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Oxidative stress and mitochondrial dysfunction in Kindler syndrome.

Zapatero-Solana E, García-Giménez JL, Guerrero-Aspizua S, García M, Toll A, Baselga E, Durán-Moreno M, Markovic J, García-Verdugo JM, Conti CJ, Has C, Larcher F, Pallardó FV, Del Rio M - Orphanet J Rare Dis (2014)

Bottom Line: Patient-derived keratinocytes showed altered levels of several oxidative stress biomarkers including MDA (malondialdehyde), GSSG/GSH ratio (oxidized and reduced glutathione) and GCL (gamma-glutamyl cysteine ligase) subunits.Consistently, confocal microscopy studies of mitochondrial fluorescent probes confirmed the mitochondrial derangement.This is the first study to describe mitochondrial dysfunction and oxidative stress involvement in KS.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Valencia, Spain. elisabet.zapatero@ciemat.es.

ABSTRACT

Background: Kindler Syndrome (KS) is an autosomal recessive skin disorder characterized by skin blistering, photosensitivity, premature aging, and propensity to skin cancer. In spite of the knowledge underlying cause of this disease involving mutations of FERMT1 (fermitin family member 1), and efforts to characterize genotype-phenotype correlations, the clinical variability of this genodermatosis is still poorly understood. In addition, several pathognomonic features of KS, not related to skin fragility such as aging, inflammation and cancer predisposition have been strongly associated with oxidative stress. Alterations of the cellular redox status have not been previously studied in KS. Here we explored the role of oxidative stress in the pathogenesis of this rare cutaneous disease.

Methods: Patient-derived keratinocytes and their respective controls were cultured and classified according to their different mutations by PCR and western blot, the oxidative stress biomarkers were analyzed by spectrophotometry and qPCR and additionally redox biosensors experiments were also performed. The mitochondrial structure and functionality were analyzed by confocal microscopy and electron microscopy.

Results: Patient-derived keratinocytes showed altered levels of several oxidative stress biomarkers including MDA (malondialdehyde), GSSG/GSH ratio (oxidized and reduced glutathione) and GCL (gamma-glutamyl cysteine ligase) subunits. Electron microscopy analysis of both, KS skin biopsies and keratinocytes showed marked morphological mitochondrial abnormalities. Consistently, confocal microscopy studies of mitochondrial fluorescent probes confirmed the mitochondrial derangement. Imbalance of oxidative stress biomarkers together with abnormalities in the mitochondrial network and function are consistent with a pro-oxidant state.

Conclusions: This is the first study to describe mitochondrial dysfunction and oxidative stress involvement in KS.

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Related in: MedlinePlus

Mitochondria distribution and function in KS keratinocytes. (a and b) Mito Tracker Red staining. Note the smeared mitochondria staining in KS keratinocytes (b) as compared to the control cells (a). (c and d) JC-1 staining. Note mitochondrial depolarization in KS keratinocytes as indicated by the decrease in the red/green fluorescence intensity ratio. (e) Quantification of JC-1 staining. Membrane potential reduction was statistically significant (*p < 0.05) after t-student test. Scale bars = 10 μm.
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Fig4: Mitochondria distribution and function in KS keratinocytes. (a and b) Mito Tracker Red staining. Note the smeared mitochondria staining in KS keratinocytes (b) as compared to the control cells (a). (c and d) JC-1 staining. Note mitochondrial depolarization in KS keratinocytes as indicated by the decrease in the red/green fluorescence intensity ratio. (e) Quantification of JC-1 staining. Membrane potential reduction was statistically significant (*p < 0.05) after t-student test. Scale bars = 10 μm.

Mentions: We sought to determine whether deranged redox status could translate into ultrastructural changes in target organelles. For this purpose, the morphology of mitochondria in patient skin biopsies and cultured keratinocytes was analyzed by electron microscopy. The Figure 3a shows a panoramic view of the KS epidermis, showing a zoom in a basal keratinocyte (Figure 3b). The ultrastructural analysis of KS skin biopsies revealed striking abnormalities in mitochondria. In fact, mitochondrial crests were irregular, dilated and did not present parallel distribution (Figure 3c), which contrasted with the well-organized mitochondrial network in normal skin biopsies (Figure 3d). Furthermore, both internal and external mitochondrial membranes had a wavy morphology and irregular thickness (Figure 3c). Similarly, the ultrastructural analysis of the cultured keratinocytes from KS patients showed mitochondrial alterations with a tendency toward the fusion of mitochondrial crests that may affect the inter-membrane space and functionality (Figure 3f) compared with control keratinocytes (Figure 3e). To analyze mitochondrial distribution, keratinocytes from healthy subjects and KS patients were incubated with the mitochondria-specific dye Mito Tracker Red and subsequently analyzed by confocal microscopy. Mitochondria in control keratinocytes formed a well-established network. In contrast, keratinocytes from KS patients showed both, a reduced and diffuse Mito Tracker Red staining consistent with a disorganized mitochondrial network (Figure 4a and b). In order to assess mitochondrial function, the membrane potential was studied in control and KS keratinocytes using the JC-1 probe. The analysis showed a significant reduction of the membrane potential in KS cells compared to controls as determined by the red-to-green JC-1 dye shift (Figure 4c-e and Additional file 2: Figure S3). Overall, our data indicate that mitochondria in KS keratinocytes are not only altered in structure, but also in their distribution and functionality.Figure 3


Oxidative stress and mitochondrial dysfunction in Kindler syndrome.

