Abstract
A GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce five dipeptide repeat proteins (DPRs). Although DPRs are thought to be neurotoxic, the molecular mechanism underlying the DPR-caused neurotoxicity has not been fully elucidated. The current study shows that poly-proline-arginine (poly-PR), the most toxic DPR in vitro, binds to and up-regulates nuclear paraspeckle assembly transcript 1 (NEAT1) that plays an essential role as a scaffold non-coding RNA during the paraspeckle formation. The CRISPR-assisted up-regulation of endogenous NEAT1 causes neurotoxicity. We also show that the poly-PR modulates the function of several paraspeckle-localizing heterogeneous nuclear ribonucleoproteins. Furthermore, dysregulated expression of TAR DNA-binding protein 43 (TDP-43) up-regulates NEAT1 expression and induces neurotoxicity. These results suggest that the increase in the paraspeckle formation may be involved in the poly-PR- and TDP-43-mediated neurotoxicity.
Highlights
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that selectively affects motor neurons[1,2]
In the current study, using glutathione S-transferase (GST) pull-down assay with GSTFLAG-tagged PR100 as a bait, we confirmed the interaction between poly-PR and endogenous or exogenous paraspeckle proteins, hnRNPF, hnRNPH1, hnRNPM, SFPQ, NONO, family with sequence similarity 98 member A (FAM98A), RNAbinding motif protein 14 (RBM14), and MATR314,16,38 (Fig. 1a, c, and S2a)
Immunocytochemical analysis showed that exogenous hnRNPF, hnRNPH1, and hnRNPM co-localized with nucleoplasm-localizing EGFPFLAG-PR100 (Fig. 1b, right panel, arrows in profile images, and Fig. S3a–c) and formed ring-like structure around poly-PR aggregates that likely correspond to the nucleolus[10] (Fig. 1b, right panel, arrowheads in profile images, and Fig. S3a–c)
Summary
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that selectively affects motor neurons[1,2]. It has been recognized that they share common pathological hallmarks, such as TAR DNA-binding protein 43 (TDP-43) aggregation[1,5], the molecular mechanism underlying neurodegeneration in ALS/FTD has not been fully elucidated. It has been reported that paraspeckles participate in RNA metabolism[18,19,20,21,22,23], the precise physiological function of paraspeckles remains unknown. It should be Official journal of the Cell Death Differentiation Association
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