Mitochondrial and autophagic alterations in skin fibroblasts from Parkinson disease patients with Parkin mutations

Results

Mitochondrial respiration

In order to assess the bioenergetic status of fibroblasts, we first performed high-resolution mitochondrial respiration analyses. The overall respiratory control ratios shown in Figure 1 B-F, are obtained from the respiration parameters illustrated in the mitochondrial respiratory flux profile (Figure 1A). In glucose, no significant differences in the respiratory control ratios were found between PRKN-PD and control fibroblasts. Even so, trends to increased basal, ATP-linked and maximal (uncoupled) respirations and a concomitant downward trend in the spare respiratory capacity were observed in PRKN-PD fibroblasts compared to controls. In galactose, PRKN-PD fibroblasts exhibited a significant decrease in basal/maximal respiratory ratio compared to the control fibroblasts. Although the rest of parameters were preserved, a downward trend in the basal respiration and ATP-linked oxygen consumption was shown. Upon changing from glycolytic to mitochondrial-challenging conditions, control but not PRKN-PD fibroblasts, significantly increased ATP-linked oxygen consumption and the basal/maximal respiratory ratio. In addition, both groups significantly decreased the spare respiratory capacity upon changing cells from glucose to galactose medium (Figure 1 A-E).

Figure 1. Mitochondrial respiratory control ratios in control and PRKN-PD fibroblasts. Illustrative mitochondrial respiration flux profile indicating respiratory control parameters (image obtained from Agilent Seahorse XF) (A), basal respiration (B), ATP-linked respiration (C), maximal respiration (D), spare respiratory capacity (E) and basal/maximal respiratory ratio (F). In glucose, no significant differences were found between PRKN-PD and control fibroblasts in the respiratory control ratios although trends to increased basal and maximal as well as ATP-linked respirations and decreased spare respiratory capacity were observed. In galactose, PRKN-PD fibroblasts exhibited a significant decrease in basal/maximal respiratory ratio compared to the control fibroblasts as well as a downward trend in the basal respiration and ATP-linked respiration. Controls but not PRKN-PD significantly increased oxygen consumption linked to ATP production and the basal/maximal respiratory ratio in galactose compared to glucose. Both, control and PRKN-PD fibroblasts significantly decreased the spare respiratory capacity upon medium change. Each cell line was seeded in triplicate per condition (n=3 for GLC and n=3 for GAL). The results were expressed as means and standard error of the mean (SEM). *= p<0.05. CTL= Control fibroblasts. GAL= 10 mM galactose medium. GLC= 25 mM glucose medium. NS= not significant. OCR= Oxygen consumption rate. PRKN-PD= Parkin-associated PD fibroblasts. Respiratory control ratios were normalized by total protein content and by citrate synthase activity as a marker of mitochondrial content.

To understand the role of mitochondrial complex I (CI) in the overall oxygen consumption, which is reported to be affected in PD, we measured oxygen consumption through the specific oxidation of CI-substrates, pyruvate and malate (PMox). Although we did not obtain statistically significant differences, PRKN-PD showed a strong non-significant downward trend in CI-stimulated oxygen consumption compared to control fibroblasts in glucose. Exposure to galactose trended to reduce CI-stimulated oxygen consumption in control, but not in PRKN-PD fibroblasts, when compared to glucose (Figure 2).

Figure 2. Complex I-stimulated oxygen consumption through pyruvate and malate oxidation measurementin control and PRKN-PD fibroblasts. No statistically significant differences were obtained between groups. In glucose, downward trends in CI-stimulated oxygen was shown in PRKN-PD compared to control fibroblasts. Exposure to galactose trended to reduce CI-stimulated oxygen consumption in control fibroblasts when compared to glucose, but not in PRKN-PD cells. The results are expressed as means and standard error of the mean (SEM). CTL= Control fibroblasts. GAL= 10 mM galactose medium. GLC= 25 mM glucose medium. NS= not significant. PRKN-PD= Parkin-associated PD fibroblasts. Oxygen consumption values were normalized by citrate synthase activity as a marker of mitochondrial content.

