Abstract
Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder (LSD) caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase enzyme. MPS IVA patients suffer from skeletal dysplasia due to the abnormal function of chondrocytes. Given the interactions of lysosomes with various intracellular organelles, it is not surprising that lysosomal dysfunction can lead to improper functioning of lysosome-interacting organelles such as mitochondria. Mitochondrial alterations have been evaluated in several LSDs; nevertheless, they have not been fully addressed in MPS IVA. In this study, we assessed the mitochondrial alterations in MPS IVA chondrocytes using a three-dimensional culture approach. Our findings revealed that MPS IVA chondrocytes exhibited an increased mitochondrial-triggered apoptosis profile, mitochondrial depolarization, and heightened oxidative stress. Additionally, the proteins associated with mitophagy, PINK1/Parkin, were significantly reduced in MPS IVA chondrocytes, whereas LC3-II and p62 were elevated. Our assessment of mitochondrial dynamics revealed increased levels of Drp1 and Fis1 along with decreased levels of Opa1. Regarding biogenesis, the mitochondrial regulators TFAM and PGC-α were upregulated in MPS IVA chondrocytes. Finally, MPS IVA chondrocytes showed a metabolic shift from mitochondrial respiration towards a glycolytic profile. Collectively, these data indicate that alterations in mitochondrial homeostasis may play a critical role in the pathogenesis of MPS IVA.
Introduction
Mucopolysaccharidosis type IVA (MPS IVA, OMIM: 253000) is a lysosomal storage disorder (LSD) caused by mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) encoding gene. GALNS enzyme (E.C. 3.1.6.4) hydrolyzes sulfate groups from the chondroitin 6-sulfate (C6S) and keratan-sulfate (KS)1. GALNS deficiency leads to the lysosomal accumulation of KS and C6S in several cells, including chondrocytes2,3. Chondrocytes are mesenchymal-derived cells that actively participate in the endochondral ossification of the bone while producing and maintaining the extracellular matrix (ECM)4. Lysosomal overloading of KS and C6S in MPS IVA results in skeletal dysplasia in patients2,5, along with pro-inflammation and pro-oxidant events3,6,7.
Although alterations in key molecular pathways, previously described in some LSDs8,9, have also been proposed to explain the cellular consequences of the C6S and KS accumulation in MPS IVA1,3,10, the precise mechanisms of the GALNS deficiency and its relationship with the intracellular events have not been fully addressed. For instance, while early cell-based evidence and latterly proteomic studies in MPS IVA fibroblasts and leukocytes suggested that cellular processes such as apoptosis remain unchanged11,12, recent studies indicated that both basal and staurosporine (STS)-induced mitochondrial-dependent apoptosis are upregulated in MPS IVA fibroblasts13, revealing new insights into MPS IVA pathogenesis. Apoptosis is a type of programmed cell death finely regulated in eukaryotic cells, which can be triggered through extrinsic and intrinsic stressors14. While extrinsic apoptosis involves the tumor necrosis factor receptor family, intrinsic apoptosis occurs via mitochondrial regulation15,16.
Mitochondria are dynamic, energy-transforming, biosynthetic, and signaling organelles that actively regulate vital processes in the cell beyond ATP synthesis17,18,19,20,21. As dynamic organelles, mitochondria can physically interact with lysosomes through membrane contact sites (MCS)22 to mediate exchanges of metabolites such as calcium, cholesterol, and iron that regulate both lysosomal and mitochondrial functions22. Besides, lysosomes are involved in mitophagy, a well-conserved mechanism in eukaryotes responsible for recycling mitochondria via autophagy23. During autophagy, a double-membraned structure termed autophagosome is formed through various autophagy-related (ATG) proteins, which participate in autophagy p62-tagged cargos encapsulation while recruiting the LC3-II protein into the autophagosome membrane24. At the latest, lysosomes fuse with the autophagosome to release hydrolases, which degrade the cargo24,25. In MPS IVA fibroblasts, autophagosome:lysosome (A:L) fusion disruption is documented26,27, and it has been associated with a reduction in both LC3-II and p62 proteins27.
As the A:L fusion seems to be primarily affected in MPS IVA, it is rational to hypothesize that mitophagy can also be affected, therefore distressing the mitochondrial homeostasis in MPS IVA. Consistently, we have identified a significant increase in the mitochondrial-dependent oxidative stress (mt-ROS) regardless of the mutation affecting the GALNS gene in MPS IVA fibroblasts28,29, further suggesting the presence of mitochondrial alterations in MPS IVA. Similarly, mitochondrial defects have also been described in MPS II30, MPS IIIB31, and MPS IIIC32, using hepatocytes, neuroblastoma clones, and mouse brains as disease models, respectively. In MPS VI, a skeletal dysplasia similar to MPS IVA33, Tessitore et al. (2009) observed an increase in the cytochrome c oxidase subunit 4 (COX-4), along with LC3-II and P62 in MPS VI fibroblasts, supporting that mitophagy could be altered34. Conversely, a significant decrease in COX-4 was recently reported by Pinheiro et al. (2024) in MPS II mouse hepatocytes, providing opposite evidence30. These apparent discrepancies may rely not only on the differences between cell models used but also on the chemical nature of the accumulated substrate, which could be triggering differential stress responses in the cell3, suggesting different pathophysiological mechanisms beyond the common lysosomal dysfunction observed across LSDs.
Only a few pieces of evidence support mitochondrial alterations in MPS IVA13,28,29, highlighting the need for in-depth studies. Most importantly, current data were collected from MPS IVA skin fibroblasts, making it difficult to comprehensively understand the effect of these alterations on primary affected cells such as chondrocytes. Although chondrocytes rely primarily on glycolysis35,36,37, mitochondrial respiration plays a vital role in their energy metabolism, regulating their mineralizing activity and extracellular matrix synthesis38,39,40. Currently, no studies are aimed at investigating the GALNS deficiency-dependent mitochondrial alterations in MPS IVA chondrocytes.
Based on this evidence, this study aimed to investigate mitochondrial alterations in primary human MPS IVA chondrocytes using a three-dimensional culture approach.
Results
Chondrocyte characterization
Several 3D-based cultures, including Na-Alg41,42, have been reported to culture MPS IVA chondrocytes; however, they differ in media composition, supplements, and time of culture1 while missing a proper characterization of chondrocyte phenotype. To study the influence of Na-Alg-based 3D cultures on chondrogenic properties of WT and MPS IVA chondrocytes, we initially assessed the effect of the Na-Alg-based 3D culture on chondrocyte cell viability and classical chondrogenic markers43. Under our experimental conditions, cell viability was higher than 90% in all experimental groups upon 28 days of 3D culture (Fig. 1A), while Col2A1 gene expression was noticed in WT and MPS IVA chondrocytes (Supplementary Fig. 1) along with positive immunostaining for type II collagen, aggrecan, and SOX9 (Fig. 1B). Besides, Na-Alg-based 3D cultured WT and MPS IVA chondrocytes resulted positive for CD49c (Range: 31% to 53.7%) and CD151 (> 80%) immunostaining in flow cytometry experiments (Fig. 1C).
