Mitochondrial integrity, MPT opening, and apoptosis.

<p><b>A) Mitochondrial calcein loading</b> by fluorescent plate reader HTS of in NPCs grown in 96 well micro plates. Relative fluorescent signal intensities (RFU) for calcein acquired after 30 min loading with Calcein AM and CoCl₂ were normalized to mitochondrial content (Mitotracker) and to cell number by Hoechst 33342 (H33342). 1 µM ionomycin was added directly before HTS analysis as negative control (Iono) (n = 8, mean ± SD, Ctrl/SNCA-Tri: 3.4/4.9, *<i>p</i> = 0.039). <b>B)</b><b>MPT-induced mitochondrial calcein loss</b> in Ctrl and SNCA-Tri NPCs after mitochondrial calcein–AM loading. Representative fluorescence microscopy images of Ctrl and SNCA-Tri NPCs loaded with calcein (green), Mitotracker (red) and CoCl₂ were assayed 1 hr. after treatment with 4 µM staurosporine under NG conditions. MPT opening results in entry of CoCl₂ into mitochondria and loss of calcein signal (nuclear counter stain: Hoechst 33342; scale bar: 100 µm). <b>Inserts:</b> Higher magnification images obtained by conventional fluorescence microscopy (Scale bar: 10 µm). <b>C) HCI automated fluorescence microscopy analysis</b> of MPT in NPCs treated with 4 µM staurosporine as under B). Images (see B) were analyzed using MetaXpress image processing software. Depicted are data of cellular calcein signal intensities normalized to mitochondrial content (Norm. RFU Calcein/RFU Mitotracker) from two replicate wells with four image sites/well per treatment condition (n = 16, mean ± SD, Ctrl/SNCA-Tri, HG: 834/457, HG+R: 1425/1011, NG: 864/574, HG+Iono: 187/190, *<i>p</i>≤0.01). <b>D)</b><b>Kinetic evaluation of MPT opening</b> and loss of mitochondrial calcein signal after induction of MTP using fluorescence plate reader based HTS analysis. NPCs treated and prepared as under B) were loaded with 4 µM stauropsporine and changes in calcein signal normalized to cell number and mitochondrial content (Δ Norm. RFU) were recorded every 1 min for 20 min (n = 8, mean ± SD, Ctrl/SNCA-Tri, HG: −0.06/−0.12, HG+R: −0.17/−0.28, HG+Iono: −0.03/−0.04, *<i>p</i>≤0.01). <b>E)</b><b>Cytochrome c immuno-cytochemistry</b> in Ctrl and SNCA-tri NPCs challenged with 200 µM paraquat (PQ) 15 min. before fixation. Shown are permeabilized cells probed with cytochrome c antibody, detected by an Alexa-488 nm labeled secondary antibody (green). Cells were counter stained with Hoechst 33342 (blue) (Scale bar: 100 µm, insert: 10 µm). <b>F)</b><b>Immunoblot analysis of cytochrome c levels</b> in sub-cellular fractions containing either cellular organelles (containing bound cytochrome c) or cytosolic proteins (with soluble cytochrome c) from NPC cell lysates (Ctrl and SNCA-Tri) treated with paraquat (PQ) as under E). Cytochrome c (14 kDa) and GAPDH (40 kDa) specific antibodies were detected by a secondary IR-dye conjugate.</p

Mitochondrial membrane potential (MMP) and energy balance.

<p><b>A) Fluorescence microscopy</b> of MMP in live NPCs from patient (SNCA-Tri) and control (Ctrl) loaded with 100 nM TMRM in normal growth medium (HG), medium plus 20 µM Rotenone (HG+R) or with 1 µM of the ionophore CCCP (HG+CCCP) as negative control (Scale bar: 10 µm). <b>B)</b><b>Plate reader based high throughput screen (HTS) of MMP</b> in live NPCs loaded with 20 µM JC-10 for 45 min. Cells were also treated with medium w/o glucose (NG). Shown are log ratios of reduced (Ex./Em. 540 nm/590 nm) to oxidized JC-10 (Ex./Em. 488 nm/520 nm) normalized to Hoechst 33342 (Log Norm. JC-10 Ratio) after 60 min. (n = 8, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD for HG+R: 202/29/194 (xE04), *<i>p</i>≤0.05; for NG: 92/30/118 (xE03) **<i>p</i>≤0.006). <b>C) Plate reader based HTS for MMP loss</b> in live NPCs prepared and analyzed as under B). Fluorescence measurements were acquired as under B) every 5 min for 10 cycles and loss of MMP with time graphed as ΔRFU/min. (n = 8, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: −0.02/−0.06/−0.01, *p≤0.05; HG+R: −0.17/−0.70/−0.22 ***p<0.001, NG: −0.08/−0.33/−0.04, *p≤0.05). <b>D)</b><b>Luminescence plate reader based HTS</b><b>of ATP levels</b> in Ctrl, SNCA-Tri and SNCA-Tri KD NPCs under the above growth conditions (HG, HG+R, NG) assayed by a coupled luciferin/luciferase assay. Depicted are ATP contents in cells treated with 20 µM rotenone (R) for 18 hrs. (n = 8, mean ± SD nMATP/ug protein in: Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 1.66/0.75/1.37, **<i>p</i> = 0.003; NG: 0.69/0.45/0.51, *<i>p</i> = 0.04). <b>E and F) Mitochondrial metabolic activity</b> studied by Seahorse XF24 analysis. <b>E)</b> Oxygen Consumption Rate (OCR) and <b>F)</b> Extracellular Acidification Rate (ECAR). Shown are relative OCR compared to basal values as a function of the sequential addition of mitochondrial inhibitors Oligomycin (1 µM), CCCP (1.5 µM) and Rotenone (Rot, 5 µM) + Antimycin A (Ant, 1 µM). Significant changes compared to basal OCR rates (*p<0.05) and differences between lines treated with and without 6-OHDA (250 µM) for 1 hr are indicated by # (#p<0.05, mean ± SEM, n≥17; from five independent experiments).</p

