Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: A Novel Ubiquitin Ligase Adaptor PTPRN Suppresses Seizure Susceptibility through Endocytosis of Na V 1.2 Sodium Channels.

doi: 10.1002/advs.202400560

Figure Lengend Snippet: Figure 5. PTPRN promotes NaV1.2 channel internalization through ubiquitin-dependent endocytosis. A) Schematic of the effect of inhibitors on the trafficking and ubiquitination of proteins. B) Left panel: Immunoblot analysis of cell surface biotinylation performed in primary cortical neurons infected with lentivirus containing shRNA-PTPRN#3 or nonsilencing lentivirus for 7 days. Total lysates (total) and biotinylated fractions (membrane) were an- alyzed by western blot analysis. Right panel: Quantification of NaV1.2 total expression and surface expression. n = 3, *p < 0.05, two-way ANOVA with Bonferroni’s multiple-comparisons test. C) Current density versus voltage relationship for HEK-293T cells expressing NaV1.2 and PTPRN in a 1:2 ratio (NaV1.2 + PTPRN) or NaV1.2 and empty vector (NaV1.2), with or without treatment using dynasore (80 μm, 2 h). n = 21 for all samples except NaV1.2 + PTPRN plus dynasore (n = 24). D) Graph depicting voltage-dependence of activation for NaV1.2 channels described in (C). The lines are the best-fitted Boltzmann curves. E) Current density versus voltage relationship for HEK-293T cells expressing NaV1.2 and PTPRN in a 1:2 ratio (NaV1.2 + PTPRN) or NaV1.2 and empty vector (NaV1.2), with or without treatment using TAK-243 (1 μm, 2 h). n = 18 and 26 for untreated NaV1.2 and NaV1.2 + PTPRN, respectively, and n = 20 and 26 for NaV1.2 and NaV1.2 + PTPRN plus TAK-243, respectively. F) Graph depicting voltage dependence of activation for NaV1.2 channels described in (E). The lines are the best-fitted Boltzmann curves. G) Current density versus voltage relationship for HEK-293T cells expressing NaV1.2 and PTPRN in a 1:2 ratio (NaV1.2 + PTPRN) or NaV1.2 and empty vector (NaV1.2), with or without treatment with Heclin (20 μm, 2 h). n = 18 and 26 for NaV1.2 and NaV1.2 + PTPRN plus Heclin, respectively, and n = 19 for both samples without treatment. H) Graph depicting voltage dependence of activation for NaV1.2 channels described in (G). The lines are the best-fitted Boltzmann curves. I) Workflow showing experimental procedures for purification of PTPRN intracellular fragment (aa 601–979, PTPRN-cyto) and TagRFP and whole cell patch-clamp recordings. J) Left panel:

Article Snippet: The resultant materials were then subjected to western blot analysis Trafficking Inhibition: To investigate the mechanism of the regulation on NaV1.2 by PTPRN, 80 μm Dynasore (Abcam), [54] 1 μm TAK243 (TargetMol),[35] 20 μm Pitstop2 (GLPBIO),[55] or 50 μm Heclin (GLPBIO)[48c] were added into the medium 2 h before the electrophysiology recording.

Techniques: Ubiquitin Proteomics, Western Blot, Infection, shRNA, Membrane, Expressing, Plasmid Preparation, Activation Assay, Patch Clamp

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: A Novel Ubiquitin Ligase Adaptor PTPRN Suppresses Seizure Susceptibility through Endocytosis of Na V 1.2 Sodium Channels.

