Journal: Neurobiology of Aging

Article Title: Combined age- and trauma-related proteomic changes in rat neocortex: a basis for brain vulnerability

doi: 10.1016/j.neurobiolaging.2011.09.029

Figure Lengend Snippet: Protein isoforms differentially expressed with age or traumatic brain injury

Article Snippet: Primary antibodies used were total CRMP2 (1:2000) and phospho-Thr514-CRMP2 (1:1000, Cell Signaling Technology, Danvers, MA); T-kininogen 1/2 (1:1000, sc-103887) and oxoglutarate dehydrogenase ( α KGD, 1:1000, sc-67238, Santa Cruz Biotechnology, Inc, Santa Cruz, CA).

Techniques: Protease Inhibitor

Journal: Neurobiology of Aging

Article Title: Combined age- and trauma-related proteomic changes in rat neocortex: a basis for brain vulnerability

doi: 10.1016/j.neurobiolaging.2011.09.029

Figure Lengend Snippet: Protein spot relative expression with age and brain injury: two-way analysis of variance

Article Snippet: Primary antibodies used were total CRMP2 (1:2000) and phospho-Thr514-CRMP2 (1:1000, Cell Signaling Technology, Danvers, MA); T-kininogen 1/2 (1:1000, sc-103887) and oxoglutarate dehydrogenase ( α KGD, 1:1000, sc-67238, Santa Cruz Biotechnology, Inc, Santa Cruz, CA).

Techniques: Expressing

Journal: Neurobiology of Aging

Article Title: Combined age- and trauma-related proteomic changes in rat neocortex: a basis for brain vulnerability

doi: 10.1016/j.neurobiolaging.2011.09.029

Figure Lengend Snippet: Age- and Injury-related CRMP2 Changes Differed With Brain Region and Epitope Detected by Immunoblot (A) Hippocampal CRMP2 decreased with injury in juvenile and adult, but not in geriatric brains. (B) Cortex CRMP2 increased in juvenile and adult, but not in geriatric brains. (C) Hippocampal phospho-Thr514-CRMP2 (pCRMP2, Thr514-CRMP2 phospho-specific antibody, Methods) appeared to decrease with injury (p < 0.05, all ages combined). (D) Cortex pCRMP2 increased in each age group compared to shams. Data normalized to β-actin in each sample and presented as mean ± SEM. Immunoblots (n = 4–5 per group) were arranged by age (all juvenile specimens on one gel, etc.) and repeated arranged by same treatment (shams of all ages on one gel, etc.); results were comparable for both approaches. No significant changes in the β–actin internal control were observed. Two-way ANOVAs [Hippocampus FCRMP2 = 11.12, p < 0.0001; FpCRMP2 = 6.40, p < 0.0002; Cortex FCRMP2 = 11.12, p < 0.0001; FpCRMP2 = 3.77, p < 0.004] were followed by one-way ANOVA with Dunnett post hoc tests; *p < 0.05 vs. Sham (within age group); §p < 0.05 vs. Adult (within treatment group).

Article Snippet: Primary antibodies used were total CRMP2 (1:2000) and phospho-Thr514-CRMP2 (1:1000, Cell Signaling Technology, Danvers, MA); T-kininogen 1/2 (1:1000, sc-103887) and oxoglutarate dehydrogenase ( α KGD, 1:1000, sc-67238, Santa Cruz Biotechnology, Inc, Santa Cruz, CA).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Inhibition of microtubule detyrosination by parthenolide facilitates functional CNS axon regeneration

doi: 10.1101/2023.04.05.535722

Figure Lengend Snippet: A) Schematic drawing illustrating the effect of hIL-6 on the PI3K/AKT/GSK3 signaling pathway. hIL-6 binding to gp130 activates phosphatidylinositol 3-kinase (PI3K), converting phosphati-dylinositol (4, 5)-bisphosphate (PIP 2 ) to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP 3 ). PIP 3 stimulates AKT. Subsequent effects on GSK3, CRMP2, and microtubule detyrosination are shown in B - L . B) Western blots of optic nerve lysates from either untreated mice (con) or animals that had received intravitreal AAV2-hIL-6 injections 3 weeks earlier. Retinal hIL-6 expression elevated inhibitory phosphorylation of GSK3α and GSK3β, while total GSK3 levels remained unaffected. Inhibitory CRMP2 phosphorylation was reduced without altering total CRMP2 levels, while detyrosinated tubulin levels were increased. βIII-tubulin (tubulin) served as a loading control. C-H) Densitometric quantification of western blots shown in B relative to βIII-tubulin and normalized to the untreated control (con). Data represent means ± SEM of samples from at least three animals per group. I) Representative images of axon tips from dissociated RGCs from mice as described in B . RGCs were cultured for 4 days in the presence of either vehicle (-) or parthenolide (par; 5 nM) and were immunohistochemically stained for βIII-tubulin (tubulin, green) and detyrosinated α-tubulin (detyr, red). White arrows indicate the last 15 μm of detyrosinated or negative axon tips after respective treatments. Scale bar: 15 μm J) Quantification of the percentages of detyrosinated tubulin-positive (detyr + ) axon tips. Par was applied at indicated concentrations. hIL-6 treatment increased tubulin detyrosination, while par blocked this effect. Data represent means ± SEM of 3 technical replicates from 2 independent experiments. P-values refer to untreated control. K) Representative images of βIII-tubulin (tubulin) positive RGCs from cultures as described in I , cultured in the presence or absence of parthenolide (par; 0.5 nM or 5 nM). Scale bar: 50 μm L) Quantification of the average neurite length per RGC in cultures depicted in K . Par was applied at indicated concentrations in combination with hIL-6. Data were normalized to untreated controls with an average neurite length of 8.4 µm per RGC and represent means ± SEM of three technical replicates from two independent experiments. Significances of intergroup differences were evaluated using Student’s t-test for quantifications shown in C-H . A one-way analysis of variance (ANOVA) followed by a Holm-Sidak or Tukey post hoc test is shown in J and L , respectively. P-values indicate statistical significance compared to the untreated (black p-values) or the vehicle-treated hIL-6 (red p-values) groups. n.s.=non-significant. Dots represent values from single animals in C-H and three technical replicates of two independent experiments, respectively, in J and L .