Phenotypic characterization of 3D-cultured chondrocytes. (A) Cell viability of WT and MPS IVA chondrocytes. The left panel shows representative epifluorescence images of 28 days of Na-Alg-based 3D cultured chondrocytes. Note that the green fluorescence signal represents live cells. The right panel shows the green relative fluorescent units (RFUs) from Na-Alg-released chondrocytes (n = 3). (B) Immunofluorescence-based detection of type II Collagen (Col II), Aggrecan (Agg), and SOX9. Note that cells were also co-stained for nuclei (Nuc) and cytoskeleton (Cyto). Scale bar: 25 μm. (C) CD49c and CD151 immunotyping via flow cytometry (n = 4). (D) mono-KS levels (n = 2). (E) Specific GALNS enzyme activity (n = 7). Data are presented as mean ± SEM. *p < 0.05, **p < 0.005, ##p < 0.0001.
Since GALNS deficiency primarily leads to aberrant lysosomal accumulation of KS and C6S1, we quantified the levels of KS in WT and MPS IVA chondrocytes through LC–MS/MS to evaluate the Na-Alg 3D-based culture influence on the KS profile. As expected, all MPS IVA chondrocytes revealed a significant increase in the levels of mono-sulfated KS (Fold change: 1.4 to 3.2) as compared to WT chondrocytes (Fig. 1D), regardless of the mutation causing GALNS deficiency (Supplementary Table 1). GALNS mutation resulted in null GALNS enzyme activity in all MPS IVA chondrocytes. In contrast, WT chondrocytes showed a specific GALNS enzyme activity of 20.2 ± 3.8 U/mg (Fig. 1E). Collectively, these data support that Na-Alg 3D-based culture preserves chondrocyte viability, their chondrogenic properties, and leads to classical KS accumulation in MPS IVA chondrocytes.
Mitochondrial membrane depolarization triggers apoptosis in MPS IVA chondrocytes
Previous studies showed a high susceptibility to apoptosis in MPS IVA fibroblasts under basal or STS-induced conditions13. To evaluate whether mitochondrial apoptosis upregulation is also observed in MPS IVA chondrocytes, we assessed the apoptosis rate by detecting PS externalization and caspase 3/7 activation in WT and MPS IVA chondrocytes. As reported in MPS IVA fibroblasts, apoptosis rate was also enhanced in all MPS IVA chondrocytes (Fig. 2A). While WT chondrocytes apoptosis was slightly increased upon STS treatment (Up to 16.7 ± 2.9%), MPS IVA chondrocytes exhibited a significant apoptosis rate increase ranging from 32.3 ± 2.2% to 56.8 ± 2.8% (Fig. 2A), suggesting that MPS IVA chondrocytes are more susceptible to undergo apoptosis upon mitochondrial dysfunction compared to WT chondrocytes. Consistently, caspase 3/7 was significantly activated in MPS IVA chondrocytes under basal and STS-induced apoptosis, further supporting a mitochondrial-triggered cell death (Fig. 2B).
Mitochondrial-triggered apoptosis evaluation in 3D-cultured chondrocytes. (A) Apoptosis evaluation in WT and MPS IVA chondrocytes under basal and staurosporine (STS)-induced conditions. The left panel shows representative cytograms displaying population distribution in four quadrants (Q1 to Q4). Q1, Q2, Q3, and Q4 show necrotic, necroptotic, apoptotic, and live cells, respectively. The right panel shows the mean of the Q3 quadrant interpreted as the percentage of apoptotic cells under each condition (n = 5). (B) Caspase 3/7 activation assays. The top panel shows representative histograms for intracellularly stained chondrocytes under basal and STS-induced conditions. The bottom panel shows the percentage of positive cells for activated caspase 3/7 intracellular staining (n = 4). (C) ΔΨm determination using the JC-1 probe. The top panel shows representative contour plots for monomer and aggregate JC-1-associated fluorescence under basal (black) and STS (red)-induced conditions. Note that under basal conditions, the red fluorescence distributed population switches towards green fluorescence upon STS treatment. The bottom panel shows each experimental condition’s mean red/green fluorescence ratio (n = 4). Data are presented as mean ± SEM. *p < 0.05, **p < 0.005, #p < 0.001, ##p < 0.0001.
In MPS IVA fibroblasts, cytochrome C (Cyt-C) release has been reported under basal conditions13. Since Cyt-C is primarily translocated from mitochondria to cytoplasm due to loss of mitochondrial membrane potential (ΔΨm)44, we rationalized that mitochondrial depolarization could be an early stage in MPS IVA pathogenesis. To assess the ΔΨm in WT and MPS IVA chondrocytes, we conducted flow cytometry experiments using the JC-1 probe, which exhibits a ΔΨm-dependent accumulation45. Mitochondrial depolarization is observed as a green fluorescence increase (resulting from monomeric JC1), while healthy mitochondria rapidly accumulate JC1, switching towards a red fluorescence45. Under our experimental conditions, basal and STS-treated MPS IVA chondrocytes had a lower aggregate:monomer JC1 ratio as compared to WT chondrocytes (Fig. 2C), supporting the idea that mitochondrial depolarization is an early pathophysiological event in MPS IVA.
MPS IVA chondrocytes are highly susceptible to undergoing a pro-oxidant profile
It has been well-documented that oxidative stress can promote mitochondrial depolarization46,47, while damaged mitochondria can propagate ROS via the overproduction of superoxide anion (O2−) and hydrogen peroxide (H2O2)46,48,49. To assess the oxidative profile in MPS IVA chondrocytes, we conducted flow cytometry experiments to determine global- and mt-ROS (Fig. 3). As expected, under basal conditions, all MPS IVA chondrocytes showed higher levels of both global- (Fold-changed: 1.6 to 2.2) and mt-ROS (Fold-changed: 1.6 to 2.4) than WT chondrocytes (Fig. 3A,B). When chondrocytes were treated with menadione, a well-known ROS inducer50, the mt-ROS production in MPS IVA (Fold change: 1.8 to 2.8, compared to menadione-treated WT, p = 0.0005) was significantly greater than that observed under basal conditions (Fig. 3A,B), indicating that MPS IVA chondrocytes have an increased susceptibility to undergo a mitochondrial-dependent pro-oxidant profile. Nitric oxide levels were not significantly different between WT and MPS IVA chondrocytes (Supplementary Fig. 2).
Oxidative profile in 3D-cultured chondrocytes. The top panels show representative histograms for H2DCFDA (A) and MitoSOX (B) fluorescence under basal and menadione-induced conditions. The bottom panels show the MFI mean for each experimental condition (n = 5). Data are presented as mean ± SEM. *p < 0.05, **p < 0.005.
GALNS deficiency-mediated lysosomal dysfunction leads to mitophagy pathway blocking in MPS IVA chondrocytes
A persistent pro-oxidant profile, as observed in MPS IVA chondrocytes, can promote chronic mitochondrial stress, resulting in autophagy and lysosomal biogenesis activation51. Since GALNS deficiency leads to lysosomal dysfunction, which affects autophagy26,27, we hypothesized that lysosomal dysfunction could also increase mitochondrial mass via mitophagy flux blocking. We initially determined lysosomal and mitochondrial mass to evaluate this presumption using LysoTracker Deep Red and NAO, respectively. Both lysosomal (Fold change: 1.2 to 1.5) and mitochondrial (Fold change: 1.2 to 1.6) mass were significantly increased in all MPS IVA chondrocytes, compared to WT chondrocytes (Fig. 4A,B). Consistently, mitochondrial mass assessment via western blot revealed an increase in the membrane mitochondrial proteins VDAC1 (Fold change: 1.8 to 2.0), COX4 (Fold change: 1.2 to 1.4), and ANT (Fold change: 1.4 to 1.7) in MPS IVA chondrocytes, compared to WT chondrocytes (Fig. 4C), strongly supporting a mitochondria mass increase.
