Journal: Brain : a journal of neurology

Article Title: The stress granule protein G3BP1 alleviates spinocerebellar ataxia-associated deficits.

doi: 10.1093/brain/awac473

Figure Lengend Snippet: Figure 1 SG assembly mediated by SA does not alter the number of aggregates or protein levels of ATXN2 and ATXN3. (A) Confocal microscopy rep resentative images from Neuro2a cells expressing pathological and non-pathological forms of ATXN2, fused with an eGFP tag (top row), treated with SA to induce SG assembly and stained with antibodies against PABP, an SG marker (middle row). (B) Representative western blot of Neuro2a lysates expressing pathological and non-pathological forms of ATXN2 and treated SA to induce SG assembly. Western blots were labelled using ATXN2, phospho-eIF2α, and β-tubulin antibodies. (C) The number of cells with ATXN2 aggregates was not significantly altered on SG assembly (n = 3 inde pendent experiments). (D) The levels of non-pathological ATXN2 (ATXN2WT) protein were not significantly altered on SG assembly. (E) The levels of pathological ATXN2 (ATXN2MUT) protein were not significantly altered on SG assembly (n = 5 independent experiments). (F) Confocal microscopy representative images from Neuro2a cells expressing pathological and non-pathological forms of ATXN3, fused with an eGFP tag (top row), treated with SA to induce SG assembly and stained with antibodies against PABP1. (G) Representative western blot of Neuro2a lysates expressing pathologic al and non-pathological forms of ATXN3 and treated SA to induce SG assembly. Western blots were labelled using ATXN3, eIF2α, phospho-eIF2α and β-tubulin antibodies. (H) The number of cells with ATXN3 aggregates was not significantly altered on SG assembly (n = 3 independent experiments). (I) The levels of non-pathological ATXN3 (ATXN3WT) protein were not significantly altered on SG assembly. (J) The levels of pathological ATXN3 (ATXN3MUT) protein were not significantly altered on SG assembly (n = 5 independent experiments). Values are expressed as mean ± SEM. Scale bar = 10 µm.

Article Snippet: Plasmids encoding for human ATXN3 contain 28 glutamines (pEGFP-C1-Ataxin3Q28; #22122; Addgene) or 84 glutamines (pEGFP-C1-Ataxin3Q84; #22123; Addgene) were a gift from Henry Paulson and both are fused with a GFP protein at the N terminus.21 Plasmids encoding for human ATXN2 containing 22 glutamines (pEGFP-Ataxin2Q22) or 104 glutamines (pEGFP-Ataxin2Q104) were kindly provided by Professor Stefan Pulst.22 The LacZ gene was cloned in our laboratory under the control of a phosphoglycerate kinase promoter,23 and the GFP construct was cloned as previously described.24 The plasmid encoding for human G3BP1 (GeneBank accession DQ893058.2) purchased from Source Bioscience, was cloned into a lentiviral vector backbone using the GatewayTM LR ClonaseTM II Enzyme Mix, Invitrogen, according to the manufacturer’s instructions.

Techniques: Confocal Microscopy, Expressing, Staining, Marker, Western Blot

Journal: Brain : a journal of neurology

Article Title: The stress granule protein G3BP1 alleviates spinocerebellar ataxia-associated deficits.

doi: 10.1093/brain/awac473

Figure Lengend Snippet: Figure 5 G3BP1 mRNA and protein levels are reduced in the SCA2 and SCA3, and its silencing in the mouse brain increases aggregation. (A) Representative western blot for protein lysates of fibroblasts from SCA2 patients and healthy controls. (B) Representative western blot for protein ly sates from fibroblast of SCA3 patients and healthy controls. (C) The levels of G3BP1 protein and (D) mRNA are significantly reduced in SCA2, compared to controls. (E) The levels of G3BP1 protein and (F) mRNA are significantly reduced in SCA3, compared to controls (healthy controls n = 3; SCA2 n = 2; SCA3 n = 5). (G) Representative western blot for protein lysates of cerebella from a transgenic SCA3 mouse model. (H) The levels of G3BP1 protein and (I) mRNA are significantly reduced in the SCA3 transgenic mice, compared to C57BL/6 wild-type animals (n = 3–5). Western blots were labelled using G3BP1, and β-tubulin antibodies. (J) Schematic representation of the injection site for the SCA2 model. Briefly, lentiviral vectors encoding ATXN2MUT and an shRNA scramble were co-injected in one hemisphere of the striatum, and in the contralateral hemisphere was co-injected ATXN2MUT and an shRNA targeting G3bp1. (K) At 4 weeks post-injection the animals were euthanized, and brain sections labelled with ATXN2 to highlight the presence of pathological aggregates. (L) The average number of ATXN2MUT aggregates is significantly increased on shG3bp1 expression, as compared to the con trol hemisphere. (M) Schematic representation of the injection site for the SCA2 model. Briefly, lentiviral vectors encoding ATXN3MUT and a shRNA scramble were co-injected in one hemisphere of the striatum and in the contralateral hemisphere it was co-injected ATXN2MUT and a shRNA targeting G3bp1. (N) At 4 weeks post-injection, the animals were euthanized and brain sections labelled with ATXN3 to highlight the presence of pathological aggregates. (O) The average number of ATXN3MUT aggregates is significantly increased on shG3bp1 expression, as compared to the control hemi sphere. (*P < 0.05; **P < 0.01; ***P < 0.001; Student’s t-test). Values are expressed as mean ± SEM.

