Journal: Brain

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

doi: 10.1093/brain/awac473

Figure Lengend Snippet: The expression of G3BP1 reduces the number of cells with aggregates of ATXN2MUT and ATXN3MUT. ( A ) Confocal microscopy representative images depicting Neuroa2 cells expressing ATX2MUT in three different experimental conditions. ( B ) Confocal microscopy representative images depicting Neuroa2 cells expressing ATX3MUT in three different experimental conditions. ( C ) The number of cells with aggregates of ATX2MUT per 100 transfected cells was significantly reduced on G3BP1 expression, compared to both control conditions. ( D ) The number of cells with aggregates of ATX3MUT per 100 transfected cells was significantly reduced on G3BP1 expression, compared to both control conditions ( n = 3 independent experiments; * P < 0.05; one-way ANOVA followed by post hoc Bonferroni multiple comparisons test). Values are expressed as mean ± SEM. Scale bar = 10 µm. ( E ) Representative western blot of Neuro2a lysates expressing pathological and non-pathological forms of ATXN2, co-transfected with lacZ or with G3BP1. Western blots were labelled using ATXN2, G3BP1 and β-tubulin antibodies. ( F ) Representative western blot of Neuro2a lysates expressing pathological and non-pathological forms of ATXN3, co-transfected with lacZ or with G3BP1. Western blots were labelled using ATXN3, G3BP1 and β-tubulin antibodies. ( G ) The levels of ATXN2WT protein are significantly reduced on G3BP1 expression, compared with control cells co-expressing lacZ. ( H ) The levels of ATXN2MUT protein are significantly reduced on G3BP1 expression, compared with control cells co-expressing lacZ. ( I ) The levels of ATXN3WT protein are significantly reduced on G3BP1 expression, compared with control cells co-expressing lacZ. ( J ) The levels of ATXN3MUT protein are significantly reduced on G3BP1 expression, compared with control cells co-expressing lacZ ( n = 5 independent experiments; * P < 0.05; ** P < 0.01; Student’s t -test).

Article Snippet: For immunochemical procedures, the following primary antibodies were used: mouse anti-ataxin-2 (1:1000, ref. 611378, BD Biosciences); mouse anti-ubiquitin (1:1000, ref. 3936S, Cell Signaling) rabbit anti-DARPP-32 (1:1000, ref. AB10518, Merck Millipore); rabbit anti-G3BP1 (1:1000, ref. 07-1801, Millipore); mouse anti-human G3BP1 (1:1000, ref. 611126, BD Biosciences); anti-G3BP1 (1:1000, ref. 05-1938; Sigma-Aldrich); mouse anti-GFAP (1:1000, ref. 644702, BioLegend); rabbit anti-HA (1:1000, ref. Ab9110, Abcam); mouse anti-calbindin D-28K (1:1000, ref. C9848, Sigma-Aldrich); mouse anti-PABP-1 (1:1000, ref. 04-1467, Millipore) and mouse β-Gal (14B7) (1:500, ref. 2372, Cell Signaling Technology).

