Insights into C9ORF72-Related ALS/FTD from Drosophila and iPSC Models

Abstract

GGGGCC (G₄C₂) repeat expansion in C9ORF72 is the most common genetic cause of ALS and FTD. An important issue is how repeat RNAs and their translation products, various dipeptide repeat (DPR) proteins, cause neurodegeneration. Drosophila has been widely used to model G₄C₂ repeat RNA and DPR protein toxicity. Overexpression of disease molecules in flies has revealed important molecular insights. These have been validated and further explored in human neurons differentiated from induced pluripotent stem cells (iPSCs), a disease-relevant model in which expanded G₄C₂ repeats are expressed in their native molecular context. Approaches that combine the genetic power of Drosophila and the disease relevance of iPSC-derived patient neurons will continue to unravel the underlying pathogenic mechanisms and help identify potential therapeutic targets in C9ORF72-ALS/FTD.

Keywords: DNA damage; DPR protein; RAN translation; autophagy; nucleocytoplasmic transport; repeat expansion.

Figure 1. Schematic Representation of Different Drosophila Models of G₄C₂ Repeat Expansion

Major features of the DNA constructs used for each model are presented. In all Drosophila G₄C₂ models, the UAS-GAL4 system is used to overexpress G₄C₂ repeats with different lengthes, and these models share common SV40 3′UTR containing a polyadenylation signal. In the (G₄C₂)₃₀-EGFP model, repeats are interrupted in the middle by a six base pair sequence and followed by the EGFP coding region containing the ATG initiation codon. The (G₄C₂)₃₆ construct contains uninterrupted repeats, while the (G₄C₂)₃₆-RO construct harbors stop codons between every 12 repeats. The intronic (G₄C₂)₁₆₀ construct mimics the human C9ORF72 locus, as 160 copies of repeat sequence are flanked by C9ORF72 intronic sequences and adjacent exons. In the (G₄C₂)₅₈-GFP model, 58 copies of repeats are upstream to the GFP codon region but without the ATG start codon. The (G₄C₂)₄₈ construct contains stop codons in each frame 5′ to the repeat sequence. The three columns on the right list DPR proteins detected in these models when phenotypes are observed; the proposed mechanism of toxicity; and the respective references.

Figure 2. The Effect of Poly(A) Tail on the Subcellualr Localization and Toxicity of Expanded G₄C₂ Repeats

In (G₄C₂)_n-polyA models (top panel), repeat RNA is transported to the cytoplasm, where it is translated into DPR proteins. Diffuse G₄C₂ RNA is detected mostly in the cytoplasm, and higher toxicity correlate with high levels of DPR protein production. In intronic (G₄C₂)_n models (bottom panel), repeat RNA accumulates in the nucleus as RNA foci and less DPR protein is produced. Thus, only low levels of toxicity are observed.

Figure 3. Overview of the Nucleocytoplasmic Transport Defects in C9ORF72-ALS/FTD

This cartoon summarizes the identified nuclear cytoplasmic transport defects in Drosophila, yeast and C9ORF72 iPSC-derived neurons. G₄C₂ quadruplex RNA disrupts the RAN gradient and the nuclear import of proteins by binding to RanGAP, which catalyzes the conversion of Ran-GTP to Ran-GDP. Impairment of nuclear export caused by repeat RNA or DPR proteins leads to nuclear retention of RNA.

Figure 4. Insights from C9ORF72 Patient-Derived Human Neurons

Currently, patient-derived human neurons can be obtained by direct conversion of patient cells to neurons or by reprograming of the patient cells into induced pluripotent stem cells (iPSCs) followed by differentiation into neurons (iPSC-derived neurons). Several disease-relevant cellular phenotypes have been identified in studies of neurons derived from C9ORF72 patient. These include defects in autophagy and lysosome biogenesis, glutamate excitotoxicity, abnormalities in neuronal excitability, increased DNA damage, dysregulation of gene expression networks, vesicle trafficking defects, and nucleolar stress. In other instances, patient-derived neurons were used to confirm phenotypes observed first in other experimental systems. Examples include nucleocytoplasmic transport defects discovered in fly models, as well as pre-mRNA splicing and RNP granule transport defects and cell-to-cell transmission of di-peptide repeat (DPR) proteins discovered in other cell models.