This work was supported by SERIKA FUND (KA), KAKENHI Grant numbers JP19K06933 (KA) and JP20H05345 (KA).

All fish work was conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the Institutional Animal Care and Use Committee (approval identification number 24-2) of the National Institute of Genetics (Japan), which has an Animal Welfare Assurance on file (assurance number A5561-01) at the Office of Laboratory Animal Welfare of the National Institutes of Health (NIH, USA).
1. Construction of BACs for expression of optogenetic TDP-43 gene from the mnr2b ...

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The mnr2b-BAC-mediated expression of opTDP-43h and EGFP-TDP-43z in zebrafish provides a unique opportunity for live imaging of TDP-43 phase transition in the spinal motor neurons. The optical transparency of body tissues of zebrafish larvae allows for the simple and noninvasive optogenetic stimulation of opTDP-43h. Comparisons between single spinal motor neurons over time demonstrated that the light-dependent oligomerization of opTDP-43h causes its cytoplasmic clustering, which is reminiscent of ALS pathology.</...

Ribonucleoprotein (RNP) granules control a myriad of cellular activities in the nucleus and cytoplasm by assembling membrane-less partitions via liquid-liquid phase separation (LLPS), a phenomenon in which a homogeneous fluid demixes into two distinct liquid phases1,2. The dysregulated LLPS of RNA-binding proteins that normally function as RNP granule components promote abnormal phase transition, leading to protein aggregation. This process has been implicated in neurodevelopmental and neurodegenerative diseases3,4,5. The precise evaluation of a causal relationship between aberrant LLPS of RNA-binding proteins and disease pathogenesis is crucial for determining whether and how LLPS can be exploited as an effective therapeutic target. LLPS of RNA-binding proteins is relatively easy to study in vitro and in unicellular models but is difficult in multicellular organisms, especially in vertebrates. A critical requirement for analyzing such LLPS in individual cells within a tissue environment is to stably express a probe for the imaging and manipulation of LLPS in a disease-vulnerable cell type of interest.
Amyotrophic lateral sclerosis (ALS) is an ultimately fatal neurological disorder in which motor neurons of the brain and spinal cord are selectively and progressively lost due to degeneration. To date, mutations in more than 25 genes have been associated with the heritable (or familial) form of ALS, which accounts for 5%-10% of total ALS cases, and some of these ALS-causing genes encode RNA-binding proteins consisting of RNPs, such as hnRNPA1, TDP-43, and FUS6,7. Moreover, the sporadic form of ALS, which accounts for 90%-95% of total ALS cases, is characterized by the cytoplasmic aggregation of TDP-43 deposited in degenerating motor neurons. A major characteristic of these ALS-associated RNA-binding proteins is their intrinsically disordered regions (IDRs) or low-complexity domains that lack ordered three-dimensional structures and mediate weak protein-protein interactions with many different proteins that drive LLPS7,8. The fact that ALS-causing mutations often occur in the IDRs has led to the idea that aberrant LLPS and phase transition of these ALS-related proteins may underlie ALS pathogenesis9,10.
Recently, the optoDroplet method, a Cryptochrome 2-based optogenetic technique that allows the modulation of protein-protein interactions by light, was developed to induce phase transition of proteins with IDRs11. As this technique has been extended successfully to TDP-43, it has begun to uncover the mechanisms underlying pathological phase transition of TDP-43 and its associated cytotoxicity12,13,14,15. In this protocol, we outline a genetic method to deliver an optogenetic TDP-43 to ALS-vulnerable cell types, namely, spinal motor neurons in zebrafish using the BAC for the mnr2b/mnx2b gene encoding a homeodomain protein for motor neuron specification16,17. The high translucency of zebrafish larvae allows for simple, noninvasive light stimulation of the optogenetic TDP-43 that triggers its phase transition in the spinal motor neurons. We also present a basic workflow for the live imaging of the zebrafish spinal motor neurons and image analysis using the freely available Fiji/ImageJ software to characterize the responses of the optogenetic TDP-43 to the light stimulation. These methods allow for an investigation of TDP-43 phase transition in an ALS-vulnerable cellular environment and should help to explore its pathological consequences at cellular and behavioral levels.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>ZEISS</td>
			<td>Axioimager Z1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence stereomicroscope</td>
			<td>Leica</td>
			<td>MZ16FA</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass base dish</td>
			<td>IWAKI</td>
			<td>3910-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>MEE</td>
			<td>CN-25C</td>
			<td></td>
		</tr>
		<tr>
			<td>LED panel</td>
			<td>Nanoleaf Limited</td>
			<td>Nanoleaf AURORA smarter kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Mupid-2plus</td>
			<td>TAKARA</td>
			<td>AD110</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoBond BAC100</td>
			<td>MACHEREY-NAGEL</td>
			<td>740579</td>
			<td></td>
		</tr>
		<tr>
			<td>NuSieve GTG Agarose</td>
			<td>LONZA</td>
			<td>50181</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Olympus</td>
			<td>XLUMPlanFL N 20&#215;/1.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>ZEISS</td>
			<td>Plan-Neofluar 5x/0.15</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical power meter</td>
			<td>HIOKI</td>
			<td>3664</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical sensor</td>
			<td>HIOKI</td>
			<td>9742-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red solution 0.5%</td>
			<td>Merck</td>
			<td>P0290-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeSTAR GXL DNA Polymerase</td>
			<td>TAKARA</td>
			<td>R050A</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Six-well dish</td>
			<td>FALCON</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrometer probe BLUE-Wave</td>
			<td>StellerNet Inc.</td>
			<td>VIS-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe needle</td>
			<td>TERUMO</td>
			<td>NN-2725R</td>
			<td></td>
		</tr>
		<tr>
			<td>TaKaRa Ex Taq</td>
			<td>TAKARA</td>
			<td>RR001A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricane</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Zebrafish BAC clone CH211-172N16</td>
			<td>BACPAC Genomics</td>
			<td>CH211-172N16</td>
			<td></td>
		</tr>
	</tbody>
</table>

