Measuring RAN Peptide Toxicity in C. elegans

Summary

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

Abstract

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington’s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Introduction

The inappropriate expansion of DNA repeat sequences is the genetic basis for several neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Huntington’s disease (HD)¹. While there are established cellular and animal models for these diseases, mechanisms underlying these conditions are not well defined. For example, HD is caused by the expansions of a CAG repeat sequence in the coding sequence for the Huntingtin protein Htt². Because CAG encodes the amino acid glutamine, the CAG repeat expansion results in the insertion of a polyGlutamine, or polyQ, sequence within....

Protocol

1. Generating codon-varied RAN peptide expression constructs

1. Design the individual RAN peptide coding sequence utilizing synonymous codons to eliminate the fundamental repetitive DNA/RNA structure but preserve the overlying amino acid sequence.
2. Order the custom codon sequences commercially at the repeat lengths needed for the studies (typically 5–100 repeats). Include a HindIII restriction site at the 5' end and a BamHI restriction site at the 3' end to facilitate cloning into C. elegans expression vectors, such as pPD95.79.
   NOTE: If synthesis of larger constructs proves difficult, smaller building block sequences ca....

Results

We used the assays described here to evaluate the effect of different gene inhibitions on the toxicity of RAN dipeptides that are found in ALS patients with a G₄C₂ repeat expansion. Using the growth assay to measure developmental toxicity, we analyzed the effects of several genetic knockout mutants identified in a genome-wide RNAi screen suppressors of muscle-expressed PR50-GFP toxicity. While expression of PR50-GFP alone resulted in a completely penetrant growth arrest, loss of function mutations i.......

Discussion

Here we report methods that can be used to assay RAN peptide toxicity modeled in the muscle or in the neurons of C. elegans. While neurodegenerative proteins have an age onset phenotype in human patients, they can also exhibit developmentally toxicity when overexpressed in model systems. Overexpression has significant interpretive limitations, but it also provides a powerful starting point for genetic or pharmacological screens aimed at identifying genes or drugs that can reverse toxic phenotypes. This is especi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35mm x 10mm Petri Dish, Sterile	CELLTREAT Scientific Products	50-202-036	Nematode growth plates and RNAi
AGAR GRANULATED 2KILOGRAM	BD DIAGNOSTIC SYSTEMS	DF0145070	Nematode growth plates and RNAi
AGAROSE ULTRAPURE	LIFE TECHNOLOGIES	16500500	Microinjection to generate RAN peptide transgenic strains
CARBENICILLIN 5G	THERMO SCI FAIRLAWN CHEMICALS	BP26485	Nematode growth plates and RNAi
COVER GLASSES NO 1 22MM 1OZ/PK	THERMO SCI ERIE	12542B	Imaging for commissure assay
FEMOTIPS DISPSBL MICROINJ 20CS	EPPENDORF NORTH AMERICA BIOTOOLS	E5242952008	Microinjection to generate RAN peptide transgenic strains
FF COV GLASS NO1 40X22MM 1OZPK	THERMO SCI ERIE	125485C	Microinjection to generate RAN peptide transgenic strains
Fisherbrand Superfrost Plus Microscope Slides	THERMO SCI ERIE	12-550-15	Imaging for commissure assay
Gibco Bacto Peptone	Gibco	DF0118-17-0	Nematode growth plates and RNAi
HALOCARBON OIL 700	SIGMA-ALDRICH INC	H8898-50ML	Microinjection to generate RAN peptide transgenic strains
IPTG BIOTECH 10G	THERMO SCI FAIRLAWN CHEMICALS	BP162010	Nematode growth plates and RNAi
Leica Advanced Fluorescence imaging software	Leica Microsystems	LAS-AF	Image acquisition software for video speed analysis and commissure assay
Leica Immersion type N (Oil)	W NUHSBAUM INC	NC9547002	Imaging for commissure assay
LEVAMISOLE HYDROCHLORIDE 10GR	THERMO SCI ACROS ORGANICS	AC187870100	Imaging for commissure assay
MICROLOADER TIPS 2 X 96 PCS	EPPENDORF NORTH AMERICA BIOTOOLS	E5242956003	Microinjection to generate RAN peptide transgenic strains
PETRI DISH, 60X15MM,500/CS	CORNING LIFE SCIENCES PLASTIC	FB0875713A	Nematode growth plates and RNAi
TISSUE CULT PLATE 24WEL 50/CS	CORNING LIFE SCIENCES DL	87721	Nematode growth plates and RNAi