Reduced hn RNPA 3 increases C9orf72 repeat RNA levels and dipeptide‐repeat protein deposition
2016; Springer Nature; Volume: 17; Issue: 9 Linguagem: Inglês
10.15252/embr.201541724
ISSN1469-3178
AutoresKohji Mori, Yoshihiro Nihei, Thomas Arzberger, Qihui Zhou, Ian R. Mackenzie, Andreas Hermann, Frank Hanisch, Frits Kamp, Brigitte Nuscher, Denise Orozco, Dieter Edbauer, Christian Haass,
Tópico(s)Neurogenetic and Muscular Disorders Research
ResumoArticle26 July 2016free access Source DataTransparent process Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide-repeat protein deposition Kohji Mori Corresponding Author orcid.org/0000-0003-2629-0723 Biomedical Center (BMC), Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Yoshihiro Nihei Biomedical Center (BMC), Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Thomas Arzberger German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Center for Neuopathology and Prion Research, Ludwig-Maximilians-University Munich, Munich, Germany Department of Psychiatry and Psychotherapy, Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Qihui Zhou German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Ian R Mackenzie Department of Pathology, University of British Columbia and Vancouver General Hospital, Vancouver, Canada Search for more papers by this author Andreas Hermann Deptartment of Neurology and Center for Regenerative Therapies Dresden (CRTD), Technical University Dresden, Dresden, Germany German Center for Neurodegenerative Diseases (DZNE), Dresden, Germany Search for more papers by this author Frank Hanisch Department of Neurology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany Department of Neurology, Vivantes Humboldt-Klinikum, Berlin, Germany Search for more papers by this author German Consortium for Frontotemporal Lobar DegenerationFor the German Consortium for Frontotemporal Lobar Degeneration: Adrian Danek, Janine Diehl-Schmid, Klaus Fassbender, Hans Förstl, Johannes Kornhuber and Markus Otto Search for more papers by this author Bavarian Brain Banking AllianceFor the Bavarian Brain Banking Alliance: Andres Ceballos-Baumann, Marianne Dieterich, Regina Feuerecker, Armin Giese, Hans Klünemann, Alexander Kurz, Johannes Levin, Stefan Lorenzl, Thomas Meyer, Georg Nübling, Sigrun Roeber and Adrian Danek Search for more papers by this author Frits Kamp Biomedical Center (BMC), Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Brigitte Nuscher Biomedical Center (BMC), Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Denise Orozco German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Dieter Edbauer orcid.org/0000-0002-7186-4653 German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Munich Cluster for System Neurology (SyNergy), Munich, Germany Search for more papers by this author Christian Haass Corresponding Author orcid.org/0000-0002-4869-1627 Biomedical Center (BMC), Ludwig-Maximilians-University Munich, Munich, Germany German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Munich Cluster for System Neurology (SyNergy), Munich, Germany Search for more papers by this author Kohji Mori Corresponding Author orcid.org/0000-0003-2629-0723 Biomedical Center (BMC), Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Yoshihiro Nihei Biomedical Center (BMC), Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Thomas Arzberger German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Center for Neuopathology and Prion Research, Ludwig-Maximilians-University Munich, Munich, Germany Department of Psychiatry and Psychotherapy, Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Qihui Zhou German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Ian R Mackenzie Department of Pathology, University of British Columbia and Vancouver General Hospital, Vancouver, Canada Search for more papers by this author Andreas Hermann Deptartment of Neurology and Center for Regenerative Therapies Dresden (CRTD), Technical University Dresden, Dresden, Germany German Center for Neurodegenerative Diseases (DZNE), Dresden, Germany Search for more papers by this author Frank Hanisch Department of Neurology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany Department of Neurology, Vivantes Humboldt-Klinikum, Berlin, Germany Search for more papers by this author German Consortium for Frontotemporal Lobar DegenerationFor the German Consortium for Frontotemporal Lobar Degeneration: Adrian Danek, Janine Diehl-Schmid, Klaus Fassbender, Hans