Mitochondrial respiratory chain enzymatic activities

We next wanted to determine if the differences observed in mitochondrial respiration analysis between PRKN-PD and control fibroblasts were reflected in the enzymatic activities of the Mitochondrial respiratory chain (MRC) complexes. Although no significant differences were obtained between groups, the same pattern exhibited in CI-stimulated oxygen consumption was reflected in CI enzymatic activity, where PRKN-PD showed a strong downward trend compared to control fibroblasts in glucose. Exposure to galactose significantly reduced CI-enzymatic activity of controls, but not PRKN-PD fibroblasts, when compared to glucose, in accordance to CI-stimulated oxygen consumption results. In addition, exposure to galactose significantly decreased CIV enzymatic activity in PRKN-PD fibroblasts when compared to glucose (Figure 3 A-C).

Figure 3. Mitochondrial respiratory chain (MRC) enzymatic activities in control and PRKN-PD fibroblasts. Enzymatic activities of the complexes I (A), II+III (B) and CIV (C) of the MRC. No significant differences were obtained between groups. Exposure to galactose significantly reduced CI-enzymatic activity of control fibroblasts and significantly decreased CIV enzymatic activity of PRKN-PD fibroblasts when compared to glucose. The results were expressed as means and standard error of the mean (SEM). *= p<0.05. CTL= Control fibroblasts. GAL= 10 mM galactose medium. GLC= 25 mM glucose medium. NS= not significant. PRKN-PD= Parkin-associated PD fibroblasts. Enzymatic activity values were normalized by citrate synthase activity as a marker of mitochondrial content.

Oxidative stress

Lipid peroxidation was measured as an indicator of ROS-derived oxidative damage. As illustrated in Figure 4, PRKN-PD exhibited an upward trend in lipid peroxidation compared to control fibroblasts in glucose, while exposure to galactose significantly reduced oxidative stress levels in PRKN-PD compared to glucose.

Figure 4. Oxidative stress measured through lipid peroxidation in control and PRKN-PD fibroblasts. In glucose, PRKN-PD exhibited an upward trend in lipid peroxidation compared to control fibroblasts while exposure to galactose significantly reduced oxidative stress levels in PRKN-PD compared to glucose. The results are expressed as means and standard error of the mean (SEM). *= p<0.05. CTL= Control fibroblasts. GAL= 10 mM galactose medium. GLC= 25 mM glucose medium. PRKN-PD= Parkin-associated PD fibroblasts.

Mitochondrial membrane potential

Given that a correct mitochondrial polarization is crucial for mitochondrial integrity, we measured mitochondrial membrane potential in control and PRKN-PD fibroblasts. No significant differences in mitochondrial membrane potential were obtained between groups. However, upon galactose exposure, control fibroblasts trended to enhance mitochondrial membrane potential as compared to glucose, while PRKN-PD fibroblasts remained preserved (Figure 5).

Figure 5. Mitochondrial membrane potential in control and PRKN-PD fibroblasts. Mitochondrial membrane potential is represented as the ratio of red vs. green fluorescence signals of JC-1 representing the cells with correctly polarized vs the cells with depolarized mitochondria. No significant differences in mitochondrial membrane potential were obtained between groups. Upon galactose exposure, control fibroblasts trended to enhance mitochondrial membrane potential as compared to glucose, while PRKN-PD fibroblasts remained unchanged. The results are expressed as means and standard error of the mean (SEM). AU= Arbitrary units. CTL= Control fibroblasts. GAL= 10 mM galactose medium. GLC= 25 mM glucose medium. JC-1: 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazol-carbocyanine iodide. NS= not significant. PRKN-PD= Parkin-associated PD fibroblasts.

Mitochondrial network complexity

Mitochondrial function is a process intimately associated with changes in mitochondrial dynamics [37]. For this reason, we assessed mitochondrial length Aspect ratio (AR) and branching Form factor (FF) in control and PRKN-PD fibroblasts and found it was comparable between groups in both media (Supplementary Figure 1A and B).

Mitochondrial content

We next evaluated mitochondrial content through the assessment of Mitochondrial DNA (mtDNA) copy number, Citrate synthase (CS) activity and mitochondrial network and found that all parameters were preserved between groups in both media (Figure 6 A-C).