Techniques: Expressing, Confocal Microscopy, Transfection, Western Blot

Journal: Brain

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

doi: 10.1093/brain/awac473

Figure Lengend Snippet: The NFT2-like domain of G3BP1 is important in the modulation of aggregation and protein levels of ATXN2MUT and ATXN3MUT. ( A ) Schematic representation of G3BP1 structural domains and the respective constructs with deleted NFT2 domain and deleted RRM domain. The delta symbol indicates deletion; PxxP = proline-rich region; RGG box = arginine and glycine rich box. ( B ) Neuro2a cells were transfected with full-length G3BP1, G3BP1-ΔRRM or G3BP1-ΔNTF2, and non-transfected (NT). Protein lysates were analysed through western blot depicting the expression of G3BP1 truncated forms with different molecular weight. Western blots were labelled using G3BP1, and β-tubulin antibodies. ( C ) Confocal microscopy representative images depicting Neuroa2 cells expressing ATXN2MUT and lacZ or G3BP1 or G3BP1-ΔNTF2 or G3BP1-ΔRRM. The expression of ATXN2MUT leads to the formation of aggregates (arrowheads). ( D ) Confocal microscopy representative images depicting Neuro2a cells expressing ATXN3MUT and lacZ or G3BP1 or G3BP1-ΔNTF2 or G3BP1-ΔRRM. The expression of ATXN3MUT leads to the formation of aggregates (arrowheads). Scale bar = 10 µm. ( E ) The number of cells with aggregates of ATX2MUT per 100 transfected cells with G3BP1-ΔRRM was significantly reduced compared with the lacZ control condition and increased compared to full-length G3BP1 condition. The expression of G3BP1-ΔNTF2 leads to a significant increase in the number of cells with aggregates compared to all the other experimental conditions ( n = 4 independent experiments; ## P < 0.01 to ATXN2MUT + lacZ; **** P < 0.0001 to ATXN2MUT + G3BP1; ++++ P < 0.0001 to ATXN2MUT + G3BP1-ΔRRM; one-way ANOVA, followed by post hoc Bonferroni multiple comparisons test). ( F ) The number of cells with aggregates of ATX3MUT per 100 transfected cells with G3BP1-ΔRRM was significantly reduced compared with the lacZ control condition and increased compared to full-length G3BP1 condition. The expression of G3BP1-ΔNTF2 leads to a significant increase in the number of cells with aggregates compared to ATXN3MUT + G3BP1 and ATXN3MUT + G3BP1-ΔRRM ( n = 4 independent experiments; ## P < 0.01 to ATXN3MUT + lacZ; **** P < 0.0001 to ATXN3MUT + G3BP1; ++ P < 0.01 to ATXN3MUT + G3BP1-ΔRRM; one-way ANOVA, followed by post hoc Bonferroni multiple comparisons test). ( G ) Representative western blot of Neuro2a lysates expressing ATXN2MUT, co-transfected with lacZ or G3BP1-ΔRRM or G3BP1-ΔNTF2. ( H ) The levels of ATXN2MUT protein are significantly reduced on G3BP1-ΔRRM expression compared to the other experimental conditions, whereas the expression of G3BP1-ΔNTF2 leads to a significant increase in ATXN2MUT protein levels, compared to the other conditions ( n = 4 independent experiments; ** P < 0.01 to ATXN2MUT + lacZ; #### P < 0.0001 to ATXN2MUT + G3BP1-ΔRRM; one-way ANOVA, followed by post hoc Bonferroni multiple comparisons test). ( I ) Representative western blot of Neuro2a lysates expressing ATXN3MUT, co-transfected with lacZ or G3BP1-ΔRRM or G3BP1-ΔNTF2. ( J ) The levels of ATXN3MUT protein are significantly reduced on G3BP1-ΔRRM expression compared to the other experimental conditions, whereas the expression of G3BP1-ΔNTF2 leads to a significant increase in ATXN3MUT protein levels, compared to ATXN3MUT + G3BP1-ΔRRM ( n = 4 independent experiments; * P < 0.05 to ATXN3MUT + lacZ; ** P < 0.01 to ATXN3MUT + lacZ; ### P < 0.001 to ATXN3MUT + G3BP1-ΔRRM; one-way ANOVA, followed by post hoc Bonferroni multiple comparisons test). Values are expressed as mean ± SEM.

Article Snippet: For immunochemical procedures, the following primary antibodies were used: mouse anti-ataxin-2 (1:1000, ref. 611378, BD Biosciences); mouse anti-ubiquitin (1:1000, ref. 3936S, Cell Signaling) rabbit anti-DARPP-32 (1:1000, ref. AB10518, Merck Millipore); rabbit anti-G3BP1 (1:1000, ref. 07-1801, Millipore); mouse anti-human G3BP1 (1:1000, ref. 611126, BD Biosciences); anti-G3BP1 (1:1000, ref. 05-1938; Sigma-Aldrich); mouse anti-GFAP (1:1000, ref. 644702, BioLegend); rabbit anti-HA (1:1000, ref. Ab9110, Abcam); mouse anti-calbindin D-28K (1:1000, ref. C9848, Sigma-Aldrich); mouse anti-PABP-1 (1:1000, ref. 04-1467, Millipore) and mouse β-Gal (14B7) (1:500, ref. 2372, Cell Signaling Technology).

Techniques: Construct, Transfection, Western Blot, Expressing, Molecular Weight, Confocal Microscopy