Förstl, Johannes Kornhuber and Markus Otto Search for more papers by this author Bavarian Brain Banking AllianceFor the Bavarian Brain Banking Alliance: Andres Ceballos-Baumann, Marianne Dieterich, Regina Feuerecker, Armin Giese, Hans Klünemann, Alexander Kurz, Johannes Levin, Stefan Lorenzl, Thomas Meyer, Georg Nübling, Sigrun Roeber and Adrian Danek Search for more papers by this author Frits Kamp Biomedical Center (BMC), Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Brigitte Nuscher Biomedical Center (BMC), Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Denise Orozco German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Dieter Edbauer orcid.org/0000-0002-7186-4653 German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Munich Cluster for System Neurology (SyNergy), Munich, Germany Search for more papers by this author Christian Haass Corresponding Author orcid.org/0000-0002-4869-1627 Biomedical Center (BMC), Ludwig-Maximilians-University Munich, Munich, Germany German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Munich Cluster for System Neurology (SyNergy), Munich, Germany Search for more papers by this author Author Information Kohji Mori 1,†, Yoshihiro Nihei1, Thomas Arzberger2,3,4, Qihui Zhou2, Ian R Mackenzie5, Andreas Hermann6,7, Frank Hanisch8,9, , , Frits Kamp1, Brigitte Nuscher1, Denise Orozco2, Dieter Edbauer2,10 and Christian Haass 1,2,10 1Biomedical Center (BMC), Ludwig-Maximilians-University Munich, Munich, Germany 2German Center for Neurodegenerative Diseases (DZNE), Munich, Germany 3Center for Neuopathology and Prion Research, Ludwig-Maximilians-University Munich, Munich, Germany 4Department of Psychiatry and Psychotherapy, Ludwig-Maximilians-University Munich, Munich, Germany 5Department of Pathology, University of British Columbia and Vancouver General Hospital, Vancouver, Canada 6Deptartment of Neurology and Center for Regenerative Therapies Dresden (CRTD), Technical University Dresden, Dresden, Germany 7German Center for Neurodegenerative Diseases (DZNE), Dresden, Germany 8Department of Neurology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany 9Department of Neurology, Vivantes Humboldt-Klinikum, Berlin, Germany 10Munich Cluster for System Neurology (SyNergy), Munich, Germany †Present address: Department of Psychiatry, Osaka University Graduate School of Medicine, Suita, Osaka, Japan *Corresponding author. Tel: +49 89 4400 46549; E-mail: [email protected] *Corresponding author. Tel: +81 6 6879 3051; E-mail: [email protected] EMBO Rep (2016)17:1314-1325https://doi.org/10.15252/embr.201541724 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Intronic hexanucleotide (G4C2) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat-dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G4C2 repeat RNA. We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci. Synopsis FTLD/ALS-associated repeat expansions in C9orf72 are translated into dipeptide repeat proteins. Reduction of repeat-binding hnRNPA3 increases levels of the repeat RNA and enhances production of dipeptide repeat proteins and RNA foci. Reduction of nuclear hnRNPA3 increases levels of the C9orf72 repeat RNA. Reduction of nuclear hnRNPA3 increases RNA foci formation and enhances generation and deposition of dipeptide repeat proteins. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased dipeptide repeat protein deposition. Introduction Unusual translation of bi-directionally transcribed intronic hexanucleotide repeats in the absence of ATG initiation codons in all reading frames leads to the generation of five distinct dipeptide repeat proteins (DPR), namely poly-GA, poly-GP, poly-GR, poly-AP, and poly-PR 12345. DPR accumulate in disease characterizing p62-positive and TDP-43-negative inclusions 1234. The G4C2 repeat RNA may trap RNA binding proteins and thus inhibit their normal function 678. Indeed, we recently identified a number of G4C2 repeat binding proteins including hnRNPA1, A2 and A3 6. Strikingly, mutations in hnRNPA1 and A2 cause multisystem proteinopathy and the sites of the pathogenic mutations are conserved in hnRNPA3 9. Moreover, hnRNPA3 accumulates in a subset of p62-positive inclusions and may be reduced within hippocampal neurons of C9orf72 mutation carriers 6. We therefore investigated if a loss of hnRNPA3 may modulate DPR production and deposition. Results hnRNPA3 modulates repeat RNA and poly-GA levels Knockdown of hnRNPA3 but not of hnRNPA1, hnRNPA2 and hnRNPH