Lysosomal mass, mitochondrial mass, and mitophagy evaluation in 3D-cultured chondrocytes. (A) and (B) show lysosomal and mitochondrial mass evaluation via flow cytometry. The left panels display representative histograms for LysoTracker- and NAO-associated fluorescence. The right panels show the mean of relative lysosomal and mitochondrial mass for each MPS IVA chondrocyte with respect to WT chondrocytes (n = 5). The dotted line refers to WT chondrocyte levels. (C) Mitochondrial mass assessment via western blot. The mitochondrial-associated membrane proteins VDAC1, ANT, and COX4 were assessed. The upper panel shows a representative western blot, while the lower panel displays the band intensity quantification for each target (n = 4). (D) Mitophagy evaluation. The left and right top panels show the mitophagy markers PINK1, Parkin, TOMM40, p62, and LC3I/II under basal and CCCP-induced mitophagy conditions. Note two bands for PINK1 corresponding to precursor (PINK1-P) and cleavage (PINK1-C) forms. The bottom panels show band intensity quantification for each marker evaluated (n = 4) under each condition. Uncropped and unedited western blot membranes are shown in Supplementary Figs. 3, 4. Data are presented as mean ± SEM. *p < 0.05, **p < 0.005, #p < 0.001, ##p < 0.0001.
To assess whether a defective mitophagy flux can explain the mitochondrial mass increase observed in MPS IVA chondrocytes, we evaluated the expression levels of LC3, p62, TOMM40, PINK1, and Parkin via western blot under basal and CCCP-induced conditions (Fig. 4D). Under basal conditions, p62 (Fold change: 1.2 to 1.3) and LC3-II (Fold change: 1.2 to 1.4) levels significantly increased in all MPS IVA chondrocytes. When assessing PINK1, we observed two bands corresponding to the precursor (PINK1-P) and cleaved (PINK1-C) form. While PINK1-P decreased in all MPS IVA chondrocytes (Fold change: 0.2 to 0.6), PINK1-C showed a significant reduction in MPS IVA chondrocytes 1 and 2. In contrast, MPS IVA chondrocytes resulted in a slight, non-significant PINK1-C decrease compared to WT chondrocytes. Likewise, Parkin levels dramatically decreased (Fold change: 0.2 to 0.3) in all MPS IVA chondrocytes. TOMM40 levels in MPS IVA chondrocytes were not different from WT chondrocytes. When mitophagy was induced by incubating chondrocytes in the presence of CCCP, we observed that PINK1/Parkin expression remained lower in MPS IVA chondrocytes compared to WT chondrocytes, as observed under basal conditions. CCCP treatment increased the expression of TOMM40, p62, and LC3 in all MPS IVA chondrocytes compared to WT chondrocytes. Calculation of the LC3-II/LC3-I ratio, an indicator of autophagy flux34, increased for all MPS IVA chondrocytes under basal and CCCP-induced mitophagy flux evaluation, compared to WT chondrocytes (Fig. 4E), suggesting global mitophagy impairment.
GALNS deficiency-dependent mitophagy flux impairment promotes mitochondrial dynamic dysregulation
Mitophagy is widely recognized for maintaining mitochondrial homeostasis, and its interaction with mitochondrial processes such as mitochondrial fusion and fission is well-understood52,53. To evaluate the impact of mitophagy dysfunction on mitochondrial dynamics, we assessed the protein levels of Opa1, Fis1, and Drp1 via western blot. Under our experimental conditions, the mitochondrial fission-associated proteins Fis1 (Fold change: 1.1 to 1.4) and Drp1 (Fold change: 1.4 to 1.7) resulted significantly increased in all MPS IVA chondrocytes as compared to WT chondrocytes (Fig. 5A), whereas Opa1 expression, a mitochondrial fusion-associated protein, showed a slight decrease in MPS IVA chondrocytes (Fold change: ~ 0.9) compared to WT chondrocytes; overall suggesting increased mitochondrial fragmentation. TEM evaluation consistently showed a significant increase in short mitochondria from MPS IVA chondrocytes compared to WT ones (Fig. 5B), further supporting this presumption.
Mitochondrial dynamics and biogenesis assessment in 3D-cultured chondrocytes. (A) Mitochondrial dynamics evaluation via western blot. The top panel shows a representative western blot image. The bottom panel shows band intensity analysis normalized against β-actin expression (n = 3). Note that fission-associated proteins Drp1 and Fis1 were highly expressed in MPS IVA chondrocytes compared to WT. Also, a decrease in the Opa1 large (L) fragment was noted. Opa1 short (S) fragment resulted in a discrete reduction in MPS IVA chondrocytes 1, 2, and 4. Uncropped and unedited western blot membranes are shown in Supplementary Fig. 5. (B) Mitochondrial morphology assessment via TEM. The upper panel shows a representative TEM picture of each experimental group. The bottom panel shows the quantification of long (l), intermediate (i), and short (s) mitochondria (n = 4). Scale bar: 50 μm. Uncropped and unedited TEM pictures are shown in Supplementary Fig. 6. (C) Gene expression quantification via qPCR for TFAM and PGC-1α (n = 4). Data are presented as mean ± SEM. *p < 0.05, **p < 0.005, #p < 0.001, ##p < 0.0001.
Since an increase in mitochondrial fission could involve a loss in the mitochondrial network function52, we rationalized that compensatory mechanisms should be activated in response to the increased mitochondrial fragmentation. To test this presumption, we conducted experiments to quantify the expression level of the mitochondrial biogenesis-associated genes TFAM and PGC-1α54,55. We observed a significant overexpression of both TFAM (Fold change: 1.6 to 1.9) and PGC-1α (Fold change: 4.3 to 4.9) genes in MPS IVA chondrocytes compared to WT chondrocytes (Fig. 5C), supporting that mitochondrial biogenesis is upregulated in MPS IVA chondrocytes to compensate the enhanced mitochondrial loss and potentially contributing to the mitochondrial mass increased observed (Fig. 4B,C).
Disrupted lysosome: mitochondrial crosstalk shifts the metabolic profile of MPS IVA chondrocytes
Although chondrocytes rely primarily on glycolysis-dependent metabolism to obtain ATP, mitochondrial respiration and dynamics are required for chondrocyte survival56 as they provide ATP and regulate cell death, proliferation, and redox balance57. Mitochondrial dysfunction is commonly linked to cartilage-affecting diseases such as osteoarthritis (OA)57, and significant impairments in the mitochondrial respiration of osteoarthritic chondrocytes have been reported58,59. To evaluate how the mitochondrial disturbances found in this study affect the respiration in MPS IVA chondrocytes, we lastly assessed the metabolic profile of MPS IVA chondrocytes by monitoring the OCR using the Seahorse XFp Cell Mito Stress Test. Seahorse assay is a plate-based cellular metabolism measurement approach that quantifies OCR and ECAR in real-time via serial injections of mitochondrial modulators (Fig. 6A)60,61. The assays initially monitor basal respiration, followed by quantifying the ATP production ratio upon adding the complex V inhibitor oligomycin60,61. Concomitant with ATP quantification, the assay quantifies proton leak, commonly interpreted as a mitochondrial damage sign. Later, adding the electron transport chain uncoupler FCCP simulates a high energy demand state, leading to the calculation of the maximal achievable respiration60. The spare respiratory capacity can also be calculated from FCCP-mediated OCR, and it is helpful to establish the cell’s flexibility to respond to an increase in the cell energy demand61. A third injection of Rotenone/Antimycin A is added to block the complex I and III activity60. Rotenone/Antimycin A mixture shuts down mitochondrial respiration and allows non-mitochondrial respiration to be calculated61.
