Human subjects

PD patients were recruited from the outpatient clinic for Movement Disorders of the Department of Neurology of the Leiden University Medical Center (Leiden, the Netherlands) and nearby university and regional hospitals. All participants fulfilled the U.K. Parkinsons Disease Society Brain Bank criteria for idiopathic PD. The study was approved by the medical ethics committee of the Leiden University Medical Center (P12.194/NV/ib), and written informed consent was obtained from all PD patients.

Fibroblasts were isolated at Leiden University Medical Center from skin biopsies derived from the ventral side of the upper leg and cultured under highly standardized conditions as previously described in [14]. Peripheral whole blood was collected from PD patients at Leiden University Medical Center and PBMCs were isolated at the Department of Molecular Genetics at the Erasmus Medical Center in Rotterdam. Control iPSC were obtained from the Eramsus MC iPS Core facility.

Peripheral whole blood from 24 age-matched healthy controls (age >55 years) was obtained from Sanquin Rotterdam (NVT0585.00 Mantel, NVT0585.01 Annex).

Bioinformatic analysis was performed using the Parkinsons Progression Markers Initiative (PPMI) database.

To generate erythroblasts, PD patients peripheral blood mononuclear cells (PBMC) were extracted from 10ml of freshly extracted blood with the use of Lympholyte-H (Cedarline) and Leucosep polypropylene tubes (227290, Greiner) according to manufacturers indications. Briefly, blood was diluted in PBS at a 1:2 ratio and loaded on a 15mL Lympholyte Leucosep tube. Blood was centrifuged at 800g for 25min with no brakes at 4C. Upon removal of the plasma, the PBMC enriched cell fraction was collected, washed several times with sterile PBS and upon PBMCs were cultured in StemSpan SFEM medium (Stemcell Technologies) containing 2mM Ultraglutamine (Lonza), 1% Nonessential aminoacids (NEAA), 1% penicillin/streptomycin, 50ng/ml Stem Cell Factor, 2U/ml Erythropoietin, 1uM Dexamethasone (Sigma), 10ng/ml Interleukin-3 (R&D Systems), 10ng/ml Interleukin-6 (R&D Systems), 40ng/ml IGF-1 (R&D Systems) and 50ug/ml Ascorbic Acid (Merck) for 69 days refreshing half of the medium every other day starting from day 2. Erythroblasts were isolated when reaching 6070% of the total cell population by gradient centrifugation at 1000g for 20minutes at room temperature over Percoll (GE Healthcare). Isolated erythroblasts were frozen in FBS containing 10% DMSO at 80C. Metabolic analysis was performed within 2 days after thawing.

PD patients fibroblasts used in this study were prepared and isolated at Leiden University Medical Center from skin biopsies derived from the ventral side of the upper leg and cultured under highly standardized conditions as previously described in [14]. The study was approved by the medical ethics committee of the Leiden University Medical Center, and written informed consent was obtained from all PD patients.

Fibroblasts were reprogrammed to pluripotent stem cells using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (A16517, Thermo Fisher) according to the manufacturers protocol.

Human iPSC lines were generated as previously described [18]. Briefly, to generate embryoid bodies with neuroepithelial outgrowths (EBs), iPSC colonies were dissociated with 2mg/mL collagenase IV and transferred to non-adherent plates in hESC medium.

(Dulbeccos modified Eagles medium (DMEM)/F12 (Thermo Fisher Scientific), 20% knockout serum (Thermo Fisher Scientific), 1% minimum essential medium/non-essential amino acid (NEAA, Sigma-Aldrich, St Louis, MO, USA), 7nlml1 -mercaptoethanol (Sigma-Aldrich), 1% L-glutamine (Thermo Fisher Scientific) and 1% penicillin/streptomycin (P/S, Thermo Fisher Scientific) supplemented with 10M SB-431542 (Ascent Scientific), 1M dorsomorphin (Tocris), 3M CHIR 99021 (Axon Medchem) and 0.5M Purmorphamine (Alexis) on a shaker in an incubator at 37C/5% CO2. On the second day, medium was replaced with N2B27 medium [DMEM-F12/neurobasal 50:50 (Thermo Fisher Scientific), 1% P/S, 1:100 B27 supplement lacking vitamin A (Thermo Fisher Scientific) and 1:200 N2 supplement (Thermo Fisher Scientific)] containing 10M SB-431542, 1M dorsomorphin, 3M CHIR99021 and 0,5M Purmorphamine. On day 4, N2B27 medium was replaced and supplemented with 3M CHIR99021, 0.5M Purmorphamine, and 150M Ascorbic Acid (Sigma).