significantly enhanced generation of poly-GA in HeLa cells expressing 80 G4C2 repeats under the EF1 promotor (Fig 1A and B), whereas overexpression of hnRNPA3 decreased poly-GA generation (Fig 1C and D). RT–qPCR analysis revealed a corresponding increase of G4C2 repeat RNA upon hnRNPA3 knockdown (Fig 1E). Consistent with this finding, more cells harboring repeat RNA foci were detected (Fig 1F and G). Figure 1. hnRNPA3 modulates repeat RNA and poly-GA expression A, B. Knockdown of hnRNPA3 increases poly-GA expression, while expressions of EGFP protein levels are not altered upon knockdown of hnRNPs. The control (“x0″) vector lacks the G4C2 repeats but still contains the 5′ flanking region and 3x TAG. C, D. Overexpression of hnRNPA3 suppresses poly-GA expression. n = 2 experiments performed in duplicates. E. Increased repeat RNA upon hnRNPA3 knockdown. EGFP RNA levels are only slightly increased as compared to the levels of the repeat RNA. n = 2 experiments performed in duplicates. F, G. Knockdown of hnRNPA3 increases RNA foci. n = 3 replicates; two-tailed paired t-test, Scale bar, 20 μm. Data information: All graphs are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed paired t-test (G), ANOVA with Dunnett's post-test (B, E) or ANOVA with Tukey's post-test (D). Source data are available online for this figure. Source Data for Figure 1 [embr201541724-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint hnRNPA3-mediated repression of G4C2 repeat RNA and poly-GA protein is dependent on its ability to bind RNA. Whereas ectopic hnRNPA3WT rescued repression of repeat RNA and poly-GA upon knockdown of endogenous hnRNPA3 (Figs 2A–C and EV1A and B), a mutant variant, which is unable to bind RNA 10, failed to do so (Fig 2A–C). Nuclear import of hnRNPA3 also appears to be required since an hnRNPA3 variant lacking a M9 nuclear localization signal (NLS) (mCh-A3ΔM9) fails to fully rescue repression of repeat RNA and poly-GA protein (Fig 2D–F). The remaining activity is likely due to M9-NLS independent residual nuclear import of hnRNPA3 (Fig EV1C). Although knockdown of hnRNPA2 fails to increase GA production (Fig 1A and B), hnRNPA2 overexpression restored repression of repeat RNA and poly-GA (Fig 2B and C). In line with this finding, knockdown of hnRNPA3 together with hnRNPA1 or hnRNPA2 further increased poly-GA expression suggesting that hnRNPA1 and hnRNPA2 can partially compensate hnRNPA3 function (Fig EV1D and E). Overall, these findings suggest that hnRNPA3 negatively regulates repeat RNA expression levels, a process, which requires the RNA binding capacity of hnRNPA3 as well as its nuclear import via the M9-NLS. Figure 2. RNA binding and nuclear transport are required for hnRNPA3-mediated repression of poly-GA production A–C. Rescue of repression of poly-GA and repeat RNA by wild-type (wt) hnRNPA3 and hnRNPA2 but not by the RNA binding mutant hnRNPA3DxD. (B) n = 3 experiments performed in duplicates; (C) n = 2 experiments performed in duplicates. D–F. Rescue of repression of poly-GA and repeat RNA by hnRNPA3WT but not the M9-NLS deletion mutant. (E) n = 3 experiments performed in duplicates; (F) n = 4 experiments performed in duplicates. Data information: All graphs are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 ANOVA with Tukey's post-test. See also Fig EV1. Source data are available online for this figure. Source Data for Figure 2 [embr201541724-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. hnRNPA3 modulates GA expression level A, B. hnRNPA3 knockdown using an independent siRNA targeting hnRNPA3 (siA3#6) increases poly-GA expression and is rescued by mCherry-fused hnRNPA3. n = 4 experiments in duplicates. C. Partial cytoplasmic distribution of mCherry-hnRNPΔM9 lacking the M9 nuclear localization signal (NLS). mCherry-fused hnRNPA3WT, its RNA binding mutant (DxD), and hnRNPA2 are almost completely nuclear. Scale bar, 10 μm. n = 2. D, E. Double knockdown of hnRNPA3 and either hnRNPA1 or hnRNPA2 augments poly-GA expression. n = 4 experiments in duplicates. Data information: Graphs show mean ± SEM. *P < 0.05, ***P < 0.001; ANOVA with Dunnett's post-test (B, E). Source data are available online for this figure. Download figure Download PowerPoint Reduction of hnRNPA3 leads to enhanced poly-GA, poly-GP, and poly-GR production in HeLa cells and primary hippocampal neurons In the above experiments, we only detected the most efficiently translated GA protein 4. To investigate whether production