, bursts of ROS could lead to the impairment of autophagic flux with lysosomal dysfunction[22]. Moreover, it has been reported that moderate autophagy can inhibit the apoptosis of rat NPCs in vitro experiments[23, 24]. Thus, modification on autophagy of NPCs may be a promising therapeutic strategy for IVDD. Mitofusin 2 (Mfn2) is an outer mitochondrial membrane protein that motivates mitochondrial fusion and involves in the homeostasis of mitochondrial dynamics[25]. Mfn2 is also demonstrated to be a multifunctional protein involved in various signal pathways beyond mitochondrial fusion, including in the regulation of cellular autophagy[26, 27]. It was reported that Mfn2 decreases during aging in skeletal muscle, resulting in the consequence of autophagy inhibition and accumulation of damaged mitochondria[28]. However, little is known about the role of Mfn2 in the pathogenesis of IVDD. Here, we attempt to explore the role of Mfn2 during IVDD. We investigated the effects of Mfn2 knockdown and Mfn2 overexpression on rat NPCs under oxidative stress as well as the potential mechanism. Moreover, we further assessed the effects of Mfn2 in vivo and evaluated the therapeutic potential of Mfn2 overexpression in a rodent model of IVDD. Materials and methods Ethics statement All surgical interventions, treatments and postoperative animal care procedures were performed in accordance with the Animal Care and Use Committee of Wenzhou Medical University (wydw2014-0129). Human nucleus pulposus collection Human NP tissues were classified according to the Pfirrmann grade system[29] and harvested from the IVDD patients who received spinal surgeries: 2 males and 1 females, age range from 26 to 45 years (36.3±9.6), grade II; 2 males and 2 females, age range from 31 to 52 years (43.8±9.3), grade III; 2 males and 3 females, age range from 45 to 63 years (55.0±6.9), grade IV; 1 males and 3 females, age range from 52 to 66 years (59.5±6.2), grade V. All of these patients signed an informed consent form allowing the researchers to use the NP tissues obtained during spinal surgery. The NP samples were cut into small pieces, followed by digestion in 0.1% type II collagenase (Gibco) for approximately 4 h at 37°C. After being washed for three times with phosphate-buffered saline (PBS), the digested tissues were resuspended in completed DMEM/ F12 (1:1) medium supplemented with 15% foetal bovine serum (FBS) and 1% 3

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antibiotics (penicillin/streptomycin), and then seeded into culture flasks. Rat nucleus pulposus cell isolation and culture Thirty male Sprague–Dawley rats (5-6 weeks old) were euthanized with an overdose of 10% chloral hydrate. The lumbar discs were collected integrally under sterile conditions and the NP tissues were separated from discs with a dissecting microscope. Subsequently, NP cells were extracted from NP tissues as described above and cultured in DMEM/ F12 (1:1) medium supplemented with 15% FBS and 1% antibiotics for 3 weeks at 37 °C and 5% CO2. Cells from passages 1 to 3 were used for our experiments. Lentivirus and siRNA transfection When reaching 40–60% confluence, the NPCs were transfected with lentivirus (GeneChem, China) at a multiplicity of infection (MOI) of 100. After 12 hours of transfection, the culture medium was changed every other day. When confluent, the transfected NPCs were passaged for further experiments. To inhibit Parkin expression, rat NPCs (70% confluence) were treated with Parkin small interfering RNAs (siRNAs) (Bioneer, South Korea) according to the manufacturer instruction. Sequences of the Mfn2-shRNA and Parkin-siRNA used were reported in Supplemental Table S1. Western blot assay Cells were lysed in RIPA buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF) followed by 15 min centrifugation at 12000 r.p.m. at 4 °C. The concentrations of the samples were measured using BCA protein assay kit (Beyotime, China). Then the samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad, USA). After blocking with 5% nonfat milk for 1.5 h, the membranes were incubated with primary antibodies specific to Mfn2 (1:1000, #11925 CST (Cell Signaling Technologies, USA)), Cleaved-caspase3 (1:1000, #9661 CST), β-actin (1:1000, #3700 CST), P62 (1:1000, ab56416 Abcam, UK), PINK1 (1:1000, #6946 CST), Parkin (1:1000, #4211 CST), LC3 (1:1000, #83506 CST) , Bcl-2 (1:1000, #3498 CST), Bax (1:1000, #14796 CST), Bak (1:1000, #12105 CST) and Cytochrome C (1:1000, #11940 CST) overnight at 4 °C, followed by incubation with the respective secondary antibodies for 2 h at room temperature. Finally, the intensity of the protein bands was quantified with Image Lab 3.0 software (Bio-Rad). TUNEL staining DNA fragmentation was detected with the terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) method (Roche, CA). After being fixed with 4% paraformaldehyde for 1 h, NPCs were incubated with 3% H2O2 and 0.1% Triton X-100 for 10 minutes. Cells were then washed with PBS, and co-stained with TUNEL inspection fluid and 4’,6-diamidino-2-phenylindole (DAPI) according to manufacturer’s instructions. Finally, three random microscopic fields per slide were observed under a fluorescence microscope (Olympus Inc, Japan). Transmission electron microscopy NPCs were fixed in 2.5% glutaraldehyde overnight, post-fixed in 2% osmium tetroxide for 1 h and then stained with 2% uranyl acetate for 1 h. After dehydration in an ascending series of acetone, the samples were embedded into araldite and cut into 4