Figure 6. Mitochondrial content in control and PRKN-PD fibroblasts. Mitochondrial content was measured through mtDNA copy number (A), citrate synthase enzymatic activity (B) and mitochondrial network (C). Comparable mitochondrial content was observed between groups in both media. The results are expressed as means and standard error of the mean (SEM). CTL= Control fibroblasts. GAL= 10 mM galactose medium. GLC= 25 mM glucose medium. mtDNA= mitochondrial DNA. PRKN-PD= Parkin-associated PD fibroblasts.

Autophagy

To ascertain the potential impact of PRKN mutations on autophagy we quantified p62 and LC3BII protein levels. PRKN-PD fibroblasts presented significant lower basal levels of p62 and LC3BII in both media compared to controls as shown in Figure 7A and B (0h).

Figure 7. Autophagic flux incontrol and PRKN-PD fibroblasts. p62 (A) and LC3BII (B) protein levels at basal (0h) and under bafilomycin A1 treatment (4 or 8h) in glucose and galactose media. Basal levels p62 and LC3BII were significantly decreased in PRKN-PD compared to control fibroblasts in both media. PRKN-PD fibroblasts presented significantly lower p62 and LC3BII levels after 4 and 8 hours of treatment compared to controls in glucose and the same tendency was obtained in galactose. Exposure to galactose significantly decreased basal levels of both molecules compared to glucose in controls, but not in PRKN-PD fibroblasts. Controls, but not PRKN-PD, also showed significantly reduced p62 in front of conserved LC3BII protein levels upon treatment. Asterisks above the bars indicate statistically significant differences between protein levels at basal (0h) and after of bafilomycin A1 treatment (4 or 8h) within a group. The results are expressed as means and standard error of the mean (SEM). Asterisk brackets indicate statistically significant differences between CTL and PRKN-PD fibroblasts. Bold asterisk brackets indicate statistically significant differences between media. GAL= 10 mM galactose medium. GLC= 25 mM glucose medium. PRKN-PD= Parkin-associated PD fibroblasts.

The processes of autophagosome synthesis and degradation are spatially and temporally separated. In order to determine whether the decrease in basal p62 and LC3BII protein levels was due to a reduction in autophagosome synthesis or to a later stage in the pathway such as autophagosome degradation, we treated cells with bafilomycin A1. Bafilomycin A1 clamps fusion of autophagosomes with lysosomes and thus blocks autophagosome degradation, allowing measurement of autophagosome synthesis, whereas both synthesis and degradation occur in the untreated samples. The degradation rate can be deduced by subtracting LC3BII levels in the untreated samples from those treated with bafilomycin A1 [38].

In both groups, p62 and LC3BII levels were significantly increased at 4 and 8 hours under bafilomycin A1 treatment compared to basal state, indicating some extent of autophagic flux in both media. However, in glucose, p62 and LC3BII levels at 4 and 8 hours of treatment were significantly lower in PRKN-PD compared to control fibroblasts and the same trends were observed in galactose (Figure 7 A and B; 4h and 8h). Upon exposure to galactose, controls but not PRKN-PD fibroblasts, showed significantly decreased basal levels of both molecules compared to glucose with concurrent significantly decreased p62 and conserved LC3BII protein levels upon bafilomycin A1 treatment, suggesting an enhancement of autophagic flux in these cells.

Cell growth

We finally investigated cell growth rates in control and PRKN-PD fibroblasts in glucose and galactose media to assess how PRKN mutations impact on the overall cellular health. Although not significantly, PRKN-PD fibroblasts showed downward trend in cell growth rate in both media as shown in Figure 8.

Figure 8. Cell growth rate in control and PRKN-PD fibroblasts. Although not significant, PRKN-PD fibroblasts showed downward trend in cell growth rate in both media. The results are expressed as means and standard error of the mean (SEM). AU= Arbitrary units. CTL= Control fibroblasts. GAL= 10 mM galactose medium. GLC= 25 mM glucose medium. PRKN-PD= Parkin-associated PD fibroblasts.