of the other DPRs is also increased by reduced hnRNPA3 expression, we thought to achieve higher repeat RNA levels by expressing 80 G4C2 repeats under the control of the strong CMV promotor (Fig EV2A and B). This indeed allowed detection of poly-GA, poly-GP, and poly-GR in Western blots (Fig EV2C) and by immunocytochemistry (Fig EV2D and E). Moreover, we detected p62-positive poly-GA deposits in a subset of cells (Fig 3A). Furthermore, poly-GA and poly-GR double-positive cells, but not poly-GA single-positive cells, frequently showed altered TDP-43 intracellular distribution (Figs 3B and EV3A and B) thus reproducing important pathological features of C9orf72-associated neuropathology. TDP-43 redistribution was typically associated with altered nuclear morphology, which may indicate onset of apoptosis. TDP-43 mislocalization was enhanced in cells, which express both, poly-GA and poly-GR (Figs 3B and EV3A and B). In line with the data shown in Fig 1, hnRNPA3 knockdown significantly increased (G4C2)80 repeat RNA (Fig 3C) as well as poly-GA, poly-GP, and poly-GR protein with poly-GA being the most abundant DPR protein as observed in C9orf72 patients 4 (Fig 3D–F). Furthermore, co-expression of two or three different DPRs in one cell was significantly more frequently detected in hnRNPA3-depleted cells (Fig 3G). Click here to expand this figure. Figure EV2. High (G4C2)80 expression allows poly-GA, poly-GP, and poly-GR production A. Schematic representation of the repeat RNA expression constructs under the control of the strong CMV promoter. B. (G4C2)80HIGH(+0) drives significantly more G4C2 repeat RNA transcription than that of the original (G4C2)80 construct, n = 3 experiments in duplicates. ***P < 0.001; ANOVA with Tukey's post-test. Mean ± SEM are shown. C. Upon transfection of the (G4C2)80HIGH(+0) construct, abundant poly-GA protein was detected at the expected molecular weight together with insoluble aggregates that remained on the top of the gel as shown before 4. Asterisk indicates unspecific band. D, E. All three sense DPRs show variable intracellular distribution upon transient transfection of one of the (G4C2)80HIGH constructs. Cells expressing relatively low amounts of GA show a diffuse GA distribution pattern (see E), whereas cells with high levels contain large cytoplasmic and/or nuclear GA aggregates. GP locates to the cytoplasm and nucleus and often co-aggregates with GA. GR typically shows a diffuse cytoplasmic distribution. Expression and intracellular distribution pattern of each DPR appears is not affected by different TAGs. Scale bar, 10 μm. n = 3. (E) A large image visualizes frequent DPR expressions with various patterns. Scale bar, 20 μm. Download figure Download PowerPoint Figure 3. DPR expression and deposition Poly-GA aggregates co-localize with p62-positive deposits. Redistribution of nuclear TDP-43 to the cytoplasm in a poly-GA/poly-GR double-positive cell. Note that nuclei of poly-GA/poly-GR double-positive cells are frequently disrupted. Knockdown of hnRNPA3 increases repeat RNA expression. n = 2 experiments performed in duplicates. Filter trap assay probed with anti-FLAG, anti-myc, or anti-HA antibodies reveal abundant DPR production. Relative expression level of poly-GA, poly-GP, and poly-GR normalized to GR. n = 3 experiments. Expression of all three DPRs is increased upon knockdown of hnRNPA3. Signals from knockdown with siRNA#8 were normalized to 1. n = 3 experiments. Co-expression of two/three different DPRs in single cells is increased upon hnRNPA3 knockdown. n = 3 replicates. Data information: All graphs are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed paired t-test (G), ANOVA with Dunnett's post-test (C, F, G) or ANOVA with Tukey's post-test (E). Scale bars, 20 μm. See also Figs EV2 and EV3. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. High (G4C2)80 expression results in strong DPR expression followed by TDP-43 mislocalization Representative examples of endogenous TDP-43 mislocalization in GA and GR co-expressing cells. GA deposit-positive but GR-negative cell (arrow) shows normal TDP-43 distribution (lowest panels). Nuclei were counterstained with DAPI. Scale bar, 10 μm. Poly-GA and poly-GR double-positive cells frequently show increased mislocalization of endogenous TDP-43. In three independent experiments, 697, 852, 287, 32, 767, 201, 40, 728, 207, and 64 cells per column were analyzed. ***P < 0.001; ANOVA with Tukey's post-test. Mean ± SEM