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semi-thin sections. Sections were subsequently stained with toluidine blue to locate cells and finally observed with a transmission electron microscope (Hitachi, Japan). Immunofluorescence Samples were blocked with 5% bovine serum albumin for 1 h at 37 °C. Primary antibodies against P62 (1:200), LC3 (1:100), TOMM20 (1:200, #42406 CST), Cleaved-caspase3 (1:200) and Mfn2 (1:200) were applied to the incubation of the samples overnight at 4 °C. The day after, glass plates were washed and incubated with Alexa Fluor488 or Alexa Fluor594 conjugated second antibodies for 1 h at room temperature and labeled with DAPI for 5 min. The slides were then observed with a fluorescence microscope (Olympus Inc., Japan). Surgical procedure A total of 24 male Sprague Dawley rats (8 weeks old) were randomly divided into three groups (Sham group, AV-Ctrl group and AV-Mfn2 group, n=8 per group). The experimental level of the rat lumber disc (L5/L6) was located by X-ray radiograph. Rats in AV-Ctrl group and AV-Mfn2 group underwent the following surgery according to a published report[30]. After anaesthetization with intraperitoneal injection of 10% chloral hydrate (3.6 ml/kg), the lumbar 4th–6th (L4–L6) spinous processes along with the supraspinous and interspinous ligaments were resected to induce instability of the lumbar spine. Subsequently, 3 µl of adenovirus (AV-Ctrl or AV-Mfn2) were injected into the NP cavity through the interlaminar approach by using a microliter syringe with a 27 G needle. Sham surgeries were only performed by decollement of the posterior paravertebral muscles from the L4–L6 vertebrae without lentivirus injection. All animals were allowed free unrestricted weight bearing and activity, and were monitored every day to ensure their well-being. MRI methods Magnetic resonance imaging (MRI) was performed to assess the signal and structural changes of discs in sagittal T2-weighted images with a 3.0T clinical magnet (Philips Intera Achieva 3.0MR) at 0 week and 8 weeks after surgery. The parameters of T2-weighted imaging had been described in our previous reports[31]. The MRI images were evaluated by another blinded researcher according to the Pfirrmann grading scale and the mean gray value of all pixels in the NP area was measured by ImageJ[32]. Histopathologic analysis The rats were sacrificed by an intraperitoneal overdose injection of 10% chloral hydrate and then the discs were harvested. Samples were fixed in paraformaldehyde and decalcified in neutral 10% (v/v) EDTA solution before embedding in paraffin. After that, the samples were cut into 5-µm sagittal sections. Slides of each disc were stained with safranin O and hematoxylin and eosin (H&E) to assess disc condition. Histologic scores were evaluated by a blinded manner according to a grading scale (5 for normal disc, 6–11 for moderately degenerated disc, and 12–15 for severely degenerated disc)[33]. Statistical analysis The data were expressed as the Mean ± SD. Statistical analyses were conducted by using SPSS statistical software program 18.0. Data were analyzed by one-way 5

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analysis of variance (ANOVA) followed by Tukey’s test for comparison between the control and treatment groups. Student's t test (t-test) was used for comparison between two groups. Non-parametric data (Pfirrmann grading) were analyzed by the Kruskal– Wallis H test. P < 0.05 was considered statistically significant.

