Staufen2 dysregulation in neurodegenerative disease

Abstract

Staufen2 (STAU2) is an RNA-binding protein that controls mRNA trafficking and expression. Previously, we showed that its paralog, Staufen1 (STAU1), was overabundant in cellular and mouse models of neurodegenerative diseases and amyotrophic lateral sclerosis (ALS) patient spinal cord. Here, we investigated features of STAU2 that might parallel STAU1. STAU2 protein, but not mRNA, was overabundant in spinocerebellar ataxia type 2 (SCA2), ALS/frontotemporal dementia patient fibroblasts, ALS patient spinal cord tissues, and in central nervous system tissues from SCA2 and ALS animal models. Exogenous expression of STAU2 in human embryonic kidney 293 cells activated mechanistic target of rapamycin (mTOR) and stress granule formation. Targeting STAU2 by RNAi normalized mTOR in SCA2 and C9ORF72 cellular models. The microRNA miR-217, previously identified as downregulated in SCA2 mice, targets the STAU2 3'-UTR. We now demonstrate that exogenous expression of miR-217 significantly reduced STAU2 and mTOR levels in cellular models of neurodegenerative disease. These results suggest a functional link between STAU2 and mTOR signaling and identify a major role for miR-217 that could be exploited in therapeutic development.

Keywords: RNA-binding protein; Staufen; autophagy; neurodegeneration; neurodegenerative diseases and ataxia.

Figure 1

Increased STAU2 levels and abnormal mTOR signaling in NDD patient cell lines.A, characterization of the STAU2 antibody. HEK293 cells were transfected with an STAU2 siRNA or a scrambled control siRNA (siCont) and analyzed 4 days post-transfection by Western blotting. STAU2 bands of 62 and ∼64 kDa were observed, which were reduced by the STAU2 siRNA but not siCont, indicating antibody specificity. B–F, Western blot analysis of protein extracts from CRISPR–Cas9 edited HEK293-ATXN2-Q58 KI cells (B), FBs from individuals with SCA2 mutations (three with Q35, Q42, and Q45 repeats) (C), and ALS–FTD patients with C9ORF72 expansions (D). All show increased levels of STAU2, mTOR, P-mTOR, p62, and LC3-II compared with controls. Quantified STAU2 and mTOR average fold changes are shown in E and F. G, STAU2 mRNA levels are unaltered in-patient cell lines. qRT–PCR analyses of RNA extracts from the SCA2 and C9ORF72 FB cells (C and D). H and I, spinal cord tissues from patients with C9ORF72 expansions and sALS showing increased STAU2 and mTOR levels compared with non-ALS controls (NC) on Western blots (H). Quantification of STAU2 and mTOR levels on Western blots (I). Each lane represents an individual patient’s cell line or tissue (C, D, and H). ACTB was used as a loading control, and the representative blots of three independent experiments are shown. Two-way ANOVA followed by Bonferroni tests for multiple comparisons. Data are mean ± SD, ns, p > 0.05; ∗∗∗p < 0.001. ALS, amyotrophic lateral sclerosis; FB, fibroblast; FTD, frontotemporal dementia; HEK293, human embryonic kidney 293 cell line; KI, knock-in; mTOR, mechanistic target of rapamycin; NDD, neurodegenerative disease; ns, not significant; qRT–PCR, quantitative RT–PCR; sALS, sporadic amyotrophic lateral sclerosis; STAU2, Staufen2.

