Journal: Nature Communications

Article Title: Targeting Tyro3 ameliorates a model of PGRN-mutant FTLD-TDP via tau-mediated synaptic pathology

doi: 10.1038/s41467-018-02821-z

Figure Lengend Snippet: Tyro3 mediates Gas6-induced signal to tau phosphorylation. Western blot analyses of the effect of siRNA-mediated knockdown of Tyro3 on phosphorylation of Tyro3 ( a ), Shc ( b ), PLCγ ( c ), B-Raf ( d ), PKCα ( e ), and tau ( f ). SiRNA-mediated knockdown of Tyro3 exerted suppressive effects on activation of Shc ( b ) and PLCγ ( c ), as well as on phosphorylation of tau at Ser203 and Thr220 ( e ). # p < 0.05; ## p <0.01 ( N = 5, Tukey’s HSD test). Averages and s.e.m. are shown. Phosphatase treatment of the samples substantially decreased the western blot band intensities of phosphorylated Tyro3 ( a ), Shc ( b ), PLCγ ( c ) B-Raf ( d ), PKCα ( e ), and Tau ( f ). p -values are shown in Supplementary Data

Article Snippet: In KD rescue experiments, lentiviral particles (2 × 10 5 titer units in 100 μl) for Tau-shRNA (sc-430402-V, Santa Cruz Biotechnology), scrambled shRNA (SC-108080, Santa Cruz Biotechnology), B-Raf shRNA (sc-63294-V, Santa Cruz Biotechnology), or Tyro3-siRNA (iV037848, ABM, Richmond, BC, Canada) diluted 1:4 with ACSF buffer (NaCl, 125 mM; KCl, 2.5 mM; NaH 2 PO 4 , 1.25 mM; MgCl 2 , 1 mM; CaCl 2 , 1 mM; NaHCO 3 , 26 mM; glucose, 25 mM) or plasmid vectors (10 μg) for PKCα-shRNA (TG501653, OriGene, Rockville, MD, USA), scrambled shRNA (TG501653, OriGene), or Gas6-shRNA (sc-35451-SH, Santa Cruz Biotechnology) dissolved in 100 μl of in vivo-jetPEI (201–10 G, Polyplus-transfection, Illkirch, France) were injected into the subarachnoid space of the right RSD via osmotic pump (0.15 μl/h) from 8 to 12 weeks of age.

Techniques: Phospho-proteomics, Western Blot, Knockdown, Activation Assay

Journal: Nature Communications

Article Title: Targeting Tyro3 ameliorates a model of PGRN-mutant FTLD-TDP via tau-mediated synaptic pathology

doi: 10.1038/s41467-018-02821-z

Figure Lengend Snippet: Suppression of Tyro3 signal rescues decrease in spine abundance and tau mislocalization in mutant PGRN-KI mice. a Experimental protocol for knockdown vectors. AAV-Syn-EGFP was injected into the area adjacent to M2 at 6 weeks of age. Lentivirus for expression of Tau-shRNA, scrambled shRNA, B-Raf-shRNA, or Tyro3-siRNA, or plasmid vectors for expression of PKCα-shRNA, scrambled shRNA, or Gas6-shRNA dissolved in in vivo-jetPEI, were continuously injected into the subarachnoid space of the right M2 via osmotic pump from 8 to 12 weeks of age. Dendritic spines in layer 1 of M2 were observed by two-photon microscopy with three mice at 12 weeks of age. 8 to 10 images were obtained from one mice and the average of spine parameters were used for quantitative analyses ( N = 3). The protocols for vemurafenib and Gö6976 were similar to those shown in Fig. , and two-photon microscopy was performed at 12 weeks of age. b Static spine morphology was observed by two-photon microscopy. Spine protrusion number, length, head diameter, and volume were analyzed. * p < 0.05; ** p < 0.01 ( N = 3, Student’s t -test). Averages and s.e.m. are shown. c Images were obtained by two-photon microscopy at 0, 8, and 24 h on the last day of injection. Dynamic changes in spines were analyzed by serial observation. Spine formation and elimination were counted. * p < 0.05; ** p < 0.01 ( N = 3, Student’s t -test). Averages and s.e.m. are shown. d Lentivirus for expression of shRNA-Tau was injected according to the protocol. ** p < 0.01 ( N = 3, Tukey’s HSD test). Scrambled RNA was used as a control. Averages and s.e.m. are shown. e Lentivirus for expression of shRNA-B-Raf was injected according to the protocol. ** p < 0.01 ( N = 3, Tukey’s HSD test). Scrambled RNA was used as a control. Averages and s.e.m. are shown. f Plasmid for expression of shRNA-PKCα was injected according to the protocol. * p < 0.05; ** p < 0.01 ( N = 3, Tukey’s HSD test). Scrambled RNA was used as a control. Averages and s.e.m. are shown. g Lentivirus for expression of siRNA-Tyro3 was injected according to the protocol. Scrambled RNA was used as a control. ** p < 0.01 ( N = 3, Tukey’s HSD test). Scrambled RNA was used as a control. Averages and s.e.m. are shown. h Plasmid for expression of shRNA-Gas6 was injected according to the protocol. Scrambled RNA was used as a control. ** p < 0.01 ( N = 3, Tukey’s HSD test). Scrambled RNA was used as a control. Averages and s.e.m. are shown. i Mice received oral administration of vemurafenib (32 mg/kg of BW/day) from 6 to 12 weeks of age, and two-photon microscopic analysis was performed at 12 weeks. ** p < 0.01 ( N = 3, Tukey’s HSD test). Averages and s.e.m. are shown. j Gö6976 (4.4 μM) or PBS was injected via osmotic pump into the subarachnoid space of PGRN-KI and C57BL/6J mice from 10 to 12 weeks of age, and two-photon microscopic analysis was performed at 12 weeks. ** p < 0.01 ( N = 3, Tukey’s HSD test). Averages and s.e.m. are shown

