Isogenic human iPSC Parkinson's model shows nitrosative stress-induced dysfunction in MEF2-PGC1α transcription

Abstract

Parkinson's disease (PD) is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). An association has been reported between PD and exposure to mitochondrial toxins, including environmental pesticides paraquat, maneb, and rotenone. Here, using a robust, patient-derived stem cell model of PD allowing comparison of A53T α-synuclein (α-syn) mutant cells and isogenic mutation-corrected controls, we identify mitochondrial toxin-induced perturbations in A53T α-syn A9 DA neurons (hNs). We report a pathway whereby basal and toxin-induced nitrosative/oxidative stress results in S-nitrosylation of transcription factor MEF2C in A53T hNs compared to corrected controls. This redox reaction inhibits the MEF2C-PGC1α transcriptional network, contributing to mitochondrial dysfunction and apoptotic cell death. Our data provide mechanistic insight into gene-environmental interaction (GxE) in the pathogenesis of PD. Furthermore, using small-molecule high-throughput screening, we identify the MEF2C-PGC1α pathway as a therapeutic target to combat PD.

Figure 1. Characterization of hiPSC Differentiation into A9-type DA Neurons

(A) Micrographs of hiPSC differentiation into human DA neurons (hNs). NPCs (top) are both FOXA2/LMX1A double positive and OTX2/FOXA2 double positive at 11 days in vitro (DIV). Most β-III tubulin (TUJ1)+ neurons (lower-middle) stain for TH by DIV 25. TH⁺ neurons are also LMX1A+, NURR1+ (upper-middle) and GIRK2+ (bottom) at DIV 35. Blue, DAPI; scale bar, 50 µm. (B) Quantification of TUJ1, TH/TUJ1, TH/LMX1A, and TH/GIRK2-positive cells relative to total cell number. Data from replicate experiments at DIV 35 are shown as mean + SEM; n = 6. (C–E) Electrophysiological characterization of A53T hNs and corrected controls at DIV 35. I_Na and I_K traces (n = 30, C). Cells were prepulsed to −90 mV for 150 ms and held at −60 mV with 10 mV voltage steps. Evoked action potential trains (Vh = −70 mV, n = 30, D). Spontaneous action potentials (n = 8, E). Mean resting potential, −39 ± 6 mV for A53T hN and −40 ± 3 mV (mean ± SEM) for corrected hN. Electrophysiological recordings were performed using K-gluconate internal solution. (F) α-synuclein costaining with thioflavin T in hiPSC-derived corrected (Corr) and A53T hNs. A53T mutant shows increased thioflavin T. DIV, 35; blue, DAPI; scale bar, 10 µm. (G) Quantification of thioflavin T fluorescence. Data from replicate experiments at DIV 35 shown as mean + SEM; n = 3. (H and I) Immunostaining of corrected hNs (H) and A53T mutant hNs (I) shows PS-129 colocalization with ubiquitin in neurites and cell bodies of A53T hNs (arrows). DIV, 35; blue, DAPI; scale bar, 10 µm. See also Figure S1 and Table S1.

Figure 2. A53T Mutant hNs Are Highly Susceptible to Mitochondrial Toxin-Induced Apoptosis

(A and B) Mitochondrial O₂ consumption monitored by Seahorse platform at DIV 35. After recording basal respiration, mitochondrial metabolic state 4 was induced with 1 µg/ml of the ATP synthase inhibitor oligomycin (Oligo), followed by induction of maximal respiration by the mitochondrial uncoupler FCCP in 1.5 µM increments. Maximal (Max) respiration was attained at a total FCCP concentration of 4.5 µM. Data from one of three replicate experiments are shown as mean ± SEM; n = 22 wells (A). Experimental data normalized to cell number per well (B). Neither basal respiration (reflecting cellular ATP demand) nor state 4 respiration (reflecting proton leak) differed between control and mutant cells. A53T mutant hNs displayed greatly reduced maximal respiratory capacity compared to corrected control. (C–E) A53T mutant hNs are more susceptible to apoptosis than their genetically corrected counterparts following exposure to PQ alone (C), MB alone (D), or combined exposure (E). Data represent mean + SEM. **p < 0.01 by ANOVA with posthoc Tukey; n = 6–12; DIV, 35. (F) Micrographs of TUNEL+ hNs following mitochondrial toxin exposure. DIV, 35; blue, DAPI; scale bar, 10 µm. See also Figure S2.

