Altered Mitochondrial Respiration and Other Features of Mitochondrial Function in Parkin-Mutant Fibroblasts from Parkinson's Disease Patients

Abstract

Mutations in the parkin gene are the most common cause of early-onset Parkinson's disease (PD). Parkin, an E3 ubiquitin ligase, is involved in respiratory chain function, mitophagy, and mitochondrial dynamics. Human cellular models with parkin null mutations are particularly valuable for investigating the mitochondrial functions of parkin. However, published results reporting on patient-derived parkin-mutant fibroblasts have been inconsistent. This study aimed to functionally compare parkin-mutant fibroblasts from PD patients with wild-type control fibroblasts using a variety of assays to gain a better understanding of the role of mitochondrial dysfunction in PD. To this end, dermal fibroblasts were obtained from three PD patients with homozygous whole exon deletions in parkin and three unaffected controls. Assays of mitochondrial respiration, mitochondrial network integrity, mitochondrial membrane potential, and cell growth were performed as informative markers of mitochondrial function. Surprisingly, it was found that mitochondrial respiratory rates were markedly higher in the parkin-mutant fibroblasts compared to control fibroblasts (p = 0.0093), while exhibiting more fragmented mitochondrial networks (p = 0.0304). Moreover, cell growth of the parkin-mutant fibroblasts was significantly higher than that of controls (p = 0.0001). These unanticipated findings are suggestive of a compensatory mechanism to preserve mitochondrial function and quality control in the absence of parkin in fibroblasts, which warrants further investigation.

Figure 1

Respiratory flux profiles of patient-derived and control fibroblasts, as determined by a Seahorse Extracellular Flux Analyzer with twelve consecutive measurements of oxygen consumption rate (OCR). Addition of ATP synthase inhibitor oligomycin, electron transport chain uncoupler FCCP and complex I and III inhibitors rotenone and antimycin A are indicated. (a) Respiratory flux profiles of patient-derived and control fibroblasts. Results are expressed as mean ± SEM. (b) Illutrative respiratory flux profile indicating various parameters of respiratory control. These include: OCR due to non-mitochondrial respiration (rotenone/antimycin A response); basal mitochondrial OCR (basal measurement minus rotenone/antimycin A response); ATP-linked OCR (basal measurement minus oligomycin response); OCR due to proton leak (oligomycin response minus rotenone/antimycin A response); ATP coupling efficiency (basal mitochondrial OCR divided by ATP-linked OCR); maximum OCR (FCCP response minus rotenone/antimycin A response) and spare respiratory capacity (maximum OCR divided by basal mitochondrial OCR). AU, arbitrary units; Ct, control; OCR, oxygen consumption rate; P, patient; SEM, standard error of the mean.

Figure 2

Parameters of respiratory control in patient-derived and control fibroblasts. Boxplots depict grouped patients (P) and control (Ct) values. (a) Basal mitochondrial OCR. (b) ATP-linked OCR. (c) OCR due to proton leak. (d) ATP coupling efficiency (percentage OCR due to ATP synthesis). (e) Maximum OCR. (f) Percentage spare respiratory capacity. ^∗ p < 0.05; ^∗∗ p < 0.01; °, outlier; AU, arbitrary units; OCR, oxygen consumption rate.

Figure 3

Mitochondrial network analysis of patient-derived and control fibroblasts. Mitotracker Red and live-cell microscopy were used to visualize the mitochondrial network. (a) Representative images of control fibroblasts (left) and patient fibroblasts (right). Scale bars = 20 μm. All images were assessed in regard to the degree of mitochondrial branching (form factor) and degree of mitochondrial elongation (aspect ratio). The distribution of these parameters in grouped patient-derived (P) and grouped control (Ct) fibroblasts are represented on logarithmic scale in boxplots, N = 40. (b) Comparison of form factor, which was significantly lower in patient cells than in control cells (p = 0.0304). (c) Comparison of aspect ratio, which was similar in patient and control cells (p = 0.1638). ^∗ p < 0.05; °, outlier; AU, arbitrary units; N; cells analyzed.

Figure 4

Relative Δψ _m of patient-derived and control fibroblasts. Relative Δψ _m was determined by JC-1 red : green fluorescent emission ratios for each fibroblast cell line in three experimental runs. Fibroblasts from P1 were not available; results pertain to a comparison of P2 and P3 parkin-mutant fibroblast with the three controls. Similar Δψ _m was seen for patient-derived (P) and control (Ct) fibroblasts (p = 0.3285). °, outlier; Δψ _m, mitochondrial membrane potential.

Figure 5

Cell growth in patient-derived and control fibroblasts under basal (untreated) and CCCP-stressed conditions, as assessed by a CyQUANT assay. Boxplots depict grouped patients (P) and control (Ct) measurements for three experimental runs. Patient cells demonstrated higher cell growth under basal conditions (p = 0.0001). A comparison of the magnitude of the effect of CCCP treatment within each fibroblast group (i.e., with and without cellular stress) demonstrated that the growth of patient-derived fibroblasts was significantly more suppressed by CCCP than the growth of control fibroblasts (p = 0.0013). Fibroblasts from P1 were not available; results pertain to a comparison of P2 and P3 parkin-mutant fibroblast with the three controls. ^∗∗ p < 0.01; ^∗∗∗ p < 0.001; °, outlier; AU, arbitrary units.