Article Snippet: In KD rescue experiments, lentiviral particles (2 × 10 5 titer units in 100 μl) for Tau-shRNA (sc-430402-V, Santa Cruz Biotechnology), scrambled shRNA (SC-108080, Santa Cruz Biotechnology), B-Raf shRNA (sc-63294-V, Santa Cruz Biotechnology), or Tyro3-siRNA (iV037848, ABM, Richmond, BC, Canada) diluted 1:4 with ACSF buffer (NaCl, 125 mM; KCl, 2.5 mM; NaH 2 PO 4 , 1.25 mM; MgCl 2 , 1 mM; CaCl 2 , 1 mM; NaHCO 3 , 26 mM; glucose, 25 mM) or plasmid vectors (10 μg) for PKCα-shRNA (TG501653, OriGene, Rockville, MD, USA), scrambled shRNA (TG501653, OriGene), or Gas6-shRNA (sc-35451-SH, Santa Cruz Biotechnology) dissolved in 100 μl of in vivo-jetPEI (201–10 G, Polyplus-transfection, Illkirch, France) were injected into the subarachnoid space of the right RSD via osmotic pump (0.15 μl/h) from 8 to 12 weeks of age.

Techniques: Mutagenesis, Knockdown, Injection, Expressing, shRNA, Plasmid Preparation, In Vivo, Microscopy, Control

Journal: Nature Communications

Article Title: Targeting Tyro3 ameliorates a model of PGRN-mutant FTLD-TDP via tau-mediated synaptic pathology

doi: 10.1038/s41467-018-02821-z

Figure Lengend Snippet: Suppression of Tyro3 signal rescues tau mislocalization and cognitive impairment in mutant PGRN-KI mice. a Co-staining of phosphorylated tau (Ser203 or Thr220) and PSD-95. Confocal microscopic analysis of M2 regions of background (B6, C57BL/6J), PGRN-R504X-KI (PGRN-KI), vemurafenib-treated PGRN-KI, Gö6976-treated PGRN-KI, and lentivirus-tau-shRNA–infected PGRN-KI mice. All images were acquired by confocal microscopy (LSM510META, Carl Zeiss, Germany). Z-stack images were acquired with the following parameters; objective: ×63, average: line 2; filter (Cy3 ChS1: 550–679 nm; FITC Ch1: 505–530 nm; DAPI Ch2: 420–480 nm); master gain (Cy3 ChS1: 760; FITC Ch1: 741; DAPI Ch2: 615). b Quantitative analyses of PSD95-positive dots and p-tau/PSD95 double-positive spots. All bar graphs indicate averages and s.e.m. Three mice were used for each group. The number of spots was counted in 10 image fields (100 × 100 μm) for each mouse, and the average was used for calculation of mean with s.e.m. for each group. The number of p-tau/PSD95 double-positive spots increased in PGRN-KI mice. # p < 0.05, ## p < 0.01 ( N = 6, Dunnett’s test). Averages and s.e.m. are shown. c In vivo effects of KD of B-Raf, PKCα, Tyro3, Gas6, and tau on two parameters (% time spent at target region and number of target crosses) in the Morris water maze test, performed with PGRN-KI mice ( N = 6 in each experiment) at 12 weeks. # p < 0.05, ## p < 0.01 ( N = 6, Dunnett’s test). Averages and s.e.m. are shown. d In vivo effect of KD of B-Raf, PKCα, Tyro3, Gas6, and tau on % total freezing time in the fear-conditioning test, performed with PGRN-KI mice ( N = 6 in each experiment) at 12 weeks. # p < 0.05, ## p < 0.01 ( N = 6, Dunnett’s test). Averages and s.e.m. are shown. e Tau-KD with or without vemurafenib/Gö6976 had similar effects on % time spent at target region and number of target crosses in the Morris water maze test and on % total freezing time in fear-conditioning test. Both tests were performed at 12 weeks. * p < 0.05 ( N = 6, Dunnett’s test). Averages and s.e.m. are shown. f Western blot analysis confirming shRNA-mediated knockdown of B-Raf, PKCα, and tau. Lower graphs show quantitation. P -values were determined by Tukey’s HSD test ( N = 6). Averages and s.e.m. are shown. p -values are shown in Supplementary data

Article Snippet: In KD rescue experiments, lentiviral particles (2 × 10 5 titer units in 100 μl) for Tau-shRNA (sc-430402-V, Santa Cruz Biotechnology), scrambled shRNA (SC-108080, Santa Cruz Biotechnology), B-Raf shRNA (sc-63294-V, Santa Cruz Biotechnology), or Tyro3-siRNA (iV037848, ABM, Richmond, BC, Canada) diluted 1:4 with ACSF buffer (NaCl, 125 mM; KCl, 2.5 mM; NaH 2 PO 4 , 1.25 mM; MgCl 2 , 1 mM; CaCl 2 , 1 mM; NaHCO 3 , 26 mM; glucose, 25 mM) or plasmid vectors (10 μg) for PKCα-shRNA (TG501653, OriGene, Rockville, MD, USA), scrambled shRNA (TG501653, OriGene), or Gas6-shRNA (sc-35451-SH, Santa Cruz Biotechnology) dissolved in 100 μl of in vivo-jetPEI (201–10 G, Polyplus-transfection, Illkirch, France) were injected into the subarachnoid space of the right RSD via osmotic pump (0.15 μl/h) from 8 to 12 weeks of age.

Techniques: Mutagenesis, Staining, shRNA, Infection, Confocal Microscopy, In Vivo, Western Blot, Knockdown, Quantitation Assay