Article Snippet: Blots were blocked in 5% dried milk in phosphate-buffered saline with 0.05% Tween-20 (PBS-T) (Sigma, St Louis, USA) and incubated with antibodies against S21-phosphorylated GSK3α (1:2000; Cell Signaling Technology, RRID: AB_11166651), S9-phosphorylated GSK3β (1:1000; Cell Signaling Technology, RRID:AB_2115196), total GSK3α/β (1:1000; Cell Signaling Technology, RRID: AB_10547140), T514-phosphorylated CRMP2 (1:1000; Abcam, RRID:AB_942229), total CRMP2 (1:1000; Cell Signaling Technology, RRID:AB_2094339), detyrosinated tubulin (1:1000; Millipore, RRID:AB_177350) and βIII-tubulin (1:2000; BioLegend, RRID:AB_2313773) at 4°C overnight.

Techniques: Binding Assay, Western Blot, Expressing, Cell Culture, Staining

Journal: bioRxiv

Article Title: Inhibition of microtubule detyrosination by parthenolide facilitates functional CNS axon regeneration

doi: 10.1101/2023.04.05.535722

Figure Lengend Snippet: A) Representative photographs of dissociated RGCs stained for T514-phosphorylated CRMP2 (pCRMP2; red) and βIII-tubulin (tubulin; green) after 4 d in culture. RGCs were isolated from WT mice and either incubated with 0.5 nM parthenolide (par) or the vehicle (veh). Scale bar: 50 μm. B) Quantification of pCRMP2 staining intensities of cultured RGCs as described in A . Integrated density of pCRMP2 staining was measured and normalized against values of the vehicle-treated control. Values represent means ± SEM of three independent experiments per group. In each experiment, 80 RGCs per group were analyzed in two replicate wells. Statistical analysis using Student’s t-test revealed no significant differences between the two groups.

Article Snippet: Blots were blocked in 5% dried milk in phosphate-buffered saline with 0.05% Tween-20 (PBS-T) (Sigma, St Louis, USA) and incubated with antibodies against S21-phosphorylated GSK3α (1:2000; Cell Signaling Technology, RRID: AB_11166651), S9-phosphorylated GSK3β (1:1000; Cell Signaling Technology, RRID:AB_2115196), total GSK3α/β (1:1000; Cell Signaling Technology, RRID: AB_10547140), T514-phosphorylated CRMP2 (1:1000; Abcam, RRID:AB_942229), total CRMP2 (1:1000; Cell Signaling Technology, RRID:AB_2094339), detyrosinated tubulin (1:1000; Millipore, RRID:AB_177350) and βIII-tubulin (1:2000; BioLegend, RRID:AB_2313773) at 4°C overnight.

Techniques: Staining, Isolation, Incubation, Cell Culture

Journal: bioRxiv

Article Title: Inhibition of microtubule detyrosination by parthenolide facilitates functional CNS axon regeneration

doi: 10.1101/2023.04.05.535722

Figure Lengend Snippet: A) Schematic drawing illustrating the effect of PTEN -/- on the PI3K/AKT/GSK3 signaling pathway. PTEN -/- promotes the conversion of phosphatidylinositol (4, 5)-bisphosphate (PIP 2 ) to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP 3 ). PIP 3 stimulates AKT and thus induces inhibitory GSK3 phosphorylation. As a result, the phosphorylation of CRMP2 and MAP1B is reduced. The effect on tubulin detyrosination is illustrated in B-E . B) Representative images of axon tips from dissociated RGCs of PTEN-floxed mice. Animals were either untreated (con) or had received intravitreal injections of AAV2-Cre 3 weeks before dissociation (PTEN -/- ). RGCs were cultured for 4 days in the presence of either vehicle (-) or parthenolide (par; 5 nM) and were immunohistochemically stained for βIII-tubulin (tubulin, green) and detyrosinated α-tubulin (detyr, red). White arrows indicate the last 15 μm of axon tips. Scale bar: 15 μm C) Quantification of the percentages of detyrosinated tubulin-positive (detyr + ) axon tips. Par was applied at indicated concentrations. PTEN -/- increased tubulin detyrosination, while par blocked this effect. Data represent means ± SEM of 3 technical replicates from 2 independent experiments. D) Representative images of βIII-tubulin (tubulin) positive RGCs from cultures as described in B , cultured in the presence or absence of parthenolide (par; 0.5 nM or 5 nM). Scale bar: 50 μm E) Quantification of the average neurite length per RGC in cultures depicted in D . Par was applied at indicated concentrations in combination with PTEN -/- . Data were normalized to untreated controls with an average neurite length of 1.04 µm per RGC and represent means ± SEM of three technical replicates from two independent experiments. The significance of intergroup differences was evaluated through a one-way analysis of variance (ANOVA) followed by a Holm-Sidak or Tukey post hoc test for data shown in C and E , respectively. P-values indicate statistical significance compared to the untreated (black p-values) or the vehicle-treated PTEN -/- (red p-values) groups. n.s. = non-significant. Dots represent values from three technical replicates of two independent experiments.