Mitochondrial respiration assessment in 3D-cultured chondrocytes. (A) Characteristic XF Cell Mito Stress assay profile using the Seahorse approach. Note that upon each drug injection, different parameters can be determined. (B) Real-time oxygen consumption rate (OCR) evaluation. Note that a significant decrease in the global OCR is observed in MPS IVA chondrocytes compared to WT chondrocytes (n = 6). OCR was monitored under basal conditions and sequential injection of 2 mM oligomycin, 1 mM FCCP, and 0.5 mM rotenone/antimycin. (C–G) shows the respiratory indexes derived from real-time OCR obtained in (B) Note that a significant decrease in basal respiration, maximal respiration, ATP production, and spare respiratory capacity characterizes MPS IVA chondrocytes. At the same time, a higher proton leak was observed in MPS IVA chondrocytes than in WT chondrocytes. (H) Metabolic profile was determined by calculating the ratio between ECAR and OCR. Note that MPS IVA chondrocytes show a more glycolytic profile than that observed in WT chondrocytes. (I) Extracellular lactate levels (n = 5). Data are presented as mean ± SEM. *p < 0.05, **p < 0.005, #p < 0.001, ##p < 0.0001.
Under our experimental conditions, MPS IVA chondrocytes exhibited a lower OCR level compared to WT chondrocytes (Fig. 6B). Consistently, MPS IVA chondrocytes showed a significant decrease in basal respiration (Fig. 6C; fold change: 0.5 to 0.7), maximal respiration (Fig. 6D; fold change: 0.5 to 0.8), and spare respiratory capacity (Fig. 6F; fold change: 0.5 to 0.6) compared to WT chondrocytes, suggesting that mitochondrial respiration is impaired. ATP production in MPS IVA chondrocytes 2 (Fold change: 0.6, p = 0.0369) and 4 (Fold change: 0.5, p = 0.0292) was significantly lower than that observed in WT chondrocytes, while MPS IVA chondrocytes 1 (Fold change: 0.7, p = 0.2681) and 3 (Fold change: 0.7, p = 0.0807) resulted in a non-significant ATP decrease (Fig. 6E). Besides, MPS IVA chondrocytes displayed a significant increase in proton leaking compared to WT chondrocytes (Fig. 6G, fold change: 1.6 to 2.4), further supporting mitochondrial damage. Finally, when calculating the ECAR/OCR ratio, we noticed that MPS IVA chondrocytes underwent a more glycolytic phenotype than WT chondrocytes (Fig. 6H). Consistently, lactate levels were significantly higher in MPS IVA chondrocytes (Fold change: 1.2 to 1.5) compared to WT chondrocytes (Fig. 6I). Collectively, these data support that GALNS deficiency-dependent mitochondrial alterations result in a metabolic shift in MPS IVA chondrocytes.
Discussion
As dynamic organelles, lysosomes maintain cell homeostasis through direct and indirect interactions with other intracellular organelles, such as mitochondria3,62,63. Although lysosomal dysfunction leads to impairments in mitochondrial function, thus affecting overall cell homeostasis, mitochondrial alterations have been barely studied in some LSDs28,29,30,31,32,34. In this study, we assessed the mitochondrial alterations in four MPS IVA chondrocytes using a 3D culture approach based on Na-Alg beads. Na-Alg beads have been successfully used to culture MPS IVA chondrocytes as they lead to the expression of type II collagen and KS accumulation64,65. Consistently, we found that Na-Alg beads are suitable for type II collagen, aggrecan, SOX9 expression, and mono-KS accumulation while preserving chondrocyte viability over 28 days of culture (Fig. 1).
MPS IVA fibroblasts showed enhanced apoptosis and caspase 3/7 activation13,66. In line with this evidence, we found that MPS IVA chondrocytes are characterized by a significant increase in basal and STS-induced apoptosis, as observed when evaluating PS externalization and caspase 3/7 activation via flow cytometry (Fig. 2A,B). Caspase 3 is an effector caspase that strongly correlates with early ΔΨm loss67; therefore, its activation suggests mitochondrial depolarization. Consistent with this premise, we observed that all MPS IVA chondrocytes showed a significant mitochondrial depolarization (Fig. 2C). Likewise, Brokowska et al. (2023) found cytochrome C release in MPS IVA fibroblasts, further supporting that mitochondrial depolarization is an early pathological finding in MPS IVA13. An increase in apoptosis was reported early by Simonaro et al. (2001) in MPS VI rat and cat chondrocytes. Even though the mechanism was not associated with mitochondrial alterations, the authors hypothesized that increased apoptosis was due to the dermatan sulfate overaccumulation and its effect on the NO release68.
In contrast to Simonaro et al. (2001), we did not observe an increase in the NO levels in human MPS IVA chondrocytes (Supplementary Fig. 2). This discrepancy might be due to the differences in culture conditions between both studies, as we used a 3D approach while Simonaro et al. (2001) utilized a monolayer approach. The chemical nature of the GAG accumulated could also elicit different cellular outcomes. Upcoming experiments to evaluate the effect of KS and C6S on the NO release may help elucidate this premise. Even though we did not observe the NO increase in MPS IVA chondrocytes, we found a significant increase in the global and mitochondrial oxidative stress (Fig. 3A), which agreed with previous reports in several MPS69,70, including MPS IVA6,7,28,29. ROS is extensively involved in many mitochondrial functions, such as ATP production, calcium buffering, and apoptosis regulation52. Indeed, mitochondrial depolarization can trigger higher ROS generation and respiratory dysfunction71, thus explaining the pro-oxidant phenotype observed in MPS IVA chondrocytes. Novel experiments evaluating the levels of superoxide dismutase, catalase, and thioredoxin using approaches such as immunoblotting could be carried out to support the flow cytometry-based pro-oxidant profile observed in MPS IVA chondrocytes.
Mitochondrial depolarization is a key signal that activates mitophagy to remove damaged mitochondria. Mitophagy is initially triggered by the mitochondrial damage sensor PINK1, which is translocated through the pore formed by TOMM40 in the outer mitochondrial membrane, followed by the recruitment of the amplifier Parkin72,73. Later steps in mitophagy require lysosome fusion23. Interestingly, we observed a dramatic decrease in the PINK1/Parkin axis (Fig. 4D), suggesting early mitophagy impairment. LC3-II and p62 protein expression significantly increased in MPS IVA chondrocytes, suggesting autophagosome accumulation and global autophagy blocking. Similarly, an increase in LC3-II and p62 was also reported in MPS VI fibroblasts34. Although mitophagy is poorly investigated in MPS models, it was recently tested by Tillo et al. (2022) in brown adipose tissue (BAT) of MPS IIIA mice74. MPS IIIA BAT was characterized by increased mitochondrial mass along with LC3-II levels, which agrees with our findings for mitochondrial mass (Fig. 4B,C) and mitophagy flux (Fig. 4D). Even though our results agree with several findings in other MPS models, they are opposite to previous studies conducted in MPS IVA fibroblasts26,27. For instance, although global autophagy seems to be blocked in MPS IVA fibroblasts, the mechanism appears to be mediated by a decrease in the expression of p62 and LC3-II26,27 rather than autophagosome accumulation as observed in MPS IVA chondrocytes (Fig. 4E). These apparent discrepancies might be due to differences in the cell models and culture conditions evaluated.