On day 6, EBs were slightly triturated and plated on Matrigel-coated (Matrigel - 354277, Corning) plates at a density 1015 EB per well containing smNPC expansion medium (N2B27 medium containing 3M CHIR 99021, 200M Ascorbic Acid, 0.5M Purmorphamine) and expanded for 5 passages before final differentiation. The medium was refreshed every other day.

smNPC were dissociated with Accutase at RT, diluted, and seeded on Poly-D-lysine Matrigel-coated cover glasses in a 12-well plate at the concentration of 5104 cells per well in the Patterning medium [N2B27 medium containing 1ng/mL GDNF (Peprotech), 2ng/ml BDNF (Pepotech), 200M Ascorbic Acid and 0.5M Smoothened Agonist (SAG Pepotech)]. The medium was refreshed every 2 days.

At day 8, the medium wash switched to the Maturation medium containing N2B27 medium, 2ng/ml GDNF, 2ng/ml BDNF, 1ng/mL TGF-b3 (Peprotech), 200M ascorbic Acid and 5ng/ml of ActivinA for the first feeding and 2ng/ml ActivinA for the following feedings. Medium change occurred every third day.

Erythroblasts and iPSCs were washed and resuspended in FC buffer (HBSS w/o calcium and magnesium + 0.5% BSA) and incubated with PE Mouse Anti-Human CD44 antibody (1:25, BD, 550989), FITC Mouse Anti-Human CD71 antibody (1:50, BD, 555536), or 7-AAD (Thermo Fisher, A1310) for 30min at 4C. Mitochondria were stained with Mitotracker Green FM (100nm, Cell Signaling, 9074) and active mitochondria with TMRM (100nm, Thermo Fisher, T668) for 30min at 37C. Cells were detected by flow cytometry using a LSRFortessa Cell Analyzer (BD, USA). Flowjo software (BD, USA) was used for data analysis.

Oxygen consumption rates (OCR) and extracellular acidification rate (ECAR) were measured using a XF-24 Extracellular Flux Analyzer (Agilent Technologies), as previously described [14]. Erythroblasts were seeded at a density of 2105 cells/well on Cell-Tak (Corning, 354240) coated Seahorse plates in unbuffered XF DMEM medium (Agilent Technologies) supplemented with 1mM sodium pyruvate, 2mM glutamine and 10mM glucose or galactose. Immediately after seeding, cells were centrifuged at 200g for 1minute to attach evenly to the bottom of the well and the plate was equilibrated for 30minutes at 37C in the absence of CO2. iPSCs derived from fibroblasts of PD patients and healthy controls were seeded at a density of 8103 cells/well on Seahorse plates and differentiated to dopaminergic neurons over a period of 3 weeks according to the described methodology. On an experimental day, the medium was changed to an unbuffered XF DMEM medium supplemented with 1mM sodium pyruvate, 2mM glutamine and 10mM glucose or galactose. Cells were incubated for 1h at 37C in the absence of CO2, before the Seahorse assay. For each assay, medium and reagent acidity were adjusted to pH 7.4 on the day of the assay, according to the manufacturers procedure. Optimal cell densities were determined experimentally to ensure a proportional response to FCCP (oxidative phosphorylation uncoupler).

After 3 measurements to detect the oxygen consumption ratio baseline, cells were then challenged with sequential injections of mitochondrial toxins: 1M oligomycin (Adenosine triphosphate ATP - synthase inhibitor), 1M FCCP, and 1M antimycin (complex III inhibitor). A minimum number of 5 replicates were performed for each cell line; data represent the mean of the different replicates. Basal respiration (measured as the average OCR rates at the baseline), maximal mitochondrial respiration (maximal respiration), reserve capacity (difference between maximal respiration and basal respiration), and respiration dedicated to ATP production (difference between basal respiration and oligomycin-dependent respiration) were used to investigate mitochondrial bioenergetics. Basal glycolysis, measured as extracellular acidification rate (ECAR) maximal glycolysis and reserve glycolytic capacity (difference between maximal glycolysis and basal glycolysis) were taken into account to investigate glycolytic properties.

