Journal: Open Biology

Article Title: Loss of CRMP2 O-GlcNAcylation leads to reduced novel object recognition performance in mice

doi: 10.1098/rsob.190192

Figure Lengend Snippet: CRMP2 protein level and phosphorylation by GSK3β is unchanged in Crmp2 S517A mice. ( a ) Mouse brain lysates were probed with site-specific O-GlcNAc-CRMP2 antibody (OG-CRMP2) on western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) signal was used as loading control. Replicates of five wild-type and Crmp2 S517A mice are shown. ( b ) Quantification of CRMP2 signal normalized to GAPDH signal based on blot shown in ( c ). ( c ) Western blot using P509/514-CRMP2 specific antibody and total CRMP2 antibody on mouse brain lysates. ( d ) Quantification of diphospho-CRMP2 signal normalized to total CRMP2 signal. ( e ) Western blot using quadrophospho-CRMP2 (P509/514/518/522) specific antibody, 3F4. ( f ) Quantification of 3F4 signal, normalized to total CRMP2 signal.

Article Snippet: Rabbit anti-gSer517 OG-CRMP2 (1 : 1000) raised for this study by the van Aalten laboratory Rabbit anti-CRMP2 (1 : 2000) Millipore Rabbit anti-CRMP2 (1 : 2500) Cell Signalling 9393 Rabbit anti-pan-TUC (CRMP) (1 : 10 000) Millipore ABN108 Sheep anti-P-T509/514 CRMP2 (1 : 2000) with dephospho-peptide [ ] Mouse anti-O-GlcNAc /RL2 (1 : 2000) Abcam ab2739 Mouse anti-FLAG (M2) (1 : 5000) Sigma Mouse anti-PSD95 (SAP90, DLG4) (108E10) SYSY Rabbit anti-GAPDH (FL-335) (1 : 1000) Santa Cruz sc-25778 Goat anti-GATA-2 (0.5 µg ml −1 ) R&D Systems AF2046 Mouse 3F4 (1 µg ml −1 ) Takara 29060 Mouse NeuN (A60) (1 : 2000) Millipore MAB377 Licor 680, Licor 800

Techniques: Western Blot

Journal: Open Biology

Article Title: Loss of CRMP2 O-GlcNAcylation leads to reduced novel object recognition performance in mice

doi: 10.1098/rsob.190192

Figure Lengend Snippet: CRMP2 undergoes dynamic O-GlcNAc post-translational modification at Ser517 in vivo . ( a ) Sequence alignment shows that the O-GlcNAcylation modification site on the C-terminal tail is unique to CRMP2 among other members of the CRMP family. The Ser517 O-GlcNAcylation site is in close proximity to several phosphorylation sites targeted by Cdk5 (dark green) and GSK3β (light green) kinases. ( b ) Western blotting for O-GlcNAc-CRMP2 (OG-CRMP2) on post-mortem human hippocampus samples ( n = 14, age range 17–89 years). ( c ) Correlation analysis reveals that the Pearson correlation coefficient for the OG-CRMP2 level versus age is 0.57, indicating a positive correlation. ( d ) O-GlcNAcylation of CRMP2 is post-translational. Neuro2A cells expressing Flag-tagged CRMP2 were treated with cycloheximide (CHX), a protein translation inhibitor, and subsequently supplemented with GlcNAcstatin G (GG) OGA inhibitor. Blocking translation did decrease CRMP2 protein levels, but it did not inhibit induction of O-GlcNAcylation on CRMP2. ( e ) O-GlcNAcylation of CRMP2 is inducible in vivo. Mice were infused (osmotic pump implant) with 10 mg ml −1 GG ( n = 3) or dose vehicle alone ( n = 3) at 8 µl h −1 for 24 h. Brain lysates were probed with site-specific OG-CRMP2 antibody on western blot. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Rabbit anti-gSer517 OG-CRMP2 (1 : 1000) raised for this study by the van Aalten laboratory Rabbit anti-CRMP2 (1 : 2000) Millipore Rabbit anti-CRMP2 (1 : 2500) Cell Signalling 9393 Rabbit anti-pan-TUC (CRMP) (1 : 10 000) Millipore ABN108 Sheep anti-P-T509/514 CRMP2 (1 : 2000) with dephospho-peptide [ ] Mouse anti-O-GlcNAc /RL2 (1 : 2000) Abcam ab2739 Mouse anti-FLAG (M2) (1 : 5000) Sigma Mouse anti-PSD95 (SAP90, DLG4) (108E10) SYSY Rabbit anti-GAPDH (FL-335) (1 : 1000) Santa Cruz sc-25778 Goat anti-GATA-2 (0.5 μg ml −1 ) R&D Systems AF2046 Mouse 3F4 (1 μg ml −1 ) Takara 29060 Mouse NeuN (A60) (1 : 2000) Millipore MAB377 Licor 680, Licor 800

