Chemoptogenetic ablation of neuronal mitochondria in vivo with spatiotemporal precision and controllable severity

  1. Wenting Xie
  2. Binxuan Jiao
  3. Qing Bai
  4. Vladimir A Ilin
  5. Ming Sun
  6. Charles E Burton
  7. Dmytro Kolodieznyi
  8. Michael J Calderon
  9. Donna B Stolz
  10. Patricia L Opresko
  11. Claudette M St Croix
  12. Simon Watkins
  13. Bennett Van Houten
  14. Marcel P Bruchez
  15. Edward A Burton  Is a corresponding author
  1. Department of Neurology, University of Pittsburgh, United States
  2. Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh, United States
  3. Tsinghua University Medical School, China
  4. Center for Biologic Imaging, University of Pittsburgh, United States
  5. Winchester Thurston School, United States
  6. Departments of Biological Sciences and Chemistry, Carnegie Mellon University, United States
  7. Department of Cell Biology, University of Pittsburgh, United States
  8. Department of Environmental and Occupational Health, University of Pittsburgh, United States
  9. Genome Stability Program, UPMC Hillman Cancer Center, United States
  10. Department of Pharmacology and Chemical Biology, University of Pittsburgh, United States
  11. Molecular Biosensors and Imaging Center, Carnegie Mellon University, United States
  12. Geriatric Research, Education and Clinical Center, Pittsburgh VA Healthcare System, United States
6 figures, 4 videos, 2 tables and 1 additional file

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.

The dL5**-MG2I excitation spectrum (Figure 1H) was multiplied by emission spectra from the green and red LEDs (Figure 1H) to generate energy transfer spectra. Quantitative properties of the energy …

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 …

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 …

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 …

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, …

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 …

Figure 6—figure supplement 1
Delayed CNS cell death in NeuMitoFAP zebrafish exposed to MG2I and far-red light.

Similar micrographs are shown as in Figure 6C, and quantified in Figure 6D, but including the control groups in comparison with NeuMitoFAP-MG2I larvae, before exposure, 2 hr after, and 24 hr after …

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).
RedGreenIRdL5**-MG2I (minor)dL5**-MG2I (major)
Peak λ (nm)661516877456666
Centroid λ (nm)656520880452649
FWHH (nm)1836515561
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**-MG2IGreen x dL5**-MG2I
Peak λ (nm)661524
Height0.980.013
Area19.101.15

Additional files

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