Zapatero-Solana E, García-Giménez JL, Guerrero-Aspizua S, García M, Toll A, Baselga E, Durán-Moreno M, Markovic J, García-Verdugo JM, Conti CJ, Has C, Larcher F, Pallardó FV, Del Rio M - Orphanet J Rare Dis (2014)

Mitochondria distribution and function in KS keratinocytes. (a and b) Mito Tracker Red staining. Note the smeared mitochondria staining in KS keratinocytes (b) as compared to the control cells (a). (c and d) JC-1 staining. Note mitochondrial depolarization in KS keratinocytes as indicated by the decrease in the red/green fluorescence intensity ratio. (e) Quantification of JC-1 staining. Membrane potential reduction was statistically significant (*p < 0.05) after t-student test. Scale bars = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4302591&req=5

Fig4: Mitochondria distribution and function in KS keratinocytes. (a and b) Mito Tracker Red staining. Note the smeared mitochondria staining in KS keratinocytes (b) as compared to the control cells (a). (c and d) JC-1 staining. Note mitochondrial depolarization in KS keratinocytes as indicated by the decrease in the red/green fluorescence intensity ratio. (e) Quantification of JC-1 staining. Membrane potential reduction was statistically significant (*p < 0.05) after t-student test. Scale bars = 10 μm.
Mentions: We sought to determine whether deranged redox status could translate into ultrastructural changes in target organelles. For this purpose, the morphology of mitochondria in patient skin biopsies and cultured keratinocytes was analyzed by electron microscopy. The Figure 3a shows a panoramic view of the KS epidermis, showing a zoom in a basal keratinocyte (Figure 3b). The ultrastructural analysis of KS skin biopsies revealed striking abnormalities in mitochondria. In fact, mitochondrial crests were irregular, dilated and did not present parallel distribution (Figure 3c), which contrasted with the well-organized mitochondrial network in normal skin biopsies (Figure 3d). Furthermore, both internal and external mitochondrial membranes had a wavy morphology and irregular thickness (Figure 3c). Similarly, the ultrastructural analysis of the cultured keratinocytes from KS patients showed mitochondrial alterations with a tendency toward the fusion of mitochondrial crests that may affect the inter-membrane space and functionality (Figure 3f) compared with control keratinocytes (Figure 3e). To analyze mitochondrial distribution, keratinocytes from healthy subjects and KS patients were incubated with the mitochondria-specific dye Mito Tracker Red and subsequently analyzed by confocal microscopy. Mitochondria in control keratinocytes formed a well-established network. In contrast, keratinocytes from KS patients showed both, a reduced and diffuse Mito Tracker Red staining consistent with a disorganized mitochondrial network (Figure 4a and b). In order to assess mitochondrial function, the membrane potential was studied in control and KS keratinocytes using the JC-1 probe. The analysis showed a significant reduction of the membrane potential in KS cells compared to controls as determined by the red-to-green JC-1 dye shift (Figure 4c-e and Additional file 2: Figure S3). Overall, our data indicate that mitochondria in KS keratinocytes are not only altered in structure, but also in their distribution and functionality.Figure 3

Bottom Line: Patient-derived keratinocytes showed altered levels of several oxidative stress biomarkers including MDA (malondialdehyde), GSSG/GSH ratio (oxidized and reduced glutathione) and GCL (gamma-glutamyl cysteine ligase) subunits.Consistently, confocal microscopy studies of mitochondrial fluorescent probes confirmed the mitochondrial derangement.This is the first study to describe mitochondrial dysfunction and oxidative stress involvement in KS.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Valencia, Spain. elisabet.zapatero@ciemat.es.

ABSTRACT

Background: Kindler Syndrome (KS) is an autosomal recessive skin disorder characterized by skin blistering, photosensitivity, premature aging, and propensity to skin cancer. In spite of the knowledge underlying cause of this disease involving mutations of FERMT1 (fermitin family member 1), and efforts to characterize genotype-phenotype correlations, the clinical variability of this genodermatosis is still poorly understood. In addition, several pathognomonic features of KS, not related to skin fragility such as aging, inflammation and cancer predisposition have been strongly associated with oxidative stress. Alterations of the cellular redox status have not been previously studied in KS. Here we explored the role of oxidative stress in the pathogenesis of this rare cutaneous disease.

Methods: Patient-derived keratinocytes and their respective controls were cultured and classified according to their different mutations by PCR and western blot, the oxidative stress biomarkers were analyzed by spectrophotometry and qPCR and additionally redox biosensors experiments were also performed. The mitochondrial structure and functionality were analyzed by confocal microscopy and electron microscopy.

Results: Patient-derived keratinocytes showed altered levels of several oxidative stress biomarkers including MDA (malondialdehyde), GSSG/GSH ratio (oxidized and reduced glutathione) and GCL (gamma-glutamyl cysteine ligase) subunits. Electron microscopy analysis of both, KS skin biopsies and keratinocytes showed marked morphological mitochondrial abnormalities. Consistently, confocal microscopy studies of mitochondrial fluorescent probes confirmed the mitochondrial derangement. Imbalance of oxidative stress biomarkers together with abnormalities in the mitochondrial network and function are consistent with a pro-oxidant state.

Conclusions: This is the first study to describe mitochondrial dysfunction and oxidative stress involvement in KS.

Show MeSH
Related in: MedlinePlus