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Pur-alpha regulates cytoplasmic stress granule dynamics and ameliorates FUS toxicity

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Abstract

Amyotrophic lateral sclerosis is characterized by progressive loss of motor neurons in the brain and spinal cord. Mutations in several genes, including FUS, TDP43, Matrin 3, hnRNPA2 and other RNA-binding proteins, have been linked to ALS pathology. Recently, Pur-alpha, a DNA/RNA-binding protein was found to bind to C9orf72 repeat expansions and could possibly play a role in the pathogenesis of ALS. When overexpressed, Pur-alpha mitigates toxicities associated with Fragile X tumor ataxia syndrome (FXTAS) and C9orf72 repeat expansion diseases in Drosophila and mammalian cell culture models. However, the function of Pur-alpha in regulating ALS pathogenesis has not been fully understood. We identified Pur-alpha as a novel component of cytoplasmic stress granules (SGs) in ALS patient cells carrying disease-causing mutations in FUS. When cells were challenged with stress, we observed that Pur-alpha co-localized with mutant FUS in ALS patient cells and became trapped in constitutive SGs. We also found that FUS physically interacted with Pur-alpha in mammalian neuronal cells. Interestingly, shRNA-mediated knock down of endogenous Pur-alpha significantly reduced formation of cytoplasmic stress granules in mammalian cells suggesting that Pur-alpha is essential for the formation of SGs. Furthermore, ectopic expression of Pur-alpha blocked cytoplasmic mislocalization of mutant FUS and strongly suppressed toxicity associated with mutant FUS expression in primary motor neurons. Our data emphasizes the importance of stress granules in ALS pathogenesis and identifies Pur-alpha as a novel regulator of SG dynamics.

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    Authors and Affiliations

    1. Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA, USA

      J. Gavin Daigle

    2. Division of Child Neurology, Department of Pediatrics, Children’s Hospital of Pittsburgh, University of Pittsburgh Medical Center, Pittsburgh, PA, USA

      J. Gavin Daigle, Nandini Ramesh, Ian Casci, John Monaghan & Udai Bhan Pandey

    3. Frances and Joseph Weinberg Unit for ALS Research, Department of Neuroscience, Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA

      Karthik Krishnamurthy, Kevin McAvoy & Piera Pasinelli

    4. Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA, USA

      Nandini Ramesh, Ian Casci & Udai Bhan Pandey

    5. Department of Pathology and Anatomy, Eastern Virginia Medical School, Norfolk, VA, USA

      Earl W. Godfrey

    6. Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA, USA

      Dianne C. Daniel & Edward M. Johnson

    7. Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA

      Zachary Monahan & Frank Shewmaker

    8. Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

      Udai Bhan Pandey

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    Corresponding author

    Correspondence to Udai Bhan Pandey.

    Additional information

    J G. Daigle and K. Krishnamurthy contributed equally to the work.

    Electronic supplementary material

    Below is the link to the electronic supplementary material.

    401_2015_1530_MOESM1_ESM.tif

    Supplementary material 1 Fig. 1: Quantification of Pur-alpha and FUS positive SGs. (a). ALS-Patient lymphoblastoid cells carrying FUS R518G and age/sex matched population control were stress with 0.5 mM sodium arsenite for 1 h 30 min and stained for endogenous Pur-alpha (anti-PURA), G3 PB, and DAPI. The number of cells containing SGs was counted and the number of cells containing pur-alpha positive SGs was quantified. Percentage of cells colocalized with both pur-alpha and G3BP under stress conditions was determined. Bar graph represents the average from three separate fields. Approximately, 80 % of cells contained G3BP-positive SGs (b). Control, ALS patient cells carrying FUS R518G, and FUS R521C mutations were stained with anti-FUS N-terminal and anti-G3BP. Number of cells containing FUS incorporated into SGs was divided by the total number of cells containing SGs to give a percent of cells with FUS positive SGs. N = ~200 cells. ANOVA with Tukey’s posthoc analysis was applied. (**P = 0.01). (TIFF 10113 kb)

