Metabolic rewiring caused by mitochondrial dysfunction promotes mTORC1-dependent skeletal aging

Abstract

Decline of mitochondrial respiratory chain (mtRC) capacity is a hallmark of mitochondrial diseases. Patients with mtRC dysfunction often present reduced skeletal growth as a sign of premature cartilage degeneration and aging, but how metabolic adaptations contribute to this phenotype is poorly understood. Here we show that, in mice with impaired mtRC in cartilage, reductive/reverse TCA cycle segments are activated to produce metabolite-derived amino acids and stimulate biosynthesis processes by mechanistic target of rapamycin complex 1 (mTORC1) activation during a period of massive skeletal growth and biomass production. However, chronic hyperactivation of mTORC1 suppresses autophagy-mediated organelle recycling and disturbs extracellular matrix secretion to trigger chondrocytes death, which is ameliorated by targeting the reductive metabolism. These findings explain how a primarily beneficial metabolic adaptation response required to counterbalance the loss of mtRC function, eventually translates into profound cell death and cartilage tissue degeneration. The knowledge of these dysregulated key nutrient signaling pathways can be used to target skeletal aging in mitochondrial disease.

Fig. 1.. Skeletal aging processes are accelerated in mutant mice with a defective mtRC in chondrocytes.

(A) Breeding scheme: Generation of mice with a cartilage-specific expression of Twinkle^K320E mutant helicase (CreTW) (27). Consequences of Twinkle mutant expression for mitochondrial (mt) DNA and respiration complexes are illustrated. (B) Overview of the femur head morphology during aging in Cre and CreTW mice. The loss of transparency and the increase in red color shows age-related cartilage degeneration and secondary bone formation. (C) Histomorphological analysis of safranin O–stained femur head sections. Total femur head (orange) and growth plate cartilage (dark orange) are visualized. Arrows illustrate initiation sites of growth plate cartilage degeneration and its replacement with bony tissue (unstained). Scale bars, 500 μm (B) and 100 μm (C).

Fig. 2.. Glycolysis is activated, and TCA cycle metabolites accumulate in vivo in cartilage with a defective mtRC.

(A) Scheme of glycolysis and TCA cycle. (B) Glycolytic and (C) TCA cycle–derived metabolites in femur head tissue extracts of 1-month-old Cre and CreTW mice were quantified using LC-MS–based analysis. Raw data for peak values are provided in fig. S1. (D) Visualization of differentially abundant entities within the KEGG pathways “TCA cycle” and “OXPHOS” of CreTW mice using a proteome dataset generated from femur head tissue extracts of 1-month-old Cre and CreTW mice [PXD027109 and (8)]. The number of color-coded regulated entities within each pathway is shown, with the corresponding log₂ fold changes summarized in box plot to the right. Raw data for log₂ LFQ are provided in fig. S2. (E) NAD⁺/NADH ratio determined in femur head extracts from 1-month-old Cre and CreTW mice using colorimetric quantification assays (n = 4 animals per genotype). Data presented as means ± SEM. Unpaired two-tailed Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.. Engagement of the reverse TCA cycle segments in chondrocytes with impaired mtRC.

(A) Overview of the ¹³C₆ glucose and ¹³C₅ glutamine enrichment analysis in isolated ribcage chondrocytes. Circles represent the carbon atoms in metabolites. Blue color indicates the presence of ¹³C atom originating from¹³C₆ glucose; green color originates from¹³C₅ glutamine. (B to D) Incorporation of labeled metabolites derived from¹³C₆ glucose (B) and (C) and from ¹³C₅ glutamine (D) were determined by LC-MS–based analysis. Relative fold changes of the mass isotopomer distribution (MID) between genotypes are shown {n = 6 animals per genotype [¹³C₆ glucose, (B) and (C)], n = 7 animals per genotype [¹³C₅ glutamine, (D)]}. Raw data are provided in figs. S4 and S5. Quantitative data are means ± SEM. Unpaired two-tailed Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.. Rewiring of the TCA cycle can stimulate de novo amino acid biosynthesis in vivo in femur head cartilage with mtRC dysfunction.