Article Snippet: Blots were blocked in 5% dried milk in phosphate-buffered saline with 0.05% Tween-20 (PBS-T) (Sigma, St Louis, USA) and incubated with antibodies against S21-phosphorylated GSK3α (1:2000; Cell Signaling Technology, RRID: AB_11166651), S9-phosphorylated GSK3β (1:1000; Cell Signaling Technology, RRID:AB_2115196), total GSK3α/β (1:1000; Cell Signaling Technology, RRID: AB_10547140), T514-phosphorylated CRMP2 (1:1000; Abcam, RRID:AB_942229), total CRMP2 (1:1000; Cell Signaling Technology, RRID:AB_2094339), detyrosinated tubulin (1:1000; Millipore, RRID:AB_177350) and βIII-tubulin (1:2000; BioLegend, RRID:AB_2313773) at 4°C overnight.

Techniques: Cell Culture, Staining

Journal: Communications Biology

Article Title: GSK3-CRMP2 signaling mediates axonal regeneration induced by Pten knockout

doi: 10.1038/s42003-019-0524-1

Figure Lengend Snippet: PTEN −/− induces inhibitory GSK3 phosphorylation. a Schematic drawing illustrating the PTEN/AKT/mTOR/pS6 and PTEN/AKT/GSK3 signaling pathways. PTEN depletion activates phosphatidylinositol 3-kinase (PI3K), which then converts phosphatidylinositol (4, 5) bisphosphate (PIP 2 ) into phosphatidylinositol (3, 4, 5) trisphosphate (PIP 3 ). The latter activates AKT by a phosphatidylinositol-dependent kinase (PDK) 1/2-mediated phosphorylation. AKT activates mammalian target of rapamycin (mTOR) by releasing TSC1/2 inhibition leading to phosphorylation of ribosomal protein S6 via mTOR. Moreover, it inhibits GSK3 isoforms by phosphorylation. b , c Retinal cross-sections of PTEN f/f mice after intravitreal AAV-GFP (PTEN +/+ ) or AAV-Cre injection leading to PTEN depletion (PTEN −/− ). Animals remained either untreated (con) or received ONC 5 days before tissue isolation. ONC-induced S21-phosphorylation of GSK3α (pGSK3α, red, b ) and S9-phosphorylation of GSK3β (pGSK3β, red, c ) in βIII-tubulin-positive RGCs (green) in PTEN +/+ , while sole PTEN −/− showed already a markedly stronger effect. Inhibitory GSK3 phosphorylation was absent in retinae of Gsk3α S/A , or Gsk3β S/A knockin mice, verifying antibody specificity. GCL, ganglion cell layer; INL, inner nuclear layer. Scale bars: 50 µm. d Western blots: Retinal lysates from animals as described in ( b ) and ( c ). ONC moderately increased pGSK3α and pGSK3β signals in PTEN +/+ mice compared with untreated controls, whereas PTEN −/− showed a much stronger effect. Lysates from Gsk3(α/β) S/A knockin ((α/β) S/A ) mice showed no pGSK3 signals, verifying antibody specificities. Levels of T 308 -phosphorylated AKT (pAKT) were elevated after ONC in PTEN +/+ and (α/β) S/A mice, but stronger increased by PTEN −/− . Phosphorylation of ribosomal protein S6 (pS6) was low in the PTEN +/+ retinae and further decreased by ONC, while PTEN −/− induced a strong upregulation in uninjured and injured retinae. Total GSK3α and GSK3β levels were comparable in all experimental groups. Loading control: βIII-tubulin (tubulin). e – j Quantification of Western blots in ( d ) relative to βIII-tubulin and normalized to PTEN +/+ con, or in case of pGSK3α and pGSK3β to ONC. One-way analysis of variance (ANOVA) with Tukey post hoc test was used. Treatment effects compared with PTEN +/+ con: ** p < 0.01; *** p < 0.001; n.s. = non-significant. Values represent means ± SEM of 3–8 retinae per group ( e – h : n = 3, i : PTEN +/+ con, n = 4; PTEN +/+ ONC, n = 5; PTEN −/− con, n = 8; PTEN −/− ONC, n = 5; PTEN −/− (α/β S/A ) ONC, n = 3; j : PTEN +/+ con, n = 4; PTEN +/+ ONC, n = 7; PTEN −/− con, n = 7; PTEN −/− ONC, n = 6; PTEN −/− (α/β S/A ) ONC, n = 7)

Article Snippet: Blots were blocked in 5% dried milk in phosphate-buffered saline with 0.05% Tween-20 (PBS-T) (Sigma, St Louis, USA) and incubated with antibodies against βIII-tubulin (1:2000; BioLegend, San Diego, USA, RRID:AB_2313773), S9-phosphorylated GSK3β (1:1000, RRID:AB_2115196) S21-phosphorylated GSK3α (1:2000; RRID:AB_2114897), total GSK3α/β (1:500, RRID:AB_10547140; all from Cell Signaling Technologies, Cambridge, UK), ribosomal protein S6 (1:5000 Cell Signaling Technologies RRID:AB_2181035), T 514 -phosphorylated CRMP2 (1:2000; Abcam, Cambridge, UK, RRID:AB_942229), total CRMP2 (1:500; Cell Signaling Technologies, RRID:AB_2094339), or T308-phosphorylated AKT (1:1000; Cell Signaling Technologies, RRID:AB_2629447) at 4 °C overnight.

