Amelioration of toxicity in neuronal models of amyotrophic lateral sclerosis by hUPF1

Abstract

Over 30% of patients with amyotrophic lateral sclerosis (ALS) exhibit cognitive deficits indicative of frontotemporal dementia (FTD), suggesting a common pathogenesis for both diseases. Consistent with this hypothesis, neuronal and glial inclusions rich in TDP43, an essential RNA-binding protein, are found in the majority of those with ALS and FTD, and mutations in TDP43 and a related RNAbinding protein, FUS, cause familial ALS and FTD. TDP43 and FUS affect the splicing of thousands of transcripts, in some cases triggering nonsense-mediated mRNA decay (NMD), a highly conserved RNA degradation pathway. Here, we take advantage of a faithful primary neuronal model of ALS and FTD to investigate and characterize the role of human up-frameshift protein 1 (hUPF1), an RNA helicase and master regulator of NMD, in these disorders. We show that hUPF1 significantly protects mammalian neurons from both TDP43-and FUS-related toxicity. Expression of hUPF2, another essential component of NMD, also improves survival, whereas inhibiting NMD prevents rescue by hUPF1, suggesting that hUPF1 acts through NMD to enhance survival. These studies emphasize the importance of RNA metabolism in ALS and FTD, and identify a uniquely effective therapeutic strategy for these disorders.A myotrophic lateral sclerosis (ALS) is a progressive and lethal motor neuron disease that most often arises sporadically, but can be inherited in 10-15% of patients (1). Many of the genes implicated in familial ALS (fALS) encode RNA-binding proteins, including fused in sarcoma (FUS), transactive response element DNA/RNA-binding protein of 43 kDa (TDP43), and heteronuclear ribonuclear proteins (hnRNPs) (2), emphasizing RNA-based toxicity as a fundamental mechanism contributing to motor neuron degeneration in ALS.More than 40 different mutations in the TDP43 gene (TARDBP) have now been associated with fALS (3). Disease-associated mutations in TARDBP affect the turnover (4), amount (5), and subcellular localization of TDP43 (6, 7), in many cases resulting in cytoplasmic TDP43 inclusions. Affected neurons in sporadic ALS (sALS) exhibit identical inclusions containing wild-type (WT) TDP43 (8), and WT TDP43 accumulation causes neurodegeneration in cellular and animal models (6, 9, 10), providing a pathogenic link between fALS and sALS. FUS mutations have also been linked to fALS (11,12). Unlike TDP43, FUS-related pathology is limited to fALS due to FUS mutations (13). FUS and TDP43 bind largely nonoverlapping RNA targets (14), leading to the unexpected conclusion that, despite their homology and involvement in fALS, TDP43 and FUS have distinct roles in RNA metabolism.TDP43 regulates its own expression through a negative feedback loop (15), but the mechanism by which it does so remains unclear. Conflicting data suggest that TDP43 autoregulation involves nonsense-mediated decay (NMD) (16) or exosome-mediated degradation (15). Excess TDP43 enhances splicing in the TARDBP 3′UTR, potentially targeting the transcript for NMD, whereas deficiencies in human up-frameshift prote...

DDX 3X and specific initiation factors modulate FMR 1 repeat‐associated non‐AUG‐initiated translation

“…To identify regulators of FMR1 RAN translation, we conducted a candidate-based screen using a Drosophila melanogaster model of FXTAS [66]. This model carries an EGFP transgene 3 0 to an upstream activation sequence (UAS) and the 5 0 UTR of human FMR1 with 90 CGG repeats, with EGFP in the GGC (+1, FMRpolyG) reading frame [(CGG) 90 -EGFP)]. The expression in the eye via a GMR-GAL4 driver manifests in a significant rough-eye phenotype observable at eclosion, with ubiquitin-positive aggregates of the RAN product FMRpolyG-EGFP accumulating in retinal neurons [14].…”

“…The expression in the eye via a GMR-GAL4 driver manifests in a significant rough-eye phenotype observable at eclosion, with ubiquitin-positive aggregates of the RAN product FMRpolyG-EGFP accumulating in retinal neurons [14]. For the screen, females expressing (CGG) 90 -EGFP under a GMR-GAL4 driver were crossed to males carrying germline mutations in, UAS-driven transgenes of, and UAS-driven shRNA constructs to 10 canonical eIFs, 4 RNA helicases, a ribosomal protein associated with non-canonical translation initiation [69,70], and an RNA-binding protein implicated in ALS/FTD [71] (Appendix Fig S1). We selected these candidates in a hypothesis-driven fashion based on their known functions in non-canonical translation initiation and regulation of start codon fidelity, as well their potential to modulate GC-rich secondary structures in RNA.…”

“…Of the 57 candidate lines tested, 21 acted as suppressors of toxicity, and 17 acted as enhancers (Fig 1, Appendix Fig S1). All lines were subsequently crossed to flies carrying GMR-GAL4 alone to control for toxic effects independent of (CGG) 90 -based toxicity (Appendix Fig S1). Six of the 17 toxicity-enhancing lines had no effect in the absence of (CGG) 90 -EGFP, suggesting this enhancement is specific to the presence of expanded CGG repeats.…”

“…All lines were subsequently crossed to flies carrying GMR-GAL4 alone to control for toxic effects independent of (CGG) 90 -based toxicity (Appendix Fig S1). Six of the 17 toxicity-enhancing lines had no effect in the absence of (CGG) 90 -EGFP, suggesting this enhancement is specific to the presence of expanded CGG repeats. We selected three suppressors for further analysis-belle (bel)/DDX3X, eIF4B, and eIF4H1-based on their individual functions in translation initiation.…”

RNA Degradation in Neurodegenerative Disease

“…Consistent with a potential link between NMD and ALS/FTD pathogenesis, overexpression of UPF1 or UPF2 prevents FUS- and TDP43-mediated neurodegeneration in model systems [312]. One possibility is that UPF1 overexpression in these models prevents cell death by boosting endogenous NMD, thereby enabling the pathway to properly metabolize an overabundance of NMD substrates.…”