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Results Mfn2 expression was decreased in human degenerated NP tissues and was related to the apoptosis of NPCs As shown in Fig. 1(A-C), the expression of Mfn2 in human NP samples was decreased along with the increase of IVDD degree, while the apoptosis marker protein Cleaved-caspase3 presented a significant increase in degenerated NP tissues, implying a possible link between Mfn2 and apoptosis in NPCs. Given that oxidative stress is an essential contributor in the pathophysiological process of IVDD, Tert-Butyl hydroperoxide (TBHP), an exogenous ROS donor, was applied to establish degeneration model in vitro. From CCK8 assay, TBHP treatment was observed to reduce cell viability in a dose-dependent manner, especially at the concentration of 120 µM [Fig. 1(D)]. As shown in Fig. 1(E, F), Mfn2 expression was increased with low concentrations of TBHP treatment (below 60 µM). However, a sharp decrease of Mfn2 expression was observed when it came to a higher concentration (120 µM). Besides, the time-dependent experiment showed that Mfn2 expression was first increased and then decreased after TBHP treatment [Fig. 1(G, H)]. We subsequently utilized 120 µM TBHP in our following studies since TBHP treatment at 120 µM for 4 h significantly reduced cell viability and inhibited Mfn2 expression. To determine whether Mfn2 expression is associated with NPCs apoptosis, rat NPCs were respectively transfected with lentivirus-Ctrl (LV-Ctrl), lentivirus-shMfn2 (LV-shMfn2) and lentivirus-Mfn2 (LV-Mfn2). The transfection efficiency was detected by western blot [Fig. 1(I, J)]. As shown in Fig. 1(K, L), Mfn2 knockdown exacerbated the increase of Cleaved-caspase3 induced by TBHP, while Mfn2 overexpression mitigated the release of apoptosis protein, which was consistent with the TUNEL assay results [Fig. 1(M, N)]. To assess the mitochondrial homeostasis in rat NPCs, mitochondrial membrane potential (∆ψm) and intracellular ROS were also investigated (Fig. S1). TBHP treatment significantly decreased ∆ψm while increased the ROS level, indicating the dysfunction of mitochondrial in NPCs. Mfn2 knockdown markedly aggravated mitochondrial depolarization and ROS burst in NPCs, while Mfn2 overexpression reversed these alterations. These results indicated that Mfn2 was involved in a protective mechanism against cell apoptosis and mitochondrial dysfunction during oxidative stress. Mfn2 expression affects autophagic flux in TBHP-treated rat NPCs To further investigate the potential mechanism through which Mfn2 affected NPCs, the intracellular submicroscopic structures were detected by transmission electron microscopy (TEM). As shown in Fig. 2(A), TBHP treatment made the mitochondria appeared swollen and dissolved, companied with a few autophagosomes and 6

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autophagolysosomes formation in the cytoplasm. In Mfn2 knockdown group, there were more autophagosomes and fewer autophagolysosomes in the cytoplasm, with graver destruction of mitochondrial morphological. However, Mfn2 overexpression recovered the blocked autophagic flux, as well as the morphology of mitochondria. The LC3 II/LC3 I ratio and P62/SQSTM1 were regarded as indicators of autophagy formation and has been used to monitor autophagic flux changes. Western blot analysis of P62 and LC3 also showed a compromised flux of autophagy in Mfn2 knockdown group, while Mfn2 overexpression reversed this trend [Fig. 2(B-D)], which was consistent with the immunofluorescence analysis of P62 [Fig. 2(H)]. We subsequently detected the levels of P62 and LC3 II in TBHP-treated NPCs at different time points. As shown in Fig. 2(E-G), accumulation of both P62 and LC3 II suggested that the autophagic flux in LV-shMfn2 group was blocked. However, in LV-Mfn2 group, the LC3 II/LC3 I ratio was increased in a time-dependent manner while P62 started to descend after a temporary rise, indicating the autophagic flux was restored by Mfn2 overexpression. To further confirm this finding, carbonylcyanide-m-chlorophenylhydrazone (CCCP), a mitophagy inducer, was applied. As shown in Fig. S2, CCCP visibly activated autophagy in normal NPCs, which was blocked by Mfn2 knockdown with a significant accumulation of P62 and LC3 II. These results demonstrated that Mfn2 played a crucial role in the preservation of autophagic flux in NPCs. Mfn2 overexpression stimulates an ROS-dependent mitophagy via PINK1/Parkin pathway in TBHP-treated rat NPCs Mfn2 has been shown to promote mitophagy in neurons and cardiomyocytes[27, 34]. To evaluate this possibility, we measured the level of proteins involved in mitochondrial autophagy. PINK1 (PTEN-induced putative kinase protein 1)/ Parkin pathway is widely involved in the regulation of mitophagy in mammal cells. As shown in Fig. 3(A-E), stimulation of TBHP increased PINK1 expression both in normal NPCs and Mfn2-transfected NPCs. Mfn2 overexpression significantly increased the level of Parkin and LC3II/LC3I ratio in TBHP-treated NPCs while decreased the content of P62, indicating the promotion of mitophagy. This result was further confirmed by the co-localization of LC3 and mitochondria marker TOMM20 [Fig. 3(F)], which evidenced the formation of mitophagosomes. However, compared to normal NPCs, no evident changes of Parkin expression and LC3II/LC3I ratio were observed in Mfn2-transfected NPCs without TBHP treatment, implying an essential role of ROS in Mfn2 overexpression-induced mitophagy. To this end, we applied antioxidant compound N-acetylcysteine (NAC) to eliminate the effect of ROS. Administration of NAC markedly decreased the levels of mitophagy proteins, suggesting the mitophagy induced by Mfn2 overexpression was ROS-dependent [Fig. 3(G-I)]. To further investigate the role of PINK1 and Parkin in Mfn2 overexpression-induced mitophagy, cells were pretreated with Cyclosporin A (CsA, an inhibitor of the mitochondrial permeability transition that promotes PINK1 degradation) or transfected with Parkin-siRNA. As shown in Fig. 3(J-L), CsA inhibited the increase of PINK1, Parkin and the LC3II/LC3I ratio, as well as the degradation of P62. 7