Figure 2

Central nervous system tissues from mice transgenic for ATXN2[Q127] or BAC-C9ORF72 have increased STAU2 and mTOR levels. Western blot analyses of protein extracts from cerebella of ATXN2[Q127] mice (16 weeks of age) (A and B) and cerebral hemisphere (C and D) of BAC-C9ORF72 mice (16 weeks of age) show increased STAU2, mTOR, P-mTOR, p62, and LC3-II levels compared with WT controls. E and F, protein extracts from frontal region of cerebral hemisphere of BAC-C9ORF72 mice (16 weeks of age) show increased STAU2 levels compared with WT controls on Western blot. Each lane represents an individual mouse, and three animals per group were analyzed. Protein levels were normalized to ACTB, and quantified average fold changes for STAU2 and/or mTOR are shown in B, D, and F. Blots are from three/four technical replicate experiments. G and H, Stau2 and mTor RNA levels are unaltered in neurodegenerative disease tissues. qRT–PCR analyses of Stau2 and mTor mRNAs from cerebella from ATXN2[Q127] mice (8 and 16 weeks of age; three animals per group) (G) and cerebral hemisphere from BAC-C9ORF72 mice (12 and 16 weeks of age, four animals per group) (H) compared with WT littermates. Gene expression levels were normalized to Actb. Two-way ANOVA followed by Bonferroni tests for multiple comparisons. Data are mean ± SD, ns, p > 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. ACTB, β-actin; mTOR, mechanistic target of rapamycin; ns, not significant; P-mTOR, phosphor-mTOR; qRT–PCR, quantitative RT–PCR; STAU2, Staufen2.

Figure 3

Overexpression of STAU2 induces mTOR abundance, whereas lowering STAU2 reduces mTOR levels in SCA2 and ALS–FTD cellular models.A, HEK293 cells exogenously expressing GFP-tagged STAU2 (STAU2-GFP) or GFP were analyzed 48 h post-transfection by Western blotting and showed increased levels of mTOR, P-mTOR, p62, and LC3-II. B, mTOR mRNA levels, determined by qRT–PCR, remained unchanged in cells overexpressing STAU2-GFP compared with GFP controls. RNA expression levels were normalized to ACTB. Data are mean ± SD, ns = p > 0.05, Student's t test. C, ectopic expression of STAU2 leads to formation of cytoplasmic SG-like aggregates. U2OS cells were transfected with STAU2-GFP or GFP plasmids for 48 h followed by immunostaining with the SG marker protein G3BP1. Cells expressing STAU2-GFP (lower panel), but not GFP (upper panel), form spontaneous cytoplasmic SG-like aggregates positive for G3BP1 (merged image, yellow). Scale bar represents zoom in and out 10 and 20 μM, respectively. D, quantifications of STAU2 colocalizations with G3BP1 are shown. Fifty GFP-transfected cells (control) of 176 cells and 31 STAU2-GFP-transfected cells of 105 cells were used for analysis. Data are mean ± SD, ∗∗∗p < 0.001, Student’s t test. E–H, lowering STAU2 levels by RNAi reduces mTOR activation in SCA2 and ALS–FTD cellular models. HEK293-ATXN2-Q58 KI SCA2 cells (E) or ALS–FTD-C9ORF72 FBs (C9-1 and C9-2) (F) were transfected with STAU2 siRNA for 4 days and analyzed by Western blotting. Reducing STAU2 levels in both SCA2 and ALS–FTD cells resulted in decreased levels of mTOR, P-mTOR, p62, and LC3-II compared with cells treated with control siRNA, indicating restored autophagy activity. ACTB was used as a loading control, and quantification of protein levels (E and F) is shown in G and H. The blots are from three replicate experiments. Two-way ANOVA followed by Bonferroni tests for multiple comparisons. Data are mean ± SD, ns, p > 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. I, model for STAU2 in the pathology of SCA2 and other neurodegenerative diseases. ACTB, β-actin; ALS, amyotrophic lateral sclerosis; FB, fibroblast; FTD, frontotemporal dementia; HEK293, human embryonic kidney 293 cell line; mTOR, mechanistic target of rapamycin; ns, not significant; qRT–PCR, quantitative RT–PCR; SCA2, spinocerebellar ataxia type 2; SG, stress granule; STAU2, Staufen2.

