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

Summary

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.

Abstract

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.

Introduction

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 phases^1,2. 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 diseases^3,4,5.....

Protocol

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 .......

Discussion

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.

Materials

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