optogenetic phase transition of tdp-43 in spinal motor neurons of zebrafish larvae

Abnormal protein aggregation and selective neuronal vulnerability are two major hallmarks of neurodegenerative diseases. Causal relationships between these features may be interrogated by controlling the phase transition of a disease-associated protein in a vulnerable cell type, although this experimental approach has been limited so far. Here, we describe a protocol to induce phase transition of the RNA/DNA-binding protein TDP-43 in spinal motor neurons of zebrafish larvae for modeling cytoplasmic aggregation of TDP-43 occurring in degenerating motor neurons in amyotrophic lateral sclerosis (ALS). We describe a bacterial artificial chromosome (BAC)-based genetic method to deliver an optogenetic TDP-43 variant selectively to spinal motor neurons of zebrafish. The high translucency of zebrafish larvae allows for the phase transition of the optogenetic TDP-43 in the spinal motor neurons by a simple external illumination using a light-emitting diode (LED) against unrestrained fish. We also present a basic workflow of live imaging of the zebrafish spinal motor neurons and image analysis with freely available Fiji/ImageJ software to characterize responses of the optogenetic TDP-43 to the light illumination. This protocol enables the characterization of TDP-43 phase transition and aggregate formation in an ALS-vulnerable cellular environment, which should facilitate an investigation of its cellular and behavioral consequences.

Live imaging of optogenetic and non-optogenetic TDP-43 proteins in the mnr2b+ spinal motor neurons of zebrafish larvae 
To induce TDP-43 phase transition in the spinal motor neurons in zebrafish, a human TDP-43h that is tagged with mRFP1 and CRY2olig22 at the N- and C-termini, respectively, was constructed and designated as opTDP-43h14 (Figure 1A). The opTDP-43h gene fragment was ...

Watch this Scientific Journal Video about Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae at JoVE.com

Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae

We describe a protocol to induce phase transition of TAR DNA-binding protein 43 (TDP-43) by light in the spinal motor neurons using zebrafish as a model.

Abnormal protein aggregation and selective neuronal vulnerability are two major hallmarks of neurodegenerative diseases. Causal relationships between ...

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.

optogenetic-phase-transition-tdp-43-spinal-motor-neurons-zebrafish

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JoVE Journal

Neuroscience

5.8K Aufrufe.  Tokyo Medical University. Dieses Protokoll ermöglicht es uns also, pathologische Phasenübergänge von TDP-43 zu induzieren und ihre zellulären Folgen in den spinalen Motoneuronen in vivo zu adressieren, was für das Verständnis relevant ist, warum Motoneuronen bei ALS degenerieren. Durch die hohe Transparenz der Zebrafischlarven sind die Motoneuronen im Rückenmark direkt sichtbar. So kann man das dynamische Verhalten von TDP-43 visualisieren, das Phasenübergänge in einzelnen spinalen Motoneuronen durchläuft.