measuring-ran-peptide-toxicity-in-c-elegans

Measuring RAN Peptide Toxicity in <em>C. elegans</em>

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

University of Pittsburgh

University of Pittsburgh School of Medicine

Cells respond to elevated temperatures through a well-characterized heat-shock response that enables short-term survival, long-term adaptation and mitigation of macromolecular damage. New work reveals a cell non-autonomous layer of stress-response regulation between neurons and the gonad involving serotonin.

Stress signaling: serotonin spreads systemic stress.

Misfolded proteins accumulate and aggregate in neurodegenerative disease. The existence of these deposits reflects a derangement in the protein homeostasis machinery. Using a candidate gene screen, we report that loss of RAD-23 protects against the toxicity of proteins known to aggregate in amyotrophic lateral sclerosis. Loss of RAD-23 suppresses the locomotor deficit of Caenorhabditis elegans engineered to express mutTDP-43 or mutSOD1 and also protects against aging and proteotoxic insults. Knockdown of RAD-23 is further neuroprotective against the toxicity of SOD1 and TDP-43 expression in mammalian neurons. Biochemical investigation indicates that RAD-23 modifies mutTDP-43 and mutSOD1 abundance, solubility, and turnover in association with altering the ubiquitination status of these substrates. In human amyotrophic lateral sclerosis spinal cord, we find that RAD-23 abundance is increased and RAD-23 is mislocalized within motor neurons. We propose a novel pathophysiological function for RAD-23 in the stabilization of mutated proteins that cause neurodegeneration.

Loss of RAD-23 Protects Against Models of Motor Neuron Disease by Enhancing Mutant Protein Clearance.

An extensive proteostatic network comprised of molecular chaperones and protein clearance mechanisms functions collectively to preserve the integrity and resiliency of the proteome. The efficacy of this network deteriorates during aging, coinciding with many clinical manifestations, including protein aggregation diseases of the nervous system. A decline in proteostasis can be delayed through the activation of cytoprotective transcriptional responses, which are sensitive to environmental stress and internal metabolic and physiological cues. The homeodomain-interacting protein kinase (hipk) family members are conserved transcriptional co-factors that have been implicated in both genotoxic and metabolic stress responses from yeast to mammals. We demonstrate that constitutive expression of the sole Caenorhabditis elegans Hipk homolog, hpk-1, is sufficient to delay aging, preserve proteostasis, and promote stress resistance, while loss of hpk-1 is deleterious to these phenotypes. We show that HPK-1 preserves proteostasis and extends longevity through distinct but complementary genetic pathways defined by the heat shock transcription factor (HSF-1), and the target of rapamycin complex 1 (TORC1). We demonstrate that HPK-1 antagonizes sumoylation of HSF-1, a post-translational modification associated with reduced transcriptional activity in mammals. We show that inhibition of sumoylation by RNAi enhances HSF-1-dependent transcriptional induction of chaperones in response to heat shock. We find that hpk-1 is required for HSF-1 to induce molecular chaperones after thermal stress and enhances hormetic extension of longevity. We also show that HPK-1 is required in conjunction with HSF-1 for maintenance of proteostasis in the absence of thermal stress, protecting against the formation of polyglutamine (Q35::YFP) protein aggregates and associated locomotory toxicity. These functions of HPK-1/HSF-1 undergo rapid down-regulation once animals reach reproductive maturity. We show that HPK-1 fortifies proteostasis and extends longevity by an additional independent mechanism: induction of autophagy. HPK-1 is necessary for induction of autophagosome formation and autophagy gene expression in response to dietary restriction (DR) or inactivation of TORC1. The autophagy-stimulating transcription factors pha-4/FoxA and mxl-2/Mlx, but not hlh-30/TFEB or the nuclear hormone receptor nhr-62, are necessary for extended longevity resulting from HPK-1 overexpression. HPK-1 expression is itself induced by transcriptional mechanisms after nutritional stress, and post-transcriptional mechanisms in response to thermal stress. Collectively our results position HPK-1 at a central regulatory node upstream of the greater proteostatic network, acting at the transcriptional level by promoting protein folding via chaperone expression, and protein turnover via expression of autophagy genes. HPK-1 therefore provides a promising intervention point for pharmacological agents targeting the protein homeostasis system as a means of preserving robust longevity.

The homeodomain-interacting protein kinase HPK-1 preserves protein homeostasis and longevity through master regulatory control of the HSF-1 chaperone network and TORC1-restricted autophagy in Caenorhabditis 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.

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

The inappropriate genetic expansion of various repetitive DNA sequences underlies over 20 distinct inherited diseases. The genetic context of these repeats in exons, introns and untranslated regions has played a major role in thinking about the mechanisms by which various repeat expansions might cause disease. Repeat expansions in exons are thought to give rise to expanded toxic protein repeats (i.e. polyQ). Repeat expansions in introns and UTRs (i.e. FXTAS) are thought to produce aberrant repeat-bearing RNAs that interact with and sequester a wide variety of essential proteins, resulting in cellular toxicity. However, a new phenomenon termed 'repeat-associated nonAUG dependent (RAN) translation' paints a new and unifying picture of how distinct repeat expansion-bearing RNAs might act as substrates for this noncanonical form of translation, leading to the production of a wide range of repeat sequence-specific-encoded toxic proteins. Here, we review how the model system  has been utilized to model many repeat disorders and discuss how RAN translation could be a previously unappreciated contributor to the toxicity associated with these different models.

Models and mechanisms of repeat expansion disorders: a worm's eye view.

Expanded CAG nucleotide repeats are the underlying genetic cause of at least 14 incurable diseases, including Huntington's disease (HD). The toxicity associated with many CAG repeat expansions is thought to be due to the translation of the CAG repeat to create a polyQ protein, which forms toxic oligomers and aggregates. However, recent studies show that HD CAG repeats undergo a non-canonical form of translation called Repeat-associated non-AUG dependent (RAN) translation. RAN translation of the CAG sense and CUG anti-sense RNAs produces six distinct repeat peptides: polyalanine (polyAla, from both CAG and CUG repeats), polyserine (polySer), polyleucine (polyLeu), polycysteine (polyCys), and polyglutamine (polyGln). The toxic potential of individual CAG-derived RAN polypeptides is not well understood. We developed pure C. elegans protein models for each CAG RAN polypeptide using codon-varied expression constructs that preserve RAN protein sequence but eliminate repetitive CAG/CUG RNA. While all RAN polypeptides formed aggregates, only polyLeu was consistently toxic across multiple cell types. In GABAergic neurons, which exhibit significant neurodegeneration in HD patients, codon-varied (Leu)38, but not (Gln)38, caused substantial neurodegeneration and motility defects. Our studies provide the first in vivo evaluation of CAG-derived RAN polypeptides in a multicellular model organism and suggest that polyQ-independent mechanisms, such as RAN-translated polyLeu peptides, may have a significant pathological role in CAG repeat expansion disorders.

Todd Lamitina

Graduate Program in Cell Biology and Molecular Physiology

University of Pittsburgh

Measuring RAN Peptide Toxicity in C. elegans

Todd Lamitina

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Graduate Program in Cell Biology and Molecular Physiology

University of Pittsburgh

Measuring RAN Peptide Toxicity in C. elegans

Graduate Program in Cell Biology and Molecular Physiology