Dipeptide Repeat Proteins (DPRs) in C9orf72-ALS/FTD

Dipeptide Repeat Proteins (DPRs) in C9orf72-ALS/FTD

Overview

Dipeptide repeat proteins (DPRs) are aberrant protein products generated by repeat-associated non-AUG (RAN) translation of the expanded GGGGCC (G4C2) hexanucleotide repeat in the C9orf72 gene. This repeat expansion is the most common genetic cause of both ALS and FTD, accounting for approximately 40% of familial ALS and 25% of familial FTD cases. DPRs represent a gain-of-function toxicity mechanism distinct from the C9orf72 loss-of-function and RNA foci pathology also caused by the expansion.

Five distinct DPR species are produced from sense and antisense transcription of the repeat, each with different biochemical properties and toxicity profiles. Understanding DPR biology has revealed fundamental insights into nucleocytoplasmic transport, proteostasis, and selective neuronal vulnerability in ALS/FTD. [@ash2013]

RAN Translation Mechanism

Non-canonical Translation

RAN translation is a non-canonical form of protein synthesis that occurs without the standard AUG initiation codon: [@kwon2014]

```mermaid
graph TD
A["C9orf72 G4C2 Repeat Expansion<br/>(Hundreds to thousands of repeats)"]
A --> B["Sense Transcription (5'->3')"]
A --> C["Antisense Transcription (3'->5')"]

B --> D["Sense RNA<br/>(GGGGCC)n"]
C --> E["Antisense RNA<br/>(CCCCGG)n"]

D --> F["RNA G-quadruplex and<br/>Hairpin Structures"]
E --> G["RNA Secondary<br/>Structures"]

F --> H["RAN Translation<br/>Three Reading Frames"]
G --> I["RAN Translation<br/>Two Reading Frames"]

Dipeptide Repeat Proteins (DPRs) in C9orf72-ALS/FTD

Overview

Dipeptide repeat proteins (DPRs) are aberrant protein products generated by repeat-associated non-AUG (RAN) translation of the expanded GGGGCC (G4C2) hexanucleotide repeat in the C9orf72 gene. This repeat expansion is the most common genetic cause of both ALS and FTD, accounting for approximately 40% of familial ALS and 25% of familial FTD cases. DPRs represent a gain-of-function toxicity mechanism distinct from the C9orf72 loss-of-function and RNA foci pathology also caused by the expansion.

Five distinct DPR species are produced from sense and antisense transcription of the repeat, each with different biochemical properties and toxicity profiles. Understanding DPR biology has revealed fundamental insights into nucleocytoplasmic transport, proteostasis, and selective neuronal vulnerability in ALS/FTD. [@ash2013]

RAN Translation Mechanism

Non-canonical Translation

RAN translation is a non-canonical form of protein synthesis that occurs without the standard AUG initiation codon: [@kwon2014]

The Five DPR Species

Poly-GA (Glycine-Alanine) — Sense Strand

• Most abundant DPR species in patient tissue
• Forms p62-positive, TDP-43-negative cytoplasmic inclusions
• Aggregates via beta-sheet-rich amyloid structures
• Sequesters proteasome components (Unc119, HR23 proteins)
• Impairs proteasomal degradation and ER-associated degradation (ERAD)
• Moderate toxicity in model systems
• Can spread between cells in a prion-like manner

Poly-GP (Glycine-Proline) — Sense Strand

• Detectable in CSF of C9orf72-ALS/FTD patients — serves as a pharmacodynamic biomarker
• Relatively inert biochemically
• Forms cytoplasmic inclusions in neurons
• Low intrinsic toxicity in cellular and animal models
• Most useful as a readout for therapeutic reduction of DPR production (e.g., ASO trials)

Poly-GR (Glycine-Arginine) — Sense Strand

• Most toxic DPR species (along with poly-PR)
• Arginine-rich: interacts electrostatically with nucleic acids and acidic proteins
• Localizes to the nucleolus, disrupting ribosomal RNA processing
• Disrupts liquid-liquid phase separation of membraneless organelles
• Impairs nucleocytoplasmic transport by disrupting the nuclear pore complex
• Causes DNA damage by binding to chromatin
• Induces oxidative stress and mitochondrial dysfunction
• Directly binds ribosomes and impairs translation

Poly-PA (Proline-Alanine) — Antisense Strand

• Low abundance in patient tissue
• Minimal toxicity in most experimental systems
• Forms small cytoplasmic aggregates
• Least studied of the five DPR species

Poly-PR (Proline-Arginine) — Antisense Strand

• Highly toxic — comparable to poly-GR
• Arginine-rich: shares toxicity mechanisms with poly-GR
• Localizes to the nucleolus and disrupts ribosome biogenesis
• Disrupts heterochromatin organization
• Impairs stress granule dynamics
• Binds to and disrupts nuclear pore complex function
• Causes widespread splicing dysregulation

Toxicity Mechanisms

Nucleolar Stress and Ribosome Biogenesis

The arginine-rich DPRs (poly-GR and poly-PR) preferentially accumulate in nucleoli: [@freibaum2015]

• Displace nucleolar proteins (nucleophosmin/NPM1, nucleolin/NCL, fibrillarin/FBL)
• Impair rRNA transcription by RNA polymerase I
• Disrupt rRNA processing and ribosome assembly
• Lead to p53 stabilization and activation of the nucleolar stress response
• Reduce global translation capacity

Nucleocytoplasmic Transport Disruption

