Mitochondrial dysfunction in mandibular hypoplasia, deafness and progeroid features with concomitant lipodystrophy (MDPL) patients

Abstract

Mandibular hypoplasia, Deafness and Progeroid features with concomitant Lipodystrophy is a rare, genetic, premature aging disease named MDPL Syndrome, due to almost always a de novo variant in POLD1 gene, encoding the DNA polymerase δ. In previous in vitro studies, we have already described several hallmarks of aging, including genetic damage, telomere shortening, cell senescence and proliferation defects. Since a clear connection has been reported between telomere shortening and mitochondria malfunction to initiate the aging process, we explored the role that mitochondrial metabolism and activity play in pathogenesis of MDPL Syndrome, an aspect that has not been addressed yet. We thus evaluated mtDNA copy number, assessing a significant decrease in mutated cells. The expression level of genes related to mitochondrial biogenesis and activity also revealed a significant reduction, highlighting a mitochondrial dysfunction in MDPL cells. Even the expression levels of mitochondrial marker SOD2, as assessed by immunofluorescence, were reduced. The decrease in this antioxidant enzyme correlated with increased production of mitochondrial ROS in MDPL cells, compared to WT. Consistent with these data, Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) analysis revealed in MDPL cells fewer mitochondria, which also displayed morphological abnormalities. Accordingly, we detected autophagic vacuoles containing partially digested mitochondria. Overall, our results demonstrate a dramatic impairment of mitochondrial biogenesis and activity in MDPL Syndrome. Administration of Metformin, though unable to restore mitochondrial impairment, proved efficient in rescuing nuclear abnormalities, suggesting its use to specifically ameliorate the premature aging phenotype.

Keywords: FIB/SEM; MDPL syndrome; ROS production; SOD2; autophagy; metformin; mitochondria; premature aging syndrome.

Figure 1

mtDNA copy number and quantification of mitochondrial markers in HDFs WT and MDPL. (A) Comparison of mtDNA copy number between WT and MDPL. mtDNA copy number are reported as mean ± standard deviation. ^*p < 0.05. (B) Quantification of mRNA levels of MFN1, MFN2, MFF, PARKIN, SIRT1, TFAM transcription factors, GPX1 in MDPL and control fibroblasts (WT). Data are from three independent experiments and represented as mean ± SD; (^*p < 0.05 ^**p < 0.01).

Figure 2

Ultrastructural analyses of mitochondria and their autophagic activity. (A) FIB/SEM analysis of MDPL-HDFs vs. WT fibroblasts. Healthy cells (on the left) show regular nucleus (N), several mitochondria (m) and abundant rough endoplasmic reticulum (RER). By contrast, MDPL-HDFs cells (on the right) display fewer RER cisternae, while smooth endoplasmic reticulum (SER) and Golgi apparatus are more prominent than in WT. Diseased cells also show several autophagosomes (black arrows), often containing partially digested mitochondria. Statistical analysis demonstrates significant decreased number of mitochondria, which are also significantly more damaged, than their normal counterpart (^*p < 0.05, for both parameters). (B) Representative image of immunofluorescence analysis of LC3 in WT and MDPL HDFs. (C) Western blot densitometric analysis of the LC3-II/I ratio. Data are presented as means ± SD. β-actin was used as control.

Figure 3

Functional mitochondrial evaluation of MDPL and WT HDFs. Flow cytometry quantification of (A) total reactive oxygen species using CM-H2DCFDA in HDFs WT and MDPL ones. (B) Flow cytometry quantification of mitochondrial superoxide using MitoSOX Red in HDFs WT and in MDPL ones. The error bar indicated in panels A and B is the average of two independent experiments. (C) Confocal analysis following SOD2 immunofluorescence and its quantification (^**p < 0.01). (D) JC1 staining for flow cytometry to evaluate changes in mitochondrial potential membrane and the quantification of red/green fluorescence intensity ratio.

Figure 4

Total and mitochondrial superoxide content after metformin treatment at different concentrations and timing. Flow cytometry quantification of total ROS (A) using CM-H2DCFDA in WT and in POLD1 human dermal fibroblasts after 48 and 72 hours of 40 uM, 100 uM, 500 uM, 1 and 5 mM of metformin treatment. (B) Flow cytometry quantification of mitochondrial superoxide using MitoSOX Red in WT and in POLD1 human dermal fibroblasts after 48 and 72 hours of 40 uM, 100 uM, 500 uM, 1 and 5 mM of metformin treatment.

Figure 5

Evaluation of nuclear shape organization after metformin treatment at different concentrations. (A) Representative image of nuclear shape organization observed in MDPL HDFs stained for lamin A/C (red), showing the presence of membrane invaginations (asterisks), and micronuclei (white arrows). (B) Representative image of lamin A/C immunostaining in MDPL HDFs following 48 h of treatment with 500 μM of metformin, in which a clear reduction in nuclear anomalies is evident. (C) Evaluation of aberrant nuclear alteration (% NA) in MDPL HDFs treated for 48 h with increasing doses of metformin. Each value represents the mean ± SD. of the analysis of 300 cells observed in three independent experiments (^*p < 0.05; ^**p < 0.01). Values are displayed as the average percentages of two different patients (D) Percentage of micronuclei (MN) encountered in MDPL-HDFs after 48 h of metformin treatment. The data have been obtained counting the micronuclei after Hoechst 33342 nuclear staining for fluorescence imaging. Each value represents the mean ± S.D. of the analysis of 300 cells for three independent experiments (^**p-value <0.01; ^***p-value <0.001). Values are displayed as the average percentages of two different patients. Hoechst 33342 nuclear staining (blue). Magnification 40× and 100×. Abbreviations: HDFs: human dermal fibroblasts; Met: metformin; MN: micronuclei; NA: aberrant nuclear alteration; Untreat: untreated cells.