Journal: Frontiers in Genetics

Article Title: Different Effect of Sox11 in Retinal Ganglion Cells Survival and Axon Regeneration

doi: 10.3389/fgene.2018.00633

Figure Lengend Snippet: Whole mount retinal immunohistochemistry showing AAV transduction in RGCs. (A) Confocal image of whole mounted retina showing transduction of AAV2-CMV-GFP. (B) Confocal image of whole mounted retina showing transduction of AAV2-shRNA- Pten -GFP. (C) Confocal image of flat mounted retina showing GFP expressing in RGCs. (D) RBPMS label RGCs specifically as a marker. (E) More than half of RGCs are transduced by AAV and express GFP; merge image. Scale bar = 100 μm.

Article Snippet: To downregulate Sox11, Sox11 f/f mice received intravitreal injections of AAV2-CMV-GFP-Cre [2 μl of 10 12 vg/mL, expressing Cre recombinase with GFP from a bicistronic vector (Addgene plasmid #49056), produced by Fred Gage ( )].

Techniques: Immunohistochemistry, Transduction, shRNA, Expressing, Marker

Journal: Frontiers in Genetics

Article Title: Different Effect of Sox11 in Retinal Ganglion Cells Survival and Axon Regeneration

doi: 10.3389/fgene.2018.00633

Figure Lengend Snippet: Downregulation of Sox11 facilitates RGC survival following ONC. (A) Sox11 is partially knocked out by intravitreal injection of AAV2-CMV-Cre-GFP in 4 to 6-week-old Sox11 f/f mice. The control group receives AAV2-CMV-GFP. Optic nerve crush is performed 2 weeks after viral vector injection. Twelve days after ONC, Alexa Fluor 647-conjugated cholera toxin B (CTB) is used to label regenerating axons. Two days later mice are sacrificed. (B) For the retinal whole mounts, RGCs are labeled with the pan-RGC marker RBPMS. The number of AAV2-GFP vector transduced RGCs without crush injury is 1782 ± 117.5/mm 2 . (C) Downregulation of Sox11 in RGCs greatly increases the survival of AAV2 vector transduced RGCs (double labeled by GFP and RBPMS) after ONC as compared to AAV2-CMV-GFP injection with ONC group (357 ± 7.1/mm 2 vs. 158 ± 15.7/mm 2 , n = 4, p = 0.029, Mann–Whitney U -test). (D) The number of total RGCs labeled by RBPMS without crush injury is 3389 ± 92.1/mm 2 . When comparing the total RGCs labeled by RBPMS, it shows that Sox11 partial knockout in the Sox11 f/f mice significantly increases RPBMS-positive RGC survival after ONC as compared to AAV2-CMV-GFP ONC group (1398 ± 63/mm 2 vs. 828 ± 23/mm 2 , n = 4, p = 0.028, Mann–Whitney U -test). Scale bar = 100 μm.

Article Snippet: To downregulate Sox11, Sox11 f/f mice received intravitreal injections of AAV2-CMV-GFP-Cre [2 μl of 10 12 vg/mL, expressing Cre recombinase with GFP from a bicistronic vector (Addgene plasmid #49056), produced by Fred Gage ( )].

Techniques: Injection, Plasmid Preparation, Labeling, Marker, MANN-WHITNEY, Knock-Out

Journal: Frontiers in Genetics

Article Title: Different Effect of Sox11 in Retinal Ganglion Cells Survival and Axon Regeneration

doi: 10.3389/fgene.2018.00633

Figure Lengend Snippet: Representative images of optic nerves showing regenerating axons from different groups at 2 weeks after injury. (A) Pseudocolor green was used for the CTB-labeled axons in all the optic nerve images of this study for clear visual observation (red asterisks represent the crush site). Quantification axon number at 0.5 mm (B) and 1 mm (C) from crush site reveals that there is a statistically significant increase in the number of axons following an injection of either AAV2-GFP or AAV2-Cre-GFP in the Sox11 f/f mice relative to ether the C57BL/6J control of the Sox11 f/f control. This indicates that the increased axon regeneration is due to the use of AAV2-gene delivery systems in the retina and not the partial knockout of Sox11 . A similar result was observed for the distance of longest five regenerating axons traveled (D) and the distance of longest single regenerating axons (E) . Again, the most regeneration was observed in the animals that received gene delivery in the retina by AAV2 vectors. There was a significant difference ( p < 0.05, n = 4, Mann–Whitney U -test) between the ONC only mice and the Sox11 f/f mice that received intravitreal injections of either AAV2-GFP or AAV2-Cre-GFP. No statistic difference was found between the two ONC only groups. No statistic difference was found between the Sox11 pKO and the GFP control groups. N = 4 per group. Scale bar = 200 μm.

Article Snippet: To downregulate Sox11, Sox11 f/f mice received intravitreal injections of AAV2-CMV-GFP-Cre [2 μl of 10 12 vg/mL, expressing Cre recombinase with GFP from a bicistronic vector (Addgene plasmid #49056), produced by Fred Gage ( )].