Techniques: Phospho-proteomics, Protein-Protein interactions, Inhibition, Injection, Isolation, Knock-In, Western Blot, Control

Journal: Communications Biology

Article Title: GSK3-CRMP2 signaling mediates axonal regeneration induced by Pten knockout

doi: 10.1038/s42003-019-0524-1

Figure Lengend Snippet: PTEN −/− -induced mTOR activity depends on GSK3 inhibition. a Schematic drawing illustrating the outcome of experiments depicted in Figs. 5 and . b Retinal flat mounts isolated from PTEN +/+ /GSK3(α/β) +/+ (wt), GSK3β S/A (β S/A ), PTEN −/− , and PTEN −/− /GSK3β S/A (PTEN −/− / β S/A ) mice which were either left untreated (con) or subjected to optic nerve crush (ONC). Five days after surgery, retinae were fixed and stained for phosphorylated ribosomal protein S6 (pS6; red) and βIII-tubulin (tubulin; green in lower row to visualize RGCs). Scale bar: 50 μm. c Quantification of pS6-positive RGCs in flat mounts as depicted in ( b ). Compared with wt animals, pS6 levels were markedly increased after PTEN −/− and significantly compromised in animals with additional GSK3β S/A . Values represent means ± SEM of 3–5 retinae per group (untreated controls: PTEN +/+ , n = 4; β S/A , n = 4; PTEN −/− , n = 3; PTEN −/− /β S/A , n = 4; mice 5 days after ONC: PTEN +/+ , n = 4; β S/A , n = 5; PTEN −/− , n = 3; PTEN −/− /β S/A , n = 4). d Western blot analysis for phosphorylated AKT (pAKT) and pS6 of retinal lysates from animals as described in ( b ). βIII-tubulin (tubulin) served as a loading control. e , f Densitometric quantification of Western blots as depicted in ( d ) relative to βIII-tubulin and normalized to PTEN −/− con. Values represent means ± SEM of 3–8 retinae per group ( e : untreated controls: PTEN +/+ , n = 6; β S/A , n = 3; PTEN −/− , n = 6; PTEN −/− /β S/A , n = 3; mice 5 days after ONC: PTEN +/+ , n = 6; β S/A , n = 5, PTEN −/− , n = 6; PTEN −/− /β S/A , n = 3; f : untreated controls: PTEN +/+ , n = 8; β S/A n = 3; PTEN −/− , n = 7; PTEN −/− /β S/A , n = 3; mice 5 days after ONC: PTEN +/+ , n = 7; β S/A n = 3; PTEN −/− , n = 4; PTEN −/− /β S/A , n = 6). Significances of intergroup differences were evaluated using two-way analysis of variance (ANOVA) with Tukey or Holm-Sidak post hoc test. Treatment effects to PTEN +/+ con, or as indicated: * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. = non-significant

Article Snippet: Blots were blocked in 5% dried milk in phosphate-buffered saline with 0.05% Tween-20 (PBS-T) (Sigma, St Louis, USA) and incubated with antibodies against βIII-tubulin (1:2000; BioLegend, San Diego, USA, RRID:AB_2313773), S9-phosphorylated GSK3β (1:1000, RRID:AB_2115196) S21-phosphorylated GSK3α (1:2000; RRID:AB_2114897), total GSK3α/β (1:500, RRID:AB_10547140; all from Cell Signaling Technologies, Cambridge, UK), ribosomal protein S6 (1:5000 Cell Signaling Technologies RRID:AB_2181035), T 514 -phosphorylated CRMP2 (1:2000; Abcam, Cambridge, UK, RRID:AB_942229), total CRMP2 (1:500; Cell Signaling Technologies, RRID:AB_2094339), or T308-phosphorylated AKT (1:1000; Cell Signaling Technologies, RRID:AB_2629447) at 4 °C overnight.

Techniques: Activity Assay, Inhibition, Isolation, Staining, Western Blot, Control

Journal: eLife

Article Title: Inhibition of microtubule detyrosination by parthenolide facilitates functional CNS axon regeneration

doi: 10.7554/eLife.88279

Figure Lengend Snippet: ( A ) Schematic drawing illustrating the effect of hIL-6 on the PI3K/AKT/GSK3 signaling pathway. hIL-6 binding to gp130 activates phosphatidylinositol 3-kinase (PI3K), converting phosphatidylinositol (4, 5)-bisphosphate (PIP 2 ) to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP 3 ). PIP 3 stimulates AKT. Subsequent effects on GSK3, CRMP2, and microtubule detyrosination are shown in B–L . ( B ) Western blots of optic nerve lysates from untreated mice (con) or animals that had received intravitreal AAV2-hIL-6 injections 3 weeks earlier. Retinal hIL-6 expression elevated inhibitory phosphorylation of GSK3α and GSK3β, while total GSK3 levels remained unaffected. Inhibitory CRMP2 phosphorylation was reduced without altering total CRMP2 levels, while detyrosinated tubulin levels were increased. βIII-tubulin (tubulin) served as a loading control. ( C–H ) Densitometric quantification of western blots shown in B relative to βIII-tubulin and normalized to the untreated control (con). Data represent means ± SEM of samples from at least three animals per group. ( I ) Representative images of axon tips from dissociated RGCs from mice, as described in B . RGCs, were cultured for 4 days in the presence of either vehicle (-) or parthenolide (par; 5 nM) and were immunohistochemically stained for βIII-tubulin (tubulin, green) and detyrosinated α-tubulin (detyr, red). White arrows indicate the last 15 μm of detyrosinated or negative axon tips after respective treatments. Scale bar: 15 μm ( J ) Quantification of the percentages of detyrosinated tubulin-positive (detyr + ) axon tips. Par was applied at indicated concentrations. hIL-6 treatment increased tubulin detyrosination, while par blocked this effect. Data represent means ± SEM of 3 technical replicates from 2 independent experiments. P-values refer to untreated control. ( K ) Representative images of βIII-tubulin (tubulin) positive RGCs from cultures as described in I , cultured in the presence or absence of parthenolide (par; 0.5 nM or 5 nM). Scale bar: 50 μm ( L ) Quantification of the average neurite length per RGC in cultures depicted in K . Par was applied at indicated concentrations in combination with hIL-6. Data were normalized to untreated controls with an average neurite length of 8.4 µm per RGC and represent means ± SEM of three technical replicates from two independent experiments. Significances of intergroup differences were evaluated using Student’s t-test for quantifications shown in C-H . A one-way analysis of variance (ANOVA) followed by a Holm-Sidak or Tukey post hoc test is shown in J and L , respectively. P-values indicate statistical significance compared to the untreated (black p-values) or the vehicle-treated hIL-6 (red p-values) groups. n.s.=non-significant. Dots represent values from single animals in C-H and three technical replicates of two independent experiments, respectively, in J and L .