doi: 10.1002/advs.202400560

Figure Lengend Snippet: Figure 7. PTPRN-NaV1.2 axis regulates neuronal intrinsic excitability and seizure susceptibility. A) Schematic diagram showing stereotaxic injection of shRNA-Scn2a AAV in adult PTPRN-KO mice. B) Infection of AAV expressing shRNA-Scn2a and EGFP in the hippocampal DG region 14 days after injection. Scale bar, 125 μm. C) Validation of Scn2a knockdown efficiency by RT-PCR analysis of Scn2a in hippocampal tissues obtained from mice infected with AAV-containing shRNA-Scn2a or control AAV for 14 days. The levels of mRNA were normalized to those of 𝛽-Actin. n = 3, **p < 0.01, unpaired two-tailed Student’s t-test. D) Top panel: Validation of Scn2a knockdown efficiency by western blot analysis using hippocampi from mice infected with AAV-containing shRNA-Scn2a or control AAV for 14 days. Bottom panel: Quantification of the results by normalizing the protein levels of NaV1.2 to those of 𝛽-Actin. n = 3, *p < 0.05, unpaired two-tailed Student’s t-test. E) Left panel: Representative current-clamp recordings of DG granule cells from PTPRN-WT and PTPRN-KO mice infected with AAV containing shRNA-Scn2a or control AAV for 14 days. For comparison, the cells were held at -80 mV of membrane potential. Right panel: The mean number of APs generated in response to depolarizing current pulses. *p < 0.05, ***p < 0.001, ****p < 0.0001, ####p < 0.0001, two-way ANOVA with Bonferroni’s multiple-comparisons test. F) Typical APs of DG granule cells from mice described in (E). G) Phase plane plots associated with APs in (F). H) The average AP threshold (left) and peak rising phase dV/dt (right). *p < 0.05, ####p < 0.0001, $$$$p < 0.0001, one-way ANOVA with Bonferroni’s multiple-comparisons test. I) Graph depicting the seizure progression in PTPRN-WT and PTPRN-KO mice infected with AAV containing shRNA-Scn2a or control AAV for 14 days, illustrated as mean maximum seizure stage reached by 15, 30, 45, 60, and 75 min after KA administration. n = 6, $p < 0.05, *p < 0.05, ***p < 0.001, ****p < 0.0001, ###p < 0.001, $$$p < 0.001, two-way ANOVA with Bonferroni’s multiple-comparisons test. J) Graph showing time taken to reach each stage of seizure after administration of KA in mice described in (I). n = 6, $$p < 0.01, ****p < 0.0001, two-way ANOVA with Bonferroni’s multiple-comparisons test. K) Number of class V seizures within 1 h after KA administration. n = 6, ***p < 0.001, ###p < 0.001, one-way ANOVA with Bonferroni’s multiple-comparisons test. L) Incidence of maximum seizure stage reached during the course of the experiments in (I). n = 6, ****p < 0.0001, ####p < 0.0001, $$$$p < 0.0001, Chi-square test. Data are represented as mean ± s.e.m.

Article Snippet: The resultant materials were then subjected to western blot analysis Trafficking Inhibition: To investigate the mechanism of the regulation on NaV1.2 by PTPRN, 80 μm Dynasore (Abcam), [54] 1 μm TAK243 (TargetMol),[35] 20 μm Pitstop2 (GLPBIO),[55] or 50 μm Heclin (GLPBIO)[48c] were added into the medium 2 h before the electrophysiology recording.

Techniques: Injection, shRNA, Infection, Expressing, Biomarker Discovery, Knockdown, Reverse Transcription Polymerase Chain Reaction, Control, Two Tailed Test, Western Blot, Comparison, Membrane, Generated

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: A Novel Ubiquitin Ligase Adaptor PTPRN Suppresses Seizure Susceptibility through Endocytosis of Na V 1.2 Sodium Channels.

doi: 10.1002/advs.202400560

Figure Lengend Snippet: Figure 8. Proposed mechanism for PTPRN regulation of NaV1.2 channels and neuronal intrinsic plasticity. Elevated neuronal activity augments the recruitment of NEDD4L by PTPRN to NaV1.2 sodium channels. This interaction promotes NEDD4L-mediated ubiquitination, subsequently leading to the endocytosis of NaV1.2 channels. The presented regulatory mechanism provides a feedback response to heightened activity within the nervous system, thereby aiding the tuning of neuronal intrinsic plasticity.

Article Snippet: The resultant materials were then subjected to western blot analysis Trafficking Inhibition: To investigate the mechanism of the regulation on NaV1.2 by PTPRN, 80 μm Dynasore (Abcam), [54] 1 μm TAK243 (TargetMol),[35] 20 μm Pitstop2 (GLPBIO),[55] or 50 μm Heclin (GLPBIO)[48c] were added into the medium 2 h before the electrophysiology recording.

Techniques: Activity Assay, Ubiquitin Proteomics