Materials and Methods

Subjects and PRKN mutational screening

We recruited seven PD patients carrying mutations in the PRKN gene (PRKN-PD, n=7), and thirteen unrelated healthy subjects (CTL, n=13) among patients and relatives visiting the Movement Disorders outpatient clinic from the Hospital Clínic of Barcelona (Barcelona, Spain). PRKN-PD patients were diagnosed according to the UK Brain Bank criteria [50] and the mutational screening of the PRKN gene was performed as previously described [51]. Clinical and epidemiological data of PRKN-PD patients is summarized in Table 1. Gender and age-paired control group included six males and seven female subjects with an age range of 35-86 years and with a mean age of 58 years. Subjects with comorbidities, mitochondrial disorders, and those consuming mitochondrial toxic drugs were excluded from the study [52]. The study was approved by the ethics committee of the Hospital Clínic of Barcelona, following the guidelines of Helsinki declaration, and subjects were included in the study after signing the informed consent form.

Table 1. Clinical and epidemiological data of patients and control subjects providers of skin biopsy.

Subject	Mutation	Gender	Age of disease onset	Age at skin punch biobsy	Treatment
PRKN-PD 1	Heterozygous PRKN val15met	Male	47	57	L-Dopa
PRKN-PD 2	Homozygous PRKN exon 2-3-4 deletion	Male	35	69	L-Dopa
PRKN-PD 3	Homozygous PRKN exon 5-6 deletion	Female	27	63	L-Dopa
PRKN-PD 4	Compound heterozygous PACRG exon 1 deletion/ PRKN exon 6 deletion	Female	8	35	L-Dopa
PRKN-PD 5	Compound heterozygous PRKN exon 2 duplication/ exon 6 deletion	Male	20	49	L-Dopa
PRKN-PD 6	Homozygous PRKN exon 3 deletion	Male	25	48	L-Dopa
PRKN-PD 7	Compound heterozygous PRKN exon 6 deletion/ exon 8 deletion	Female	38	44	L-Dopa
No differences in mean age and gender were found between PRKN-PD and CTL subjects. Control group included six males and seven female subjects with an age range of 35-86 years and with a mean age of 58 years. CTL= healthy control subjects (not affected by PD or any PRKN mutation). PACRG=Parkin coregulated gene. PRKN-PD= Parkin-associated Parkinson’s disease. Val15Met= Methionine to Valine substitution at position 15.

Cell culture

Fibroblasts were obtained by a 6 mm of diameter punch skin biopsy from the alar surface of the non-dominant arm of the subjects. Fibroblasts were cultured first in DMEM with high glucose (25 mM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) L-glutamine and penicillin/ streptomycin at 37ºC and 5% CO₂ until 80-90% optimal confluence was reached. Each cell line was then exposed in parallel to 25 mM glucose (standard) or 10 mM galactose (mitochondrial-challenging) media for 24 hours [28,53–55]. Fibroblasts were harvested for further analyses as reported elsewhere [56]. All functional assays were performed in cells between passage 5 and 10.

Mitochondrial respiration analysis

Mitochondrial oxygen consumption rates

Oxygen consumption rates (OCRs) were measured in intact adherent fibroblasts through Agilent Seahorse XFe24 Analyzer (Seahorse Bioscience). Briefly, 35,000-40,000 fibroblasts per well were seeded in customized 24-well Seahorse cell culture plates and left to adhere overnight in 250 uL of growth medium. Each cell line was seeded in triplicate per condition (n=3 for glucose and n=3 for galactose). After 24 hours, growth medium was removed and wells were washed and replaced with Seahorse XF Base Medium (Seahorse Bioscience) containing either 25 mM glucose or 10 mM galactose plus 1 mM sodium pyruvate and 1 mM glutamine. Afterwards, plates were incubated for 30 min at 37 °C without CO₂ according to manufacturer’s protocol. In order to determine the different respiratory control ratios, oxygen consumption was measured under basal conditions and after the addition of oligomycin, which blocks ATP synthase allowing the assessment of the natural proton leak across the inner mitochondrial membrane (IMM). This was followed by the addition of the uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) to measure the maximal respiratory capacity. FCCP is an ionophore that directly transports protons across the IMM bypassing the ATP synthase proton channel thus leading to a rapid consumption of oxygen without the ATP generation. Finally, complex I and III inhibitors, rotenone and antimycin-A, were added to assess unspecific non-mitochondrial respiration (all reagents from Sigma-Aldrich). In addition, spare respiratory capacity and basal/maximal respiratory ratio were calculated as the maximal after basal OCR subtraction expressed in percentage, and the ratio between basal and maximal OCRs, respectively. All respiration values were normalized to total cell protein content determined in each well through the bicinchoninic acid assay (BCA) assay according to manufacturer’s protocol (Thermo Scientific) and CS activity (see mitochondrial respiratory chain enzymatic activities method section).