are shown. Download figure Download PowerPoint Similar findings were made in primary rat hippocampal neurons. shRNA knockdown of hnRNPA3 led to an increased accumulation of poly-GA-positive punctae in (G4C2)80 expressing cells indicating enhanced DPR aggregation and deposition (Fig 4A and B). Indeed, filter trap analysis confirmed the enhanced accumulation of insoluble poly-GA aggregates upon reduction of hnRNPA3 (Fig 4C and D). Figure 4. Knockdown of hnRNPA3 induces neuronal DPR accumulation Rat hippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA (shA3) or a control shRNA (shCtrl) and tagRFP. Three days after transduction (DIV3 + 3), neurons were transfected with (G4C2)80HIGH(+0) and analyzed at (DIV3 + 7). Neurons were fixed, immunostained, and imaged by confocal microscopy. Double immunofluorescence for poly-GA aggregates (green) and tagRFP (red). Nuclei were labeled with DAPI. Scale bar represents 20 μm. Efficient knockdown of endogenous rat hnRNPA3 with its targeting shRNA. Poly-GA aggregates were detected in a filter trap assay using an anti-Flag antibody. The amounts of poly-GA aggregates were quantified and are presented as the fold change of signals from neurons treated with the control shRNA or the repeat construct. Means ± SD of three independent experiments are shown. *P < 0.05 by a Student's t-test. n = 3 replicates. Source data are available online for this figure. Source Data for Figure 4 [embr201541724-sup-0004-SDataFig4.pdf] Download figure Download PowerPoint Reduction of hnRNPA3 leads to enhanced formation of nuclear RNA foci in fibroblasts derived from patients with C9orf72 repeat extensions To test whether reduced hnRNPA3 also leads to enhanced C9orf72-associated phenotypes under physiological condition without ectopic expression of C9orf72 repeat extensions, we used fibroblast lines derived from three independent and unrelated patients with confirmed C9orf72 repeat extensions (Fig 5A; see also Materials and Methods for further clinical characterization). Since we could not detect DPR expression in primary patient-derived cells including neurons derived from induced pluripotent stem cells using several different monoclonal antibodies, we focused on enhanced formation of RNA foci, which consistently accumulate upon hnRNPA3 knockdown in HeLa cells (see Fig 1F and G). Two independent siRNAs efficiently knocked down hnRNPA3 and lowered hnRNPA3 protein levels in all three fibroblast lines (Fig 5B), where RNA foci composed of the repeat RNA could be detected by in situ hybridization (Fig 5C). As a result of lowered hnRNPA3, we observed an approximately two fold increase of cells containing RNA foci (Fig 5D). In addition, hnRNPA3 knockdown significantly increased the number of foci per individual cell (Fig 5E). These findings were independently confirmed using lentiviral-mediated knockdown (Fig 5F and G). Taken together, these results confirm regulation of the GGGGCC repeat RNA by hnRNPA3 on an endogenous level in patient-derived primary cells. Figure 5. Reduction of hnRNPA3 leads to enhanced formation of nuclear RNA foci in fibroblasts derived from patients with C9orf72 repeat extensions Fibroblasts from three individual patients with confirmed C9orf72 repeat expansions were investigated. Knockdown of hnRNPA3 with two independent siRNA results in selective depletion of hnRNPA3 in fibroblasts from 3 C9orf72 repeat carriers. Actin was used as a loading control. Knockdown of hnRNPA3 increases RNA foci in cells derived from C9orf72 carriers. No G4C2 repeat RNA foci are detected in fibroblasts without C9orf72 repeat expansions (WT). Nucleoli were stained with anti-nucleolin antibodies (green), and nuclei were stained with DAPI (blue). Scale bar, 10 μm. Quantification of the relative frequency of RNA foci-positive cells (RNA foci positivity; fold change). n = 3 experiments for each case. The average foci number of non-treated (N/T) fibroblast was normalized to 1. Color code labels values obtained in fibroblasts derived from the three individual patients. Bars indicate mean, individual points indicate mean values obtained from a single experiment (49–214 cells were counted in a single experiment), and error bars indicate 95% CI. *P < 0.05, ***P < 0.001; ANOVA with Tukey–Kramer HSD test. Quantification of the number of RNA foci per RNA foci-positive cell (fold change). n = 3 experiments for each case. The average foci number of non-treated (N/T) fibroblast was normalized to 1. Color