Article Snippet: Blots were blocked in 5% dried milk in phosphate-buffered saline with 0.05% Tween-20 (PBS-T; Sigma, St Louis, USA) and incubated with antibodies against S21-phosphorylated GSK3α (1:2000; Cell Signaling Technology, RRID: AB_659836 ), S9-phosphorylated GSK3β (1:1000; Cell Signaling Technology, RRID: AB_2115196 ), total GSK3α/β (1:1000; Cell Signaling Technology, RRID: AB_10547140 ), T514-phosphorylated CRMP2 (1:1000; Abcam, RRID: AB_942229 ), total CRMP2 (1:1000; Cell Signaling Technology, RRID: AB_2094339 ), detyrosinated tubulin (1:1000; Millipore, RRID: AB_177350 ) and βIII-tubulin (1:2000; BioLegend, RRID: AB_2313773 ) at 4 °C overnight.

Techniques: Binding Assay, Western Blot, Expressing, Cell Culture, Staining

Journal: bioRxiv

Article Title: Inhibition of microtubule detyrosination by parthenolide facilitates functional CNS axon regeneration

doi: 10.1101/2023.04.05.535722

Figure Lengend Snippet: A) Schematic drawing illustrating the effect of hIL-6 on the PI3K/AKT/GSK3 signaling pathway. hIL-6 binding to gp130 activates phosphatidylinositol 3-kinase (PI3K), converting phosphati-dylinositol (4, 5)-bisphosphate (PIP 2 ) to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP 3 ). PIP 3 stimulates AKT. Subsequent effects on GSK3, CRMP2, and microtubule detyrosination are shown in B - L . B) Western blots of optic nerve lysates from either untreated mice (con) or animals that had received intravitreal AAV2-hIL-6 injections 3 weeks earlier. Retinal hIL-6 expression elevated inhibitory phosphorylation of GSK3α and GSK3β, while total GSK3 levels remained unaffected. Inhibitory CRMP2 phosphorylation was reduced without altering total CRMP2 levels, while detyrosinated tubulin levels were increased. βIII-tubulin (tubulin) served as a loading control. C-H) Densitometric quantification of western blots shown in B relative to βIII-tubulin and normalized to the untreated control (con). Data represent means ± SEM of samples from at least three animals per group. I) Representative images of axon tips from dissociated RGCs from mice as described in B . RGCs were cultured for 4 days in the presence of either vehicle (-) or parthenolide (par; 5 nM) and were immunohistochemically stained for βIII-tubulin (tubulin, green) and detyrosinated α-tubulin (detyr, red). White arrows indicate the last 15 μm of detyrosinated or negative axon tips after respective treatments. Scale bar: 15 μm J) Quantification of the percentages of detyrosinated tubulin-positive (detyr + ) axon tips. Par was applied at indicated concentrations. hIL-6 treatment increased tubulin detyrosination, while par blocked this effect. Data represent means ± SEM of 3 technical replicates from 2 independent experiments. P-values refer to untreated control. K) Representative images of βIII-tubulin (tubulin) positive RGCs from cultures as described in I , cultured in the presence or absence of parthenolide (par; 0.5 nM or 5 nM). Scale bar: 50 μm L) Quantification of the average neurite length per RGC in cultures depicted in K . Par was applied at indicated concentrations in combination with hIL-6. Data were normalized to untreated controls with an average neurite length of 8.4 µm per RGC and represent means ± SEM of three technical replicates from two independent experiments. Significances of intergroup differences were evaluated using Student’s t-test for quantifications shown in C-H . A one-way analysis of variance (ANOVA) followed by a Holm-Sidak or Tukey post hoc test is shown in J and L , respectively. P-values indicate statistical significance compared to the untreated (black p-values) or the vehicle-treated hIL-6 (red p-values) groups. n.s.=non-significant. Dots represent values from single animals in C-H and three technical replicates of two independent experiments, respectively, in J and L .

Article Snippet: Blots were blocked in 5% dried milk in phosphate-buffered saline with 0.05% Tween-20 (PBS-T) (Sigma, St Louis, USA) and incubated with antibodies against S21-phosphorylated GSK3α (1:2000; Cell Signaling Technology, RRID: AB_11166651), S9-phosphorylated GSK3β (1:1000; Cell Signaling Technology, RRID:AB_2115196), total GSK3α/β (1:1000; Cell Signaling Technology, RRID: AB_10547140), T514-phosphorylated CRMP2 (1:1000; Abcam, RRID:AB_942229), total CRMP2 (1:1000; Cell Signaling Technology, RRID:AB_2094339), detyrosinated tubulin (1:1000; Millipore, RRID:AB_177350) and βIII-tubulin (1:2000; BioLegend, RRID:AB_2313773) at 4°C overnight.