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Compared with the Ctrl-siRNA group, Parkin-siRNA markedly reduced the ratio of LC3-II/LC3-I but increased P62 expression [Fig. 3(M-O)]. These results were consistent with the double-labeled immunofluorescence staining of LC3 and TOMM20 [Fig. 3(P)], suggesting that Mfn2 overexpression could promote mitophagy in TBHP-treated rat NPCs through PINK1/Parkin pathway. CQ reverses Mfn2 overexpression-induced protective effects in rat NPCs under oxidative stress To determine whether the effects of Mfn2 overexpression on cell apoptosis were related to mitophagy-lysosome pathway, chloroquine (CQ), a classical inhibitor of autophagic flux, was used in our study. As expected, CQ aggravated the accumulation of p62 and LC3II, indicating that the autophagy flux was impaired [Fig. 4(A-C)]. The apoptosis-related proteins including B-cell lymphoma-2 (BCL-2) families (Bax, Bak and Bcl-2, control the intrinsic apoptotic pathway), Cytpchrome C (marker of mitochondria dysfunction) and Cleaved-caspase3 were subsequently detected by western blot. As shown in Fig. 4(D-J), the protective effects of Mfn2 overexpression against apoptosis were significantly inhibited or completely abolished by CQ. Double-labeled immunofluorescence staining of Cleaved-caspase3 and p62 showed the similar changes in the rat NPCs [Fig. 4(K)]. These results suggested that the beneficial effects of Mfn2 overexpression against apoptosis were depend on the mechanism of autophagic flux restoration. AV-Mfn2 injection ameliorates IVDD in vivo To further confirm the positive effect of Mfn2 overexpression on NPCs in vivo, a rodent IVDD model was built. We detected the levels of Mfn2 and P62 in discs with double-labeled immunofluorescence staining at 8 weeks after surgery. As shown in Fig. 5(A-C), Mfn2 was markedly decreased in IVDD group with AV-Ctrl injection compared to sham group, accompanied with increasing P62 accumulation. AV-Mfn2 injection significantly improved the expression of Mfn2 and ameliorated the accumulation of P62. Besides, AV-Mfn2 transfection significantly increased the expression of LC3 II and decreased the rate of cell apoptosis in rat degenerated discs [Fig. 5(D-F]. By MRI analysis we found that AV-Mfn2 group showed higher T2-weighted signal intensities (the white arrow) and lower Pfirrmann grade scores in comparison with the IVDD group at 8 weeks post-surgery [Fig. 6(A, B)]. The MRI T2 disc intensity was also detected as a semi-quantitative measurement [Fig. 6(C)]. Hematoxylin and eosin (H&E) staining and safranin O staining were also applied to evaluate the morphological changes of discs. As shown in Fig. 6(D), NP tissues in surgical discs were gradually lost and replaced with disorganized fibrocartilaginous tissue in the IVDD group, accompanied with disordered border between AF and NP. The NPCs were reduced and aggregated into clusters, indicating serious degeneration of the discs. Massive loss of proteoglycans in NP tissues could be observed in IVDD group according to the safranin O staining. However, AV-Mfn2 injection prominently alleviated the IVDD process, as shown by the existence of more structured NP tissue, less irregular AF, more numerous NPCs and more abundant proteoglycan matrix. In addition, a lower histological score of operation discs was observed in AV-Mfn2 group 8