Journal: Brain

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

doi: 10.1093/brain/awac473

Figure Lengend Snippet: The Ser149 phosphorylation site is important in G3BP1 action on ATXN2 and ATXN3 mutant proteins. ( A ) Confocal microscopy representative images depicting Neuroa2 cells expressing ATXN2MUT and wild-type G3BP1 or G3BP1(S149A) or G3BP1(S149D). In the cells expressing wild-type G3BP1 or the phosphomimetic G3BP1(S149D), there are no aggregates of ATXN2MUT (arrows), contrasting with the cells expressing the phospho-dead G3BP1(Ser149A), where ATXN2MUT aggregates are observed (arrowheads). ( B ) Confocal microscopy representative images depicting Neuro2a cells expressing ATXN3MUT and wild-type G3BP1 or G3BP1(S149A) or G3BP1(S149D). In the cells expressing wild-type G3BP1 or the phosphomimetic G3BP1 (S149D) there are no aggregates of ATXN3MUT (arrows), contrasting with the cells expressing the phospho-dead G3BP1(Ser149A), where ATXN3MUT aggregates are observed (arrowheads). Scale bar = 20 µm. ( C ) Representative western blot of Neuro2a lysates expressing ATXN2MUT, co-transfected with wild-type G3BP1 or G3BP1(S149A) or G3BP1(S149D). Western blots were labelled using ATXN2, G3BP1 and β-tubulin antibodies. ( D ) Representative western blot of Neuro2a lysates expressing ATXN3MUT, co-transfected with wild-type G3BP1 or G3BP1(S149A) or G3BP1(S149D). Western blots were labelled using ATXN3, G3BP1 and β-tubulin antibodies. ( E ) The levels of ATXN2MUT protein are significantly increased in cells expressing the phospho-dead G3BP1(Ser149A), compared to ATX2MUT + G3BP1 ( n = 3 independent experiments; * P < 0.05; one-way ANOVA, followed by post hoc Bonferroni multiple comparisons test). ( F ) No significant alterations were found in the ATX2MUT mRNA levels between all the experimental conditions. ( G ) The levels of ATXN3MUT protein are significantly increased in cells expressing the phospho-dead G3BP1(Ser149A), compared to the other two conditions, whereas the cells expressing the phosphomimetic G3BP1(Ser149D) are significantly reduced, compared to the other two conditions. ( H ) No significant alterations were found in the ATX3MUT mRNA levels between all the experimental conditions ( n = 3 independent experiments; * P < 0.05 to ATXN3MUT + G3BP1; ## P < 0.01 to ATXN3MUT + G3BP1(Ser149A); one-way ANOVA, followed by post hoc Bonferroni multiple comparisons test). Values are expressed as mean ± SEM.

Article Snippet: For immunochemical procedures, the following primary antibodies were used: mouse anti-ataxin-2 (1:1000, ref. 611378, BD Biosciences); mouse anti-ubiquitin (1:1000, ref. 3936S, Cell Signaling) rabbit anti-DARPP-32 (1:1000, ref. AB10518, Merck Millipore); rabbit anti-G3BP1 (1:1000, ref. 07-1801, Millipore); mouse anti-human G3BP1 (1:1000, ref. 611126, BD Biosciences); anti-G3BP1 (1:1000, ref. 05-1938; Sigma-Aldrich); mouse anti-GFAP (1:1000, ref. 644702, BioLegend); rabbit anti-HA (1:1000, ref. Ab9110, Abcam); mouse anti-calbindin D-28K (1:1000, ref. C9848, Sigma-Aldrich); mouse anti-PABP-1 (1:1000, ref. 04-1467, Millipore) and mouse β-Gal (14B7) (1:500, ref. 2372, Cell Signaling Technology).

Techniques: Mutagenesis, Confocal Microscopy, Expressing, Western Blot, Transfection

Journal: Brain

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

doi: 10.1093/brain/awac473

Figure Lengend Snippet: 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 lysates 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 control 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 hemisphere. (* P < 0.05; ** P < 0.01; *** P < 0.001; Student’s t- test). Values are expressed as mean ± SEM.

Article Snippet: For immunochemical procedures, the following primary antibodies were used: mouse anti-ataxin-2 (1:1000, ref. 611378, BD Biosciences); mouse anti-ubiquitin (1:1000, ref. 3936S, Cell Signaling) rabbit anti-DARPP-32 (1:1000, ref. AB10518, Merck Millipore); rabbit anti-G3BP1 (1:1000, ref. 07-1801, Millipore); mouse anti-human G3BP1 (1:1000, ref. 611126, BD Biosciences); anti-G3BP1 (1:1000, ref. 05-1938; Sigma-Aldrich); mouse anti-GFAP (1:1000, ref. 644702, BioLegend); rabbit anti-HA (1:1000, ref. Ab9110, Abcam); mouse anti-calbindin D-28K (1:1000, ref. C9848, Sigma-Aldrich); mouse anti-PABP-1 (1:1000, ref. 04-1467, Millipore) and mouse β-Gal (14B7) (1:500, ref. 2372, Cell Signaling Technology).