(A) ¹³C₆ glucose- (glycine, alanine, and aspartate) and ¹³C₅ glutamine–dependent (proline) metabolite enrichment analysis into amino acid biosynthesis pathways using LC-MS–based analysis. Key enzymes are highlighted (scheme, left). Fold changes of the MID between genotypes is given (right, n = 6 animals per genotype for ¹³C₆ glucose tracing; n = 7 animals per genotype for ¹³C₅ glutamine tracing experiments). Raw data are provided in figs. S4 and S5. (B) KEGG pathway enrichment analysis of differentially abundant entities in femur head tissue extracts from 1-month-old Cre and CreTW mice as determined by mass spectrometry analysis; proteome dataset PXD027109 (8). Terms are ranked according to the mean log₂FC (FC, fold change) and −log₁₀ P value. The number of color-coded regulated entities within each pathway is shown, with the corresponding log₂ fold changes of the KEGG mmu01230 pathway Biosynthesis of amino acids summarized in box plot to the right. (C) Mean log₂ fold change (log₂FC, FDR 0.05) of the replicates and significance values are given for the entities of the KEGG category Biosynthesis of amino acids. The direction of fold changes is color-coded: Red indicates entities with increased abundance, while blue represents entities with decreased abundance [(PXD027109 and (8)]. (D) Characterization of the amino acid content in femur head tissue extracts from 1-month-old Cre and CreTW mice using LC-MS–based analysis. Fold changes between Cre and CreTW mice are shown (n = 4 animals per genotype). Raw data are provided in fig. S1. Quantitative data are means ± SEM. Unpaired two-tailed Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.. Metabolic perturbations are sensed by the mTORC1 signaling pathway in femur head cartilage of CreTW chondrocytes.

The phosphoproteome of isolated femur head cartilage from 1-month-old Cre and CreTW mice was compared using mass spectrometry. (A) The corresponding volcano plot is shown. Differentially phosphorylated entities (blue) and entities associated with the mTOR signaling pathway (red) are highlighted (four biological replicates per group, three animals per group). (B) A Venn diagram of enriched mTOR targets with individual phosphorylation sites is shown. (C) Immunoblot analysis of regulators and targets of mTORC1 in femur head cartilage extracts from 1- month-old Cre and CreTW mice. ACTA1 was used as a loading control. (D) Fold changes between Cre and CreTW mice are shown (n = 5 animals per genotype). (E) Isolated ribcage chondrocytes from 1-month-old Cre and CreTW mice were cultured in standard growth medium (DMEM) or in amino acid depletion (−aa) medium for 2 and 16 h. Phosphorylation of mTORC1 substrates (S6K1, (p)4E-BP1) was characterized by immunoblot analysis. Hypophosphorylated (α) and hyperphosphorylated forms (β- & -γ) of 4E-BP1 are indicated (15). (F) Fold changes in S6K1 phosphorylation between Cre and CreTW chondrocytes are shown (n = 5 biological samples per genotype). (G) Immunoblot analysis of mTORC1 pathway activation in chondrogenic cells cultured in standard growth medium (DMEM, DMEM/F12 10%,FCS 1% PS) or in −aa medium for 2 hours before 2 hours of stimulation with aspartate (D), phenylalanine (F), glycine (G), isoleucine (I), proline (P), valine (V), or the amino acid mixture (DFGIPV mix). Leucine (L) and standard growth medium (DMEM) were used as stimulation control. (H) Fold changes in S6K1 protein phosphorylation are shown (n = 3 biological samples per genotype). Quantitative data are means ± SEM, Unpaired two-tailed Student’s t test [(D) and (F)] or one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test (H), *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.. Biosynthesis processes are stimulated in cartilage with mtRC dysfunction.