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than IVDD group at 8 weeks after surgery [Fig. 6(E)]. Taken together, these findings suggested that Mfn2 overexpression could ameliorates IVDD in vivo. Discussion As a multifunctional protein, Mfn2 is implicated in a variety of biological processes under both physical and pathological conditions, including mitochondrial fusion and transport, cell metabolism, apoptosis and autophagy[35]. Abnormal expression of Mfn2 leads to kinds of diseases, such as neurodegeneration, sarcopenia, metabolic diseases and heart diseases[36-39]. Although Mfn2 has long been associated with various diseases, to our knowledge, this is the first report revealing a role of Mfn2 in the pathogenesis of IVDD. We reported that Mfn2 expression markedly reduced in NP tissues with the development of IVDD, whereas it presented a different trend in TBHP-treated rat NPCs. According to a previous study, Mfn2 is phosphorylated by Jun N-terminal kinase (JNK) during cellular stress, which leads to ubiquitin-mediated proteasomal degradation of Mfn2 by the Ubiquitin-Proteasome System (UPS)[40]. We suggest that Mfn2 is upregulated as an adaptive response to oxidative stress at the early stage of IVDD, while excessive ROS generation accelerates the degradation of Mfn2 in severely degenerative discs. Mounting evidence reveals that autophagy is involved in the pathogenesis of IVDD[24, 41]. Nevertheless, the role of autophagic flux in the procession of IVDD has not been fully clarified. ROS has been shown to activate autophagy as a self-protective mechanism in response to oxidative stress[42, 43]. It was also reported that there are more autophagic vacuoles in degenerative NPCs than in normal NPCs[44]. However, several in vitro studies have shown that severe oxidative stress impairs autophagic flux[22, 45]. According to our previous reports, autophagy dysfunction characterized by blocked autophagic flux was observed both in rat degenerated NPCs and human osteoarthritis chondrocytes[20, 21], which is consistent with our observation in TBHP-treated rat NPCs and degenerated rat NP tissues. In this study, the abundance of P62 in NPCs was increased after TBHP treatment, even though the ratio of LC3II/LC3I was upregulated, indicating a blocked autophagic flux after the initiation of autophagy. In our consideration, the increasing autophagosomes observed in degenerative NPCs were mainly attributed to the disruption of autophagic flux that impedes the degradation of autophagosomes. Therefore, we believe that in spite of autophagy activation by ROS during IVDD, excessive oxidative stress will disrupt autophagic flux in NPCs and eventually accelerate the procession of IVDD. Interestingly, in the present study we found that Mfn2 and P62 are negatively correlated, indicating that disrupted autophagic flux might be associated with Mfn2 repression in TBHP-treated NPCs. This negative correlation between Mfn2 and P62 was also shown in the in vivo experiment. Coincidentally, our discovery is similar to the opinion from a cardiovascular research asserting that Mfn2 repression retards the fusion of autophagosome and lysosome, leading to the disruption of autophagy flux in cardiomyocytes[34]. These results reveal a possible role of Mfn2 repression as the cause underlying the disruption of autophagic flux during IVDD in NPCs. 9