While PINK1/Parkin-dependent mitophagy relies on permanently recruiting Parkin to the mitochondria, PINK1/Parkin-independent mitophagy is an additional pathway to remove dysfunctional mitochondria23,73. PINK1/Parkin-independent mitophagy involves several proteins, such as BNIP3, which inhibits Opa1 and activates Drp1 to promote mitochondrial fragmentation73,75. In line with this, we found that MPS IVA chondrocytes were characterized by a significant increase in Fis1 and Drp1 (Fig. 5A), suggesting that PINK1/Parkin-independent mitophagy may be activated in MPS IVA chondrocytes. Consistently, Opa1 decreased slightly (Fig. 5A), further supporting our presumption. We have hypothesized that the PINK1/Parkin-independent mitophagy pathway is activated; nevertheless, the persistent lysosomal dysfunction may impair the A:L fusion, leading to autophagosome accumulation76, as observed in MPS IVA chondrocytes when inducing mitochondrial dysfunction via CCCP (Fig. 4D). Novel experiments addressing the role of BNIP3 along with other PINK1/Parkin-independent mitophagy-related proteins such as NIX and FUNDC123,73 will dissect the precise mitophagy mechanism in MPS IVA chondrocytes.
Regardless of the mitophagy pathway involved, the findings for mitochondrial mass increase (Fig. 4B,C) and autophagosome accumulation (Fig. 4D) strongly suggest an impaired mitophagy activation. In remodeling the mitochondrial network, mitochondrial fragmentation enables mitophagy and biogenesis17,23,40. As expected, we found that both TFAM and PGC-α, genes involved in mitochondrial biogenesis77 were significantly upregulated in MPS IVA chondrocytes (Fig. 5C), uncovering a potential compensatory mechanism to the enhanced mitochondrial fragmentation (Fig. 5B). As TFAM promotes mtDNA transcription and replication52, mtDNA quantification could also be evaluated in future experiments to support the mitochondrial biogenesis increase observed in MPS IVA chondrocytes. Nevertheless, biogenesis upregulation is insufficient to restore the mitochondrial respiration in MPS IVA chondrocytes (Fig. 6). Instead, it may potentially contribute to the increased mitochondrial mass observed (Fig. 4B,C) and the overloading of damaged mitochondria, which ultimately leads to impaired cellular energy production. Similar to our findings, a study published by Ivanova et al. (2019) showed overexpression of TFAM in human Gaucher peripheral blood mononuclear cells (PBMCs), which resulted in normalization to WT levels upon enzyme replacement therapy (ERT)78. The effect of ERT and gene therapy (GT) on the mitochondrial biogenesis in MPS IVA chondrocytes needs to be addressed.
Although chondrocytes primarily use glycolysis to meet energy needs, oxidative phosphorylation is critical for chondrocyte metabolism79,80. Mitochondrial dysfunction is a key biochemical feature preceding chondrocyte senescence79,81. In this regard, under our experimental conditions, we found a significant impairment in the mitochondrial respiration of all MPS IVA chondrocytes (Fig. 6A), which shifts the metabolic chondrocyte profile toward a more glycolytic profile (Fig. 6H). Although mitochondrial respiration has not been assessed in another LSD chondrocyte model, it has been reported in human Gaucher PBMCs78, MPS IIIB neuroblastoma cells31, and MPS II hepatocytes30, further supporting that the lysosome-mitochondria axis is a key feature in several LSDs. Interestingly, in OA pathogenesis, it has been shown that OA chondrocytes undergo metabolic changes involving enhanced glycolysis dependence to produce ATP and decreased mitochondrial respiration and tricarboxylic acid cycle59,82. This metabolic shift towards a more glycolytic metabolism (predominant lactate production; Fig. 6I) seems to contribute to ECM degeneration via microenvironment acidification mediated by an increase in the extracellular levels of lactate83. Metalloproteinase 9 (MMP-9) overexpression in MPS IVA patients has been documented and associated with ECM abnormalities84. Since MMP-9 is a specific ECM metalloproteinase that can be overexpressed85 and cleaved by cathepsin K to achieve an active enzyme state in acidic environments86, we postulated that a metabolic shift in MPS IVA chondrocytes towards a glycolytic profile could contribute to the cartilage abnormalities observed in MPS IVA patients. Further evidence should be provided to confirm this premise.
In conclusion, we have characterized, for the first time, mitochondrial alterations in MPS IVA chondrocytes using a 3D-culture approach. Mitophagy pathway impairment seems to contribute, at least in part, to a dysfunctional mitochondrial accumulation resulting in a pro-oxidant profile and mitochondrial respiration abnormalities, which lead to a metabolic shift in MPS IVA chondrocytes towards a glycolytic profile (Fig. 7). Novel studies involving a more significant number of MPS IVA chondrocytes will provide a better understanding about the variability between individuals carrying different mutations on the GALNS gene. Likewise, addressing the impact of these mitochondrial alterations on critical cellular processes such as mito-inflammation will undoubtedly reveal new insights in MPS IVA pathogenesis, paving the way to attempt novel therapeutic alternatives beyond ERT and GT.
Proposed model of mitochondrial alterations in MPS IVA chondrocytes. (A) Under physiological conditions, mitochondrial damage (i.e., mitochondrial depolarization; 1) induces recruitment of PINK1, which leads to the accumulation of Parkin to mediate mitochondrial ubiquitination and complex formation with p62 (2). PINK1/Parkin-independent mitophagy can also be activated (3). Then, LC3 associates with the p62/ubiquitin-targeted mitochondria (4), resulting in mitochondria-containing autophagosome formation. Mitochondria-containing autophagosome formation is also achieved via PINK1/Parkin-independent mitophagy. Lysosomal fusion to mitochondria-containing autophagosomes forms autophagolysosomes (A:L) (6), resulting in mitochondrial degradation through lysosome-dependent hydrolase release (7). (B) In MPS IVA, mitochondrial damage (8) could activate PINK1/Parkin-independent mitophagy (9) over PINK1/Parkin-dependent mitophagy (10). PINK1/Parkin-independent mitophagy correlates with increased Drp1/Fis1 expression, enhancing mitochondrial fragmentation and decreasing Opa1 expression to limit mitochondrial fusion (11). Although p62 can bind LC3-II, dysfunctional lysosomes prevent A:L formation (12), later preventing mitochondrial removal (13). Mitophagy impairment leads to dysfunctional mitochondrial accumulation (14), promoting a pro-oxidant profile, decreasing mitochondrial respiration, and triggering apoptosis (15). Mitochondrial respiration (OXPHOS) decreases and shifts chondrocytes’ metabolism towards a glycolytic profile, increasing lactate production (16). Consequent extracellular acidification could promote metalloproteinase (MMP)-9 expression and cartilage abnormalities (17). Mitochondrial biogenesis (18) is stimulated as a compensatory mechanism through TFAM and PGC-1α overexpression (19). Nevertheless, permanent mitophagy impairment leads to a further mitochondrial mass increase (20) concomitant with dysfunctional mitochondrial accumulation due to the chondrocytes’ inability to remove old/damaged mitochondria and/or perform mitochondrial network remodeling (21). This figure was created in Biorender.com.