code labels values obtained in fibroblasts derived from the three individual patients. Bars indicate mean, individual points indicate mean values obtained from a single experiment (49–214 cells were counted in a single experiment), and error bars indicate 95% CI. *P < 0.05, **P < 0.01; ANOVA with Tukey–Kramer HSD test. Representative presentation of increased RNA foci in C9orf72 carriers upon lentiviral-mediated hnRNPA3 knockdown. Scale bar, 10 μm. Quantification of the number of RNA foci (fold change). Three cases were analyzed. Single points indicate an average obtained from 29 to 30 cells per case. Color code labels values obtained in fibroblasts derived from the three individual patients. Mean ± SEM. **P < 0.01; two-tailed t-test. Source data are available online for this figure. Source Data for Figure 5 [embr201541724-sup-0005-SDataFig5.pdf] Download figure Download PowerPoint Nuclear hnRNPA3 reduction correlates with poly-GA accumulation in C9orf72 patients Enhanced generation of poly-GA deposits and RNA foci in primary neurons and patient's fibroblasts upon hnRNPA3 knockdown prompted us to investigate the link between reduced hnRNPA3 expression and DPR deposition in brains of patients with C9orf72 repeat extensions. To do so, we performed double immunofluorescence with anti-hnRNPA3 and anti-GA antibodies in hippocampal sections, where hnRNPA3-related pathology was most prominent and abundant DPR pathology was observed 346. Two examiners independently analyzed sections from 34 C9orf72 cases. In C9orf72 patients, we frequently detected individual neurons with reduced nuclear hnRNPA3 as well as partial co-localization of hnRNPA3 with poly-GA aggregates (Fig 6A and B). When we grouped the cases according to their hnRNPA3 expression levels split at the median, we observed a significant increase in GA deposition in neurons with low hnRNPA3 expression (Fig 6C). Approximately 50% higher levels of poly-GA deposits in cases with low hnRNPA3 levels are comparable to the effect of hnRNPA3 knockdown of RNA foci in patient fibroblasts (Fig 5). This is also in line with the data derived from cultured cells, where lowering of hnRNPA3 leads to higher levels of poly-GA. Figure 6. Nuclear hnRNPA3 reduction correlates with poly-GA accumulation in patients with C9orf72 repeat expansions Double immunofluorescence staining with anti-GA (green) and anti-hnRNPA3 (red) antibodies of the granular layer of the dentate gyrus of a control case and three C9orf72 mutation cases. In C9 mutation cases with low nuclear hnRNPA3 expression (#8 & #1), more poly-GA aggregates were observed as in cases with high nuclear hnRNPA3 (#7). Inserts show examples of co-localization of poly-GA and hnRNPA3 aggregates. Scale bar, 10 μm. Granular layer neurons of a C9orf72 mutation case with (white arrows) or without (orange arrows) nuclear clearance of hnRNPA3. Scale bar, 10 μm. Poly-GA aggregates are more frequent in C9orf72 mutation cases with lower nuclear hnRNPA3 levels than in those cases with higher nuclear hnRNPA3 levels (divided by median of nuclear hnRNPA3 intensities in 34 C9orf72 mutation cases into two subgroups). Bar graph indicates mean values. Error bars indicate 95% CI. Single points indicate mean from three micrographs per case. Note that the difference in GA positivity between both groups remains significant (P = 0.0086) after removal of the highest outlier in the low nuclear A3 group; two-tailed t-test. Download figure Download PowerPoint Discussion Reduced hnRNPA3 increases the levels of the G4C2 repeat RNA in three independent tissue culture systems. As a consequence, DPR production and deposition is elevated as well. This occurs independent of promotor effects, as two different promotors were used to drive ectopic expression G4C2 repeats and controls with an irrelevant reporter protein were included as well. Moreover, in patient-derived fibroblasts, we also observed an accumulation of RNA foci similar to the results in HeLa cells. These findings suggest that binding of hnRNPA3 to the repeat RNA reduces its stability and thus decreases its levels. Binding of hnRNPA3 to the repeat RNA may lead to a reduction of its secondary structure, which could facilitate its degradation. In line with the emerging evidence that C9orf72 repeat extensions disturb nuclear transport 111213, these findings may suggest that C9orf72 repeat-associated toxicity affects nuclear import of hn
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