Techniques: Modification, In Vivo, Sequencing, Phospho-proteomics, Western Blot, Expressing, Blocking Assay

Journal: Open Biology

Article Title: Loss of CRMP2 O-GlcNAcylation leads to reduced novel object recognition performance in mice

doi: 10.1098/rsob.190192

Figure Lengend Snippet: Characterization of Crmp2 S517A mice reveals bodyweight phenotype ( a , b ). ( a ) Comparison of the body weights of WT and Crmp2 S517A mice at 12–25 weeks old. Data are expressed as the mean ± s.e.m. (WT, n = 8; Crmp2 S517A , n = 10) (male, n = 9; female, n = 9). Repeated measures ANOVA for the whole dataset, test of between-subject effect, genotype, F = 11.026, p = 0.005. ( b ) Comparison of the body weights of WT and Crmp2 S517A mice at 20 weeks old. Data are expressed as the mean ± s.d.; paired, two-tailed t -test (female, p = 0.0199; WT, n = 6; Crmp2 S517A , n = 7; male, p = 0.0125; WT, n = 6; Crmp2 S517A , n = 6). Histological analysis of brain samples from WT and Crmp2 S517A mice ( c – g ). Mean ± s.d.; WT, n = 5; Crmp2 S517A , n = 6. ( c ) Representative photomicrographs (left panels) show coronal sections stained with cresyl-violet (Nissl staining) in WT and Crmp2 S517A mice. NeuN immunolabelling (right panels) reveals Crmp2 S517A has no effect on neuronal viability in the dentate gyrus. ( d ) Ventricular volume (mm 3 ) for left and right lateral ventricles and third ventricle. WT (3.592 ± 1.155) and Crmp2 S517A (4.160 ± 1.252); n.s. ( e ) Cortical thickness (μm), measurement was taken at four points along the cortex in each animal. WT (1197 ± 175.4) and Crmp2 S517A (1127 ± 93.24); n.s. ( f ) Western blot shows comparable levels of O-GlcNAc CRMP2 (OG-CRMP2) in several brain regions in WT and Crmp2 S517A mice relative to total CRMP levels. ( g ) Western blot indicates presence of CRMP2 O-GlcNAcylation in cytoplasmic and synaptic cellular fractions.

Article Snippet: Rabbit anti-gSer517 OG-CRMP2 (1 : 1000) raised for this study by the van Aalten laboratory Rabbit anti-CRMP2 (1 : 2000) Millipore Rabbit anti-CRMP2 (1 : 2500) Cell Signalling 9393 Rabbit anti-pan-TUC (CRMP) (1 : 10 000) Millipore ABN108 Sheep anti-P-T509/514 CRMP2 (1 : 2000) with dephospho-peptide [ ] Mouse anti-O-GlcNAc /RL2 (1 : 2000) Abcam ab2739 Mouse anti-FLAG (M2) (1 : 5000) Sigma Mouse anti-PSD95 (SAP90, DLG4) (108E10) SYSY Rabbit anti-GAPDH (FL-335) (1 : 1000) Santa Cruz sc-25778 Goat anti-GATA-2 (0.5 μg ml −1 ) R&D Systems AF2046 Mouse 3F4 (1 μg ml −1 ) Takara 29060 Mouse NeuN (A60) (1 : 2000) Millipore MAB377 Licor 680, Licor 800

Techniques: Comparison, Two Tailed Test, Staining, Western Blot