A mitochondrial inside-out iron-calcium signal reveals drug targets for Parkinson's disease

Abstract

Dysregulated iron or Ca²⁺ homeostasis has been reported in Parkinson's disease (PD) models. Here, we discover a connection between these two metals at the mitochondria. Elevation of iron levels causes inward mitochondrial Ca²⁺ overflow, through an interaction of Fe²⁺ with mitochondrial calcium uniporter (MCU). In PD neurons, iron accumulation-triggered Ca²⁺ influx across the mitochondrial surface leads to spatially confined Ca²⁺ elevation at the outer mitochondrial membrane, which is subsequently sensed by Miro1, a Ca²⁺-binding protein. A Miro1 blood test distinguishes PD patients from controls and responds to drug treatment. Miro1-based drug screens in PD cells discover Food and Drug Administration-approved T-type Ca²⁺-channel blockers. Human genetic analysis reveals enrichment of rare variants in T-type Ca²⁺-channel subtypes associated with PD status. Our results identify a molecular mechanism in PD pathophysiology and drug targets and candidates coupled with a convenient stratification method.

Keywords: CP: Neuroscience.

Figure 1.. Iron promotes MCU oligomerization

(A–D) HEK cells were treated with 500 μM Fe²⁺ for 22 h, stimulated with thrombin, and mitochondrial (Rhod-2) and cytosolic Ca²⁺ levels (Calcium Green) were measured. (A and C) Representative Ca²⁺ traces. (B and D) Quantification of the peak fluorescent intensity (Fmax) normalized to the baseline (F0) within each cell. n = 20 cells from four independent coverslips. (E) HEK cells were treated with 5 mM Fe²⁺ for 22 h, run in Native-PAGE, and blotted. Right: Qualification of the band intensity normalized to the total protein amount measured by a BCA assay. n = 5. (F) Similar to (E), but with a range of Fe²⁺ concentrations added to the media. n = 4. Data point shows mean ± SEM. (G) Elution profiles of MCU from SEC samples. (H) HEK cells were treated with 500 μM Co²⁺ for 22 h and run in Native-PAGE. n = 4. (I) HEK cells treated with 500 μM Co²⁺ for 22 h were stimulated with thrombin, and mitochondrial Ca²⁺ levels (Rhod-2) were measured. n = 20 cells from four independent coverslips. (B), (D), (E), (H), and (I) Two-tailed Welch’s t Test. (J and K) Fluorescence-detection SEC profiles. See also Figure S1.

Figure 2.. MCU Binds to Fe²⁺

(A) HEK cells were treated with or without 500 μM Fe²⁺ for 21 h, then IPed with anti-MCU, and Fe²⁺ concentrations in the IP samples were detected (normalized to the total protein amount). Two-tailed paired t test. n = 4. (B) Different concentrations of Fe²⁺ were added to the purified MCU complex in vitro and after wash the total iron level in the complex was detected by ICP-MS. Mean ± SEM. n = 3. (Ci) Structural visualization of the matrix domain of MCU (green) bound to Fe²⁺ (orange) via these sites (pink) generated by PyMol. (Cii) MCU^−/− HEK cells transfected as indicated were treated with 500 μM Fe²⁺ for 20 h, then IPed with anti-Flag, and Fe²⁺ concentrations in the IP samples were detected. Two-tailed paired t test. n = 4. (Ciii) ICP-MS measurement of the total iron level in IPed MCU-Flag from cells as in (Cii), normalized to the total protein amount. n = 3. (D) Representative blots of IP with anti-Flag using cell lysates as indicated, run in Native- or SDS-PAGE. n = 4. (E) MCU^−/− HEK cells transfected as indicated and treated with or without 500 μM Fe²⁺ for 22 h were stimulated with thrombin, and mitochondrial Ca²⁺ levels (Rhod-2) were measured. Representative Ca²⁺ traces. (F and G) Based on traces like in (E), the peak fluorescent intensity normalized to baseline (F) or efflux rate (G) is quantified. n = 17 cells from four independent coverslips. (H) Cells as in (D) and (E) were lysed and blotted. Normalized to tubulin. n = 4. (D–H) Two-tailed Welch’s t test. Relative to “MCU-WT” or “MCU-3A” without treatment.

