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. 2020 Mar 18;12(6):5195-5208.
doi: 10.18632/aging.102941. Epub 2020 Mar 18.

Peroxisomal abnormalities and catalase deficiency in Hutchinson-Gilford Progeria Syndrome

Xiaojing Mao  1 , Pratima Bharti  1 , Abhirami Thaivalappil  1 , Kan Cao  1
Affiliations

Peroxisomal abnormalities and catalase deficiency in Hutchinson-Gilford Progeria Syndrome

Xiaojing Mao et al. Aging (Albany NY). .

Abstract

Peroxisomes are small, membrane-enclosed eukaryotic organelles that house various enzymes with metabolic functions. One important feature in both Hutchinson-Gilford Progeria Syndrome (HGPS) and normal aging is the elevated levels of Reactive Oxygen Species (ROS), which are generated from metabolic pathways with the capacity to cause oxidative damage to macromolecules within the cells. Although peroxisomal bioreactions can generate free radicals as their byproducts, many metabolic enzymes within the peroxisomes play critical roles as ROS scavengers, in particular, catalase. Here, we observed impaired peroxisomes-targeting protein trafficking, which suggested that the poorly assembled peroxisomes might cause high oxidative stress, contributing to the premature senescent phenotype in HGPS. We then investigated the ROS clearance efficiency by peroxisomal enzymes and found a significantly decreased expression of catalase in HGPS. Furthermore, we evaluated the effects of two promising HGPS-treatment drugs Methylene Blue and RAD001 (Everolimus, a rapamycin analog) on catalase in HGPS fibroblasts. We found that both drugs effectively reduced cellular ROS levels. MB, as a well-known antioxidant, did not affect catalase expression or activity. Interestingly, RAD001 treatment significantly upregulated catalase activity in HGPS cells. Our study presents the first characterization of peroxisomal function in HGPS and provides new insights into the cellular aspects of HGPS and the ongoing clinical trial.

