Dear Editor,

The reduced proliferation and increased senescence of nucleus pulposus cells (NPCs) are associated with the aging process of intervertebral discs (IVDs), consequently contributing to intervertebral disc degeneration (IVDD),^1-3 and also defective expression of the anti-aging protein Klotho (KL) is linked to various age-related diseases, including IVDD.^4-7 Our findings indicate that reduced KL expression in NPCs is a key factor leading to altered autophagy, mitochondrial dysfunction, and cellular senescence, offering new insights into the molecular mechanisms behind IVD degeneration.

Given the role of KL protein in cellular senescence, its potential function in IVDD has been proposed (Supplementary file 1, Note S1). We examined gene expression of KL and extracellular matrix (ECM) components in two datasets (GSE122429 and GSE186542) obtained from human NPCs and IVD tissues (Supplementary file 2, Method S1).⁸ NPCs differentiated from embryonic or pluripotent stem cells showed an increase of KL and COL2A1 (Figures 1a–d and S1a–f), indicating potential for intervertebral disc repair (Note S2). By contrast, the gene expression analysis of degenerated IVD tissues exhibited a significant decrease of KL expression, underscoring its vital role in maintaining healthy discs (Figures 1e–g and S1g–i; Note S2).⁹ Additionally, in vivo mice studies showed that aged IVDs were correlated with decreased KL and its co-receptor FGF-23 (Supplementary file 3, Method S2, Figures 1h and S2a), as well as increased senescent markers (Figures 1i and S2b), highlighting the impact of KL-associated cellular aging on IVDD (Note S3).

We further explored the cellular dynamics of mouse NPCs (mNPCs) from the intervertebral lumbar region of older mice (Figure S3a). One-year-old mice showed reduced regenerative capacity, with decreased growth rates, irregular morphology, and diminished clonogenic potential at passage 5, while mNPCs from 1-month-old mice exhibited strong growth potential and regular morphology from passage 0 to 5 (Figure 1j–n). NPCs derived from older mice also showed the increased activity of senescence-associated β-galactosidase (SA-β-GAL) and other senescence markers such as p53, p21, and p16, along with higher IL1-β levels (Figure 1o, p and r). KL and ACAN expression decreased with further passages in older NPCs, and matrix metalloproteinase-13 (MMP-13) levels increased, unlike in younger mice's NPCs (Figure 1q). A complementary analysis using the GSE113199 dataset supported these findings, showing that IL1-β treatment reduced KL, ACAN, and COL2A1 expression (Figures 1s–v and S3b–d). Further details are in Note S4.

Moreover, in vitro cell passaging experiments with human NPCs (hNPCs) demonstrated a reduction in the number of cell population doublings (NCPD) and increased cell population doubling time (CPDT) (Supplementary file 4, Method S3, Figure 2a). Early passage cells (P0 to P5, termed EA-hNPCs) showed relatively high KL and FGF-23 protein expression compared to late passage cells (P10 to P13, termed LA-hNPCs) (Figure 2b). More details are in Note S5.

We conducted an in-depth analysis of the neural network involving LA-hNPCs and healthy EA-hNPCs, using training data from NCPD, CPDT, and KL protein expression (Supplementary file 5, Method S4). The results confirmed that the model accurately predicted and classified KL expression as stable in EA-hNPCs but significantly decreased in LA-hNPCs, correlating with changes in proliferative capacity (Figures 2c and d and S4–S7, Table S1). Further exploration is in Note S6.

The proliferation capability of hNPCs during in vitro culture was assessed by examining cell morphology, BrdU incorporation, and clonogenic ability in EA- and LA-NPCs. Pre-mature cellular senescence was evaluated by SA-β-GAL activity, autophagy flux activity, mitochondrial bioenergetics analysis, and inflammatory markers like IL1β levels with other functional assays (Supplementary file 6, Method S5–S12, Table S2). Indeed, LA-hNPCs exhibited cell morphological changes, increased senescence and apoptosis, elevated IL1β levels, reduced proliferation rates, and altered ECM expression, resulting in premature senescence and IVDD phenotypes (Figure 2e–l). LA-hNPCs also showed decreased autophagic activity and increased mitochondrial ROS levels, with a noticeable shift in bioenergetics profiling (Figures 2m–r and S8a and b). Additionally, bioenergetic profiling in LA-hNPCs represented significant alterations, with an increase in oxidative phosphorylation and glycolytic flux (Figures 2s–z and S8c–e), suggesting a shift in energy metabolism in response to the rising nutritional demands. Details are further in Note S7.

KL knockdown in hNPCs led to diminished cell growth, increased mitochondrial ROSs, and altered respiration (Supplementary file 7, Method S13, Figure 3a–i). Furthermore, reduction of KL expression resulted in decreased autophagy, accelerated senescence, and disrupted ECM balance (Figure 3j–q). These findings highlight the critical role of KL in cellular homeostasis and preventing IVDD. More discussions are in Note S8.

Additionally, recombinant KL (rKL) treatment exhibited growth potential by enhancing cell proliferation, decreasing oxidative stress, and boosting mitochondrial function in LA-hNPCs (Supplementary file 8, Method S14, Figures 4a–g and S9a–d). Further results are discussed in Note S9.

In 3D cell culture analyses, EA-hNPCs spheroids grew steadily, while LA-hNPCs spheroids lost growth potential (Method S15, Figure 4h). Interestingly, rKL treatment restored growth activity in LA-hNPCs spheroids. rKL treatment also stimulated antioxidant effects and increased KL expression and autophagy activity (Figures 4i and j and S10a and b), suggesting diverse therapeutic potentials for rKL in controlling spheroid growth dynamics, reducing oxidative stress, and promoting autophagic processes. This is further discussed in Note S10.

Furthermore, co-treatment with rKL and chloroquine (CQ), an autophagy inhibitor, demonstrated that the protective function associated with KL was impaired due to reduced autophagy, increased mitochondrial ROS, and disrupted mitochondrial homeostasis (Figure 4k–u). Blocking autophagy also reduced rKL-induced ECM secretion and promoted cellular senescence, suggesting a potential role of rKL in therapeutic effects via autophagy regulation. Further results are in Note S11.

In conclusion, our comprehensive study showed a significant KL role in autophagy, along with metabolic changes in senescent NPCs, including impaired mitochondrial function and increased glycolysis (Supplementary file 9, Note S12, Figure S12). These findings emphasise that targeting KL-associated pathways could offer therapeutic strategies to delay or prevent IVDD. Notably, rKL therapy emerges as a promising approach for tackling IVDD and potentially alleviating other age-related diseases.

AUTHOR CONTRIBUTIONS

M.E.B. conceptualised, wrote the manuscript, and created all original figures. D.S.M. analysed data and edited the manuscript. T.H.L. supported the experiments and edited the manuscript. J.S.H. supported the experiments and edited the manuscript. N.N.Q. and R.F.H. supported the experiments. J.Y. edited the manuscript. W.K. edited the manuscript. D.K.L. edited the manuscript. S.P.Y. edited the manuscript. J.H.B. edited the manuscript, provided financial support. D.R.K. conceptualised, provided financial support, edited all figures, and contributed to writing the manuscript. All authors have read and agreed to the published version of the manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no competing interests

FUNDING INFORMATION

This study was supported by grants from the Basic Science Research Program through the National Research Foundation of Korea (RS-2023-00219399, RS-2023-00238051).

ETHICS STATEMENT

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Gyeongsang National University (GNU-240702-M0130) and conducted in accordance with the IACUC guidelines of Gyeongsang National University, Republic of Korea.