Mitochondrial complex I-stimulated oxygen consumption

To assess oxygen consumption under the stimulation of mitochondrial CI, 1 million of fibroblasts were obtained and resuspended in ice-cold respiration MiR05 medium. High-resolution respirometry was performed in digitonin-permeabilized cells using Oroboros^TM Oxygraph-2k system® (Innsbruck, Austria) and oxidation of CI substrates, pyruvate and malate (PMox), was monitored following manufacturer’s protocol [56,57]. Results were normalized to cell number and CS activity (see mitochondrial respiratory chain enzymatic activities method section).

Mitochondrial respiratory chain enzymatic activities

In order to study mitochondrial respiratory chain (MRC) function, enzymatic activities of mitochondrial complexes I (CI), II+III (CII+III) and IV (CIV) were spectrophotometrically measured at 37ºC in fibroblasts, as reported elsewhere [58,59]. CS activity was also spectrophotometrically determined at 37ºC in fibroblasts, as it is considered a reliable marker of mitochondrial content [60]. Mitochondrial content was further confirmed by alternative methods (see mitochondrial content method section).

All enzymatic assays were performed following national standardized methods and were run in parallel with internal quality controls [58]. Changes in absorbance were registered in a HITACHI U2900 spectrophotometer through the UV-Solution software v2.2 and were expressed as nanomoles of consumed substrate or generated product per minute and milligram of protein (nmol/minute·mg protein). All enzymatic activities were normalized by CS activity.

Lipid peroxidation

Lipid peroxidation levels are indicative of the ROS-derived oxidative damage in cell lipid compounds. Lipid peroxidation was quantified using the BIOXYTECH® LPO-586™ colorimetric assay (Oxys International Inc., CA, USA). Specifically, the levels of malondialdehyde (MDA) and 4-hydroxyalkenal (HAE) which are peroxides derived from fatty acid oxidation, were quantified in duplicate through spectrophotometry. The results were next normalized by protein content and expressed as μM MDA+ HAE/mg protein, as previously reported [61].

Mitochondrial membrane potential

Mitochondrial membrane potential of fibroblasts was assessed in a BD FACSCalibur^TM cell analyzer (BD Biosciences) by JC-1 potentiometric dye and both, green (~525 nm) and red (~590 nm) fluorescent emissions of each cell population were simultaneously monitored by flow cytometry, as reported elsewhere [62,63]. Results were obtained as percentage of cells with specific fluorescence indicating polarized or depolarized mitochondria. We calculated the red/green ratio indicating the ratio between correctly polarized and depolarized mitochondria, whose decrease indicates mitochondrial depolarization.

Mitochondrial network complexity analysis

Mitochondrial content and mitochondrial network complexity were assessed by immunochemistry and confocal microscopy as reported elsewhere [56,64]. A minimum of 3 fibroblasts from each subject were visualized and analysed using the Image J software and a macro of instructions was used to perform semi-automatic quantitation [65]. Mitochondrial network of each cell was subjected to particle analysis and the following parameters were assessed: aspect ratio (AR) (major axis/minor axis), form factor (FF), which was calculated as the inverse of the circularity (4π·area/perimeter²) and, mitochondrial network or content (total number of mitochondria/total cell area). AR and FF values correspond to mitochondrial length and branching, respectively, and are considered parameters of mitochondrial health. AR and FF values of 1 are indicative of circular unbranched mitochondria which are a sign of pathologic mitochondrial isolation. As mitochondria elongate and become more branched, AR and FF values increase indicating mitochondrial health.