Keywords: ROS; catalase; peroxisome; progeria; rapamycin.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1
Characterization of the peroxisomes in HGPS fibroblasts. (A) Peroxisomes localization indicated by PMP70 and catalase immunofluorescence staining in HGPS patient-derived dermal fibroblasts and its normal counterpart. Bar = 25μm. (B) The peroxisomes density per cell was indicated by the quantification of PMP70 puncta per square unit in normal and HGPS fibroblast cells. More than 100 cells from 3 independent experiments were analyzed and the data was represented in Tukey box plot. Boxes show the 25th, 50th, and 75th percentiles and the dots indicate the outliers. (C) Confocal fluorescence microscopy analysis of normal and HGPS fibroblasts transfected with GFP-SCP2, and probed with PMP70 antibody. Bar = 25μm. (D) Box plot of the portion of PMP70 puncta not colocalized with SCP2 (peroxisomal ghosts) in each cell. More than 100 cells from 3 independent experiments were analyzed. Boxes show the 25th, 50th, and 75th percentiles. Kolmogorov-Smirnov test was used to compare the distributions of these two samples. (E, F) Quantitative RT-PCR analysis of the relative expression of Pex5 and Pex7 in normal and HGPS fibroblasts. (G) Western blot analysis of Pex5 and Pex7 in normal and HGPS fibroblasts at passage 21. All experiments were performed using mid-passage cells between p15 to p25. All experiments were repeated at least three times and representative data were shown as indicated. *, p < 0.05, n.s., not significant.
Figure 2
Figure 2
Characterization of the ROS-scavenging enzymes in HGPS peroxisomes. (A) Quantitative RT-PCR analysis of the relative expression of catalase in normal and HGPS fibroblasts. ****, p < 0.0001. (B) Western blot analysis of Lamin A/C, progerin and catalase expression in normal and HGPS fibroblasts at passage 25. (C) Catalase activity assay measured by the decomposed H2O2 per mg total protein from normal and HGPS fibroblasts. *, p < 0.05. (D) Catalase activity normalized with the protein expression level in normal and HGPS fibroblasts. n.s., not significant. (E) Quantitative RT-PCR analysis of the relative expression of Glutathione peroxidase in normal and HGPS fibroblasts. n.s., not significant. (F) Glutathione peroxidase activity in normal and HGPS fibroblasts. All experiments were performed using mid-passage cells between p15 to p25. All experiments were repeated at least three times and representative data were shown as indicated.
Figure 3
Figure 3
Peroxisomal defects in normal fibroblasts expressing progerin. (A) Normal human dermal fibroblast cells were infected by DsRed-LA and DsRed-Pg lentiviruses. The exogenous Lamin A and progerin expression were detected by DsRed fluorescence in the nuclei. Peroxisomes localization was indicated by PMP70 and catalase immunofluorescence staining. Bar = 25μm. (B) Quantification of PMP70 puncta per square unit in fibroblasts overexpressing lamin A and progerin. More than 200 cells from 3 independent experiments were analyzed and the data was represented in Tukey box plot. Boxes show the 25th, 50th, and 75th percentiles and the dots indicate the outliers. (C) Relative fold change of ROS activity measured by DCFDA flow cytometry analysis in fibroblasts overexpressing lamin A and progerin. *, p < 0.05. (D) Quantitative RT-PCR analysis of the relative expression of catalase in fibroblasts overexpressing lamin A and progerin. ***, p < 0.001. (E) Normalized catalase activity in fibroblasts overexpressing lamin A and progerin. n.s., not significant. All experiments were performed using mid-passage cells between p15 to p25. All experiments were repeated at least three times and representative data were shown as indicated.
Figure 4
Figure 4
Catalase overexpression alleviates oxidative stress in normal and HGPS fibroblasts. (A, B) Quantitative RT-PCR analysis showed the relative expression of catalase in normal and HGPS fibroblasts infected by pBABE-CAT and control pBABE-puro retroviral vectors (Vehicle), respectively. **, p < 0.01. (C, D) Relative fold change of ROS activity measured by DCFDA flow cytometry analysis in normal and HGPS fibroblasts overexpressing catalase. *, p < 0.05, **, p < 0.01. (E) Western blot analysis showed catalase expression in normal and HGPS fibroblasts infected with pBABE-CAT and control vectors (cell passage number = 18). (F) Western blot analysis showed Nrf2 and p16 expression in normal and HGPS fibroblasts infected with pBABE-CAT and control vectors (cell passage number = 18). (G, H) Quantification of Nrf2 and p16 relative expression from Figure 4F. *, p < 0.05, **, p < 0.01, n.s., not significant. All experiments were performed using mid-passage cells between p15 to p25. All experiments were repeated at least three times and representative data were shown as indicated.
Figure 5
Figure 5
Methylene Blue (MB) and RAD001 reduce cellular ROS and RAD001 activated catalase activity in HGPS cells. Normal and HGPS fibroblasts were treated with 100nM Methylene Blue and RAD001 for 2 weeks. (A, B) The relative fold change of ROS activity was measured by DCFDA flow cytometry analysis. One-way ANOVA followed by Dunnett's multiple comparisons test was used to compare the effect of Methylene Blue and RAD001 treatment with the control group (Vehicle). *, p < 0.05, **, p < 0.01, ***, p < 0.001. (C) Relative mRNA expression of catalase was detected by quantitative RT-PCR analysis. Two-way ANOVA followed by Dunnett's multiple comparisons test was used to compare the mRNA expression of Methylene Blue and RAD001 treated cells with the control group within each block (normal and HGPS). n.s., not significant, **, p < 0.01. (D) Catalase expression level was detected by Western blot analysis (cell passage number = 19). (E) Normalized catalase activity. Two-way ANOVA followed by Dunnett's multiple comparisons test was used to compare the catalase activity of Methylene Blue and RAD001 treated cells with the control group within each block (normal and HGPS). n.s., not significant, **, p < 0.01. All experiments were repeated at least three times and representative data were shown as indicated.
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