Journal: Neuroscience

Article Title: Modulation of hippocampal synapse maturation by activity-regulated E3 ligase via non-canonical pathway.

doi: 10.1016/j.neuroscience.2017.08.057

Figure Lengend Snippet: Fig. 2. Effect of neuronal activity on polyubiquitination and subsequent stabilization of Rnf2. (A) Neuronal activity triggers self-polyubiquitination of Rnf2. Hippocampal neurons (DIV7) from Ube3A+/+ and Ube3A/ mice were stimulated with 60 mM KCl for 2 h. Rnf2 was immunoprecipitated from hippocampal lysate and polyubiquitination was assessed by western blot analysis using antibody recognizing polyubiquitin conjugates. Amount of Rnf2 immunoprecipitated was assessed by western blot analysis of immunoprecipitated sample using Rnf2 antibody. Representative photomicrograph showing neuronal activity enhanced polyubiquitination of Rnf2 in wild type hippocampal neurons and this enhancement of Rnf2 polyubiquitination was further increased in Ube3A deficient hippocampal neurons in response to neuronal activity. n = 3. (B) Neuronal activity enhances Rnf2 protein level by self-polyubiquitination. Total hippocampal lysate from above experiment (represented in ‘‘A”) was assessed for Rnf2 abundance by western blot analysis using antibody specific for Rnf2. The western blot was normalized by Tuj1. The representative photomicrograph showed that neuronal stimulation increased total Rnf2 protein level in hippocampal neurons obtained from wild type animals. This enhancement of Rnf2 was further increased in hippocampal neurons obtained from Ube3A null mice in response to neuronal activity. Enhanced polyubiquitination of Rnf2 in Ube3A deficient mice shown in (A) and subsequent increase in total Rnf2 level in Ube3A deficient neuron shown in (B) suggests that stabilization of Rnf2 occurred via self-polyubiquitination in response to neuronal activity. (C) Quantitation of western blot data. Neuronal activity induced Rnf2 expression in hippocampal neurons obtained from wild type mice (1.493 ± 0.063 fold increase) and Ube3A null mice (1.569 ± 0.085 fold increase). The activity-induced enhancement of Rnf2 expression was higher in hippocampal neurons obtained from Ube3A null mice (2.495 ± 0.257 fold increase) as compared to unstimulated hippocampal neurons obtained from wild type mice. The additional increase (1.67 ± 0.116 fold increase) of total Rnf2 level in stimulated hippocampal neurons obtained from Ube3A null mice as compared to wild type mice indicates stabilization of Rnf2 via self-polyubiquitination. Data represent mean ± SEM; *p < 0.03 for stimulated vs unstimulated wild type neurons, **p < 0.02 for stimulated vs unstimulated Ube3A null neurons, ***p < 0.01 for stimulated wild type vs stimulated Ube3A null neurons; n = 5, One-Way ANOVA; yp < 0.006 for stimulated Ube3A null neurons vs unstimulated wild type neurons, n = 5, Two-Way ANOVA.

Article Snippet: shRNA cassette for Rnf2 was cloned into MluI and ClaI sites of the lentiviral vector, pLVTHM (Addgene Plasmid # 12247).

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Quantitation Assay, Expressing

Journal: Neuroscience

Article Title: Modulation of hippocampal synapse maturation by activity-regulated E3 ligase via non-canonical pathway.

doi: 10.1016/j.neuroscience.2017.08.057

Figure Lengend Snippet: Fig. 3. Localization of Rnf2 in brain and synaptic compartment. (A) Expression of Rnf2 in developing rat brain. in situ hybridization for Rnf2 mRNA in sagittal section of a P7, P14 and P21 rat brains. Sense as well as anti-sense probes were in vitro transcribed and labeled with DIG-oligonucleotide prior to in situ hybridization. Scale bar: as indicated in photomicrograph. (B) Developmental expression pattern of Rnf2. Lysates from hippocampal neuronal culture at different developmental stages (DIV0, DIV4, DIV7, DIV14 and DIV21) were analyzed by western blot using Rnf2 antibody and the western blot data was normalized by GAPDH expression. (C) Quantitation of western blot data. Rnf2 is expressed all throughout during these developmental stages but expression is reduced at DIV21 (0.34-fold decrease for DIV0 vs DIV21, p < 0.013 and 0.43-fold decrease for DIV4 vs DIV21, p < 0.007) as compare to DIV0 and DIV4. Data represent mean ± SEM; n = 6, t-test. (D) Localization of endogenous Rnf2 in synaptic compartment. Synaptoneurosome was prepared from P14 rat hippocampus by density gradient centrifugation and characterized by western blot analysis using antibodies specific for proteins enriched at the synapse (PSD95 and Synaptophysin), protein enriched in astrocyte (GFAP) and protein enriched in the nucleus (Histone H1). Tuj1 was used for normalization (Crude = total lysate including both nuclear and cytoplasmic proteins, Synaptoneurosome = synaptic proteins). Representative photomicrographs showing localization of Rnf2 at the synaptic compartment. n = 3.

