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Mitochondrial Bioenergetics Is Altered in Fibroblasts From Patients With Sporadic Alzheimer's Disease

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Mitochondrial Bioenergetics Is Altered in Fibroblasts From Patients With Sporadic Alzheimer's Disease

María J Pérez et al. Front Neurosci.

Abstract

The identification of an early biomarker to diagnose Alzheimer's disease (AD) remains a challenge. Neuropathological studies in animal and AD patients have shown that mitochondrial dysfunction is a hallmark of the development of the disease. Current studies suggest the use of peripheral tissues, like skin fibroblasts as a possibility to detect the early pathological alterations present in the AD brain. In this context, we studied mitochondrial function properties (bioenergetics and morphology) in cultured fibroblasts obtained from AD, aged-match and young healthy patients. We observed that AD fibroblasts presented a significant reduction in mitochondrial length with important changes in the expression of proteins that control mitochondrial fusion. Moreover, AD fibroblasts showed a distinct alteration in proteolytic processing of OPA1, a master regulator of mitochondrial fusion, compared to control fibroblasts. Complementary to these changes AD fibroblasts showed a dysfunctional mitochondrial bioenergetics profile that differentiates these cells from aged-matched and young patient fibroblasts. Our findings suggest that the human skin fibroblasts obtained from AD patients could replicate mitochondrial impairment observed in the AD brain. These promising observations suggest that the analysis of mitochondrial bioenergetics could represent a promising strategy to develop new diagnostic methods in peripheral tissues of AD patients.

Keywords: Alzheimer's disease; OPA1; biomarker; fibroblasts; mitochondria.

Figures

Figure 1
Figure 1
AD cultured fibroblasts present a decrease in mitochondrial length. (A) Cells were loaded with MitoGreen to determinate mitochondrial morphology. Bar = 10 μm. (B) Quantification of the mitochondrial length average obtained from microscopy analysis (see Methods). Data are mean ± SE, n = 3 (technical replicates for each subject). *p < 0.001 indicate differences between groups calculated by Student t-test. (C) Distribution of mitochondrial length in cultured fibroblasts from AD and control patients. Mitochondrial lengths were grouped in short, medium and long size. Data are mean ± SE, n = 3 (technical replicates for each subject). *p < 0.005, **p < 0.003 indicates differences between groups calculated by Student t-test between short, medium and long mitochondria from AD and control patients.
Figure 2
Figure 2
Defects in mitochondrial fusion regulation in fibroblasts obtained from AD patients. (A) The levels of mitochondrial fission protein DRP1, in AD and control patients were determined by Western blot (see Methods). (B) Quantitative analysis of DRP1 relative expression. Data are mean ± SE. (C) Levels of mitochondrial fusion protein MFN1 are indicating the protein splicing variants (arrows). (D) Quantification of the MFN1 splicing products. *p < 0.05 indicates differences between groups calculated by Student t-test, data are mean ± SE. (E) Levels of mitochondrial fusion protein OPA1 showing isoforms and proteolytic products of OPA1 (arrows). (F,G) Quantification of OPA1 content and isoforms pattern. *p < 0.05, **p < 0.01 indicate differences between groups calculated by Student t-test, data are mean ± SE.
Figure 3
Figure 3
Fibroblasts of AD patients showed an increase in reactive oxygen species (ROS). (A) Representative fluorescent images of TMRM show mitochondrial membrane potential levels in fibroblasts of control and AD patients. Bar = 10 μm. (B) The graph represents a quantitate data of mitochondrial potential levels as arbitrary units. Data are mean ± SE, n = 3 (technical replicates for each subject). (C) Representative fluorescent images of DCF intensity. Bar = 10 μm. (D) Graph shows quantitative data of ROS levels in each patient's cell type. Data are mean ± SE, n = 3 (technical replicates for each subject). *p < 0.05 indicated differences between groups calculated by Student t-test. (E) The graph shows total ATP levels (pmol) normalized by μg of protein extracted from control and AD fibroblasts. Data are mean ± SE, n = 3. *p < 0.05 indicate differences between groups calculated by Student t-test.
Figure 4
Figure 4
Thapsigargin induces cytosolic calcium handling defects in AD fibroblasts. (A) Representative images of Fluo3 intensity showing relative basal cytosolic calcium levels in fibroblasts of control and AD patients. Bar = 10 μm. (B) Quantification of relative cytosolic calcium levels as arbitrary units. Data are mean ± SE, n = 3 (technical replicates for each subject). (C) Representative trends of cytosolic calcium levels over 25 min in AD and control fibroblasts. After 5 min the cells were treated with 10μM of thapsigargin (arrow). Data are mean ± SE, n = 3 (technical replicates for each subject). (D) Relative cytosolic calcium levels 1 min after treatment with thapsigargin (peak of calcium increase). Data are mean ± SE. *p < 0.05 indicated differences between groups calculated by Student t-test.
Figure 5
Figure 5
Calcium overload induces mitochondrial impairment in AD fibroblasts. (A) Determination of mitochondrial length changes after 1 min treatment with thapsigargin. Data are mean ± SE, n = 3 (technical replicates for each subject). (B) Mitochondrial membrane potential levels determinate with TMRM of each patient after 10 μM thapsigargin treatment. Data are mean ± SE, n = 3 (technical replicates for each subject). *p < 0.05 indicate differences before and after thapsigargin treatment calculated by Student t-test. (C) Fibroblasts were treated with 10 μM FCCP for 30 min before the experiment was performed. Quantification of the intensity of Fluo3 showing cytosolic calcium levels over 25 min. After 5 min of basal fluorescence detection, the cells were treated with 10μM of thapsigargin (arrow). Data are mean ± SE, n = 3 (technical replicates for each subject). (D) Quantification of cytosolic calcium levels after 1 min treatment with thapsigargin. Data are mean ± SE. *p < 0.05 indicated differences with or without FCCP, calculated by Student t-test.
Figure 6
Figure 6
Comparison of mitochondrial bioenergetics profile in young, healthy-aged and AD patient fibroblasts. (A) The graph represents the mitochondrial membrane potential levels as arbitrary units. Data are mean ± SE, n = 3 (technical replicates for each subject). (B) Quantification of total ROS levels in each patient as arbitrary units. Data are mean ± SE, n = 3 (technical replicates for each subject). *, ** p < 0.05 indicated differences with between groups calculated by one-way ANOVA test. (C) Cytosolic calcium levels after 1 min treatment with thapsigargin. Data are mean ± SE. *p < 0.05 indicated differences between groups calculated by one-way ANOVA test. (D) Total ATP levels (pmol) normalized by μg of protein. Data are mean ± SE, n = 3 (technical replicates for each subject). (E) Changes in the protein expression levels of DRP1, MFN1, and OPA1 were assessed by Western Blot in cell extracts from young, aged-match healthy and AD fibroblasts. A representative image of actin western blot is show as internal control (see additional data in Figure S1). (F) Quantitative analysis for DRP1 expression levels. Data are mean ± SE. (G) Quantitative data indicates splicing products for MFN1 expression. *p < 0.05 indicates differences between groups calculated by one-way ANOVA test, data are mean ± SE. (H) Quantification shows total OPA1 expression. *p < 0.05 indicate differences calculated by one-way ANOVA test, data are mean ± SE. (I) The mitochondrial length was visualized using a MitoGreen. Bar = 10 μm. (J) Measurement of mitochondrial length (average) in all cell groups. Data are mean ± standard error (SE), n = 3 (technical replicates for each subject). *p < 0.05 indicate differences between groups calculated by one-way ANOVA test.

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