Materials and methods
Chondrocyte culture and phenotype characterization
Chondrocyte culture
De-identified human cartilage-derived chondrocytes were obtained from Nemours Children’s Health BioBank (Institutional Review Board # 2,107,509 and #750,932). Chondrocyte characteristics are shown in Supplementary Table 1. Chondrocytes were cultured in a monolayer in a 75 cm2 flask using chondrocyte growth media (CGM; Lonza, Walkersville, MD) until reaching 80% confluence and then harvested by gentle trypsinization using Chondrocyte Reagent Pack Subculture Reagents (Lonza). Pelleted chondrocytes were resuspended in 1 mL of 1.2% sodium alginate (Na-Alg; Sigma-Aldrich, St. Louis, MO) at a density of 400,000 cells/mL. The homogenized chondrocytes were carefully transferred into a 5 mL sterile syringe, aided by a Pasteur pipette, and seeded by passing the cell suspension through a 20G needle. The cells were dropped into Petri dishes filled with a crosslinker solution composed of 102 mM CaCl2 solution while stirring and incubated at room temperature (RT) for 15 min. The crosslinker solution was carefully removed, and the chondrocyte-containing beads were washed four times with 0.9% NaCl. Chondrocyte-containing beads were further washed once with chondrocyte differentiation media (CDM; Lonza), then transferred to ultra-low adherence plates (Thermo Fisher Scientific, Waltham, MA) filled with CDM and kept at 37 ºC and 5% CO2. CDM was replaced by a fresh one 24 h later. Finally, chondrocyte-containing beads were cultured for 28 days in CDM supplemented with 70 mM ascorbic acid (Lonza)64 and 100 ng/mL bone morphogenic protein-2 (BMP-2; StemCell Technologies, Vancouver, Canada)87. When needed, chondrocytes were released from alginate beads by immersing beads in a dissociation solution composed of 0.055 M Sodium Citrate, 0.15 M NaCl, pH 6.8, at 37 ºC for 10 min while gently shaking. Chondrocytes were pelleted at 300 × g for 5 min, washed in Hanks’ balanced salt solution (1X HBSS; Gibco, Waltham, MA), and used in downstream experiments. The cells were periodically tested for Mycoplasma spp infection using PCR, as described by Siegl et al. (2023)88. All the cultures were negative throughout the experiments (data not shown). Chondrocytes in passages 2 to 6 were used in all experiments.
Cell viability assessment of sodium alginate bead-encapsulated chondrocytes
The cell viability of chondrocyte-containing beads was evaluated by using the LIVE/DEAD Viability/Cytotoxicity Assay Kit (Invitrogen, Carlsbad, CA) following the protocol described by Visscher et al. (2019)89 with slight modifications. Briefly, chondrocyte-containing beads were placed in a 1.5 mL tube and stained with 0.25 mM Propidium Iodide and 2 mM Calcein AM at 37 ºC in 1X HBSSCM (HBSS calcium, magnesium, Gibco) for 1.5 h. The beads were then washed in 1X HBSSCM and pictured in a fluorescent microscope (Axio Observer Z1 microscope, Zeiss, Germany) using Extended Depth of Focus (EDF). The resulting images were 3D projected and processed using ImageJ2 (https://imagej.net/software/imagej2/)90. After image acquisition, the beads were dissociated, and chondrocytes were transferred to 96-well black flat-bottom microplates (Corning; Corning, NY) for cell viability quantification. The plate was read in a FLUOstar Omega microplate reader to detect green (live cells; Exc/Emi: 485/520 nm) and red (dead cells; Exc/Emi: 544/620 nm) fluorescence. The percentage of live cells was expressed as the relative fluorescent units (RFUs) detected in the green filter divided by the total RFUs (Signal in the green and red filters).
Type I and II collagen expression
Wild-type (WT) and MPS IVA chondrocytes were characterized by quantifying type I and II collagen expression via relative qPCR. Chondrocytes were released from Na-Alg beads and washed five times with HBSS 1X. The pelleted cells were used for RNA isolation aided by the Monarch Total RNA Miniprep Kit (New England Biolabs, Ipswich, MA). High-capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) was used to synthesize cDNA following the manufacturer’s instructions. Finally, the cDNA was used for qPCR assays using type I (Col1a2; Hs01028956_m1) and II (Col2a1; Hs00264051_m1) collagen TaqMan probes (Applied Biosystem). Expression levels were determined via double delta Ct analysis by normalizing Col1a2 and Col2a1 expression against the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Hs02786624_g1) and beta-actin (β-Act; Hs01060665_g1). All the experiments were performed in a QuantStudio 7 Flex Real-Time PCR instrument (Applied Biosystems).
Immunofluorescence assays
Expression of type II collagen, SOX9, and aggrecan was evaluated through immunofluorescence assays. Briefly, chondrocyte-containing beads were fixed in 4% paraformaldehyde in 1X HBSSCM at RT for 30 min91 and then dissociated as previously described. Pelleted chondrocytes were washed twice with 1X PBS and seeded in 50 mg/mL polyD lysine-coated (Gibco) 8-well glass (Millipore Sigma, Burlington, MA). The cells were allowed to attach for one hr at RT. The chondrocytes were then covered with 0.1% Triton X-100 in PBS at RT for 10 min. After two washes with 1X PBS at RT for 5 min each, the cells were incubated at RT for one hr with a blocking solution comprising 1X PBS, 5% bovine serum albumin (BSA; Sigma-Aldrich), and 10% goat serum (Gibco). Unconjugated primary rabbit antibodies targeting type II collagen (PA5-99159; Invitrogen), SOX9 (MA5-41174; Invitrogen), and aggrecan (13880–1-AP; Invitrogen) were prepared at a 1:50 dilution in 1X PBS plus 5% BSA and added to the chondrocytes. After overnight incubation at 4 ºC, chondrocytes were washed three times with 0.2% Tween 20 (Thermo Fisher Scientific) in 1X PBS and incubated at RT for one hr with Alexa Fluor™ 594-labeled goat anti-Rabbit IgG secondary antibody (A-11012; Invitrogen) at 1:200 dilution in 1X PBS plus 5% BSA. Alexa Fluor 488 Phalloidin (Invitrogen) at 1:1000 dilution was added during secondary antibody incubation for cytoskeleton staining. Three further washes were performed with 0.2% Tween 20 (Thermo Fisher Scientific) in 1X PBS to remove unbound antibodies. Finally, one drop of ProLong Glass Antifade Mountant with NucBlue Stain (Invitrogen) was added to each well, and a cover glass was placed on the stained chondrocytes. Chondrocytes processed as detailed above, without primary antibody incubation, were included to assess the background. After overnight curing at RT, the cells were visualized and imaged in a fluorescence microscope (Axio Observer Z1 microscope, Zeiss, Germany). Image analysis was performed using ImageJ2 (https://imagej.net/software/imagej2/)90.
Immunotyping characterization
Chondrocytes were further characterized by detecting the surface expression of CD49c and CD151 using flow cytometry as previously described43. Briefly, chondrocyte-containing beads were dissociated, as stated previously, and washed twice with 1X HBSS. The chondrocytes were resuspended in 1% BSA in 1X PBS and incubated at 4 ºC for 30 min with CD49c-APC (17–0494-42; Invitrogen) or CD151-APC (17–1519-42; eBioscience, San Diego, CA) following the manufacturer’s recommendations. Mouse IgG1 kappa-APC (17–4714-81; Invitrogen) was included as an isotype control. After incubation, the chondrocytes were washed twice, resuspended in 150 mL of 1% BSA in 1X PBS, and transferred to a 96-well plate. Propidium iodide (PI; Molecular Probes, Eugene, OR) was added at a 1:1000 dilution to exclude dead cells during flow cytometry experiments. Flow cytometry experiments were conducted in a Novocyte 3000 (Agilent Technologies, Santa Clara, CA). At least 10,000 events were recorded in the P1 region of the side scatter (SSC) and forward scatter (FSC) plots. APC signal was collected with a 675/30 nm bandpass filter. Raw data was analyzed in FlowJo v10.10 (Becton Dickinson, Franklin Lakes, NJ).