(A) SUnSET assay to sense de novo translation rates in isolated ribcage chondrocytes of 1-month-old Cre and CreTW mice. Slot blot analysis of cells treated with puromycin (Puro). Cycloheximide (CHX) served as control to inhibit translational elongation. Puromycin-incorporation into peptides was determined by immunoblot (left). Fold changes between puromycin-treated Cre and CreTW chondrocytes are shown (graph, n = 7 animals per genotype). (B) Scheme of ¹³C₆ glucose carbon enrichment analysis from oxaloacetate into carbamoylaspartate and uridine nucleotide synthesis and ¹³C₅ glutamine carbon enrichment into triacylglycerides (TAG) of cultured Cre and CreTW chondrocytes (scheme). (C) Fold changes of the indicated isotopomers between genotypes are given (n = 6 animals per genotype). Raw data are provided in figs. S4 and S5. (D) Mass spectrometry analysis of fatty acyl chain species within the pool of esterified triacylglycerides (TAGs) in femur head cartilage extracts from 1-month-old Cre and CreTW mice. Fold changes are shown (n ≥ 3 animals per genotype). (E) Fold change enrichment of the palmitic (C16:0) and stearic acids (C18:0) between genotypes is given (n = 6 animals per genotype). (F) Immunoblot analysis of SREBP1 levels in cultured chondrocytes. Quantification of full-length (125 kDa) and cleaved (70 kDa) SREBP1 normalized to ACTA1. (G) qPCR analysis of lipogenic genes. Quantitative data are normalized to the mean of Acta1/Mapk7 and are displayed as log₂ fold change (n = 6 animals per genotype). (H) Holotomographic imaging of primary ribcage chondrocytes (top). Lipid droplets are marked with arrows. Scale bar, 10 μm. Oil Red O visualization of neutral lipids. Scale bars, 100 μm. Quantification shows the Oil Red O–positive area normalized to the total area (μm²), (n = 5 biological replicates). Quantitative data are means ± SEM. Unpaired two-tailed Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7.. Autophagy, organelle organization, and core matrisome composition are disturbed in chondrocytes with mtRC dysfunction.

(A) pS6 (Ser240/244) and THBS1 immunostaining on plane-matched paraffin sections of femur head cartilage from 1-month-old Cre and CreTW mice. Enlarged regions of interest (squares) are shown. The ratio of pS6⁺ cells within the THBS1⁺ area was determined (n ≥ 4 animals per genotype). (B) SQSTM1/p62 and THBS1 immunostaining on plane-matched paraffin sections of femur head cartilage from 1-month-old Cre and CreTW mice. Enlarged regions of interest (squares) are shown. The ratio of SQSTM1/p62⁺ cells within the THBS1⁺ area was determined (n = 4 animals per genotype). (C) Alcian blue/hematoxylin and eosin–stained sections of 1-month-old femur heads from Cre or CreTW mice. Electron microscopy analysis of chondrocytes in the THBS1⁺ region from femur head cartilage of 1-month-old Cre and CreTW mice. Mitochondria, ER, Golgi, and lipid organization is shown. (D) Venn diagram of differentially regulated matrisome-associated and core matrisome entities in the proteome (PXD027109) of femur head cartilage from 1-month-old Cre and CreTW mice. Quantitative data are means ± SEM. Unpaired two-tailed Student’s t test, **P < 0.01, ***P < 0.001. Scale bars, 100 μm (A) and (B).

Fig. 8.. NMN supplementation ameliorates cell death of chondrocytes with mtRC dysfunction.

Chondrocytes from 1-month-old Cre and CreTW mice were cultured for 3 days in −aa medium in the absence (−) or presence of rapamycin or increasing concentrations of NMN. (A) Cell morphology was studied by microscopy and (B) cell death by flow cytometry. (C) The proportion of AnxA5-Cy2⁺/SytoxBlue⁻ and AnxA5-Cy2⁺/SytoxBlue⁺ dying cells is given (n = 3 liters per group). Quantitative data are means ± SEM. One-way ANOVA with Bonferroni’s multiple comparisons test, **P < 0.01. Scale bar, 50 μm.

Fig. 9.. Molecular consequences of mitochondria dysfunction in cartilage for skeletal aging processes.

The reverse TCA cycle is activated to replenish carbon loss due to dysfunctional electron transport chain (ETC) in CreTW cartilage and increases amino acid levels to activate the mTORC1 signaling pathway. mTORC1 hyperactivation disturbs organelle and ECM organization associated with age-related chondrocyte death and premature cartilage degeneration.