    401_2015_1530_MOESM2_ESM.tif

    Supplementary material 2 Fig. 2: Pur-alpha localizes to SGs in mammalian neuronal cells. (a). Neuroblastoma cells (N2A) were stained with Dapi (blue), anti-HA7 (green), and anti-G3BP (red). In unstressed conditions, G3BP was diffuse in the cytoplasm. (b). Under stress (0.5 mM sodium arsenite), G3BP decorated cytoplasmic stress granules. (c). N2A cells transiently transfected with HA-Pur-alpha. Pur-alpha was localized to the cytoplasm. Pur-alpha overexpression did not induce G3BP-positive SGs under unstressed conditions. (d). N2A cells transiently expressing HA-Pur-alpha under stress conditions (0.5 mM sodium arsenite) displayed Pur-alpha in cytoplasmic puncta that colocalized with G3BP in the cytoplasm. Arrowheads indicate cytoplasmic SGs. Arrows indicate SGs containing HA-Pur-alpha. (TIFF 33410 kb)

    401_2015_1530_MOESM3_ESM.tif

    Supplementary material 3 Fig. 3: Pur-alpha co-localizes with SGs in primary motor neurons. Primary motor neurons were cultured in normal medium conditions (unstressed) and in medium conditions containing 0.5 mM sodium arsenite for 90 min. Under unstressed conditions endogenous Pur-alpha (green) and G3BP (red) appeared diffuse in the cytoplasm. MAP2 chicken antibody (a neuronal marker) was used to label the neuronal processes. Under stress conditions, Pur-alpha was present in large cytoplasmic puncta which colocalized with SG marker G3BP (depicted as large yellow puncta in the merge image). Arrows indicate Pur-alpha incorporation into SGs. (TIFF 11896 kb)

    401_2015_1530_MOESM4_ESM.tif

    Supplementary material 4 Fig. 4: Pur-alpha gets incorporated into SGs under heat stress. Neuroblastoma cells (N2A) untransfected, transfected with HA-Pur-alpha WT, and TET-inducible FUS-R521C-GFP cells were cultured in DMEM at 37 °C on Poly-D Lysine coated coverslips (unstressed). Duplicate groups were cultured in DMEM at 42 °C for 3 h 30 min (stressed) then fixed. Immunoflourescense was performed with anti-G3BP (red), anti-HA7 to illuminate HA-Pur-alpha expressing cells (green), and FUS R521C-GFP expressing cells (green). In the Pur-alpha expressing cells colocalization of Pur-alpha (green) and G3BP positive SGs (red) = yellow puncta, in FUS-R521C-GFP (green) and G3BP positive SGs (red) = yellow granules. SGs are indicated with arrows with tails. SGs containing Pur-alpha or FUS are pointed out with arrowheads. (TIFF 41170 kb)

    401_2015_1530_MOESM5_ESM.tif

    Supplementary material 5 Fig. 5: Pur-alpha is a component of in SGs formed under H 2 0 2 stress. Neuroblastoma cells (N2A) untransfected, transfected with HA-Pur-alpha WT, and TET-inducible FUS-R521C-GFP cells were cultured in DMEM at 37 °C (unstressed). Duplicate groups were cultured in 1 mM H2O2 in DMEM for 1 h 30 min then fixed. Immunofluorescence was performed with anti-G3BP (red), anti-HA7 to illuminate HA-Pur-alpha expressing cells (green), and FUS R521C-GFP expressing cells (green). In the Pur-alpha expressing cells colocalization of Pur-alpha (green) and G3BP positive SGs (red) = yellow puncta, in FUS-R521C-GFP (green) and G3BP positive SGs (red) = yellow granules. SGs are indicated with arrows with tails. SGs containing Pur-alpha or FUS are pointed out with arrowheads. (TIFF 39456 kb)

    401_2015_1530_MOESM6_ESM.tif

    Supplementary material 6 Fig. 6: Pur-alpha is trapped in SGs containing ALS- linked FUS R521C. Mammalian neuroblastoma cells (N2a) stably expressing TET inducible GFP-FUS R521C were plated on poly-D lysine coverslips then treated with 300 ng of doxycycline (DOX) for 24 h to induce expression of FUS. DOX was included in control un-transfected cells and was present in the medium throughout the experiment. Both groups were treated with 0.5 mM sodium arsenite for 90 min to induce SGs. After SGs were formed, cells washed with DMEM and allowed to recover in DMEM + DOX for 90 min at 37 °C. Following the recovery time period Pur-alpha (red) and G3BP (white) in control cells appeared diffuse with few small puncta remaining in the cytoplasm. In cells expressing GFP FUS (green), Pur-alpha (red) remains in G3BP positive SGs that were not resolve when stress was removed (Depicted as Yellow/white puncta in the cytoplasm). Unresolved SGs showing co-localization of FUS, Pur-alpha, and G3BP are indicated with arrows. (TIFF 17169 kb)