Reprogrammed iPSCs cells cultured in an 8-chamber slide were fixed with 4% PFA for 15min at room temperature. After incubation in ice-cold methanol for 10min cells were permeabilized in 0.1% Triton in PBS for 10min and blocked using 1% BSA in PBS/0.05% Tween-20 for 30min. Next, cells were incubated with primary antibodies diluted in blocking buffer overnight at 4C - Mouse Anti-Human TRA1-81 (1:75, Abcam, AB16289#20), Rabbit Anti-Human OCT4 (1:250, Abcam, AB19857#8), Goat Anti-Human NCAM (1:100, R&D, AF2408), Goat-Anti Human SOX17 (1:100, R&D, AF1924) or Mouse Anti-Human Beta-Tubulin (1:1000, Merck, T8660) primary Chicken Anti-Human MAP2 (1:2000, Abcam, AB5392), Mouse Anti-Human TH (1:200, Millipore, MAB318). After washing with PBS cells were incubated with respective secondary Goat Anti-Mouse Alexa Fluor 546 (1:500, Invitrogen, A21045), Goat Anti-Rabbit Alexa Fluor 488 (1:500, Invitrogen, A11008#8a), Donkey Anti-Goat Alexa Fluor 488 (1:500, Invitrogen, A11055) or Goat Anti-Mouse Dylight 594 (1:500, Jackson, 115-515-166#7) antibodies diluted in blocking buffer for 1h at room temperature. Nuclei were stained with Hoechst 33342 (1:1000 in PBS, Thermo Fisher) for 10min. Cells were next washed with PBS, mounted with ProLong Diamond Antifade Mountant (P36965, Thermo Fisher), and imaged using a Leica Stellaris5 confocal microscope.

Blood transcriptome data from the Parkinson Progressive Markers Initiative (PPMI) cohort (PPMI website: https://ida.loni.usc.edu/pages/access/geneticData.jsp#441) were obtained. The libraries were prepared using the NEB/Kapa (NEBKAP) based library prep, with second-strand synthesis. RNA sequencing was performed at Hudson Alphas Genomic Services Lab on an Illumina NovaSeq6000, generating 100 million 125bp paired reads per sample. The Salmon files were imported into R using Tximport. To identify differentially expressed genes between PD groups and controls, the DESeq2 package was used. Normalized counts were subjected to Rlog transformation to improve distances/clustering for the principal component analysis (PCA). The cohort of subjects was divided into subgroups based on the delta-UPDRS-III (MDS-Unified Parkinsons Disease Rating Scale, UPDRS-III at last visit - UPDRS-III at first visit) of PD subjects: those with a delta-UPDRS-III less than 0 (defined as mild) and those with a delta-UPDRS-III greater than 0 (defined as severe), as well as controls (CTRL). A threshold of significance at FDR<0.05 was applied.

Gene Set Enrichment Analysis (GSEA) was conducted on an unfiltered, ranked list of genes. The analysis involved various terms from the Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome Pathway Databases, Hallmark Gene Set Collection, and WikiPathways (GSEA website: http://www.gsea-msigdb.org/gsea/msigdb/collections.jsp). Pathway information was obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) available at the Molecular Signatures Database (http://www.broadinstitute.org/gsea/msigdb/index.jsp) or from the Hallmark Gene Set Collection (http://www.gsea-msigdb.org/gsea/msigdb/collections.jsp). Gene set enrichment with FDR<0.1 was considered significant. Genes in each PD group were ranked based on the level of differential expression using a signal-to-noise metric and a weighted enrichment statistic.

Transcriptomic analysis was performed using R Studio version 4.2.3. The experiments were conducted with a minimum of three independent biological replicates. GraphPad Prism version 9 (GraphPad Software, La Jolla California USA) was used for all statistical analyses and graphical representations. P values were denoted as *P<0.05, **P<0.01, ***P<0.001, and were considered significant. In the absence of indications, comparisons should be considered non-significant. Comparisons between two groups were analyzed using unpaired two-tailed Students t-tests, and comparisons between more than two groups were analyzed using either one-way ANOVA followed by Dunnetts (comparison of PD means vs. healthy subjects) or Tukeys (comparison of all the means) posthoc test for multiple comparisons.

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Orthogonal analysis of mitochondrial function in Parkinson's disease patients | Cell Death & Disease - Nature.com