Article Snippet: Plasmids encoding for human ATXN3 contain 28 glutamines (pEGFP-C1-Ataxin3Q28; #22122; Addgene) or 84 glutamines (pEGFP-C1-Ataxin3Q84; #22123; Addgene) were a gift from Henry Paulson and both are fused with a GFP protein at the N terminus.21 Plasmids encoding for human ATXN2 containing 22 glutamines (pEGFP-Ataxin2Q22) or 104 glutamines (pEGFP-Ataxin2Q104) were kindly provided by Professor Stefan Pulst.22 The LacZ gene was cloned in our laboratory under the control of a phosphoglycerate kinase promoter,23 and the GFP construct was cloned as previously described.24 The plasmid encoding for human G3BP1 (GeneBank accession DQ893058.2) purchased from Source Bioscience, was cloned into a lentiviral vector backbone using the GatewayTM LR ClonaseTM II Enzyme Mix, Invitrogen, according to the manufacturer’s instructions.

Techniques: Western Blot, Transgenic Assay, Injection, shRNA, Expressing, Control

Journal: Brain : a journal of neurology

Article Title: The stress granule protein G3BP1 alleviates spinocerebellar ataxia-associated deficits.

doi: 10.1093/brain/awac473

Figure Lengend Snippet: Figure 6 G3BP1 expression reduces the number of aggregates and the loss of neuronal markers in lentiviral mouse models of SCA2 and SCA3. Mice were stereotaxically injected into the striatum either with lentiviral particles encoding for mutant forms of ATXN2 or ATXN3, or co-injected with len tiviral particles encoding for the mutant form and G3BP1. (A) Schematic representation of the injection site and lentiviral vectors injected in the mouse model of SCA2. Animals were bilaterally injected and euthanized 12 weeks after the injection for tissue collection. (B) Schematic representation of the injection site and lentiviral vectors injected in the mouse model of SCA3. Animals were bilaterally injected and euthanized 4 weeks after the injection for tissue collection. (C) Brain sections from the lentiviral mouse model of SCA2 were analysed through immunohistochemistry using ATXN2 and DARPP-32 antibodies. Images show aggregates of ATXN2MUT (arrowheads; scale bar = 20 µm) and the loss of staining (line) of the neuronal marker DARPP-32 (scale bar = 200 µm). (D) The hemisphere expressing G3BP1 presented a reduced number of ATXN2MUT aggregates, compared to the control hemisphere (n = 5; ***P < 0.0001; Student’s t-test). (E) G3BP1 expression rescues neuronal marker loss, compared to the contralateral hemisphere only expressing ATXN2MUT (n = 5; ***P < 0.0001; Student’s t-test). (F) Representative images of immunohistochemistry brain sections, from the lentiviral mouse model of SCA3. The figures show ubiquitinated ATXN3MUT aggregates (dots; scale bar = 20 µm) and the neuronal marker DARPP-32 loss of staining (scale bar = 200 µm). (G) The expression of G3BP1 led to a significant reduction in the number of ubiquitinated ATXN3MUT aggregates, com pared to the control condition (n = 7; ***P < 0.0001; Student’s t-test). (H) G3BP1 expression rescues neuronal marker loss, as compared to controls (n = 7; ***P < 0.0001; Student’s t-test). Values are expressed as mean ± SEM.

Article Snippet: Plasmids encoding for human ATXN3 contain 28 glutamines (pEGFP-C1-Ataxin3Q28; #22122; Addgene) or 84 glutamines (pEGFP-C1-Ataxin3Q84; #22123; Addgene) were a gift from Henry Paulson and both are fused with a GFP protein at the N terminus.21 Plasmids encoding for human ATXN2 containing 22 glutamines (pEGFP-Ataxin2Q22) or 104 glutamines (pEGFP-Ataxin2Q104) were kindly provided by Professor Stefan Pulst.22 The LacZ gene was cloned in our laboratory under the control of a phosphoglycerate kinase promoter,23 and the GFP construct was cloned as previously described.24 The plasmid encoding for human G3BP1 (GeneBank accession DQ893058.2) purchased from Source Bioscience, was cloned into a lentiviral vector backbone using the GatewayTM LR ClonaseTM II Enzyme Mix, Invitrogen, according to the manufacturer’s instructions.

