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.