DPRs severely impair nuclear-cytoplasmic transport: [@jovii2015]

• Poly-GR and poly-PR bind FG-repeat nucleoporins in the nuclear pore complex
• Disruption of the Ran-GTP/GDP gradient across the nuclear envelope
• Impaired nuclear import of RNA-binding proteins including TDP-43 and FUS
• Defective mRNA export from the nucleus
• This mechanism directly links DPR toxicity to TDP-43 pathology — the defining feature of ALS

Phase Separation Disruption

Arginine-rich DPRs disrupt the biophysics of liquid-liquid phase separation: [@zu2013]

• Arginine residues engage in electrostatic and cation-pi interactions with RNA and aromatic residues
• This alters the material properties of condensates, converting them from liquid to gel-like states
• Affects stress granules, nucleoli, Cajal bodies, and splicing speckles
• Low-complexity domain proteins are particularly vulnerable to DPR-mediated disruption

DNA Damage

DPRs, particularly poly-GR, cause DNA damage: [@zhang2018]

• Poly-GR binds chromatin and interferes with DNA repair machinery
• Increased double-strand breaks detected in C9orf72/FTD neurons
• Impaired recruitment of ATM/ATR DNA damage response kinases
• Poly-GR interacts with and sequesters components of the DNA repair machinery
• Accumulation of R-loops (RNA:DNA hybrids) at repeat expansions

Proteostasis Disruption

DPRs impair multiple arms of the proteostasis network: [@chew2019]

• Poly-GA directly sequesters and inhibits the 26S proteasome
• DPR aggregates overwhelm the autophagy-lysosomal pathway
• VCP/p97-dependent clearance is impaired
• HSP70/HSP40 chaperone systems are titrated away by DPR aggregates
• Leads to secondary accumulation of other aggregation-prone proteins

DPR Spreading and Propagation

DPRs can propagate between cells through prion-like mechanisms: [@gendron2017]

• Poly-GA forms amyloid structures that can seed aggregation in recipient cells
• Cell-to-cell transfer via unconventional secretion and exosomes
• Trans-synaptic spreading may explain the anatomical progression of C9orf72-ALS/FTD
• Poly-GA inclusions recruit and template native poly-GA in a dose-dependent manner

Relationship to TDP-43 Pathology

DPR pathology and TDP-43 pathology are both present in C9orf72-ALS/FTD but show important differences: [@cook2020]

• DPR inclusions are largely TDP-43-negative and vice versa
• DPR pathology is widespread (cerebellum, hippocampus, neocortex) while TDP-43 pathology correlates better with neurodegeneration
• DPRs may initiate the disease cascade, with TDP-43 pathology as a downstream consequence
• Nucleocytoplasmic transport disruption by DPRs leads to cytoplasmic TDP-43 mislocalization
• TDP-43 loss-of-function then drives cryptic exon splicing and neuronal dysfunction

Animal and Cellular Models

Drosophila Models

• Expression of poly-GR or poly-PR causes severe neurodegeneration
• Eye and motor neuron phenotypes model ALS/FTD features
• Genetic screens identify modifiers of DPR toxicity

Mouse Models

• AAV-mediated expression of (G4C2)66 repeats produces DPR pathology
• Poly-GA transgenic mice develop p62/ubiquitin-positive inclusions
• BAC transgenic mice carrying the full C9orf72 expansion show all five DPRs
• Some models show TDP-43 pathology downstream of DPR accumulation

iPSC-Derived Neurons

• C9orf72-patient iPSC motor neurons show DPR accumulation
• Nucleocytoplasmic transport defects detectable in culture
• ASO treatment reduces DPR levels and rescues cellular phenotypes
• CRISPR correction of the repeat expansion eliminates DPR production

Therapeutic Targeting

Antisense Oligonucleotides (ASOs)

• ASOs targeting the sense strand C9orf72 repeat RNA reduce DPR production
• BIIB078 (tofersen-like ASO for C9orf72) was in clinical trials
• Poly-GP in CSF serves as a pharmacodynamic biomarker for ASO efficacy
• Challenge: antisense DPRs (poly-PA, poly-PR) require separate targeting

Small Molecule Approaches

• Compounds that bind G-quadruplex RNA structures to block RAN translation
• TMPyP4 and other G-quadruplex ligands reduce DPR production in cell models
• Metformin activates PKR, paradoxically reducing RAN translation
• Small molecules that enhance DPR clearance through autophagy

Gene Therapy

• CRISPR-based excision of the G4C2 repeat expansion
• Gene replacement strategies to maintain C9orf72 protein function
• RNA interference approaches targeting repeat-containing transcripts

Research Questions

See Also

• [C9orf72](/genes/c9orf72)
• [ALS](/diseases/amyotrophic-lateral-sclerosis)
• [Frontotemporal Dementia](/diseases/frontotemporal-dementia)
• [C9orf72 loss](/mechanisms/dopaminergic-neuron-vulnerability)
• [nucleocytoplasmic transport](/ideas/payload-nucleocytoplasmic-transport-modulation-therapy)
• [proteostasis](/mechanisms/proteostasis-network)
• [TDP](/proteins/tdp-43-protein)
• [proteasome](/experiments/proteasome-ubiquitin-system-dysfunction-parkinsons)
• [proteasomal degradation](/mechanisms/dopaminergic-neuron-vulnerability)
• [ER](/mechanisms/dopaminergic-neuron-vulnerability)

References