Techniques: Expressing, Injection, Mutagenesis, Immunohistochemistry, Staining, Marker, Control

Journal: Brain : a journal of neurology

Article Title: The stress granule protein G3BP1 alleviates spinocerebellar ataxia-associated deficits.

doi: 10.1093/brain/awac473

Figure Lengend Snippet: Figure 8 G3BP1 expression mitigates motor deficits and neuropathological abnormalities in an SCA3 transgenic mouse model. Transgenic mice ani mals expressing a truncated form of the ATXN3 protein containing 69 glutamines were stereotaxically injected in the cerebellum with lentiviral par ticles encoding for GFP (control group) or with G3BP1 (treated group). Mice, at 4 weeks of age, were first tested 1–2 days before injection and then repeatedly tested every 3 weeks until 9 weeks post-injection, to be euthanized at 10 weeks post-surgery. (A–C) Representative plots of mice motor per formance at 9 weeks post-injection. (A) Mice injected with G3BP1 significantly improved motor performance (assessed by the rotarod test), as they re main more time at the rotating rod compared to control mice treated with GFP or non-injected (NI) mice. (B) Mice injected with G3BP1 significantly reduced the time needed to cross the water-filled tank and reach the platform, compared to control mice treated with GFP or non-injected animals. (C) Footprint analysis showed that mice injected with G3BP1 improved overlap measures, compared to controls treated with GFP or non-injected ani mals. (D) Representative images of immunohistochemistry brain sections from mice cerebellum either injected with lentiviral particle encoding for GFP (control) or with G3BP1. Upper panel: ATXN3 aggregates assessed by HA-tag immunoreactivity (arrowhead; scale bars = 50, 100 and 200 µm). Lower panel: Purkinje cells were assessed by calbindin immunoreactivity (scale bars = 100 and 200 µm). (E) G3BP1 expression significantly reduced the number of HA-ATXN3 aggregates (arrowhead), compared to control mice injected with GFP or non-injected. (F) G3BP1 expression significantly pre served the number of Purkinje cells within lobe IX, compared to non-injected and GFP injected controls (n = 6–7; *P < 0.05; one-way ANOVA followed by post hoc Bonferroni multiple comparisons test). Values are expressed as mean ± SEM.

Article Snippet: Plasmids encoding for human ATXN3 contain 28 glutamines (pEGFP-C1-Ataxin3Q28; #22122; Addgene) or 84 glutamines (pEGFP-C1-Ataxin3Q84; #22123; Addgene) were a gift from Henry Paulson and both are fused with a GFP protein at the N terminus.21 Plasmids encoding for human ATXN2 containing 22 glutamines (pEGFP-Ataxin2Q22) or 104 glutamines (pEGFP-Ataxin2Q104) were kindly provided by Professor Stefan Pulst.22 The LacZ gene was cloned in our laboratory under the control of a phosphoglycerate kinase promoter,23 and the GFP construct was cloned as previously described.24 The plasmid encoding for human G3BP1 (GeneBank accession DQ893058.2) purchased from Source Bioscience, was cloned into a lentiviral vector backbone using the GatewayTM LR ClonaseTM II Enzyme Mix, Invitrogen, according to the manufacturer’s instructions.

Techniques: Expressing, Transgenic Assay, Injection, Control, Immunohistochemistry

Journal: eLife

Article Title: SIRT6 is a DNA double-strand break sensor

doi: 10.7554/eLife.51636

Figure Lengend Snippet: ( A–C ) Imaging and AUC for SIRT6-GFP in cells with or without Olaparib. ( A ) Live imaging recruitment upon UV laser-induced damage (LID) shown by SIRT6-GFP in U2OS +/– Olaparib. Representative experiment examining SIRT6 recruitment to LID (n[+Ola]=23, n[–Ola]=23). ( B ) SIRT6 accumulation in same experiment as panel ( A ). ( C ) Average area under the curve (AUC) for cells +/– Olaparib in three replicate experiments. Error bars are the standard error of the mean (SEM) (n[+Ola]=38, n[–Ola]=39, p>0.05). ( D–F ) Imaging and AUC for SIRT6-GFP accumulation in shControl, shKu80 or shMRE11 Hela cells. ( D ) Average AUC from three experiments. Error bars are the SEM (shControl: n = 50; shKu80: n = 50, p<0.0005; shMRE11: n = 52, p>0.05). Accumulation of SIRT6-GFP ( E ) and imaging ( F ) from a representative experiment examining SIRT6 recruitment after LID (n[shControl]=28; n[shKu80]=30, n[shMRE11]=30). ( G–I ) MRE11-Cherry accumulation in response to LID in SIRT6 WT and KO U2OS cells. MRE11-Cherry imaging ( G ) and accumulation ( H ) in a representative experiment (n[WT]=20, n[KO]=16). ( I ) Mean AUC for three replicate experiments. Error bars are the SEM (n[WT]=36, n[KO]=33, p<0.0005). ( J–L ) Ku80-GFP accumulation in response to LID in SIRT6 WT and KO U2OS cells. Ku80-GFP imaging ( J ) and accumulation ( K ) in a representative experiment (n[WT]=17, n[KO]=17). ( L ) Mean AUC for three replicate experiments. Error bars are the SEM (n[WT]=33, n[KO]=33, p>0.05).

