The nuclear ubiquitin ligase adaptor SPOP is a conserved regulator of C9orf72 dipeptide toxicity.

A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation of the C9orf72 repeat produces dipeptide repeat proteins (DPRs). Previously, we showed that the DPRs PR50 and GR50 are highly toxic when expressed in Caenorhabditis elegans, and this toxicity depends on nuclear localization of the DPR. In an unbiased genome-wide RNA interference (RNAi) screen for suppressors of PR50 toxicity, we identified 12 genes that consistently suppressed either the developmental arrest and/or paralysis phenotype evoked by PR50 expression. All of these genes have vertebrate homologs, and 7 of 12 contain predicted nuclear localization signals. One of these genes was spop-1, the C. elegans homolog of SPOP, a nuclear localized E3 ubiquitin ligase adaptor only found in metazoans. SPOP is also required for GR50 toxicity and functions in a genetic pathway that includes cul-3, which is the canonical E3 ligase partner for SPOP Genetic or pharmacological inhibition of SPOP in mammalian primary spinal cord motor neurons suppressed DPR toxicity without affecting DPR expression levels. Finally, we find that knockdown of bromodomain proteins in both C. elegans and mammalian neurons, which are known SPOP ubiquitination targets, suppresses the protective effect of SPOP inhibition. Together, these data suggest a model in which SPOP promotes the DPR-dependent ubiquitination and degradation of BRD proteins. We speculate the pharmacological manipulation of this pathway, which is currently underway for multiple cancer subtypes, could also represent an entry point for therapeutic intervention to treat C9orf72 FTD/ALS.

Measuring RAN Peptide Toxicity in C. elegans.

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.

Nuclear localized C9orf72-associated arginine-containing dipeptides exhibit age-dependent toxicity in C. elegans.

A hexanucleotide repeat expansion mutation in the C9orf72 gene represents a prevalent genetic cause of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Non-canonical translation of this repeat gives rise to several distinct dipeptide protein species that could play pathological roles in disease. Here, we show in the model system Caenorhabditis elegans that expression of the arginine-containing dipeptides, but not alanine-containing dipeptides, produces toxic phenotypes in multiple cellular contexts, including motor neurons. Expression of either (PR)50 or (GR)50 during development caused a highly penetrant developmental arrest, while post-developmental expression caused age-onset paralysis. Both (PR)50- and (GR)50-green fluorescent protein tagged dipeptides were present in the nucleus and nuclear localization was necessary and sufficient for their toxicity. Using an inducible expression system, we discovered that age-onset phenotypes caused by (PR)50 required both continual (PR)50 expression and an aged cellular environment. The toxicity of (PR)50 was modified by genetic mutations that uncouple physiological aging from chronological aging. However, these same mutations failed to modify the toxicity of (GR)50, suggesting that (PR)50 and (GR)50 exert their toxicity through partially distinct mechanism(s). Changing the rate of physiological aging also mitigates toxicity in other C. elegans models of ALS, suggesting that the (PR)50 dipeptide might engage similar toxicity mechanisms as other ALS disease-causing proteins.