Figure 1.
A) Phase contrast microscopy 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) Mitochondrial and nuclear morphology of NPCs visualized by fluorescence microscopy using Mitotracker Red CMX Ros (red) and Hoechst 33342 (blue) (Scale bar: 10 µm). C) Stem cell marker expression. 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). D) Representative α-synuclein protein expression 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, ***p≤0.001, t-test; from two independent experiments). E) ICC of α-synuclein protein expression in adherent NPCs 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). F) Colocalization of subcellular α-synuclein distribution with mitochondria in adherent NPCs labeled with Mitotracker Red CMX Ros (red) and probed for α-syn as under E) (Scale bar: 5 µm).
Figure 2.
A) Cell cycle analysis 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, *p = 0.047). B) Survival under nutritional and toxicant stress. 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). C) Cell viability assayed by plate reader based high throughput screen (HTS) 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, *p≤0.05). D) Cell viability assayed by 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%, *p = 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%, **p = 0.004).
Figure 3.
Mitochondrial membrane potential (MMP) and energy balance.
A) Fluorescence microscopy 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) Plate reader based high throughput screen (HTS) of MMP 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), *p≤0.05; for NG: 92/30/118 (xE03) **p≤0.006). C) Plate reader based HTS for MMP loss 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). D) Luminescence plate reader based HTS of ATP levels 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, **p = 0.003; NG: 0.69/0.45/0.51, *p = 0.04). E and F) Mitochondrial metabolic activity studied by Seahorse XF24 analysis. E) Oxygen Consumption Rate (OCR) and F) 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).
Figure 4.
Protein biosynthesis and proteasome function.
A) Mitochondrial protein biosynthesis and protein import. 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) Time resolved peroxisomal and mitochondrial protein biosynthesis. Fluorescent protein expression patterns as under A), but imaged at 8 and 18 hrs post viral transduction. C) Proteasome activity measured by fluorescence microscopy 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). D) Proteasome activity measured by flow cytometry 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, *p = 0.041).
Figure 5.
Reactive oxygen species (ROS) production.
A) Fluorescence microscopy 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) Plate reader based HTS 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, *p≤0.046, **p≤0.009, ***≤0.001). C) ROS production rates 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, *p≤0.010, **p≤0.007, ***p≤0.001). D) Mitochondrial superoxide production rates 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,*p≤0.038, **p≤0.007).
Figure 6.
Mitochondrial integrity, MPT opening, and apoptosis.
A) Mitochondrial calcein loading 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, *p = 0.039). B) MPT-induced mitochondrial calcein loss 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). Inserts: Higher magnification images obtained by conventional fluorescence microscopy (Scale bar: 10 µm). C) HCI automated fluorescence microscopy analysis 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, *p≤0.01). D) Kinetic evaluation of MPT opening 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, *p≤0.01). E) Cytochrome c immuno-cytochemistry 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). F) Immunoblot analysis of cytochrome c levels 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.
Figure 7.
Apoptosis sensitivity and caspase activation.
A) Caspase 3 activity 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) Kinetics of caspase 3/7 activity 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).