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

Journal: Brain

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

doi: 10.1093/brain/awac473

Figure Lengend Snippet: 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 lentiviral 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, compared 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: For immunochemical procedures, the following primary antibodies were used: mouse anti-ataxin-2 (1:1000, ref. 611378, BD Biosciences); mouse anti-ubiquitin (1:1000, ref. 3936S, Cell Signaling) rabbit anti-DARPP-32 (1:1000, ref. AB10518, Merck Millipore); rabbit anti-G3BP1 (1:1000, ref. 07-1801, Millipore); mouse anti-human G3BP1 (1:1000, ref. 611126, BD Biosciences); anti-G3BP1 (1:1000, ref. 05-1938; Sigma-Aldrich); mouse anti-GFAP (1:1000, ref. 644702, BioLegend); rabbit anti-HA (1:1000, ref. Ab9110, Abcam); mouse anti-calbindin D-28K (1:1000, ref. C9848, Sigma-Aldrich); mouse anti-PABP-1 (1:1000, ref. 04-1467, Millipore) and mouse β-Gal (14B7) (1:500, ref. 2372, Cell Signaling Technology).

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

Journal: Brain

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

doi: 10.1093/brain/awac473

Figure Lengend Snippet: Overexpression of lentiviral vectors encoding G3BP1 in the brain of wild-type mice did not produce neuronal marker loss or inflammation. Mice at 8–12 weeks of age were stereotaxically injected into the striatum (bilaterally) either with PBS or with lentiviral particles encoding for human G3BP1 and euthanized for tissue collection 4 weeks after injection. ( A ) Schematic representation of the injection site in the striatum. ( B ) Immunohistochemistry images analysis of DARPP-32 depletion volume (dashed line; top ; scale bar = 200 µm) and G3BP1 ( bottom ; scale bar = 50 µm) labelling in brain sections from mice injected with PBS and in the contralateral hemisphere injected with lentiviral particles encoding for G3BP1. ( C ) The total area of DARPP-32 depletion, in mice brain sections, was reduced on injection of lentiviral particles encoding for G3BP1 when compared to the injection with PBS ( n = 4; * P < 0.05; Student's t- test). ( D ) We also labelled GFAP, a marker of astrogliosis, in brain sections from mice injected with PBS and in the contralateral hemisphere injected with lentiviral vectors encoding G3BP1. ( E ) The quantification of GFAP immunoreactivity did not detect significant differences between both hemispheres. Scale bar = 200 µm. Values are expressed as mean ± SEM.

Article Snippet: For immunochemical procedures, the following primary antibodies were used: mouse anti-ataxin-2 (1:1000, ref. 611378, BD Biosciences); mouse anti-ubiquitin (1:1000, ref. 3936S, Cell Signaling) rabbit anti-DARPP-32 (1:1000, ref. AB10518, Merck Millipore); rabbit anti-G3BP1 (1:1000, ref. 07-1801, Millipore); mouse anti-human G3BP1 (1:1000, ref. 611126, BD Biosciences); anti-G3BP1 (1:1000, ref. 05-1938; Sigma-Aldrich); mouse anti-GFAP (1:1000, ref. 644702, BioLegend); rabbit anti-HA (1:1000, ref. Ab9110, Abcam); mouse anti-calbindin D-28K (1:1000, ref. C9848, Sigma-Aldrich); mouse anti-PABP-1 (1:1000, ref. 04-1467, Millipore) and mouse β-Gal (14B7) (1:500, ref. 2372, Cell Signaling Technology).

Techniques: Over Expression, Marker, Injection, Immunohistochemistry

Journal: Brain

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

doi: 10.1093/brain/awac473

Figure Lengend Snippet: G3BP1 expression mitigates motor deficits and neuropathological abnormalities in an SCA3 transgenic mouse model. Transgenic mice animals expressing a truncated form of the ATXN3 protein containing 69 glutamines were stereotaxically injected in the cerebellum with lentiviral particles 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 performance at 9 weeks post-injection. ( A ) Mice injected with G3BP1 significantly improved motor performance (assessed by the rotarod test), as they remain 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 animals. ( 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 preserved 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: For immunochemical procedures, the following primary antibodies were used: mouse anti-ataxin-2 (1:1000, ref. 611378, BD Biosciences); mouse anti-ubiquitin (1:1000, ref. 3936S, Cell Signaling) rabbit anti-DARPP-32 (1:1000, ref. AB10518, Merck Millipore); rabbit anti-G3BP1 (1:1000, ref. 07-1801, Millipore); mouse anti-human G3BP1 (1:1000, ref. 611126, BD Biosciences); anti-G3BP1 (1:1000, ref. 05-1938; Sigma-Aldrich); mouse anti-GFAP (1:1000, ref. 644702, BioLegend); rabbit anti-HA (1:1000, ref. Ab9110, Abcam); mouse anti-calbindin D-28K (1:1000, ref. C9848, Sigma-Aldrich); mouse anti-PABP-1 (1:1000, ref. 04-1467, Millipore) and mouse β-Gal (14B7) (1:500, ref. 2372, Cell Signaling Technology).

Techniques: Expressing, Transgenic Assay, Injection, Immunohistochemistry