PPAR-gamma agonist pioglitazone recovers mitochondrial quality control in fibroblasts from PITRM1-deficient patients

Abstract

Introduction: Biallelic variants in PITRM1 are associated with a slowly progressive syndrome characterized by intellectual disability, spinocerebellar ataxia, cognitive decline and psychosis. The pitrilysin metallopeptidase 1 (PITRM1) is a mitochondrial matrix enzyme, which digests diverse oligopeptides, including the mitochondrial targeting sequences (MTS) that are cleaved from proteins imported across the inner mitochondrial membrane by the mitochondrial processing peptidase (MPP). Mitochondrial peptidases also play a role in the maturation of Frataxin, the protein affected in Friedreich's ataxia. Recent studies in yeast indicated that the mitochondrial matrix protease Ste23, which is a homologue of the human insulin-degrading enzyme (IDE), cooperates with Cym1 (homologue of PITRM1) to ensure the proper functioning of the preprotein processing machinery. In humans, IDE could be upregulated by Peroxisome Proliferator-Activated Receptor Gamma (PPARG) agonists. Methods: We investigated preprotein processing, mitochondrial membrane potential and MTS degradation in control and patients' fibroblasts, and we evaluated the pharmacological effect of the PPARG agonist Pioglitazone on mitochondrial proteostasis. Results: We discovered that PITRM1 dysfunction results in the accumulation of MTS, leading to the disruption and dissipation of the mitochondrial membrane potential. This triggers a feedback inhibition of MPP activity, consequently impairing the processing and maturation of Frataxin. Furthermore, we found that the pharmacological stimulation of PPARG by Pioglitazone upregulates IDE and also PITRM1 protein levels restoring the presequence processing machinery and improving Frataxin maturation and mitochondrial function. Discussion: Our findings provide mechanistic insights and suggest a potential pharmacological strategy for this rare neurodegenerative mitochondrial disease.

Keywords: cerebellar ataxia; mitochondrial disease; neurodegenaration; pioglitazone; proteostasis.

Proposed mechanism of action of pioglitazone on PITRM1^MUTcells. The beneficial effects of pioglitazone have been confirmed in the current study on fibroblasts from PITRM1 patients: pioglitazone enhances PPARG protein level, which in turn upregulates IDE and PITRM1 restoring mitochondrial health. Created with BioRender.com.

FIGURE 1

PITRM1 pathogenic variants c.548G>A, c.2239dupG and c.2795C>T impact protein stability. (A) In silico reconstruction of PITRM1 structure for wild-type (WT) and mutated PITRM1 amino acid sequences. Patients’ variants are relatively distant from the catalytic site (blue circle) docked with β-amyloid peptide (purple). (B) PITRM1 mRNA expression measured by qPCR did not show significant changes in PT1, PT2, and PT3 vs. CTRLs cells. (C) Western blot analysis of proteins separated by SDS–PAGE showed a marked reduction of PITRM1 amount in all fibroblasts suggesting protein instability. Quantifications of Western blot are shown in (D). All data are expressed as fold change compared to controls; statistical analyses were performed with 1-way Anova followed by Tukey multiple comparisons test (B), or with unpaired t-test (D); **p < 0.005.

FIGURE 2

Mitochondrial membrane potential is reduced in PITRM1-mutated fibroblasts. (A) Healthy control cells stained with JC1 presented mainly red fluorescent aggregates indicating the preservation of the mitochondrial membrane potential. Fibroblasts derived from PITRM1-mutated patients presented a diffuse green fluorescence confirming the presence of defective mitochondrial membrane potential. Magnification 20x. Quantification of red or green fluorescence is shown in (B). Statistical analysis was performed with 2-way Anova followed by Sidak’s multiple comparisons test; **p < 0.005.

