Chemoptogenetic ablation of neuronal mitochondria in vivo with spatiotemporal precision and controllable severity
- Department of Neurology, University of Pittsburgh, United States
- Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh, United States
- Tsinghua University Medical School, China
- Center for Biologic Imaging, University of Pittsburgh, United States
- Winchester Thurston School, United States
- Departments of Biological Sciences and Chemistry, Carnegie Mellon University, United States
- Department of Cell Biology, University of Pittsburgh, United States
- Department of Environmental and Occupational Health, University of Pittsburgh, United States
- Genome Stability Program, UPMC Hillman Cancer Center, United States
- Department of Pharmacology and Chemical Biology, University of Pittsburgh, United States
- Molecular Biosensors and Imaging Center, Carnegie Mellon University, United States
- Geriatric Research, Education and Clinical Center, Pittsburgh VA Healthcare System, United States
Figures
Figure 1 with 3 supplements
Generation of NeuMitoFAP zebrafish.
(A) When the fluorogen MG2I (chemical structure shown on left) is bound to the fluorogen-activating protein (FAP) dL5** (right), excitation by far-red light causes generation of singlet oxygen. (B) …
Figure 1—figure supplement 1
High-power LED light source to deliver far-red light to larval zebrafish.
(A) A LED light source (100W, 660 nm; Chanzon, Amazon.com) was mounted on a CPU heatsink and fan for cooling. The LED/fan assembly was suspended beneath an optical glass window on a custom stand, so …
Figure 1—figure supplement 2
Energy transfer spectra between LED sources and dL5**-MG2I.
Figure 1—figure supplement 3
LED light source did not cause appreciable heating of water bath.
(A) A waterproof resin-coated negative temperature coefficient thermistor was used to provide continuous monitoring of bath temperature. The resistance of the thermistor was determined in a voltage …
Figure 2 with 4 supplements
Acute loss of neurological function in NeuMitoFAP zebrafish exposed to MG2I and far-red light.
(A) Design of experiments shown in panels (B - D). Experimental groups: WT zebrafish (black); WT zebrafish exposed to MG2I (green); NeuMitoFAP zebrafish (blue); NeuMitoFAP zebrafish exposed to MG2I …
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Figure 2—source data 1
Source data for Figure 2C.
- https://cdn.elifesciences.org/articles/51845/elife-51845-fig2-data1-v1.xlsx
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Figure 2—source data 2
Source data for Figure 2E.
- https://cdn.elifesciences.org/articles/51845/elife-51845-fig2-data2-v1.xlsx
Figure 2—figure supplement 1
Normal morphology of NeuMitoFAP zebrafish exposed to MG2I and light.
Transmitted light micrographs of NeuMitoFAP zebrafish: tThe right column shows larvae that were exposed to MG2I from 3 dpf; the left column shows untreated zebrafish. The first row shows larvae at …
Figure 2—figure supplement 2
The motor phenotype of NeuMitoFAP-MG2I zebrafish is dependent on far-red light exposure energy.
(A) Visual motor responses were evaluated in Tg(eno2:gal4FF); Tg(UAS:COX4-COX8-dL5-mCer3) (‘NeuMitoFAP’) zebrafish treated with MG2I. Motor response to cycles of green light (200 Lux; 10 min) and …
Figure 2—figure supplement 3
The motor phenotype of NeuMitoFAP-MG2I zebrafish exposed to 60 J/cm2 far-red light is not dependent on light power.
(A) Visual motor responses were evaluated in NeuMitoFAP (red) and non-transgenic (green) zebrafish treated with MG2I, identically to Figure 2—figure supplement 1. Each group of 12 zebrafish was …
Figure 2—figure supplement 4
Real-time changes in motor function of NeuMitoFAP-MG2I zebrafish during far-red light exposure.
24 zebrafish from each experimental group (non-transgenic and NeuMitoFAP zebrafish, not exposed to chemical or treated with MG2I; same color key as Figure 2B) were placed in a 96 well plate at 5dpf. …
Figure 3 with 2 supplements
Acute neuronal depolarization in NeuMitoFAP zebrafish exposed to MG2I and far-red light.
(A) Whole-cell patch clamp recordings were made from posterior lateral line ganglion (PLLG) sensory neurons. The inset figure shows the preparation and experimental design (the arrow shows a …
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Figure 3—source data 1
Source data for Figure 3B-D.
- https://cdn.elifesciences.org/articles/51845/elife-51845-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Far-red light-induced depolarization of lateral line ganglion neurons in NeuMitoFAP zebrafish treated with MG2I.
The graphs show membrane potential values (y-axis) of lateral line ganglion sensory neurons over time (x-axis) in whole cell patch clamp recordings from multiple zebrafish in each experimental …
Figure 3—figure supplement 2
Severity of lateral line ganglion neuron depolarization in NeuMitoFAP-MG2I zebrafish is dependent on far-red light dose.
Whole-cell patch clamp recordings from lateral line ganglion neurons were made exactly as in Figure 4, with the exception that illumination at λpeak=661nm was stopped after 5 min (black; 13.5 J/cm2) …
Figure 4 with 2 supplements
Disruption of mitochondrial function in NeuMitoFAP zebrafish exposed to MG2I and far-red light.
(A) ATP concentration was measured in lysates (each containing 5 whole zebrafish larvae) using a bioluminescent assay and normalized to protein content. Data points show ATP/protein in individual …
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Figure 4—source data 1
Source data for Figure 4A.