Article Snippet: shRNA cassette for Rnf2 was cloned into MluI and ClaI sites of the lentiviral vector, pLVTHM (Addgene Plasmid # 12247).

Techniques: Expressing, In Situ Hybridization, In Vitro, Labeling, Western Blot, Quantitation Assay, Gradient Centrifugation

Journal: Neuroscience

Article Title: Modulation of hippocampal synapse maturation by activity-regulated E3 ligase via non-canonical pathway.

doi: 10.1016/j.neuroscience.2017.08.057

Figure Lengend Snippet: Fig. 4. Role of Rnf2 in glutamatergic synapse development. (A) Hippocampal neurons were transduced with lentivirus co-expressing GFP along with non-targeting shRNA (control RNAi) or effective shRNA against Rnf2 (Rnf2 RNAi) or ineffective shRNA against Rnf2 (ineffective Rnf2 RNAi). Representative photomicrograph showing efficacy of Rnf2 shRNA that was assessed by western blot analysis. The western blot data was normalized by GAPDH level. (B) Quantification of the western blot data. Hippocampal neurons infected with lentivirus expressing effective shRNA against Rnf2 showed 88.2 ± 12.26% decrease in Rnf2 protein expression. However, ineffective shRNA against Rnf2 did not show any reduction in Rnf2 expression. n = 5, *p < 0.04, t-test. (C). Immunostaining for PSD95 (Red), Synapsin I (Blue) of hippocampal neurons transduced with lentivirus co-expressing GFP and shRNAs (Control RNAi or Rnf2 RNAi or ineffective Rnf2 RNAi). Hippocampal neurons were transduced at DIV3-4 and immunostained at DIV21. Glutamatergic synapses were defined by overlapping PSD95 (Red) and Synapsin I (Blue) puncta on a neuronal dendrite (Green). Density of overlapping puncta (white) per micrometer of dendrite representing individual synapse was measured from confocal images using custom written program in Matlab. Representative photomicrographs showing high magnification images (obtained using 63 objective with 2 optical zoom) from hippocampal neurons infected with lentivirus expressing non-targeting shRNA (Control RNAi), effective Rnf2 shRNA (Rnf2 RNAi) and ineffective Rnf2 shRNA (ineffective Rnf2 RNAi) respectively. Part of neuron represented in high magnification images is marked in red. High-magnification images were used for synapse density measurement. Scale: as indicated in the photomicrograph. (D). Quantitation of synapse density measured from hippocampal neurons transduced with non-targeting control (Control RNAi), shRNA targeting Rnf2 (Rnf2 RNAi), ineffective shRNA targeting Rnf2 (ineffective Rnf2 RNAi). n = 19 neurons (45 high-magnification images), n = 16 neurons (32 high- magnification images) and n = 18 neurons (38 high-magnification images) transduced with lentivirus expressing non-targeting shRNA, effective shRNA against Rnf2 and ineffective shRNA against Rnf2 respectively were used to measure synapse density from three independent experiments. Rnf2 RNAi leads to 47.897 ± 3.315% increase in synapse density. Data represent mean ± SEM; *p < 0.03; One-way ANOVA. (E) Effect of Rnf2 knockdown in dendritic branching. Dendritic branching of hippocampal neurons used for synapse density measurement was evaluated by sholl analysis. The sholl analysis graph shows dendritic crossing of circles drawn at 10-lm radius interval from the soma. n = 15 neurons from three independent experiments were used for the sholl analysis. One-way ANOVA was used to measure statistical significance. p < 0.58 (Control RNAi vs Rnf2 RNAi) and p < 0.3 (Control RNAi vs ineffective Rnf2 RNAi), Kolmogorov- Smirnov-test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: shRNA cassette for Rnf2 was cloned into MluI and ClaI sites of the lentiviral vector, pLVTHM (Addgene Plasmid # 12247).