MPS IVA chondrocyte genotype and phenotype evaluation
GALNS exon sequencing
GALNS exon sequencing was performed to determine the mutation causing GALNS deficiency. Genomic DNA was processed by the Nemours Biobank and Molecular Analysis Core using standard Sanger sequencing and operating procedures.
GALNS enzyme activity
Total protein extract from WT and MPS IVA chondrocytes was used to assess specific GALNS enzyme activity using 4-methylumbelliferyl-β-d-galactopyranoside-6-sulfate (Toronto Chemicals Research, North York, ON, Canada), following our previously reported protocols28,29.
Lysosomal mass determination
According to our previous protocols, the lysosomal mass accumulation was screened through flow cytometry using LysoTracker Deep Red (Invitrogen)28,29,92. Flow cytometry experiments were conducted as aforementioned.
Glycosaminoglycan (GAG) evaluation
GAGs were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a 1290 Infinity LC system with a 6460 triple quad mass spectrometer (Agilent Technologies)93. Chondroitinase B, heparitinase, and keratanase II enzymes were used to digest the polysaccharides and isolate disaccharides from WT and MPS IVA chondrocytes. GAG levels were normalized against total protein concentration determined via Pierce BCA Protein Assay (Thermo Fisher Scientific).
Mitochondrial disturbances evaluation
Apoptosis assays
Apoptosis evaluation was carried out by detecting phosphatidyl serine (PS) externalization and caspase 3/7 activation by using Dead Cell Apoptosis Kits with Annexin V for Flow Cytometry (Molecular Probes) and CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit (Molecular Probes), respectively. Chondrocyte-containing beads incubated with 1 μM STS in CDM at 37ºC for six h were included as positive controls13. Then, the chondrocytes were released from the beads and stained as recommended by the manufacturer. The cells were then harvested, transferred to a 96-well plate, and used for flow cytometry. PS externalization was detected via Alexa fluor 488-conjugated Annexin V using an Exc/Emi of 488/530 nm and activated caspase 3/7 signal according to the manufacturer’s instructions. Necrotic cells were detected by the signal of either propidium iodide or SYTOX AADvanced by using 615/20 nm or 647/30 bandpass filters, respectively. Flow cytometry was conducted as previously described.
Mitochondrial membrane potential
Mitochondrial membrane potential was determined by using the MitoProbe JC-1 Assay Kit (Molecular Probes) according to the manufacturer’s instructions via flow cytometry. Briefly, the chondrocytes were released from the beads and incubated in the absence and presence of 50 nM CCCP (carbonyl cyanide m-chlorophenyl hydrazone)-containing media at 37ºC for five min. STS was also included as a positive control by following the protocol described for apoptosis assays. The chondrocytes were further incubated with 200 mM JC-1 at 37ºC for 30 min. After one wash with 1X HBSS, the cells were used for flow cytometry assays. Fluorescence JC-1 probe emission shift from green towards red was recorded using 488 nm excitation with fluorescein isothiocyanate (FITC, 530/30 nm) and phycoerythrin (PE, 572/28 nm) bandpass filters, respectively, as suggested by the manufacturer. CCCP-treated samples were used to perform compensation.
Oxidative stress
Global and mitochondrial-derived oxidative stress were evaluated by using Carboxy-H2DCFDA (Exc/Emi: 488/530 nm; Invitrogen) and MitoSOX Mitochondrial Superoxide Indicator (Exc/Emi: 488/572 nm; Invitrogen), respectively, by following our previously reported protocols28,29,92. Briefly, chondrocytes were released from Na-Alg beads and incubated with either 25 mM H2DCFDA-containing HBSS at 37ºC for 30 min or 5 mM MitoSOX-containing HBSS at 37ºC for 15 min. Chondrocytes incubated with 25 mM Menadione (Sigma-Aldrich) at 37ºC for 30 min were included as positive controls50. Chondrocytes were washed once with HBSS and used for flow cytometry experiments.
Nitric oxide-related species
Nitric oxide species were evaluated via DAF-FM Diacetate (Exc/Emi: 488/530 nm; Invitrogen) and Griess reaction through Griess Reagent Kit (Invitrogen). Briefly, Na-Alg beads were dissociated, and chondrocytes were later incubated with 10 mM DAF-FM Diacetate in HBSS at 37ºC for 30 min. The chondrocytes were washed once with 1X HBSS and used for flow cytometry assays as described previously. For Griess reaction, 500 mL culture supernatant was initially incubated with 0.018 U/L of nitrate reductase (NR (NAD[P]H) from Aspergillus niger, Sigma-Aldrich) in the presence of 57 mM potassium phosphate (K2HPO4·3H2O, pH: 7.4, Sigma-Aldrich) 0.005 mM Flavin Adenine Dinucleotide Solution (FAD, Sigma-Aldrich) and 0.2 mM β-Nicotinamide Adenine Dinucleotide Phosphate (β-NADPH, Sigma-Aldrich) at RT for three hrs to reduce nitrate to nitrites. The media was mixed with 20 mL Griess reagent and 130 mL deionized water. After 30 min of incubation at RT, the samples were spectrophotometrically read at 548 nm. Optical density (OD) readings were converted to nitrite concentrations by plotting OD against a sodium nitrite standard per the manufacturer’s instructions. Chondrocyte-containing beads incubated with 250 ng/mL Lipopolysaccharide (LPS from Escherichia coli 026:B6; eBioscience)-containing media were included as positive controls94,95.
Mitochondrial mass
Mitochondrial mass was initially evaluated by staining chondrocyte-containing beads with 200 nM Nonyl Acridine Orange (NAO, Exc/Emi: 488/530 nm; Invitrogen) in CDM at 37ºC for one hr. The beads were washed twice with 1X HBSS and dissociated as previously detailed. The cells were later analyzed via flow cytometry. Besides, mitochondrial mass was also evaluated through western blot by detecting cytochrome c oxidase IV (COX-4), voltage-dependent anion-selective channel 1 (VDAC1), and adenine nucleotide translocator (ANT) protein expression30 using standard protocols. Briefly, total protein was extracted using RIPA lysis buffer (Thermo Fisher Scientific) supplemented with 1X Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific), 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich). 25 mg of protein was resolved on a 4–20% polyacrylamide gel (Mini-PROTEAN TGX, Bio-Rad, Hercules, CA) at 90 V for two hrs. The transfer was performed into a 0.2 mm pore nitrocellulose membrane (Bio-Rad) at 350 mA for 60 min. A pre-stained protein ladder (Bio-Rad) was loaded onto the gel and used as a molecular weight reference. After one hr of membrane blocking, the membrane was probed for primary rabbit anti-COX-4 (PA5-29,992, 1:500, Invitrogen), anti-VDAC1 antibodies (PA1-954A, 1:500, Invitrogen), and anti-ANT (PA5-109,394, 1:500, Invitrogen) at 4ºC overnight while gently shaking. Anti-β-actin (PA1-16,889, 1:1000, Invitrogen) and anti-β-tubulin (80,713–1-RR100UL, 1:1000, Prointech, Rosemont, IL) antibodies were included as a loading control. The membrane was then incubated with goat anti-Rabbit IgG Secondary HRP-conjugated Antibody (A27036, 1:5000, Invitrogen) at RT for one hr. The membrane was additionally incubated with Precision Protein StrepTactin-HRP Conjugate (1,610,380, 1:10,000, Bio-Rad) to detect the protein ladder. Finally, the signal was developed using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) for 5 min and visualized in a C-DiGit Blot Scanner (LI-COR, Lincoln, NE). Relative protein expression was determined by measuring the relative density of COX4, VDAC1, and ANT normalized to β-actin or β-tubulin, aided by Image Studio Software (LI-COR).