Mitochondrial content

The following three approaches were used to quantify mitochondrial content in fibroblasts:

Mitochondrial DNA (mtDNA) copy number

Total DNA from fibroblasts was isolated through the standard phenol–chloroform extraction procedure as previously reported [66]. To assess mitochondrial DNA (mtDNA) content, fragments of the highly conserved mitochondrial 12S rRNA gene and the constitutive nuclear ribonuclease P gene (RNase P) were amplified in triplicates by multiplex qRT-PCR using Applied Biosystems technology [67]. mtDNA content was expressed as the ratio between the 12S rRNA and RNase P genes.

Citrate synthase enzymatic activity

CS activity was spectrophotometrically determined in fibroblasts (see mitochondrial respiratory chain enzymatic activities method section).

Determination of mitochondrial network

Immunochemistry and confocal microscopy were performed to quantify mitochondrial network or content and expressed as the total number of mitochondria per total cell area (see mitochondrial network complexity analysis method section) [68]. In general, higher mitochondrial network values are considered sign of healthy mitochondria.

Autophagic flux analysis

Where indicated, cells were treated with 0.1 µM bafilomycin A1 (Sigma) for 4 and 8 hours at 37°C in the presence of 5% CO₂. Bafilomycin A1 is a proton pump inhibitor that neutralizes lysosomal pH preventing fusion of autophagosomes with lysosomes and thus allowing monitoring of autophagosome synthesis [38]. Briefly, fibroblasts were lysed with RIPA buffer (Sigma) plus protease inhibitor cocktail (Thermo scientific) followed by shaking and centrifugation at 16,100 g at 4°C for 10 minutes. Soluble fractions were kept at -80 °C until western blot analysis. Electrophoresis and blotting were performed, as reported elsewhere [56,62]. Blots were probed with anti-SQSTM1/p62 (Abcam) and anti-LC3B (Cell Signaling) antibodies. LC3BII is considered a marker of autophagosome number and p62 is an ubiquitin-binding scaffold protein that labels molecules and organelles that need to be degraded acting as a cargo receptor that is recruited to autophagosomes through LC3BII interaction [69]. Total protein content was obtained through SYPRO Ruby Protein Blot Stain, according to manufacturer’s protocol (Molecular Probes). The intensity of signals was quantified by densitometric analysis (Image Quant TL Software, GE Healthcare). Results were expressed as p62 and LC3BII protein levels normalized by the total cell protein content [70].

Cell growth

Cell growth rate was manually determined through cell counting with the Neubauer chamber using trypan blue staining at the times of seeding and harvesting the cells [71]. Cell growth rate was calculated by applying the following formula:

Formula.

Statistical analysis

Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS, version 19) software (IBM SPSS Statistics; SPSS Inc). Differences amongst groups were sought by non-parametric tests after filtering for outlier values in the datasets. Specifically, Kruskal-Wallis and Mann–Whitney U statistical tests for independent samples were used when required. Significance was accepted for asymptotic 2-tailed p-values below 0.05 (for a confidence interval of α= 95%). Results were expressed as means ± the standard error of the mean (SEM).

Acknowledgements

The authors thank the patients who participated in the study and their family members. We also want to thank the research team of Silvia Ginés from the Neurosciences Research Institute at the UB, for the kindly donation of two control fibroblast cell lines. We also want to thank the program: Suports a Grups de Recerca de la Generalitat de Catalunya 2017-2019 (grant number SGR 2017/893). We also thank Anna Bosch, Elisenda Coll and Maria Calvo from the Advanced Optical Microscopy department of the University of Barcelona for their support with confocal microscopy.

Conflicts of Interest

The authors report no actual or potential conflict of interest including any financial, consultant, institutional or other relationships with other people or organizations within three years of beginning the work submitted that could inappropriately influence this work.

Funding

This work was supported by funds from Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos III (ISCIII) (grant number PI11/00462), the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), initiatives of Instituto Carlos III (ISCIII) and FEDER, and Fundació Privada Cellex (CP042187).

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