Apoptosis sensitivity and caspase activation.

<p><b>A) Caspase 3 activity</b> in cell lysates from adherent NPCs either left untreated or treated with 20 µM rotenone (R) for 18 hrs and then exposed to 1 uM staurosporine for 120 min before analysis. HTS analysis for caspase 3 activity from cell lysates was by activation of the fluorescent caspase substrate 7-amino-4-methylcoumarin (AMC) (Ex./Em. 340/440 nm) (n = 9, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD, HG: 33/69/42, HG+R: 42/129/87, NG: 55/138/85, *p≤0.050, **p≤0.0035; from three independent experiments). <b>B)</b><b>Kinetics of caspase 3/7</b><b>activity</b> in permeabilized NPCs pretreated as described under B) and assayed 15 min after staurosporine treatment. Changes in caspase 3 activity are depicted as ΔµM AMC fluorescence/min + mg cellular protein (detected by Bradford protein assay) (n = 9, mean ± SEM).</p

Reactive oxygen species (ROS) production.

<p><b>A) Fluorescence microscopy</b> of live adherent NPCs untreated (HG) or treated with 100 µM TBHP (HG+TBHP), loaded with CM-H₂DCFDA and imaged under controlled exposure conditions (10 sec fluorescent light exposure before image acquisition). Hoechst 33342 was used as counter stain (Scale bar: 20 µm). <b>B) Plate reader based HTS</b> of ROS levels in adherent NPC in 96-well plates and treated as under A). Relative CM-H₂DCFDA fluorescence intensities (RFU) were normalized to Hoechst 33342 (H33342) (n = 12, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.5/1/0.75, HG+R: 0.7/1.3/0.6, NG: 0.4/1.1/0.7, *<i>p</i>≤0.046, **p≤0.009, ***≤0.001). <b>C) ROS production rates</b> by HTS plate reader analysis of CM-H₂DCFDA fluorescence development over time (Δ RFU CM-H₂DCFDA/sec + H33342) in cells exposed to TBHP as under A), measured with normal medium (HG) with or without rotenone (R) and in medium without glucose (NG) (n = 12, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 22/75/68, HG+R: 177/367/178, NG: 80/353/184, *<i>p</i>≤0.010, **<i>p</i>≤0.007, ***<i>p</i>≤0.001). <b>D) Mitochondrial superoxide production</b><b>rates</b> assayed by HTS plate reader analysis of the mitochondrial targeted fluorescent superoxide indicator MitoSOX. Depicted are changes in relative fluorescence units normalized to H33342) (Δ RFU MitoSOX/min + H33342) (n = 4, mean ± SD, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.28/1.2/0.3, HG+R: 2.1/5.5/3.7, NG: 2.3/5.2/0.8,*<i>p</i>≤0.038, **<i>p</i>≤0.007).</p

NPC viability.

