Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS

Abstract

Algorithms designed to identify canonical yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbour a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here we define pathogenic mutations in PrLDs of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and in one case of familial amyotrophic lateral sclerosis. Wild-type hnRNPA2 (the most abundant isoform of hnRNPA2B1) and hnRNPA1 show an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a 'steric zipper' motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant steric zipper motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.

Figure 1. Identification of novel disease mutations in MSP and ALS

a. Family 1 pedigree indicating individuals affected by dementia, myopathy, PDB, and ALS. The causative mutation was p.D290V/302V in hnRNPA2B1. b. Family 2 pedigree indicating individuals affected by myopathy and PDB. The causative mutation was p.D262/314V in hnRNPA1. c. The pedigree of a family with ALS. The causative mutation was p.D262/314N in hnRNPA1. d–e. Sequence alignment of hnRNPA2/B1 (d) and hnRNPA1 (e) orthologs showing evolutionary conservation of the mutated aspartate and surrounding residues. f. Sequence alignment of 4 human paralogs of the hnRNP A/B family in which the disease-affected residue and surrounding residues are highly conserved.

Figure 2. Cytoplasmic pathology of hnRNPA2/B1 and hnRNPA1

a–d. Immunohistochemical analysis of hnRNPA2B1 (red) in normal muscle (a), muscle biopsy from patient II5 from family 1 (b), a patient with MSP caused by VCP mutation (R155H) (c), a patient with sporadic IBM (d). hnRNPA2/B1 (red) was cleared from DAPI-stained nuclei (blue) and accumulated in cytoplasmic inclusions (b–d). e–g. Immunohistochemical analysis of hnRNPA1 (red) in a normal muscle (e), a muscle biopsy from patient IV9 from family 2 (f), a patient with MSP caused by VCP mutation (R155H) (g). hnRNPA1 was cleared from nuclei and accumulated in cytoplasmic inclusions (f, g). h. In muscle tissue from patient IV9 (family 2), hnRNPA2/B1 was cleared from nuclei and accumulated in cytoplasmic inclusions. (i–l) Immunohistochemical analysis of TDP-43 (red) in a normal muscle (i), muscle biopsy from patient II5 from family 1 (j), muscle biopsy from patient IV9 from family 2 (k), and a patient with MSP caused by VCP mutation (R155H) (l). TDP-43 (red) was cleared from DAPI-stained nuclei (blue) and accumulated in cytoplasmic inclusions (j–l). m–p. Immunohistochemical analysis of hnRNPA2/B1 and TDP-43 colocalization in a patient with sporadic IBM. DAPI (m), TDP-43 (n), hnRNPA2/B1 (o) and merged images (p) are shown. hnRNPA2/B1 (red) and TDP-43 (green) were cleared from nuclei (arrowheads) in an atrophic muscle fiber, but found in nuclei of neighboring unaffected fibers (arrows). Scale bars represent 50 µM.

Figure 3. The disease mutations impact a PrLD in hnRNPA2/B1 and hnRNPA1

a–b. FoldIndex predicts an extended intrinsically unfolded regions (grey curve less than zero) in the C-termini of hnRNPA2 and hnRNPA1. These regions were also predicted to be prion-like according to the algorithm of Alberti et al (red curve less than zero), and narrowly missed the cutoff for the prion propensity by algorithm of Toombs et al (green curve below the dashed green line). All curves represent averages of 41 consecutive windows of 41 amino acids, corresponding to the criteria of Toombs et al The disease mutations were predicted to make these domains more prionogenic (insets). c–d. ZipperDB detected 6-amino-acid stretches (underlined in e–f) within the core PrLDs for which the disease mutations increased the predicted amyloid fibril–forming potential beyond the Rosetta threshold. e–f. Domain architecture of hnRNPA2 and hnRNPA1 shows the RNA-recognition motifs (RRM1 and RRM2), the C-terminal glycine-rich domain, and an M9 nuclear localization signal. The PrLDs are centered in the C-terminal glycine-rich domain. Highly similar predictions were made for the minor isoforms of hnRNPA2B1 (hnRNPB1) and hnRNPA1 (hnRNPA1 isoform b).

Figure 4. Disease mutations accelerate hnRNPA2 and hnRNPA1 fibrillization

a. Synthetic hexapeptides A2 wild-type (NYNDFG) or mutant (NYNVFG) were incubated at 25°C for 2h. Fibrillization was monitored by ThT fluorescence. b. EM of A2 wild-type or mutant hexapeptides after 10min at 25°C. Bar, 0.1µm. c–d. Fibrillization analysis of A1 wild-type (SYNDFG) or mutant (SYNVFG) as in (a–b). e. Full-length hnRNPA2 WT, hnRNPA2 D290V, or hnRNP2^Δ287–292 was incubated at 25 °C with agitation for 0–12h. At various times, the amount of aggregated hnRNPA2 was determined. Values represent means ± SEM (n=3). f. EM of hnRNPA2 fibrillization reactions after 0, 4, and 12h at 25°C. Note the absence of fibers after 4h for hnRNPA2 wild-type. Bar, 0.5µm. g–h. Fibrillization of full-length hnRNPA1 wild-type, hnRNPA1-D262V, hnRNPA1-D262N, or hnRNPA1^Δ259–264 monitored as in (e, f).

Figure 5. hnRNPA2 recruitment to SGs is accelerated by disease mutation

a–b. HeLa cells were transfected with Flag-tagged wild-type or mutant hnRNPA2 and stained with anti-Flag (green), anti-eIF4G (red), and DAPI (blue). Arrows indicate hnRNPA2- and eIF4G–positive SGs. b. HeLa cells were transfected as in (a), treated with 0.5 mM sodium arsenite for the indicated time, and immunostained as in (a). The percentage of cells displaying hnRNPA2-positive SGs at indicated time points following treatment with arsenite cells are plotted. Data represent mean ± SEM (n=3) (*** P<0.001). c. HeLa cells were transfected and stimulated as in (b), and sequentially extracted with RIPA and urea buffer. Immunoblotting was conducted with anti-Flag and anti-TDP-43 antibodies.

Figure 6. Mutant hnRNPA2 forms cytoplasmic inclusions in Drosophila

a. Adult flies were dissected to expose the dorsal longitudinal indirect flight muscle and stained with Texas Red-phalloidin (red), and DAPI (blue). Flies expressing human wild-type hnRNPA2 under control of the MHC-GAL4 driver showed mild degeneration, whereas flies expressing mutant human hnRNPA2 show severe degeneration affecting all muscles. Flies expressing hnRNPA2^Δ287–292 show muscle histology similar to flies expressing wild-type hnRNPA2. b. wild-type hnRNPA2 localizes exclusively to nuclei, whereas hnRNPA2-D290V also accumulates extensively in cytoplasmic inclusions. hnRNPA2^Δ287–292 localizes exclusively to nuclei. c. Thoraces of adult flies were dissected and sequential extractions were performed to examine the solubility profile of hnRNPA2. d. Quantification of the blot shown in c. Data represent mean ± SEM (n=3) (*P < 0.05, ***P < 0.001 by two-way ANOVA with Bonferroni’s post hoc test).