FIGURE 3

Presequence processing is compromised in PITRM1-mutated fibroblasts. (A) Schematic representation of the TC-FlAsH-TFAM-Kate reporter vector. (B) Schematic drawing showing the experimental rationale: degradation of MTS should impede detection of the FlAsH signal, whereas its accumulation should result in enhanced FlAsH-TC fluorescence. (C) Representative images showing that fibroblasts of PITRM1-mutated patient P3 accumulate undegraded MTS (green fluorescence) compared to CTRL B; magnification 40×; quantification is shown in (D). (E) Representative Western blot analysis of TFAM-Kate processing; PITRM1-mutated fibroblasts show reduced levels of TFAM-Kate mature protein compared to CTRL B. GAPDH was used as a loading control. Quantification is shown in (F). Statistical analysis was performed with unpaired t-test; **p < 0.005; ***p < 0.001.

FIGURE 4

Frataxin processing is significantly reduced in PITRM1^MUT fibroblasts. (A) Respresentative Western blot analysis of Intermediated Frataxin (iFXN) and mature Frataxin (mFXN) in controls and PITRM1^MUTcell lines. Quantification of (B) total mFXN, (C) total iFXN and (D) (mFXN/iFNX) are shown. VDAC1 was used as loading controls. In (C) and (D) data are expressed as fold change compared to controls; statistical analyses were performed with unpaired t-test; *p < 0.05, **p < 0.005.

FIGURE 5

H₂O₂ and Insulin exacerbate defective mitochondrial proteostasis in PITRM1^MUT fibroblasts. (A) Representative western blot analysis of PITRM1, IDE, PMPCB, and Frataxin (FXN) in fibroblasts derived from a control (CTRL M) and patient 1 (PT1) exposed to H₂O₂ and Insulin for 48 h. Densitometric evaluations expressed as fold change compared to untreated CTRL M are reported in (B), GAPDH was used as loading control. (C) TMRM staining in CTRL M and PT1 exposed to H₂O₂ or Insulin; magnification 40×. (D) Quantitative analysis of mitochondrial morphology (elongated versus fragmented mitochondria) in each experimental setting shown in (C). Colour codes as in (B). Statistical analyses were performed, for each gene, with RM-1-way Anova followed by Fisher LSD test (C), or with 2-way Anova followed by Sidak’s multiple comparisons test (D); *p < 0.05, **p < 0.005.

FIGURE 6

Pioglitazone treatment restores preprotein processing and mitochondrial membrane potential in PITRM1^MUT fibroblasts. (A) Representative Western blot analysis of pioglitazone (PG)-treated compared to vehicle (VEH)-treated fibroblasts. (B) Quantifications on the immunoblot signals normalised on VDAC1 signal and expressed as fold change compared to untreated CTRLs. PG upregulates IDE and PITRM1 levels that increase MTS degradation, restoring preprotein processing and Frataxin maturation. (C) Evaluation of mitochondrial membrane potential (JC1 fluorescence) in CTRLs and PITRM1^MUT fibroblasts exposed to vehicle or Pioglitazone for 48 h. Healthy control cells presented mainly red fluorescent aggregates indicating the preservation of the mitochondrial membrane potential. PITRM1^MUT fibroblasts presented with a diffuse green fluorescence indicating the presence of a defective mitochondrial membrane that is restored after pioglitazone treatment. Magnification: 20×. Statistical analysis was performed for each gene with 1-way Anova followed by Tukey’s multiple comparisons test (B), or with 2-way Anova followed by Sidak’s multiple comparisons test (D); *p < 0.05, **p < 0.005.

FIGURE 7

Pioglitazone enhances PITRM1 levels via PPARG. (A) Representative Western blot analysis of PPARG levels in CTRL and PITRM1^MUT fibroblasts; quantification is shown in (B). (C) MtDNA quantification in control versus mutated cells w/ or w/o pharmacological treatment with Pioglitazone; colour codes as in (B). (D) Representative Western blot analysis of OXPHOS complex subunits in CTRLs and PITRM1^MUT fibroblasts, and quantification in (E): no major differences were observed after treatment suggesting that Pioglitazone does not stimulate mitochondrial biogenesis in fibroblasts. Colour codes as in (B). (F) Prediction of PPARG binding site with putative enhancer element close to PITRM1 TSS by EPD database (https://epd.epfl.ch/cgibin/get_doc?db=hgEpdNew&format=genome&entry=PITRM1_1). Statistical analysis was performed with 1-way Anova followed by Tukey’s multiple comparisons test; *p < 0.05.