<p><b>A) Cell cycle analysis</b> by propidium-iodine (PI) staining and flow cytometry analysis of Ctrl and SNCA-Tri NPCs with staining grouped by cell cycle phase (G0/1, S and G2/M), showing a reduced percentage of SNCA-Tri NPCs in the S phase (n = 3, mean ± SD, *<i>p</i> = 0.047). <b>B)</b><b>Survival under nutritional and toxicant stress.</b> NPCs propagated in medium without glucose (NG) untreated or treated with 20 µM rotenone (R) or 20 µM paraquat (PQ). Survival curves (every 12 hours) for the Ctrl, SNCA-Tri and SNCA-Tri KD cell lines after analysis of adherent cell count (ImageJ). Percentage of surviving cells with time (hrs) (n = 3, mean ± SEM). <b>C)</b><b>Cell viability assayed by plate reader based high throughput screen (HTS)</b> of NPCs untreated (HG), treated with 20 µM rotenone (HG+R) or without glucose (NG) for 18 hrs. Live cells were stained with 1 µM of the RedOx indicator C₁₂-Resazurin/Alamar Blue for 15 min before analysis. Graphed are endpoint fluorescence units (RFU) normalized to total cellular protein/well (ug protein) (n = 3, mean ± SEM, *<i>p</i>≤0.05). <b>D)</b><b>Cell viability assayed by</b> flow cytometry evaluation of apoptosis and cell death in live NPCs treated as under A). Cells stained with C₁₂-Resazurin for cell viability and with Sytox-Green. Graphed are percentages of metabolic active NPCs, determined by Resarufin (Ex./Em. 563/587 nm) fluorescence (viable), apoptotic cells (cell membrane asymmetry detected by an Annexin-V Alexa-660 nm conjugated antibody) (n = 3, mean ± SD, Ctrl/SNCA-Tri: 5.3%/24.4%, *<i>p</i> = 0.027) or cell death (nuclear fragmentation, detected by Sytox-Green, Ex./Em. 488/530 nm) (n = 3, mean ± SD, Ctrl/SNCA-Tri: 5.3%/24.4%, **<i>p</i> = 0.004).</p

Protein biosynthesis and proteasome function.

<p><b>A) Mitochondrial protein biosynthesis and protein import.</b> Fluorescent protein expression patterns in confluent adherent NPC cultures (PC: Phase Contrast) transduced with two baculoviral vectors expressing fluorescent proteins targeted to either the peroxisomal (Perox.; Green) or the mitochondrial (Mito.; Red) compartment. Shown are fluorescent protein expression patterns in live confluent Ctrl and SNCA-Tri cell lines grown under normal growth conditions (HG) and evaluated 20 hrs post transduction (Scale bar: 200 µm, 5 µm). <b>B) Time resolved peroxisomal and mitochondrial protein biosynthesis</b>. Fluorescent protein expression patterns as under A), but imaged at 8 and 18 hrs post viral transduction. <b>C)</b><b>Proteasome activity measured by fluorescence microscopy</b> of adherent NPCs cultured with 20 µM rotenone alone or with 10 µM of the proteasome inhibitor MG132. Depicted are fixed cells stained with 5 µM of the aggresome/proteasome specific dye Bodipy TMR-AHX3L3VS (red). Hoechst 33342 was used as nuclear counter stain (blue) (Scale bar: 20 µm). <b>D)</b><b>Proteasome activity measured by flow cytometry</b> evaluation of cells treated and stained as under B). Charted are the aggresome propensity factors (APF) of NPCs calculated from the mean RFU (MRFU) of Bodipy-TMR fluorescence (APF = 100×[MRFU MG132 treated−MRFU untreated]/MRFU MG132 treated (n = 3, mean ± SD, APF Ctrl/SNCA-Tri: 51/120, *<i>p</i> = 0.041).</p

NPC characterization.

<p><b>A) Phase contrast microscopy</b> of α-synuclein gene triplication (SNCA-Tri), control (Ctrl) and α-synuclein knockdown (SNCA-Tri KD) iPSC-derived NPC lines (Scale bar: 50 µm) shows normal cell morphology. <b>B)</b><b>Mitochondrial and nuclear morphology</b> of NPCs visualized by fluorescence microscopy using Mitotracker Red CMX Ros (red) and Hoechst 33342 (blue) (Scale bar: 10 µm). <b>C)</b><b>Stem cell marker expression</b>. Immuno-cytochemistry on fixed NPCs detecting cytoplasmic Nestin expression pattern with secondary Alexa 588 conjugated antibody (orange) by fluorescence microscopy (Scale bar: 100 µm). Insert: Immuno-cytochemistry for the nuclear stem cell marker SOX1, detected by a secondary Alexa-488 conjugated antibody (green) (Scale bar: 20 µm). Nuclear counter stain by Hoechst 33342 (blue). <b>D) Representative α-synuclein protein expression</b> patterns (left) by immunoblot of protein lysates from a control line (Ctrl), the SNCA-Tri NPC line and the corresponding α-synuclein knock down line (SNCA-Tri KD) with β-actin serving as loading control. Right: Quantification of β-actin normalized α-synuclein expression levels (n = 4, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: 12.4/5.9/8.3, ***<i>p≤</i>0.001, t-test; from two independent experiments). <b>E) ICC of α-synuclein protein expression in adherent NPCs</b> detected by a polyclonal α-syn antibody and visualized by Alexa-488 conjugated secondary antibody (green). DAPI nuclear counterstain (blue); (Scale bar 20 µm). Insert: Higher magnification image (Scale bar: 10 µm). <b>F)</b><b>Colocalization of subcellular</b><b>α-synuclein distribution with mitochondria</b> in adherent NPCs labeled with Mitotracker Red CMX Ros (red) and probed for α-syn as under E) (Scale bar: 5 µm).</p