    401_2015_1530_MOESM7_ESM.tif

    Supplementary material 7 Fig. 7: Pur-alpha shRNA knockdown blocks the formation of TIAR-positive stress granules. HEK293T cells expressing Pur-alpha shRNA-GFP were stressed with 0.5 mM sodium arsenite for 1.5 h. Cells were stained with anti-TIAR (Red). Cytoplasmic SGs can be seen in adjacent untransfected cells but are absent in cells expressing Pur-alpha shRNA- GFP (green). (TIFF 31674 kb)

    401_2015_1530_MOESM8_ESM.tif

    Supplementary material 8 Fig. 8: Pur-alpha shRNA knockdown reduces the mRNA levels of SG components (a) qPCR was performed using primers specific to endogenous Pur-alpha as well as GAPDH. (b) qPCR was performed using primers specific to endogenous FMR1 as well as GAPDH. (c) qPCR was performed using primers specific to endogenous G3BP1 as well as GAPDH. (d) qPCR was performed using primers specific to endogenous TIAL1 as well as GAPDH. Relative mRNA levels were quantified and normalized to GAPDH. Graph shows fold changes in mRNA expression. ANOVA was performed with Tukey’s post hoc analysis. Error bars represent ± SEM. n.s = not significant. (*P < 0.05, **P = 0.01, ***P = 0.001). (TIFF 8819 kb)

    401_2015_1530_MOESM9_ESM.tif

    Supplementary material 9 Fig. 9: Pur-alpha shRNA knockdown inhibits the formation of SGs under heat stress. HEK293T cells expressing Pur-alpha shRNA –GFP were stressed with 42 °C for 4 h. Cells were fixed then stained with Anti-G3BP (Red) and Dapi (blue). Cytoplasmic SGs in neighboring untransfected cells are indicated with arrows. (TIFF 16643 kb)

    401_2015_1530_MOESM10_ESM.tif

    Supplementary material 10 Fig. 10: Pur-alpha knockdown in HEK293T cells does not affect G3BP protein levels. (a). HEK293T cells stably expressing pur-alpa shRNA were used to generate total lysates ~ 35,000 cells per well. Western blot analysis reveals a G3BP positive band at ~ 70 kDa. (b) Band quantification was taken from three separate blots and normalized to tubulin loading control. Average band intensities were analyzed with ANOVA revealing no significant differences across groups. (TIFF 13212 kb)

    401_2015_1530_MOESM11_ESM.tif

    Supplementary material 11 Fig. 11: Pur-alpha shRNA knockdown does not affect the levels of endogenous FUS mRNA and protein levels. HEK293T cells stably expressing Pur-alpha shRNA–GFP constructs. (a). Total lysates were made from 35,000 cells and resolved by SDS PAGE. Western blots were probed with anti-FUS (N-term) and anti-tubulin. FUS protein levels were unaltered in Pur-alpha shRNA samples compared to the scrambled shRNA control. (b). qPCR was performed using primers specific to endogenous FUS as well as GAPDH. Relative mRNA levels were quantified and normalized to GAPDH. Graph shows fold changes in FUS mRNA levels. ANOVA was performed with Tukey’s post hoc analysis. Error bars represent ± SEM. n.s = not significant. (TIFF 13212 kb)

    401_2015_1530_MOESM12_ESM.tif

    Supplementary material 12 Fig. 12: Pur-alpha shRNA does not influence formation of p-bodies under stress conditions. HEK293T cells expressing Pur-alpha shRNA constructs were stressed with 0.5 mM sodium arsenite then labeled with anti-GW182 [p-body marker]. (a) Immunofluorescence revealed p-bodies were present in control cells expressing scrambled shRNA as well as in cells expressing Pur-alpha shRNA. (b) Quantification indicates that there was no difference in number of p-bodies per cell in Pur-alpha shRNA groups compared to controls. ANOVA was performed with Tukey’s post hoc multiple comparison analysis. The graph shows the average number of p-bodies per cell. n = 300 per/group. (n.s = not significant) error bars represent SEM. Arrows indicate p-bodies counted in the quantification. (TIFF 31936 kb)

    401_2015_1530_MOESM13_ESM.tif

    Supplementary material 13 Fig. 13: Pur-alpha shRNA knockdown does not affect endogenous FUS distribution. HEK293T cells expressing Pur-alpha shRNA were labeled for endogenous hFUS [anti-FUS-N-terminal]. FUS (red) was primarily located in the nucleus in control cells expressing scrambled shRNA. In cells expressing Pur-alpha shRNA-GFP (green), the localization did not change. (TIFF 27587 kb)