Article Snippet: Notes: the pQCXIP-Ku80-GFP-LacR plasmid used in this assay contains Ku80 that was acquired from Addgene (cat. #46958) and contains the D158G mutation.

Techniques: Imaging

Journal: eLife

Article Title: SIRT6 is a DNA double-strand break sensor

doi: 10.7554/eLife.51636

Figure Lengend Snippet: ( A, B ) Initiation of DDR by ATM-LacR-Cherry (n = 33, p<0.005), NBS1-LacR-Cherry (n = 82, p<0.05), MRE11-LacR-Cherry (n = 136, p<0.005) and Ku80-LacR-GFP (n = 52, p>0.05) in the tethering system. Co-localization percentage with γH2AX (compared to GFP-LacR (n = 310)) ( A ) and immunofluorescence ( B ) are shown. ( C, D ) Recruitment of SIRT6-GFP/SIRT6-Cherry to LacO sites by NBS1-LacR-Cherry (n = 87, p<0.0005), MRE11-LacR-Cherry (n = 31, p<0.005), Ku80-LacR-GFP (n = 45, p<0.005) and GFP-LacR (n = 85). Co-localization percentage with SIRT6 (compared to GFP-LacR ( C ) and immunoflorescence ( D ) are shown. Averages are for 3–5 experiments. Error bars show the SEM.

Article Snippet: Notes: the pQCXIP-Ku80-GFP-LacR plasmid used in this assay contains Ku80 that was acquired from Addgene (cat. #46958) and contains the D158G mutation.

Techniques: Immunofluorescence

Journal: eLife

Article Title: SIRT6 is a DNA double-strand break sensor

doi: 10.7554/eLife.51636

Figure Lengend Snippet: ( A ) Schematic representation of the Tethering assay with inhibition of signaling using Wortmannin. ( B–D ) Co-localization of γH2AX ( B ), 53BP1 ( C ), and BRAC1 ( D ) with SIRT6-LacR-GFP or GFP/Cherry-LacR after 24 hr with or without Wortmannin. Data are averages for three experiments (error bars are SEMs). ( B ) Co-localization with γH2AX (0 μM: n[SIRT6] = 43, n[GFP] = 41; 1 μM: (n[SIRT6] = 42, n[GFP] = 42; 10 μM: (n[SIRT6] = 42, n[GFP]=41). ( C ) Co-localization with 53BP1 (0 μM: n[SIRT6] = 32, n[GFP] = 32; 1 μM: n[SIRT6] = 30, n[GFP] = 32; 10 μM: n[SIRT6] = 33, n[GFP] = 31). ( D ) Co-localization with BRCA1 (0 μM: n[SIRT6] = 30, n[GFP] = 32; 1 μM: n[SIRT6] = 31, n[GFP] = 31; 10 μM: n[SIRT6] = 32, n[GFP] = 31). ( E ) Co-localization of Ku80-Flag or MRE11-Flag with SIRT6-LacR-GFP or GFP/Cherry-LacR after 24 hr with or without Wortmannin. Data are averages for three experiments (error bars are SEMs). For Ku80-Flag — 0 μM: n[SIRT6] = 45, n[Cherry] = 53; 10 μM: n[SIRT6] = 45, n[Cherry] = 58. For MRE11-Flag — 0 μM: n[SIRT6] = 50, n[Cherry] = 56; 10 μM: n[SIRT6] = 60, n[Cherry] = 51. *, p<0.05; **, p<0.005; ***, p<0.0005; ****, p<0.00005).

Article Snippet: Notes: the pQCXIP-Ku80-GFP-LacR plasmid used in this assay contains Ku80 that was acquired from Addgene (cat. #46958) and contains the D158G mutation.

Techniques: Inhibition

Journal: eLife

Article Title: SIRT6 is a DNA double-strand break sensor

doi: 10.7554/eLife.51636

Figure Lengend Snippet:

Article Snippet: Notes: the pQCXIP-Ku80-GFP-LacR plasmid used in this assay contains Ku80 that was acquired from Addgene (cat. #46958) and contains the D158G mutation.

Techniques: Recombinant, DNA Binding Assay