Techniques: Binding Assay, Western Blot, Expressing, Phospho-proteomics, Control, Cell Culture, Staining

Journal: bioRxiv

Article Title: Inhibition of microtubule detyrosination by parthenolide facilitates functional CNS axon regeneration

doi: 10.1101/2023.04.05.535722

Figure Lengend Snippet: A) Schematic drawing illustrating the effect of PTEN -/- on the PI3K/AKT/GSK3 signaling pathway. PTEN -/- promotes the conversion of phosphatidylinositol (4, 5)-bisphosphate (PIP 2 ) to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP 3 ). PIP 3 stimulates AKT and thus induces inhibitory GSK3 phosphorylation. As a result, the phosphorylation of CRMP2 and MAP1B is reduced. The effect on tubulin detyrosination is illustrated in B-E . B) Representative images of axon tips from dissociated RGCs of PTEN-floxed mice. Animals were either untreated (con) or had received intravitreal injections of AAV2-Cre 3 weeks before dissociation (PTEN -/- ). RGCs were cultured for 4 days in the presence of either vehicle (-) or parthenolide (par; 5 nM) and were immunohistochemically stained for βIII-tubulin (tubulin, green) and detyrosinated α-tubulin (detyr, red). White arrows indicate the last 15 μm of axon tips. Scale bar: 15 μm C) Quantification of the percentages of detyrosinated tubulin-positive (detyr + ) axon tips. Par was applied at indicated concentrations. PTEN -/- increased tubulin detyrosination, while par blocked this effect. Data represent means ± SEM of 3 technical replicates from 2 independent experiments. D) Representative images of βIII-tubulin (tubulin) positive RGCs from cultures as described in B , cultured in the presence or absence of parthenolide (par; 0.5 nM or 5 nM). Scale bar: 50 μm E) Quantification of the average neurite length per RGC in cultures depicted in D . Par was applied at indicated concentrations in combination with PTEN -/- . Data were normalized to untreated controls with an average neurite length of 1.04 µm per RGC and represent means ± SEM of three technical replicates from two independent experiments. The significance of intergroup differences was evaluated through a one-way analysis of variance (ANOVA) followed by a Holm-Sidak or Tukey post hoc test for data shown in C and E , respectively. P-values indicate statistical significance compared to the untreated (black p-values) or the vehicle-treated PTEN -/- (red p-values) groups. n.s. = non-significant. Dots represent values from three technical replicates of two independent experiments.

Article Snippet: Blots were blocked in 5% dried milk in phosphate-buffered saline with 0.05% Tween-20 (PBS-T) (Sigma, St Louis, USA) and incubated with antibodies against S21-phosphorylated GSK3α (1:2000; Cell Signaling Technology, RRID: AB_11166651), S9-phosphorylated GSK3β (1:1000; Cell Signaling Technology, RRID:AB_2115196), total GSK3α/β (1:1000; Cell Signaling Technology, RRID: AB_10547140), T514-phosphorylated CRMP2 (1:1000; Abcam, RRID:AB_942229), total CRMP2 (1:1000; Cell Signaling Technology, RRID:AB_2094339), detyrosinated tubulin (1:1000; Millipore, RRID:AB_177350) and βIII-tubulin (1:2000; BioLegend, RRID:AB_2313773) at 4°C overnight.

Techniques: Phospho-proteomics, Cell Culture, Staining

Journal: Communications Biology

Article Title: GSK3-CRMP2 signaling mediates axonal regeneration induced by Pten knockout

doi: 10.1038/s42003-019-0524-1

Figure Lengend Snippet: PTEN −/− induces inhibitory GSK3 phosphorylation. a Schematic drawing illustrating the PTEN/AKT/mTOR/pS6 and PTEN/AKT/GSK3 signaling pathways. PTEN depletion activates phosphatidylinositol 3-kinase (PI3K), which then converts phosphatidylinositol (4, 5) bisphosphate (PIP 2 ) into phosphatidylinositol (3, 4, 5) trisphosphate (PIP 3 ). The latter activates AKT by a phosphatidylinositol-dependent kinase (PDK) 1/2-mediated phosphorylation. AKT activates mammalian target of rapamycin (mTOR) by releasing TSC1/2 inhibition leading to phosphorylation of ribosomal protein S6 via mTOR. Moreover, it inhibits GSK3 isoforms by phosphorylation. b , c Retinal cross-sections of PTEN f/f mice after intravitreal AAV-GFP (PTEN +/+ ) or AAV-Cre injection leading to PTEN depletion (PTEN −/− ). Animals remained either untreated (con) or received ONC 5 days before tissue isolation. ONC-induced S21-phosphorylation of GSK3α (pGSK3α, red, b ) and S9-phosphorylation of GSK3β (pGSK3β, red, c ) in βIII-tubulin-positive RGCs (green) in PTEN +/+ , while sole PTEN −/− showed already a markedly stronger effect. Inhibitory GSK3 phosphorylation was absent in retinae of Gsk3α S/A , or Gsk3β S/A knockin mice, verifying antibody specificity. GCL, ganglion cell layer; INL, inner nuclear layer. Scale bars: 50 µm. d Western blots: Retinal lysates from animals as described in ( b ) and ( c ). ONC moderately increased pGSK3α and pGSK3β signals in PTEN +/+ mice compared with untreated controls, whereas PTEN −/− showed a much stronger effect. Lysates from Gsk3(α/β) S/A knockin ((α/β) S/A ) mice showed no pGSK3 signals, verifying antibody specificities. Levels of T 308 -phosphorylated AKT (pAKT) were elevated after ONC in PTEN +/+ and (α/β) S/A mice, but stronger increased by PTEN −/− . Phosphorylation of ribosomal protein S6 (pS6) was low in the PTEN +/+ retinae and further decreased by ONC, while PTEN −/− induced a strong upregulation in uninjured and injured retinae. Total GSK3α and GSK3β levels were comparable in all experimental groups. Loading control: βIII-tubulin (tubulin). e – j Quantification of Western blots in ( d ) relative to βIII-tubulin and normalized to PTEN +/+ con, or in case of pGSK3α and pGSK3β to ONC. One-way analysis of variance (ANOVA) with Tukey post hoc test was used. Treatment effects compared with PTEN +/+ con: ** p < 0.01; *** p < 0.001; n.s. = non-significant. Values represent means ± SEM of 3–8 retinae per group ( e – h : n = 3, i : PTEN +/+ con, n = 4; PTEN +/+ ONC, n = 5; PTEN −/− con, n = 8; PTEN −/− ONC, n = 5; PTEN −/− (α/β S/A ) ONC, n = 3; j : PTEN +/+ con, n = 4; PTEN +/+ ONC, n = 7; PTEN −/− con, n = 7; PTEN −/− ONC, n = 6; PTEN −/− (α/β S/A ) ONC, n = 7)