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As far as we know, Mfn2 is also involved in the modulation of mitophagy in neurons and cardiomyocytes[27, 34]. Once mitochondrial dysfunction occurs, collapse of ∆ψm stabilizes PINK1 in the depolarized mitochondria. Mfn2 that located on outer mitochondrial membrane attracts and binds the cytosolic ubiquitin ligase Parkin to PINK1, inducing degradation of these damaged mitochondria. Our group has already reported that PINK1/Parkin pathway of mitophagy acts as a protective mechanism during IVDD process[23, 31]. In this study, we found that Mfn2 overexpression can promote mitophagy in rat NPCs through regulating Parkin. Importantly, the activation of mitophagy induced by Mfn2 overexpression is ROS-dependent, as no obvious changes of mitophagy-related proteins were observed in Mfn2-transfected NPCs without TBHP treatment. Meanwhile, antioxidant NAC blocked the increase of mitophagy-related proteins induced by Mfn2 overexpression, which reconfirmed the role of ROS in Mfn2 overexpression-induced mitophagy. CsA is a specific inhibitor of the mitochondrial permeability transition, which can suppress the depolarization of ∆ψm and accelerate PINK1 degradation[40]. In the present study, administration of both CsA and Parkin-siRNA decreased the mitophagy-related protein levels, verifying the dominant role of PINK1/Parkin pathway in Mfn2 overexpression-induced mitophagy. Based on our observations and previous document reports, we summarize the probable mechanism of how Mfn2 affects mitophagy in NPCs: Excessive ROS production induced by exogenous stimuli causes the depolarization of ∆ψm and blocks the degradation of PINK1, leading to the amassment of PINK1 on the outer mitochondrial membrane. Activated PINK1 phosphorylates Mfn2 as a Parkin receptor on damaged mitochondria. Thus, the downstream proteins of mitophagy can identify the damaged mitochondria. As described above, phosphorylated Mfn2 can be degraded by the Ubiquitin-Proteasome System (UPS), hence a deficiency of Mfn2 is induced, resulting in several disorders in NPCs during serious oxidative stress. It is wildly known that neither defective mitophagy nor excessive mitophagy is beneficial to cellular homeostasis. Here, we show that Mfn2 overexpression in NPCs motivates mitophagy in an ROS-dependent manner, which targets at the cells undergoing oxidative stress rather than the normal ones. Furthermore, Mfn2 overexpression in vivo with adenovirus injection did not show any negative influence on rats in terms of lifespan, weight, and mobility, indicating the security of AV-Mfn2 transfection as a therapy strategy for IVDD. However, there are several limitations associated with our study. Firstly, the exact mechanism of how ROS affect Mfn2 expression in NPCs was not confirmed by experiment. In addition, it is a pity that we could not transfect LV-Mfn2 into human degenerated NPCs and assess the therapeutic effect due to the lack of human disc tissues. Moreover, it is not clear whether there are other pathways that links Mfn2 to autophagy. In summary, we discovered the involvement of Mfn2 in the pathogenesis of IVDD. Mfn2 repression in NP tissues during IVDD may be a determinant for the aggravation of autophagy impairment, leading to mitochondria dysfunction and apoptotic cell death. Mfn2 overexpression can restore the blocked autophagy flux and promote an ROS-dependent mitophagy via PINK1/Parkin pathway. These findings should be 10

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valuable to better understand the molecular mechanisms of IVDD and provide an innovative therapeutic target for IVDD.

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Acknowledgements This work was supported by grants from the Zhejiang Public Service Technology Research Program/Social Development (LGF18H060008), Major Scientific and Technological Project of Medical and Health in Zhejiang Province (WKJ-ZJ-1527), National Natural Science Foundation of China (81871806).

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Conflict of Interest The authors declare no conflict of interest.

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Author Contributions YC and JLL performed the experiments, acquired data and drafted the article; JXC and CAH analyzed and interpreted the data; ZJZ and JLW provided reagents and materials tools; KW collected relevant papers in this field; XYW designed the experiments, and revised the manuscript before submitting.

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Figure legends:

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

Fig. 2 Mfn2 expression affects autophagic flux in TBHP-treated rat NPCs. (A) The submicroscopic structures of NPCs were detected by TEM (Scale bar: 0.5 µm). (Asterisk: normal mitochondria; Lightning: impaired mitochondria; Arrow: autophagosome; Triangle: autophagolysosome). (B-D) The protein expression of P62 and LC3 in lentivirus-transfected NPCs after TBHP treatment. (E-G) The protein expression of P62 and LC3 in NPCs at different time point after TBHP treatment. (H) The immunofluorescence analysis of P62 (Scale bar: 25 µm). All data represent mean ± S.D. (n = 4). *P<0.05, **P<0.01, & P <0.0001