Figure 3.. PD models mirror Fe²⁺-treated HEK cells

(A) Postmortem brains were run in Native- or SDS-PAGE and blotted. Blots and details are in Figure S3A. HC: healthy control. (B) The total iron level (y axis) in each brain fraction was measured by ICP-MS (normalized to wet weight, average of three repeats), and plotted with the band intensity of MCU oligomers in Native-PAGE (x axis) from the same brain fraction. Linear fit. (C) iPSC-derived neurons were stimulated with thrombin and mitochondrial (Rhod-2) and cytosolic Ca²⁺ levels (Calcium Green) were measured in the somas. Left: Representative Ca²⁺ traces. n = 15 cell bodies from three independent coverslips. (D) iPSC-derived neurons were lysed and blotted as indicated. Normalized to the total protein amount. n = 4. (E) Left: Confocal single section images of live neurons as in (C) and (D) stained with RPA (quenched by Fe²⁺) and MitoTracker Green (Mito-Green), or Mito-FerroGreen (stains Fe²⁺). Scale bars, 10 μm. Middle: Quantification of the RPA fluorescent intensity in each soma from inverted images normalized to MitoTracker Green, a membrane-potential-independent dye whose intensity was similar between control and PD neurons (p = 0.2524). n = 26 (WT) and 28 (PD) cell bodies from three coverslips. Right: Quantification of the Mito-FerroGreen fluorescent intensity in each soma subtracting background. n = 55 cell bodies from three coverslips. Two-tailed Welch’s t test. See also Figure S3.

Figure 4.. Iron functions upstream of calcium in PD neurons

(A) Similar to Figure 3, iPSC-derived neurons, with or without treatment of 100 μM DFP for 24 h, were stimulated with thrombin, and mitochondrial Ca²⁺ (Rhod-2) was measured. n = 15 cell bodies from three independent coverslips. Control data without DFP treatment are the same as in Figure 3C. (B) The DA neuron number (average of both sides) was counted in the PPL1 cluster. Drug treatment was started from adulthood (day 1). Scale bar, 20 μm. n = 6, 9, 8, 7 (from left to right) flies. (C) Drug treatment was started from embryogenesis. n = 35, 33, 40, 34 flies (from left to right), three independent experiments. (D) Representative confocal images of neurites transfected as indicated. Scale bar, 10 μm. Quantification of the percentage of total neurons with deformed mitochondria (defined as a neuron without any mitochondrion in neurites >2.5 μm in length). n = 4 coverslips. (A) and (D) One-way ANOVA post hoc Tukey test.(B) and (C) Two-way ANOVA post hoc Tukey test. (E) Schematic representation of iron-triggered mitochondrial calcium influx in PD. (F) A PD-SNCA-A53T iPSC-derived neuron was transfected as indicated and immunostained with anti-Myc. Scale bar, 10 μm. (G) Confocal live images of neurons transfected as indicated. Scale bar, 10 μm. Quantification of the intensity of GCaMP6f normalized to miRFP670 within the same cell body. n = 15 cell bodies from three coverslips. Two-tailed Welch’s t test. See also Figure S3.

Figure 5.. Miro senses calcium to mediate several PD relevant phenotypes

(A) Representative still images from live Mito-dsRed and GFP-Miro1 recordings in axons of indicated genotypes, following 100 μM Antimycin A treatment. Scale bar, 10 μm. (B and C) Degradation rate profiles of GFP-Miro1 (B) or Mito-dsRed (C) normalized to the same axonal region at 0 min. n = 5 axons (one axon per coverslip). Comparison with “0 min.” One-way ANOVA post hoc Dunnett’s test. (D) iPSC-derived neurons were transfected as indicated and GFP-Miro1 was detected by an ELISA normalized to the total protein amount. n = 4. Two-tailed Welch’s t test within each condition. (E) The DA neuron number. Scale bar, 20 μm. n = 7, 4, 6, 5 (from left to right). (F) n (from left to right) = 49, 47, 39, 47 (day 14); 48, 45, 37, 44 (day 20); three independent experiments. (E) and (F) Two-way ANOVA post hoc Tukey test. See also Figure S3.

Figure 6.. HTP screens identify Ca²⁺-related drug hits for PD

(A) Schematic representation of a custom-designed drug screen for Miro1 in PD fibroblasts. (B) Pathway analysis identifies Ca²⁺ as a shared factor in the primary hit-Miro1 network. See also Figures S4 and S5.

Figure 7.. Benidipine rescues PD relevant phenotypes

(A and B) iPSC-derived neurons treated as indicated were immunostained with anti-TH (A) or TUNEL and Dapi (B). Scale bars, 100 μm. n = 20 images from three independent coverslips. p values are compared with the far-left bar, except indicated otherwise. One-way ANOVA post hoc Tukey test. (C) DA neurons in the PPL1 cluster. Scale bar, 20 μm. n = 4, 6, 7, 4 (from left to right). (D) n = 59, 57, 54, 57 flies (from left to right), three independent experiments. (C and D) Drug treatment was started from adulthood (day 1). Two-way ANOVA post hoc Tukey test. (E) Miro1 protein levels were measured using ELISA in PBMCs treated with DMSO or 40 μM CCCP for 6 h. Dot plot with mean ± SEM. n = 80 controls and 107 PD. Two-tailed Welch’s t test. (F) PBMCs from four PD patients were treated with 40 μM CCCP for 6 h, or pretreated with 10 μM Benidipine or MR3 for 18 h and then with 40 μM CCCP for another 6 h, and Miro1 protein was detected using ELISA. Two-tailed paired t test. (G) Schematic representation of this study. See also Figures S6 and S7.