So this protocol enables us to induce pathological phase transitions of TDP-43 and address their cellular consequence in the spinal motor neurons in vivo, which is relevant for understanding why motor neurons degenerate in ALS. Due to the high transparency of zebrafish larvae, the motor neurons in the spinal cord are directly visible. So one can visualize the dynamic behavior of TDP-43 undergoing phase transitions in single spinal motor neurons.
This method offers a novel animal model of ALS. We believe that any method that can prevent the light induced TDP-43 phase transition and aggregation in this ALS model may be a potential candidate for ALS therapy. Other ALS related proteins with intrinsically disordered regions can be expressed to explore neurotoxicity associated with the abnormal phase transitions.
The key to success in this approach is to express the optogenetic TDP-43 in the spinal motor neurons at the nontoxic level. BAC transgenesis is a time consuming step, but once you create a proper transgenic fish, the following steps are relatively easier and straightforward. To begin, turn on the light emitting diode, or LED panel, by using the associated application installed on a tablet or phone.
Next, place the spectrometer probe into an empty well of a 6-well dish. Then using the application, adjust the LED light to a wavelength peaking at 456 nm. Place the optical sensor of an optical power meter in the empty well and adjust the power of the LED light.
Then place the dish with the LED panel setting into an incubator at 28 C.For imaging of the zebra fish larvae expressing optogenetic TDP-43, dechorionate the double-transgenic fish and anesthetize them in E3 buffer containing 250 g/mL of Tricane. Next, preheat 1%low melting temperature agarose containing 250 g/mL of ethyl 3-aminobenzoate methanesulfonate at 42 C.Then put a drop of the agarose on the glass base dish. The diameter of the dome-shaped agarose drop on the glass dish should be 8 to 10 mm.
Next, using a Pasteur pipette, add the anesthetized fish to the agarose on the glass based dish. Minimize the amount of E3 buffer added to the agarose along with the fish. Then, mix by pipetting a few times.
During solidification of the agarose, maintain the fish on its side using a syringe needle, such that the spinal cord is in an appropriate horizontal position. After the agarose has solidified, add a couple drops of E3 buffer onto the dome-shaped agarose-mounted fish. Using a confocal microscope equipped with a 20 times water immersion objective lens, acquire serial confocal z-sections of the spinal cord.
Include the cloaca on the ventral side of the fish in the regions of interest as a reference to identify and compare the spinal segments across the time points. As soon as the imaging is complete, use a syringe needle to carefully crack the agarose and remove the fish from the agarose. Keep the amount of time the fish is embedded in the agarose as short as possible, although agarose embedding for less than 30 minutes does not affect the viability of the fish.
Add 7.5 mL of E3 buffer to one well of a 6-well dish and place the imaged double-transgenic fish into that well. Then, place the dish on the LED panel, keeping the dish and LED panel 5 mm apart with a spacer, and turn on the blue LED light. Keep some double-transgenic fish in a separate 6-well dish covered with aluminum foil for unilluminated control fish.
After the desired illumination time, imaged the spinal cord of the illuminated fish as demonstrated earlier. Cytoplasmic relocation of the opTDP-43h in single spinal motor neurons was visualized by separating the z-series images acquired at 48. and 72 hours post fertilization into each EGFP-TDP-43z and opTDP-43h channel.
From max intensity projection images of EGFP-TDP-43z, a single isolated spinal neuron identifiable at both 48. and 72 hours post fertilization was selected. Regions of interest covering the somas of the motor neurons were set by tracing the edge of the EGFP signal at 48.
and 72 hours post fertilization. The normalized fluorescent intensity along the major axis of the soma was plotted. And at 48 hours post fertilization, the patterns of both the signals largely overlapped each other.
In contrast, at 72 hours, the peak for opTDP-43h signal was found in the region where the EGFP-TDP-43z signal was low, indicating the cytoplasmic relocation of opTDP-43h. To quantify the opTDP-43h and EGFP-TDP-43z signals before and after blue light stimulation, some slices projection images for each channel of the same z-series images were produced at 48. and 72 hours post fertilization.
Of the four mnr2b+cells that were investigated, cell 1, notably displayed a saturated EGFP-TDP-43z signal. Relative amounts of opTDP-43h to EGFP-TDP-43z were calculated by dividing the RFP value by the GFP value. Cells 2 to 4 displayed decreasing opTDP-43h levels after blue light illumination.
It is important to minimize the time the fish are embedded in the agarose to keep them healthy. By adjusting the intensity and duration of LED illumination, we control TDP-43 phase transition in a temporary regulated manner, which can help dissect progression of pathological TDP-43 phase transition in the motor neuron.