    401_2015_1530_MOESM14_ESM.tif

    Supplementary material 14 Fig. 14: Pur-alpha over-expression promotes the formation of SG at earlier time points compared to control. Neuroblastoma cells, Control, expressing HA-Pur-alpha, and expressing FUS R521C-GFP were treated with 0.5 mM sodium arsenite. Time-lapse induction of SGs was conducted with treatments stopped after 10 min, 20 min, 30 min, 45, mins, and 1 h. Fixed cells were stained with anti-G3BP (red). In untreated conditions (time 0), G3BP appeared diffuse in the cytoplasm. SG (G3BP positive granules) began to form at 20 min time-point in control and FUS R521C conditions. In Pur-alpha over expression SG appear to form as early as 10 min an earlier time point compared to control. (TIFF 27720 kb)

    401_2015_1530_MOESM15_ESM.tif

    Supplementary material 15 Fig. 15: FUS R521C expression stalls SG disassembly and over-expression of pur-alpha accelerates SG turnover. Neuroblastoma cells (untransfected control, HA-Pur-alpha transient expression, and FUS R521C-GFP inducible expression) were stressed with 0.5 mM sodium arsenite for 1 h. Stress treatment was removed, washed with DMEM, then replaced with fresh DMEM. Groups were then allowed to recover for 2 h 30 min at 37 °C. (a). Schematic depicts the formation of SGs over time. Cells treated with 0.5 mM sodium arsenite for 1 h showed a robust induction of SGs. Once the stress is removed (replaced with fresh DMEM) SGs begin do dissociate. After 2 h and 30 min SGs have dissolved in control cells. (b) Immunoflourescense shows control cells with disassembled (diffuse red staining in the cytoplasm) SGs and in some cells SG still remaining (red puncta). HA-Pur-alpha expressing cells (green) SGs have dissolved. Cells containing FUS R521C (green) FUS is incorporated into SG (yellow) and these granules do not dissociate after 2 h 30 min post stress. Co-localization is indicated with arrows. (c). At 60× magnification, Z-projections at three separate fields were imaged. Number cells containing > 1 SG were quantified and divided by the total number of DAPI positive cells to obtain percentage of cells containing SGs. ANOVA was performed with Tukey’s multiple comparisons post hoc analysis. N = ~ 250 (Error bars represent ± SEM. (*P < 0.05, **P = 0.01). (TIFF 41168 kb)

    401_2015_1530_MOESM16_ESM.tif

    Supplementary material 16 Fig. 16: Identification of the prion-like domain in Pur-alpha. Using a predictive algorithm [54], which has been used to screen other RNA binding proteins for their prion-like properties, we analyzed the Pur-alpha sequence for the presence of prion like domain (s) [48, 54]. We used the full-length amino acid sequence of human Pur-alpha to determine whether the protein contains functional domains with prion-like properties. For comparison, we show that a large portion of the FUS protein is predicted to possess prion-like properties. (TIFF 8125 kb)

    401_2015_1530_MOESM17_ESM.tif

    Supplementary material 17 Fig. 17: Primary motor neurons expressing FUS R521G have significantly smaller soma compared to control. Mouse primary motor neurons exogenously expressing FUS R521G, FUS WT, FUS WT + Pur-alpha, and FUS R521G + Pur-alpha were measured using ImageJ. Horizontal and vertical dimensions were measured to quantify the neuron soma size. Student t test was performed between groups. Error bars represent ± SEM (*P < 0.05). (TIFF 5067 kb)

    401_2015_1530_MOESM18_ESM.tif

    Supplementary material 18 Fig. 18: Expression of Pur-alpha reduces total number of SGs as well as FUS-positive SGs. (a) Representative confocal images of FUS R521G alone (top panel) and Pur-alpha co-expressing neurons (bottom panel) labeled for G3BP1 after sodium arsenite stress. Co-localization of G3BP1 with mutant FUS is shown by white arrows. Scale = 5 µm (b). SG numbers were significantly reduced in cells co-expressing Pur-alpha (* P = 0.039) (c) FUS positive SGs are significantly less in the cells co-expressing Pur-alpha compared to mutant FUS cells (*P0.034). N = 9. (TIFF 1175 kb)

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    Daigle, J.G., Krishnamurthy, K., Ramesh, N. et al. Pur-alpha regulates cytoplasmic stress granule dynamics and ameliorates FUS toxicity. Acta Neuropathol 131, 605–620 (2016). https://doi.org/10.1007/s00401-015-1530-0

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