Characterization of HNRNPA1 mutations defines diversity in pathogenic mechanisms and clinical presentation
Danique Beijer, Hong Joo Kim, Lin Guo, Kevin O’Donovan, Inès Mademan, Tine Deconinck, Kristof Van Schil, Charlotte M. Fare, Lauren E. Drake, Alice F. Ford, Andrzej Kochański, Dagmara Kabzińska, Nicolas Dubuisson, Peter Van den Bergh, Nicol C. Voermans, Richard J.L.F. Lemmers, Silvère M. van der Maarel, Devon Bonner, Jacinda B. Sampson, Matthew T. Wheeler, Anahit Mehrabyan, Steven Palmer, Peter De Jonghe, James Shorter, J. Paul Taylor, Jonathan Baets
Danique Beijer, Hong Joo Kim, Lin Guo, Kevin O’Donovan, Inès Mademan, Tine Deconinck, Kristof Van Schil, Charlotte M. Fare, Lauren E. Drake, Alice F. Ford, Andrzej Kochański, Dagmara Kabzińska, Nicolas Dubuisson, Peter Van den Bergh, Nicol C. Voermans, Richard J.L.F. Lemmers, Silvère M. van der Maarel, Devon Bonner, Jacinda B. Sampson, Matthew T. Wheeler, Anahit Mehrabyan, Steven Palmer, Peter De Jonghe, James Shorter, J. Paul Taylor, Jonathan Baets
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Research Article Genetics Neuroscience

Characterization of HNRNPA1 mutations defines diversity in pathogenic mechanisms and clinical presentation

Abstract

Mutations in HNRNPA1 encoding heterogeneous nuclear ribonucleoprotein (hnRNP) A1 are a rare cause of amyotrophic lateral sclerosis (ALS) and multisystem proteinopathy (MSP). hnRNPA1 is part of the group of RNA-binding proteins (RBPs) that assemble with RNA to form RNPs. hnRNPs are concentrated in the nucleus and function in pre-mRNA splicing, mRNA stability, and the regulation of transcription and translation. During stress, hnRNPs, mRNA, and other RBPs condense in the cytoplasm to form stress granules (SGs). SGs are implicated in the pathogenesis of (neuro-)degenerative diseases, including ALS and inclusion body myopathy (IBM). Mutations in RBPs that affect SG biology, including FUS, TDP-43, hnRNPA1, hnRNPA2B1, and TIA1, underlie ALS, IBM, and other neurodegenerative diseases. Here, we characterize 4 potentially novel HNRNPA1 mutations (yielding 3 protein variants: *321Eext*6, *321Qext*6, and G304Nfs*3) and 2 known HNRNPA1 mutations (P288A and D262V), previously connected to ALS and MSP, in a broad spectrum of patients with hereditary motor neuropathy, ALS, and myopathy. We establish that the mutations can have different effects on hnRNPA1 fibrillization, liquid-liquid phase separation, and SG dynamics. P288A accelerated fibrillization and decelerated SG disassembly, whereas *321Eext*6 had no effect on fibrillization but decelerated SG disassembly. By contrast, G304Nfs*3 decelerated fibrillization and impaired liquid phase separation. Our findings suggest different underlying pathomechanisms for HNRNPA1 mutations with a possible link to clinical phenotypes.

Authors

Danique Beijer, Hong Joo Kim, Lin Guo, Kevin O’Donovan, Inès Mademan, Tine Deconinck, Kristof Van Schil, Charlotte M. Fare, Lauren E. Drake, Alice F. Ford, Andrzej Kochański, Dagmara Kabzińska, Nicolas Dubuisson, Peter Van den Bergh, Nicol C. Voermans, Richard J.L.F. Lemmers, Silvère M. van der Maarel, Devon Bonner, Jacinda B. Sampson, Matthew T. Wheeler, Anahit Mehrabyan, Steven Palmer, Peter De Jonghe, James Shorter, J. Paul Taylor, Jonathan Baets

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Figure 5

Localization and stress granule dynamics of the hnRNPA1 mutants.

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Localization and stress granule dynamics of the hnRNPA1 mutants. (A and ...
(A and B) HeLa cells were transiently transfected with WT or mutant EGFP-tagged hnRNPA1 and subjected to arsenite stress (0.5 mM sodium arsenite, 30 minutes). Cells were fixed and stained with eIF3η (red) and DAPI (blue). Confocal images were taken for partition coefficient analysis. Scale bar: 10 μm (original magnification, ×10 for the insets). Error bars represent mean ± SD (n = 55, 43, 40, and 58 cells for WT, P288A, G304Nfs*3, and *321Eext*6, respectively). ****P < 0.0001, by ordinary 1-way ANOVA with Dunnett’s multiple comparisons test. (C) U2OS cells expressing tdTomato-tagged endogenous G3BP1 were transiently transfected with WT or mutant EGFP-tagged hnRNPA1 and subjected to heat shock (43°C, 30 minutes; orange shading) and allowed to recover at 37°C for 2 hours. Line graph represents the percentage of cells with visible tdTomato-G3BP1 puncta over time. Error bars represent mean ± SEM (n = 10, 17, 23, and 18 videos for WT, P288A, G304Nfs*3, and *321Eext*6, respectively). Blue shaded area indicates time points at which P288A mutant was statistically significantly different from WT. **P < 0.01 by 2-way ANOVA with Dunnett’s multiple comparisons test. (D and E) U2OS cells expressing tdTomato-tagged endogenous G3BP1 were transiently transfected as in C and subjected to heat shock (43°C, 60 minutes; orange shading) and allowed to recover at 37°C for 90 minutes. White dotted lines (D) delineate hnRNPA1-positive cells; scale bar indicates 10 μm. Line graph (E) represents the percentage of cells with visible tdTomato-G3BP1 puncta over time (n = 56, 34, 48, and 35 videos for WT, P288A, G304Nfs*3, and *321Eext*6, respectively). Blue shaded area indicates time points at which each mutant was statistically significantly different from WT. *P < 0.05, **P < 0.01 (colors correspond to the respective mutants), by 2-way ANOVA with Dunnett’s multiple comparisons test.