Figure 3. A53T Mutant hNs Manifest Altered Transcriptional Network Activity

(A) Expression profiling of A53T mutant and corrected (Corr) hNs at DIV 35. Genes whose expression was altered ≥ 2-fold (p < 0.05 significance) were clustered based upon functional correlation using GenePattern (Broad Institute), and the resulting heatmap is depicted (red, up; blue, down; data from three replicate experiments). A53T mutant hNs had a strikingly different genetic profile from corrected counterparts. (B–D) Pathway enrichment analysis was performed on all genes whose expression was altered (7,018 genes; p < 0.05 significance) in A53T hNs relative to corrected using NextBio and was filtered based on regulatory motifs using the Broad Institute Molecular Signatures Database (BroadMSigDB). Enrichment score was assigned by NextBio based on the method of Kupershmidt et al. (2010). Transcription factors were grouped based on their contribution to apoptosis (B), cell survival (C), or DA neuronal transcription (D). Transcription factor with the highest enrichment score was MEF2 (denoted in red). (Blue circle) Genes altered in A53T relative to corrected; (red circle) genes with transcription factor binding motifs within 2 kb of the transcriptional start site; (purple overlap) genes with transcription factor binding motifs within 2 kb of the transcriptional start site that are altered in A53T relative to corrected. See also Figure S3.

Figure 4. Accumulation of ROS/RNS in A53T hNs Aberrantly Modifies the Neuronal Prosurvival Transcription Factor MEF2C

(A) Representative micrographs of MitoSOX (red)-labeled A53T mutant and corrected hNs superimposed on phase-contrast images under basal conditions. DIV, 35; blue, DAPI; scale bar, 10 µm. (B) Quantification of superoxide anion accumulation at DIV 35 in corrected and A53T mutant hNs measured via MitoSOX labeling following pesticide exposure. A53T mutant hNs displayed increased labeling relative to corrected (Corr) at baseline and following exposure to 200 nM rotenone or 2.8 µMPQ/1 µM MB. Values are mean + SEM. **p < 0.01 by t test; n = 6. (C) Increased SOH-MEF2C detected at DIV 35 in A53T relative to corrected hNs after administration of mitochondrial toxins. SOH-protein capture using DCP-Bio1 and subsequent immunoblot for MEF2C show increased SOH-MEF2C upon exposure to H₂O₂ or PQ/MB. (D) Quantification of SOH-MEF2C. Values are mean + SEM. *p < 0.05 by ANOVA with posthoc Tukey; n = 3. (E and F) DAF-FM staining of NO levels shows higher basal values and more rapid accumulation of NO after pesticide exposure in A53T mutant relative to corrected hNs. (G) Time course of DAF-FM signal in A53T and corrected hNs following exposure to PQ/MB normalized to respective baseline levels. Inset shows that A53T mutant hNs have a 17% higher level of DAF-FM fluorescence than corrected hNs at baseline. (H) l-NAME pretreatment reduced both baseline and pesticide-evoked DAF-FM signal in A53T hNs. Pesticide exposure time, 6 hr; DIV, 35; blue, DAPI; scale bar, 10 µm. Data represent mean ± SEM. **p < 0.01 by two-way ANOVA with posthoc Tukey; n = 6. (I and J) Biotin switch at DIV 35 shows that A53T mutant hNs had substantial basal levels of SNO-MEF2C compared to corrected hNs, pesticides increased SNO-MEF2C levels (I), and l-NAME inhibited basal SNO-MEF2C formation with negative control in the absence of ascorbate (–Asc) (J). (K) Quantification of SNO-MEF2C. Values are mean + SEM. **p < 0.01, by ANOVA with posthoc Tukey; n = 3. See also Figure S4.