Mitophagy
Mitophagy was assessed via western blot following the protocol detailed for COX4 and VDAC1. Conversely, nitrocellulose membranes were probed for LC3 (1:1000, 14,600–1-AP, Prointech), SQSTM1 (1:500, PA5-20,839, Invitrogen), TOMM40 (1:500, 18,409–1-AP, Prointech), PINK1 (1:500, PA5-86,941, Invitrogen) and Parkin (1:500, PA5-13,399, Invitrogen). GAPDH (1:1000, sc47724, Santa Cruz Biotechnology Inc., Dallas, TX) and β-tubulin (1:1000, 80,713–1-RR100UL, Prointech) were used as loading controls. Chondrocytes were further incubated with 20 μM CCCP for 24 h and included as mitophagy-positive controls96.
Mitochondrial dynamics
The mitochondrial dynamics were assessed by probing nitrocellulose membrane with DRP1 (1:1000, 12,957–1-AP, Prointech), Fis1 (1:1000, PA5-22,142, Invitrogen), and Opa1 (1:1000, 27,733–1-AP, Prointech), following the protocols described for mitochondrial mass analysis.
Mitochondrial biogenesis
To evaluate mitochondrial biogenesis, the gene expression of the mitochondrial transcription factor A (TFAM, Hs00273372_s1) and the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α, Hs00173304_m1) was evaluated via qPCR by following the protocol detailed for Type I and II collagen expression.
Transmission electron microscopy (TEM)
Briefly, chondrocytes were fixed with 2% glutaraldehyde and 2% paraformaldehyde (Electron Microscopy Sciences) in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences), pH 7.4. Then, samples were embedded in 4% low-melting-point agarose (Electron Microscopy Sciences) and cut into 1mm3 cubes. The samples were further post-fixed with freshly prepared 1% osmium tetroxide and 1.5% potassium ferrocyanide in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences), pH 7.4, for 2 h and stained overnight with 1% uranyl acetate (Electron Microscopy Sciences). The samples were infiltrated with Spurr’s resin and cut on an ultramicrotome (Leica UC7, Germany), and 60 nm thick sections were collected onto 200 mesh formvar/carbon-coated copper grids. Sections were post-stained with 2% uranyl acetate in 50% methanol and Reynolds lead citrate (Electron Microscopy Sciences). Finally, the samples were imaged on a Talos L120C transmission electron microscope (Thermo Fisher Scientific) operating at 120 kV, magnification of 6700x, and acquired with a Ceta 16 M CCD (Thermo Fisher Scientific).
Mitochondrial respiration
Real-time oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were assessed using the Seahorse XFp Cell Mito Stress Test Kit (Seahorse Bioscience, Billerica, MA) following the manufacturer’s protocol. Briefly, 3 × 104 chondrocytes were released from Na-Alg beads and seeded onto a 96-well microplate (Seahorse Bioscience) in CDM overnight under standard cell culture conditions. The next day, CDM was removed and replaced with assay media composed of XF basal media pH 7.0 supplemented with 10 mM glucose, 2 mM sodium pyruvate, and 2 mM L-glutamine (Seahorse Bioscience). The 96-well plate was incubated for one hr in a non-CO2 incubator. In the meantime, the assay sensor cartridge was loaded with 2 mM oligomycin, 1 mM Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and 0.5 mM rotenone/antimycin and placed in a Seahorse XFe96 pro analyzer to perform calibration. The 96-well microplate containing chondrocytes was loaded upon calibration, and the OCR was recorded. Mitochondrial respiratory indexes were analyzed, including basal OCR, maximal respiration, spare respiratory capacity, ATP production, and proton leak. Once the OCR experiment was completed, 50 mL of RIPA lysis buffer (Thermo Fisher Scientific) supplemented with 1 mM PMSF (Sigma-Aldrich) was added to each well, and the 96-well plate was placed on ice for 30 min. Then, 20 mL of supernatant was used to quantify protein using a BCA assay. The protein levels obtained were used to normalize OCR data.
Extracellular lactate evaluation
Lactate was quantified through a Glycolysis Assay Kit (Dojindo, Kumamoto, Japan). Briefly, 100 μL of chondrocyte media was removed from 3D-cultured chondrocytes, diluted 1:10 in ddH20, and transferred into a 96-well plate. Culture media was then used to perform lactate quantification by following the manufacturer’s instructions. After 30 min of media incubation with the lactate working solution, the plate was read in a FLUOstar Omega microplate reader at 450 nm. The resulting absorbance was used to calculate the levels of lactate in MPS IVA chondrocytes normalized against lactate levels observed in WT chondrocytes.
Data analysis
Experimental data were analyzed in GraphPad Prism version 8.0.0 for Mac (GraphPad Software, GraphPad Software, Boston, MA, https://www.graphpad.com). The data is presented as mean ± standard error (SE). Data distribution was evaluated through the Shapiro–Wilk test while homoscedasticity was assessed via the Levene Test. A comparison between means was performed according to data distribution using either Student’s t-test, Mann–Whitney U test, ANOVA test, or Kruskal–Wallis’ test. Differences between groups were considered statistically significant when p < 0.05.
Data availability
Data is provided within the manuscript or supplementary information files.
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Acknowledgements
Access to the Seahorse technology was supported by the Institutional Development Award (IDeA) from the National Institute of Health’s National Institute of General Medical Sciences under grant number P20GM103446.
Funding
A.F.L. was partially supported by A Cure for Robert, Inc. This work was also supported by grants from the Austrian MPS society, A Cure for Robert, Inc., The Carol Ann Foundation, Angelo R. Cali & Mary V. Cali Family Foundation, Inc., The Vain and Harry Fish Foundation, Inc., The Bennett Foundation, Jacob Randall Foundation, and Nemours Funds. S.T. was supported by an Institutional Development Award from the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (NICHD) (1R01HD102545-01A1).
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A.F.L. conceptualized the study, designed the experimental approach, and collected and analyzed most in vitro data. S.S. assisted with the TEM assays. S.A.K. conducted GAGs processing and analysis. S.T. provided permanent supervision and feedback. A.F.L. wrote the manuscript draft. All authors contributed to the final editing of the manuscript and approved the final version.
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This study was approved by the Institutional Review Board #2107509.
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Leal, A.F., Saikia, S., Khan, S.A. et al. Three-dimensional human mucopolysaccharidosis IVA chondrocyte culture reveals significant impairments in the lysosomal-mitochondrial crosstalk. Sci Rep 15, 34140 (2025). https://doi.org/10.1038/s41598-025-04871-y
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DOI: https://doi.org/10.1038/s41598-025-04871-y