Article Snippet: Blots were blocked in 5% dried milk in phosphate-buffered saline with 0.05% Tween-20 (PBS-T) (Sigma, St Louis, USA) and incubated with antibodies against βIII-tubulin (1:2000; BioLegend, San Diego, USA, RRID:AB_2313773), S9-phosphorylated GSK3β (1:1000, RRID:AB_2115196) S21-phosphorylated GSK3α (1:2000; RRID:AB_2114897), total GSK3α/β (1:500, RRID:AB_10547140; all from Cell Signaling Technologies, Cambridge, UK), ribosomal protein S6 (1:5000 Cell Signaling Technologies RRID:AB_2181035), T 514 -phosphorylated CRMP2 (1:2000; Abcam, Cambridge, UK, RRID:AB_942229), total CRMP2 (1:500; Cell Signaling Technologies, RRID:AB_2094339), or T308-phosphorylated AKT (1:1000; Cell Signaling Technologies, RRID:AB_2629447) at 4 °C overnight.

Techniques: Phospho-proteomics, Protein-Protein interactions, Inhibition, Injection, Isolation, Knock-In, Western Blot, Control

Journal: Communications Biology

Article Title: GSK3-CRMP2 signaling mediates axonal regeneration induced by Pten knockout

doi: 10.1038/s42003-019-0524-1

Figure Lengend Snippet: PTEN −/− -induced mTOR activity depends on GSK3 inhibition. a Schematic drawing illustrating the outcome of experiments depicted in Figs. 5 and . b Retinal flat mounts isolated from PTEN +/+ /GSK3(α/β) +/+ (wt), GSK3β S/A (β S/A ), PTEN −/− , and PTEN −/− /GSK3β S/A (PTEN −/− / β S/A ) mice which were either left untreated (con) or subjected to optic nerve crush (ONC). Five days after surgery, retinae were fixed and stained for phosphorylated ribosomal protein S6 (pS6; red) and βIII-tubulin (tubulin; green in lower row to visualize RGCs). Scale bar: 50 μm. c Quantification of pS6-positive RGCs in flat mounts as depicted in ( b ). Compared with wt animals, pS6 levels were markedly increased after PTEN −/− and significantly compromised in animals with additional GSK3β S/A . Values represent means ± SEM of 3–5 retinae per group (untreated controls: PTEN +/+ , n = 4; β S/A , n = 4; PTEN −/− , n = 3; PTEN −/− /β S/A , n = 4; mice 5 days after ONC: PTEN +/+ , n = 4; β S/A , n = 5; PTEN −/− , n = 3; PTEN −/− /β S/A , n = 4). d Western blot analysis for phosphorylated AKT (pAKT) and pS6 of retinal lysates from animals as described in ( b ). βIII-tubulin (tubulin) served as a loading control. e , f Densitometric quantification of Western blots as depicted in ( d ) relative to βIII-tubulin and normalized to PTEN −/− con. Values represent means ± SEM of 3–8 retinae per group ( e : untreated controls: PTEN +/+ , n = 6; β S/A , n = 3; PTEN −/− , n = 6; PTEN −/− /β S/A , n = 3; mice 5 days after ONC: PTEN +/+ , n = 6; β S/A , n = 5, PTEN −/− , n = 6; PTEN −/− /β S/A , n = 3; f : untreated controls: PTEN +/+ , n = 8; β S/A n = 3; PTEN −/− , n = 7; PTEN −/− /β S/A , n = 3; mice 5 days after ONC: PTEN +/+ , n = 7; β S/A n = 3; PTEN −/− , n = 4; PTEN −/− /β S/A , n = 6). Significances of intergroup differences were evaluated using two-way analysis of variance (ANOVA) with Tukey or Holm-Sidak post hoc test. Treatment effects to PTEN +/+ con, or as indicated: * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. = non-significant

Article Snippet: Blots were blocked in 5% dried milk in phosphate-buffered saline with 0.05% Tween-20 (PBS-T) (Sigma, St Louis, USA) and incubated with antibodies against βIII-tubulin (1:2000; BioLegend, San Diego, USA, RRID:AB_2313773), S9-phosphorylated GSK3β (1:1000, RRID:AB_2115196) S21-phosphorylated GSK3α (1:2000; RRID:AB_2114897), total GSK3α/β (1:500, RRID:AB_10547140; all from Cell Signaling Technologies, Cambridge, UK), ribosomal protein S6 (1:5000 Cell Signaling Technologies RRID:AB_2181035), T 514 -phosphorylated CRMP2 (1:2000; Abcam, Cambridge, UK, RRID:AB_942229), total CRMP2 (1:500; Cell Signaling Technologies, RRID:AB_2094339), or T308-phosphorylated AKT (1:1000; Cell Signaling Technologies, RRID:AB_2629447) at 4 °C overnight.