Techniques: Transduction, Expressing, shRNA, Control, Western Blot, Infection, Immunostaining, Quantitation Assay, Knockdown

Journal: Neuroscience

Article Title: Modulation of hippocampal synapse maturation by activity-regulated E3 ligase via non-canonical pathway.

doi: 10.1016/j.neuroscience.2017.08.057

Figure Lengend Snippet: Fig. 5. Role of Rnf2 in functional synapse development. (A). Schematic representation showing sparsely infected or uninfected hippocampal neurons used for whole-cell patch clamp recordings. (B) Representative traces of mEPSCs from hippocampal neurons transduced with lentivirus co-expressing GFP along with non-targeting shRNA (Control RNAi) or effective shRNA targeting Rnf2 (Rnf2 RNAi) or ineffective shRNA targeting Rnf2 (ineffective RNAi) and uninfected neurons. Hippocampal neurons transduced at DIV3-4 and whole-cell patch clamp recording was performed at DIV21-25. Scale as indicated in the figure. (C). Quantitation of average mEPSC frequency for hippocampal neurons transduced with lentivirus co- expressing GFP along with Control RNAi or Rnf2 RNAi (70.229 ± 8.348% decrease) or ineffective RNAi (7.56 ± 6.82% decrease) and uninfected neurons. n = 15 neurons for Control RNAi and Rnf2 RNAi. n = 13 neurons for ineffective Rnf2 RNAi. n = 15 uninfected neurons from culture coverslip sparsely infected with effective shRNA against Rnf2. Data represent mean ± SEM; *p < 0.01 (Control RNAi vs Rnf2 RNAi) and p < 0.3 (control RNAi vs ineffective RNAi); t-test. (D). Quantitation of average mEPSC amplitude for hippocampal neurons transduced with lentivirus co- expressing GFP along with non-targeting shRNA (Control RNAi) or effective shRNA targeting Rnf2 (Rnf2 RNAi) (13.606 ± 6.259% decrease) or ineffective shRNA targeting Rnf2 (ineffective RNAi) (5.02 ± 5.6% decrease) and uninfected neurons. n = 15 neurons for Control RNAi and Rnf2 RNAi. n = 13 neurons for ineffective Rnf2 RNAi. n = 15 uninfected neurons from culture coverslip sparsely infected with effective shRNA against Rnf2. Data represent mean ± SEM; p < 0.67 (control RNAi vs Rnf2 RNAi), p < 0.3 (control RNAi vs ineffective RNAi); t-test.

Article Snippet: shRNA cassette for Rnf2 was cloned into MluI and ClaI sites of the lentiviral vector, pLVTHM (Addgene Plasmid # 12247).

Techniques: Functional Assay, Infection, Patch Clamp, Transduction, Expressing, shRNA, Control, Quantitation Assay

Journal: Nucleic acids research

Article Title: CRISPR screening uncovers nucleolar RPL22 as a heterochromatin destabilizer and senescence driver.

doi: 10.1093/nar/gkae740

Figure Lengend Snippet: Figure 5. RPL22 binds rDNA and destabilizes HP1 γ and KAP1 in senescent hMPCs. ( A ) Network diagram showing heterochromatin-associated proteins interacted with RPL22. The color key from white to red indicates coverage levels from low to high. ( B ) Western blot analysis of HP1 γ, KAP1 and H3K9me3 protein le v els in RS hMPCs. β-Tubulin was used as loading control for HP1 γ and KAP1. Histone 3 (H3) was used as loading control for H3K9me3. Data are presented as the means ± SEMs. n = 3 biological replicates. Two-tailed unpaired Student’s t test was performed. ( C ) Co-IP analysis of the e x ogenous interaction betw een RPL22 and HP1 γ or KAP1 in HEK293T cells transduced with Flag-Luc and Flag-RPL22. ( D ) Co-IP analy sis sho wing

Article Snippet: For lentiviral shRNA-mediated knock-down, shRNAs targeting KAP1 or HP1 γ mRNAs were cloned into the pLVTHM vector (Addgene # 12247).

Techniques: Western Blot, Control, Two Tailed Test, Co-Immunoprecipitation Assay, Transduction