Figure 5. MEF2C-Mediated Transcriptional Activation Is Inhibited in A53T hNs

(A) Immunoblot analysis shows decreased TH and NURR1 protein expression in A53T mutant relative to corrected hNs at DIV 35. (B and C) qRT-PCR shows significant reduction in MAP2 and PGC1α mRNA in A53T mutant relative to corrected hNs at DIV 35. Data represent mean + SEM. **p < 0.01 by t test; n = 3. (D) Activation of native PGC1α promoter and PGC1α promoter lacking the MEF2-binding site (ΔMEF2) in A53T hNs relative to corrected hNs at DIV 35. Data represent mean + SEM. **p < 0.01 by t test; n = 4. (E) MEF2-driven PGC1α promoter activity inhibited in A53T hNs relative to corrected hNs at DIV 35. l-NAME prevented 50% of this inhibition. PGC1α promoter activity attributable to MEF2 was derived from the following formula using the data in (D): (1 − (ΔMEF2/PGC1α) × 100). Data represent mean + SEM. **p < 0.01 by t test; n = 4. (F) ChIP analysis on chromatin prepared from A53T mutant versus corrected (Corr) hNs at DIV 25 revealed 4-fold reduction in MEF2C bound to PGC1α promoter. Data represent mean + SEM. *p < 0.05 by t test; n = 3. (G) l-NAME increases MEF2C binding to PGC1α promoter by ~10-fold. Data represent mean + SEM. **p < 0.01 by t test; n = 3. (H) Effect of WT MEF2C or mutant MEF2C(C39A) on PGC1α promoter activity in the presence and absence of SNOC in A53T and corrected hNs at DIV 35. Data represent mean + SEM. *p < 0.05 by ANOVA with posthoc Tukey; n = 4. See also Figure S5.

Figure 6. MEF2C and Pesticides Regulate PGC1α Protein Expression In Vivo

(A) PGC1α protein expression in WT and A53T mutant mouse brain in the presence and absence of PQ/MB exposure. (B) PQ/MB increases SNO-MEF2C expression in WT mouse brain by biotin switch assay. (C) Quantification of SNO-MEF2C in WT mouse brain. Values are mean + SEM. **p < 0.01, by t test; n = 3. (D) PGC1α protein levels are reduced in MEF2C^+/– and MEF2C conditional knockout mouse brains.

Figure 7. Activation of MEF2C Transcriptional Network Rescues A53T Mutant hNs from Mitochondrial Toxin-Evoked Death

(A) l-NAME partially blocks PQ/MB-induced death in A53T mutant hiPSC-derived hNs. **p < 0.01 by ANOVA with posthoc Tukey; n = 6. (B) TUNEL reactivity in A53T hNs exposed to mitochondrial toxins PQ and MB following overexpression of MEF2C (with or without 1 mM l-NAME) or PGC1α. WT MEF2C, mutant MEF2C(C39A), and PGC1α rescue from PQ/MB-induced apoptosis relative to empty vector (EV)-expressing cells. DIV, 25; blue, DAPI; scale bar, 10 µm. (C) Quantification of data in (C). Values are mean + SEM. **p < 0.01, *p < 0.05 by ANOVA with posthoc Tukey or Fisher PLSD; n = 6. (D) TUNEL reactivity in A53T hNs exposed to PQ and MB following pretreatment with the MEF2 activator Isoxazole (10 µM). Isoxazole rescued from rotenone- or PQ/MB-induced apoptosis. DIV, 35; blue, DAPI; scale bar, 10 µm. (E) Quantification of data in (D). Values are mean + SEM. *p < 0.05 by t test; n = 3. (F) In human A9-type DA neurons, MEF2C drives MEF2 target genes, including PGC1α expression, which in turn promotes mitochondrial biogenesis, mitochondrial respiration, and neuronal health (i). When SNCA is mutated (A53T) in familial PD, higher steady-state levels of RNS result in the S-nitrosylation of MEF2C under basal conditions. Resulting inhibition of MEF2C transcriptional activity decreases MEF2 target gene expression, including PGC1α (dashed line), with consequent impairment of mitochondrial respiration and disruption of neuronal function (ii). Combined effect of mitochondrial stress due to A53T SNCA and exposure to mitochondrial toxins produces a dramatic increase in RNS and ROS over basal conditions, resulting in increased S-nitrosylation and/or sulfonation of MEF2C and further decrease in MEF2C activity. The decreased MEF2C activity leads to further inhibition of MEF2 target gene transcription, including PGC1α, and consequently a more profound drop in mitochondrial biogenesis and respiration, culminating in neuronal cell death (width of red lines indicates magnitude of effect) (iii). See also Figure S6.