- https://cdn.elifesciences.org/articles/51845/elife-51845-fig4-data1-v1.xlsx
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Figure 4—source data 2
Source data for Figure 4B.
- https://cdn.elifesciences.org/articles/51845/elife-51845-fig4-data2-v1.xlsx
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Figure 4—source data 3
Source data for Figure 4D-E.
- https://cdn.elifesciences.org/articles/51845/elife-51845-fig4-data3-v1.xlsx
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Figure 4—source data 4
Source data for Figure 4F.
- https://cdn.elifesciences.org/articles/51845/elife-51845-fig4-data4-v1.xlsx
Figure 4—figure supplement 1
Viability of zebrafish larvae following neuromuscular paralysis with curare.
It was necessary to prevent swimming movements during oximetry measurements (Figure 4) because: (i) movements of larvae could disrupt measurement of dissolved O2 in the water; and (ii) the large …
Figure 4—figure supplement 2
No compensatory increase in glycolysis after mitochondrial targeting in NeuMitoFAP zebrafish.
Bath acidification rate (BAR) was quantified at the same time as the oxygen consumption rate measurements shown in Figure 4, using a Seahorse XF24 flux analyzer. (A) In the first 30 min of …
Figure 5 with 1 supplement
Disruption of mitochondrial structure in NeuMitoFAP zebrafish exposed to MG2I and far-red light.
(A) Confocal Z-plane projections showing mCerulean-labeled mitochondria in the lateral line nerves of live NeuMitoFAP zebrafish in the absence (upper images) or presence (lower images) of MG2I, …
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Figure 5—source data 1
Source data for Figure 5B.
- https://cdn.elifesciences.org/articles/51845/elife-51845-fig5-data1-v1.xlsx
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Figure 5—source data 2
Source data for Figure 5C.
- https://cdn.elifesciences.org/articles/51845/elife-51845-fig5-data2-v1.xlsx
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Figure 5—source data 3
Source data for Figure 5D.
- https://cdn.elifesciences.org/articles/51845/elife-51845-fig5-data3-v1.xlsx
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Figure 5—source data 4
Source data for Figure 5G.
- https://cdn.elifesciences.org/articles/51845/elife-51845-fig5-data4-v1.xlsx
Figure 5—figure supplement 1
Widespread disruption of mitochondrial morphology in NeuMitoFAP zebrafish exposed to MG2I and far-red light.
NeuMitoFAP zebrafish larvae were treated with MG2I or no chemical from 3 to 5 dpf and then exposed to 60 J/cm2 light at λpeak=661 nm, following which they were fixed immediately for electron …
Figure 6 with 1 supplement
Persistent neurological deficits and cell death in NeuMitoFAP zebrafish exposed to MG2I and far-red light.
(A) Mean swimming speed of NeuMitoFAP zebrafish treated with MG2I was quantified during the dark phase of the visual motor response (y-axis) after exposure to 60 J/cm2 far-red light (red) or no …
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Figure 6—source data 1
Source data for Figure 6D.
- https://cdn.elifesciences.org/articles/51845/elife-51845-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Delayed CNS cell death in NeuMitoFAP zebrafish exposed to MG2I and far-red light.
Videos
Video 1
Loss of motor function in NeuMitoFAP-MG2I zebrafish following far-red light exposure.
5dpf zebrafish swimming in the wells of an agarose-filled plate were illuminated from above by an infrared light, and video was recorded from below at 30 frame/s, during the dark phase of the visual …
Video 2
Normal circulation and heartbeat in NeuMitoFAP-MG2I zebrafish following far-red light exposure.
(i) Phase contrast videomicrography of 5dpf zebrafish from the same experimental groups as Video 1, illustrating identical circulation of blood cells in the vascular system from all experimental …
Video 3
Persistent motor deficits in NeuMitoFAP-MG2I zebrafish following far-red light exposure.
7dpf zebrafish are shown 48 hr after far-red light exposure at 5dpf. Methods and experimental groups are identical to Video 1. The severe neurological abnormalities seen immediately after far-red …
Video 4
Normal circulatory and cardiac development in NeuMitoFAP-MG2I zebrafish following far-red light exposure.
Phase contrast videomicrography of 7dpf zebrafish 48 hr after far-red light exposure at 5dpf. Methods and experimental groups are identical to Video 2. Despite the persistence of severe neurological …
Tables
Table 1
Peak wavelength, centroid and full width at half height (FWHH) are shown for the red, green, and infrared LED sources used in the study, in comparison with the major and minor excitation peaks of the dL5**-MG2I complex (see Figure 1H).
| Red | Green | IR | dL5**-MG2I (minor) | dL5**-MG2I (major) | |
|---|---|---|---|---|---|
| Peak λ (nm) | 661 | 516 | 877 | 456 | 666 |
| Centroid λ (nm) | 656 | 520 | 880 | 452 | 649 |
| FWHH (nm) | 18 | 36 | 51 | 55 | 61 |
Table 2
Peak wavelength, peak height and area under the curve are shown for normalized energy transfer spectra between the 661 nm LED and the dL5**-MG2I complex (‘Red x dL5**-MG2I’) and between the 516 nm LED and the dL5**-MG2I complex (‘Green x dL5**-MG2I’; see Figure 1—figure supplement 2).
| Red x dL5**-MG2I | Green x dL5**-MG2I | |
|---|---|---|
| Peak λ (nm) | 661 | 524 |
| Height | 0.98 | 0.013 |
| Area | 19.10 | 1.15 |