Techniques: Activity Assay, Inhibition, Isolation, Staining, Western Blot, Control

Journal: Communications Biology

Article Title: GSK3-CRMP2 signaling mediates axonal regeneration induced by Pten knockout

doi: 10.1038/s42003-019-0524-1

Figure Lengend Snippet: GSK3β −/− -induced S6 phosphorylation is mTOR-dependent. a Schematic illustration of results shown in experiments of b – e . b Retinal flat mounts isolated from GSK3β −/− mice that were either left untreated (con) or subjected to ONC and, additionally, received a systemic injection of either vehicle (veh) or rapamycin (rap). Five days after injury, retinae were stained for phosphorylated S6 (pS6; red) and βIII-tubulin (tubulin; green in a lower row to visualize RGCs). Scale bar: 50 μm. c Quantification of pS6-positive RGCs as depicted in b . Rapamycin abolished β −/− -induced S6 phosphorylation. Values represent means ± SEM of three retinae per group ( n = 3). d Retinae isolated from PTEN +/+ and PTEN −/− mice that were either left untreated (con) or subjected to ONC and, additionally, received a systemic injection of either veh or rap. Five days after surgery, retinae were stained for pS6 and tubulin. GFP (Cre-gfp, magenta) staining was applied to visualize transduction with GFP co-expressing AAV-Cre. Scale bar: 50 μm. e Quantification of pS6-positive RGCs as depicted in ( d ). Rapamycin abolished PTEN −/− -induced S6 phosphorylation. Values represent means ± SEM of 3–5 retinae per group (PTEN +/+ con, n = 5; PTEN +/+ con + rap, n = 3; PTEN +/+ ONC + rap, n = 3; PTEN −/− con, n = 3; PTEN −/− con + rap, n = 3; PTEN −/− ONC + rap, n = 3). Significances of intergroup differences in ( c ) and ( e ) were evaluated using two-way analysis of variance (ANOVA) with Holm-Sidak post hoc test. Treatment effects compared with β −/− veh con: *** p < 0.001, compared with β −/− veh ONC: ### p < 0.001, compared with PTEN +/+ veh con: +++ p < 0.001, or compared with PTEN −/− veh con: *** p < 0.001

Article Snippet: Blots were blocked in 5% dried milk in phosphate-buffered saline with 0.05% Tween-20 (PBS-T) (Sigma, St Louis, USA) and incubated with antibodies against βIII-tubulin (1:2000; BioLegend, San Diego, USA, RRID:AB_2313773), S9-phosphorylated GSK3β (1:1000, RRID:AB_2115196) S21-phosphorylated GSK3α (1:2000; RRID:AB_2114897), total GSK3α/β (1:500, RRID:AB_10547140; all from Cell Signaling Technologies, Cambridge, UK), ribosomal protein S6 (1:5000 Cell Signaling Technologies RRID:AB_2181035), T 514 -phosphorylated CRMP2 (1:2000; Abcam, Cambridge, UK, RRID:AB_942229), total CRMP2 (1:500; Cell Signaling Technologies, RRID:AB_2094339), or T308-phosphorylated AKT (1:1000; Cell Signaling Technologies, RRID:AB_2629447) at 4 °C overnight.

Techniques: Phospho-proteomics, Isolation, Injection, Staining, Transduction, Expressing

Journal: Communications Biology

Article Title: GSK3-CRMP2 signaling mediates axonal regeneration induced by Pten knockout

doi: 10.1038/s42003-019-0524-1

Figure Lengend Snippet: PTEN −/− stimulated neurite growth is CRMP2 dependent. a Representative pictures of retinal cell cultures plated on PDL. PTEN +/+ and PTEN −/− mice were subjected to ONC to transform RGCs into a regenerative state. Cells were cultured 5 days after that. Cultures were either untreated (−) or incubated with rapamycin (rap; 10 nM), lacosamide (LCM, 5 µM), or a combination of both. After 2 days in culture, RGCs were stained for phosphorylated S6 (pS6, red) and βIII-tubulin (tubulin, green). b Quantification of neurite growth of RGCs as described in ( a ) in the absence (PDL) or presence of CNS myelin (myelin). Some of the cultures were treated with the ROCK inhibitor Y27632 (Y27, 10 µM). Values represent means ± SEM of three ( n = 3) independent experiments with four wells as technical replicate per group and were normalized to the PTEN −/− , PDL group with an average neurite length of 6.12 µm per RGC of in total ~500 RGCs with ~30 RGCs showing neurite growth. c Quantification of the percentage of pS6-positive RGCs in cultures as described in ( a ). Values represent means ± SEM of 4 independent experiments ( n = 4). d ) Quantification of neurite length of βIII-tubulin-positive RGCs from PTEN +/+ , PTEN −/− , or PTEN −/− mice with a Gsk3α S/A (PTEN −/− /α S/A ) or Gsk3β S/A (PTEN −/− /β S/A ) knockin. Animals were subjected to ONC 5 days before culture preparation. Cultures were grown in the in the absence or presence of CNS myelin and either left untreated (−) or incubated with lacosamide (LCM, 5 µM) or Y27632 (Y27, 10 µM) and fixed after 48 h. Values represent means ± SEM of n = 4 independent experiments with four wells as technical replicate per group. Significances of intergroup differences were evaluated using two-way ( b , c ) or three-way ( d ) analysis of variance (ANOVA) with Holm-Sidak post hoc test. Treatment effects: compared with PTEN −/− , PDL: +++ p < 0.001; PTEN −/− , myelin: ### p < 0.001; or as indicated: * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. = non-significant

Article Snippet: Blots were blocked in 5% dried milk in phosphate-buffered saline with 0.05% Tween-20 (PBS-T) (Sigma, St Louis, USA) and incubated with antibodies against βIII-tubulin (1:2000; BioLegend, San Diego, USA, RRID:AB_2313773), S9-phosphorylated GSK3β (1:1000, RRID:AB_2115196) S21-phosphorylated GSK3α (1:2000; RRID:AB_2114897), total GSK3α/β (1:500, RRID:AB_10547140; all from Cell Signaling Technologies, Cambridge, UK), ribosomal protein S6 (1:5000 Cell Signaling Technologies RRID:AB_2181035), T 514 -phosphorylated CRMP2 (1:2000; Abcam, Cambridge, UK, RRID:AB_942229), total CRMP2 (1:500; Cell Signaling Technologies, RRID:AB_2094339), or T308-phosphorylated AKT (1:1000; Cell Signaling Technologies, RRID:AB_2629447) at 4 °C overnight.

Techniques: Cell Culture, Incubation, Staining, Knock-In