Illuminating ALS Motor Neurons With Optogenetics in Zebrafish

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive degeneration of motor neurons in the brain and spinal cord. Spinal motor neurons align along the spinal cord length within the vertebral column, and extend long axons to connect with skeletal muscles covering the body surface. Due to this anatomy, spinal motor neurons are among the most difficult cells to observe in vivo. Larval zebrafish have transparent bodies that allow non-invasive visualization of whole cells of single spinal motor neurons, from somas to the neuromuscular synapses. This unique feature, combined with its amenability to genome editing, pharmacology, and optogenetics, enables functional analyses of ALS-associated proteins in the spinal motor neurons in vivo with subcellular resolution. Here, we review the zebrafish skeletal neuromuscular system and the optical methods used to study it. We then introduce a recently developed optogenetic zebrafish ALS model that uses light illumination to control oligomerization, phase transition and aggregation of the ALS-associated DNA/RNA-binding protein called TDP-43. Finally, we will discuss how this disease-in-a-fish ALS model can help solve key questions about ALS pathogenesis and lead to new ALS therapeutics.

Keywords: RNA metabolism; neurodegenarative disease; optogenetics; phase transition; protein aggregation.

FIGURE 1

Large and small populations of spinal motor neurons can be manipulated with the Gal4/UAS system in zebrafish. (A) Tg[mnr2b-hs:Gal4]; Tg[UAS:EGFP] larva at 5 day post-fertilization. In the Gal4 driver Tg[mnr2b-hs:Gal4], the Gal4FF transcription factor (Asakawa et al., 2008) is expressed from the bacterial artificial chromosome (BAC) transgene carrying the mnr2b locus (encoding the Mnx-type homeobox protein, which promotes motor neuron differentiation) and drives expression of a gene downstream of upstream activation sequence (UAS) in the most of the spinal motor neurons. (B) Schematic illustration of a transverse section of the middle trunk of a 5 day-old wild-type zebrafish larva, with the dorsal side up. Fast-twitch muscle and slow muscle are shown in gray and red, respectively. A CaP innervating the ventral myotome is shown in green. sc, spinal cord. nc, notochord. Illustration modified from Bello-Rojas et al. (2019). (C) Schematic illustration of a wild-type CaP innervating the ventral myotome, from lateral view. (D) Among the spinal motor neurons, CaPs (arrows) are selectively labeled in Tg[SAIG213A] Tg[UAS:EGFP] fish. Bars are 1 mm (A) and 20 μm (D).

FIGURE 2

Optogenetic induction of TDP-43 aggregates in in vivo spinal motor neurons. (A) Structures of optogenetic TDP-43. The human TDP-43 (top) consists of 414 amino acid residues subdivided into the N-terminal domain (NTD), 2 RNA recognition motifs (RRM1 and RRM2), and a C-terminus intrinsically disordered region (IDR). The optoTDP-43 (Mann et al., 2019; Zhang et al., 2019) and opTDP-43 (Asakawa et al., 2020) constructs carry the CRY2-modules at their N- and C-terminus, respectively. mCherry (237 aa), mRFP1 (225 aa), CRY2_PHR (498 aa), and CRY2olig (498 aa) not to scale with TDP-43. (B) An agarose-embedded zebrafish embryo expressing both EGFP and opTDP-43 in CaPs (Tg[SAIG213A] Tg[UAS:opTDP-43] Tg[UAS:EGFP] triple transgenic) is illuminated with a confocal blue laser light for 3 h, 28–31 h post-fertilization (hpf). (C) The total axonal length at 48 hpf was reduced in the CaP irradiated with the blue light (Blue light) compared to the CaP grown in the dark (Dark). The figure panels are adapted from Asakawa et al. (2020). (D) An unrestrained zebrafish larva expressing both opTDP-43 and non-optogenetic EGFP-TDP-43 was irradiated with blue LED light. The spinal motor column was scanned every 24 h for 3 days (from 48 to 120 hpf). The duration of the blue light illumination is indicated in blue letters. Horizontal dashed line demarcates the ventral limit of the spinal cord. The figure panels are adapted from the study by Asakawa et al. (2020). (E) Cytoplasmic opTDP-43 foci colocalize with non-optogenetic EGFP-TDP-43 (arrows) in the spinal motor neurons at 120 hpf in (D). BL, Blue light. (F) Schematic drawing of opTDP-43 protein. (G) Blue light illumination drives CRY2olig-dependent opTDP-43 oligomerization and aggregation. A short-term illumination induces the oligomerization of opTDP-43, whereas a long-term illumination causes cytoplasmic aggregation of opTDP-43. Non-optogenetic TDP-43 is incorporated into the opTDP-43 aggregates [e.g., EGFP-TDP-43 in (E)] possibly through IDR-mediated intermolecular interactions. Cytoplasmic opTDP-43 aggregates are partially positive for immunoreactivities against ubiquitin, phosphorylation at S409/S410, and classical stress granule components (G3BP and TIAL1) (Asakawa et al., 2020). The bars indicate 250 μm (B), 20 μm (C), 1 mm (D, left), 20 μm (D, panels), and 5 μm (E).