Figure 4

Evaluation of miR-217 abundance and its regulatory function on its predicted target STAU2.A–D, miR-217 abundances are decreased in SCA2 and ALS–FTD models. qRT–PCR analyses of cerebella from BAC-ATXN2[Q72] (16 weeks of age; n = 4) and ATXN2[Q127] (14 weeks of age; n = 4) mouse cerebella show decreased miR-217 levels compared with WT littermates (A). qRT–PCR analyses of CRISPR–Cas9 edited HEK293-ATXN2-Q58 KI cells versus isogenic control cells (B), patient-derived fibroblasts (FBs) including three lines from SCA2 patients with the indicated repeat expansions in ATXN2 (Q35, Q42, and Q45 repeats) and three lines from ALS–FTD patients with C9ORF72 expansions versus five normal control (NC) FBs (C), and cerebral hemisphere from BAC-C9ORF72 mice versus WT littermates (16 weeks of age; n = 4) (D). All show significantly decreased miR-217 levels compared with controls. Gene expression levels were normalized to U6 snRNA. E–G, miR-217 reduces STAU2 expression. HEK293 cells were transfected with miR-217 mimic at indicated dosages for 4 days and were analyzed by qRT–PCR and Western blotting. Expression of miR-217 mimic results in reduction of STAU2 and FOXO3 (known miR-217 target) transcripts (E) and STAU2 protein levels (F). Gene expression (E)/protein (F) levels were normalized to ACTB/ACTB, and quantified STAU2 protein levels are shown in G. Blots are from three replicate experiments. H–J, confirmation of miRNA binding to STAU2-3′UTR. H and I, depicted are natural (WT) LUC-STAU2-3′UTR (LUC-STAU2-3′UTR^WT) and predicted miR-217 target site sequence according to TargetScan database. The mutant (Mt) construct (LUC-STAU2-3′UTR^Mt) with the same STAU2-3′UTR sequence except for mutated miR-217-binding site is described in the Experimental procedures section. J, following transient cotransfection of luciferase constructs, miR-217 mimic, and Renilla luciferase plasmid in HEK293 cells, luciferase activity assays revealed that the miR-217 mimic lowered the expression of luciferase in a dose-dependent manner for WT but not for mutant STAU2-3′UTR construct. Two-way ANOVA followed by Bonferroni tests for multiple comparisons (A, C, E, G, and J) and two-tailed unpaired Student's t tests (B and D). Data are mean ± SD, ns, p > 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. ALS, amyotrophic lateral sclerosis; FTD, frontotemporal dementia; HEK293, human embryonic kidney 293 cell line; ns, not significant; qRT–PCR, quantitative RT–PCR; STAU2, Staufen2; SCA2, spinocerebellar ataxia type 2.

Figure 5

Targeting STAU2 by miR-217 expression reduces mTOR levels in SCA2 and ALS–FTD cellular models.A, schematic depiction of the expression cassettes of the AAV vector plasmids. The AAV vector contains a GFP cassette under control of the CMV promoter and miR-217 sequence under U6 promoter. B and C, HEK293-ATXN2-Q58 KI cells (B) and ALS–FTD-C9ORF72 FBs (C9-1 and C9-2) (C) were transduced with control or miR-217 AAV particles for 5 days and analyzed by Western blotting. In both cases, miR-217 expression results in decreased STAU2 levels, as well as decreased mTOR, P-mTOR, p62, and LC3-II levels, indicating restored autophagy activity. ACTB was used as a loading control, and quantification of protein levels (B and C) is shown in D and E. The blots are from three replicate experiments. Two-way ANOVA followed by Bonferroni tests for multiple comparisons. Data are mean ± SD, ns, p > 0.05; ∗∗∗p < 0.001. AAV, adeno-associated virus; ACTB, β-actin; ALS, amyotrophic lateral sclerosis; CMV, cytomegalo virus; FB, fibroblast; FTD, frontotemporal dementia; HEK293, human embryonic kidney 293 cell line; mTOR, mechanistic target of rapamycin; ns, not significant; P-mTOR; phospho-mTOR; SCA2, spinocerebellar ataxia type 2; STAU2, Staufen2.