38 39 40 41 42 43 44

Fig. 4 CQ reverses Mfn2 overexpression-induced protective effects in rat NPCs under oxidative stress. The NPCs that transfected with LV-Mfn2 were pretreated with CQ (50µM) for 6h before TBHP stimulation. (A-C) The protein expression of P62 and LC3 in NPCs as treated above. (D-G) The protein expression of Bax, Bak and BCL-2 in NPCs as treated above. (H-J) The protein expression of Cytochrome C and Cleaved-caspase3 in NPCs as treated above. (K) Double-labeled immunofluorescence staining of P62 and Cleaved-caspase3 in NPCs as treated above

Fig. 1 Mfn2 expression was decreased in human degenerated NP tissues and was related to the apoptosis of NPCs. (A-C) The protein expression of Cleaved-caspase3 and Mfn2 in human NPCs from different degrees of IVDD tissues. (D) Cell viability of rat NPCs that treated with different concentrations of TBHP. (E, F) The protein expression of Mfn2 in rat NPCs that treated with different TBHP concentrations for 4 h. (G, H) The protein expression of Mfn2 in rat NPCs that treated with 120µM TBHP for different time. (I, J) The protein expression of Mfn2 after lentivirus transfection. (K, L) After lentivirus transfection, rat NPCs were treated with TBHP (120µM, 4h). The protein expression of Cleaved-caspase3 was then analyzed by western blot. (M, N) TUNEL assay was performed in NPCs as treated above (Scale bar: 200µm). All data represent mean ± S.D. (n = 4). *P<0.05, **P<0.01, & P <0.0001

Fig. 3 Mfn2 overexpression stimulates an ROS-dependent mitophagy via PINK1/Parkin pathway in TBHP-treated rat NPCs. (A-E) The protein expression of PINK1, Parkin, P62 and LC3 in NPCs that transfected with LV-Ctrl or LV-Mfn2 before TBHP treatment. (F) Double-labeled immunofluorescence staining of LC3 and TOMM20 in NPCs as treated above (Scale bar: 25µm). (G-I) The protein expression of PINK1, Parkin and LC3 in LV-Mfn2 NPCs with or without NAC pretreatment before TBHP stimulation. (J-L) The protein expression of PINK1, Parkin and LC3 in LV-Mfn2 NPCs with or without CsA pretreatment before TBHP stimulation. (M-O) The protein expression of PINK1, Parkin and LC3 in LV-Mfn2 NPCs with Ctrl-siRNA or Parkin-siRNA pretreatment before TBHP stimulation. (P) Double-labeled immunofluorescence staining of LC3 and TOMM20 in NPCs with different treatment (Scale bar: 25µm). All data represent mean ± S.D. (n = 4). *P<0.05, **P<0.01, & P <0.0001

1

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(Scale bar: 100µm). All data represent mean ± S.D. (n = 4). *P<0.05, **P<0.01, & P <0.0001 Fig. 5 AV-Mfn2 injection ameliorates IVDD in vivo. (A-C) Double-labeled immunofluorescence staining of Mfn2 and P62 in rat NP tissues 8 weeks after surgery (Scale bar: 1mm or 200µm). (D-F) LC3 immunofluorescence staining and TUNEL staining in rat NP tissues 8 weeks after surgery (Scale bar: 100µm or 200µm). All data represent mean ± S.D. (n =8). *P<0.05, **P<0.01, & P <0.0001

Fig. 6 AV-Mfn2 injection ameliorates IVDD in vivo. (A) T2-weighted MRI of rat lumbar discs at week 0 and week 8 after surgery (white arrows). (B) The Pfirrmann MRI grade scores in three groups at week 0 and week 8 after surgery. (C) The mean gray values of all pixels in the disc NP. (D) Representative H&E staining and safranin O staining in rat NP tissues 8 weeks after surgery (Scale bar: 1mm or 200µm). (E) The histological grades in three groups evaluated at week 8 after surgery. All data represent mean ± S.D. (n =8). *P<0.05, **P<0.01, & P <0.0001

Fig. S1 (A) Mitochondrial membrane potential (∆ψm) in NPCs was measured by JC‐ 1 staining. TRITC represents the normal ∆ψm, FITC represents the collapse of ∆ψm (Scale bar: 50 µm). (B) Images of DCFH-DA fluorescence was taken after TBHP stimulation to evaluate the intracellular ROS levels (Scale bar: 100 µm). Fig. S2 (A-C) After transfected with LV-Ctrl or LV-shMfn2, the cells were treated with CCCP (30 µM) for 1 h. The protein expression of P62 and LC3 was analyzed by western blot. All data represent mean ± S.D. (n = 4). *P<0.05, **P<0.01, & P <0.0001

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