Techniques: Labeling, Injection, Knock-Out, MANN-WHITNEY

Journal: Frontiers in Genetics

Article Title: Different Effect of Sox11 in Retinal Ganglion Cells Survival and Axon Regeneration

doi: 10.3389/fgene.2018.00633

Figure Lengend Snippet: Downregulation of Sox11 facilitates RGC survival following optic nerve crush and regeneration experiment. (A) Sox11 and Pten are knocked down by intravitreal injection of AAV2-CMV-Cre-GFP or AAV2-shRNA- Pten -GFP in 4 to 6 week-old Sox11 f/f mice. The control group receives AAV2-CMV-GFP. Optic nerve crush is performed 2 weeks after viral vector injection. Twelve days after ONC, Alexa Fluor ® 647-conjugated CTB is used to label regenerating axons. Two days after the CTB injections the animals are sacrificed, the retinas and full-length optic nerves are removed. (B) RGCs are labeled with pan-RGC marker RBPMS in retinal whole mounts. (C) Downregulation of both Sox11 and Pten resulted in a significant increase in RGC survival as compared to Pten knockdown only (1393 ± 94/mm 2 vs. 722 ± 77/mm 2 , n = 4, ∗ p < 0.05, Mann–Whitney U -test). Scale bar = 100 μm.

Article Snippet: To downregulate Sox11, Sox11 f/f mice received intravitreal injections of AAV2-CMV-GFP-Cre [2 μl of 10 12 vg/mL, expressing Cre recombinase with GFP from a bicistronic vector (Addgene plasmid #49056), produced by Fred Gage ( )].

Techniques: Injection, shRNA, Plasmid Preparation, Labeling, Marker, MANN-WHITNEY

Journal: bioRxiv

Article Title: SUSD4 controls GLUA2 degradation, synaptic plasticity and motor learning

doi: 10.1101/859587

Figure Lengend Snippet: (A) Diagram of the protein SUSD4 showing its domain organization with four extracellular Complement Control Protein (CCP) domains, one transmembrane (TM) domain and a cytoplasmic domain (C T ). (B) Quantitative RT-PCR shows an increase in Susd4 mRNA expression (relative to the housekeeping gene Rpl13a ) during postnatal development in the cerebellum and in the brainstem. Extracts were prepared from tissue samples of mice aged from 0 to 21 days (P0-21) and three months (3mo). Mean ± s.e.m. (n=3 independent experiments). (C) HA-tagged SUSD4 is found in dendrites (left panel, single plane) and in some of the distal dendritic spines (right panel, arrowheads, projection of a 1,95µm z-stack) in adult cerebellar Purkinje cells. Anti-HA and anti-GFP immunolabeling was performed on parasagittal cerebellar sections obtained from adult L7-Cre mice after stereotaxic injection of AAV particles driving the expression of HA-SUSD4 and soluble GFP. Scale bars: 10 µm (left panel) and 2µm (right panel). (D) Purkinje cells from primary mixed cerebellar culture of L7-Cre mice were transduced at 3 days in vitro (DIV3) with a HA-tagged SUSD4 expressing virus (AAV2-hSYN-DIO-HA-SUSD4-2A-eGFP) or with a control virus expressing GFP (AAV2-hSYN-DIO-eGFP), and immunostained in non-permeabilizing conditions at DIV17 for HA to localize surface SUSD4 (anti-HA, red), and in permeabilizing conditions to detect the green fluorescent protein (anti-GFP, green) and the endogenous GluD2 subunit (anti-GRID2, blue). Scale bar: 5 µm. (E) Genomic structure of the Susd4 gene. White boxes represent exons. Exon 1 is deleted in the Susd4 loss-of-function mouse model. See also Figure S2 . (F) Motor coordination and learning is deficient in adult male Susd4 -/- (KO) mice compared to age-matched Susd4 +/+ (WT) littermates. Each mouse was tested three times per day during five consecutive days on an accelerating rotarod (4 to 40 r.p.m. in 10 minutes) and the time spent on the rotarod was measured. Mean ± s.e.m. (WT n=11 and KO n=7 mice, two-way ANOVA with repeated measures, Interaction (time and genotype): ** P=0.0079, F(14, 224) = 2.22; Time: **** P<0.0001, F(14, 224) = 3.469; Genotype: P=0.0553, F(1, 16) = 4.272). (G) In situ hybridization experiments were performed on brain sections from one month-old WT and Susd4 KO mice to detect Susd4 mRNA using a probe encompassing exons 2 to 5 (See also Figure S2) . Susd4 expression was found in many regions of the brain in Susd4 +/+ (WT) mice (see also Figure S1 ) including the cerebral cortex (CTX), the cerebellum (CB), and the brainstem (BS). No labeling was found in the brain of Susd4 -/- (KO) mice. Scale bars: 500 µm.

Article Snippet: AAV2 particles were generated using a hSYN-DIO-HA-SUSD4-2A-eGFP-WPRE construct (Vector biolabs, Malvern, USA) and injected stereotaxically in cerebella of adult mice expressing the CRE recombinase in cerebellar Purkinje cells (PCs) using the L7Cre mice.

Techniques: Quantitative RT-PCR, Expressing, Immunolabeling, Injection, In Vitro, In Situ Hybridization, Labeling

Journal: bioRxiv

Article Title: SUSD4 controls GLUA2 degradation, synaptic plasticity and motor learning

doi: 10.1101/859587

Figure Lengend Snippet: (A) The number of GLUA2 clusters (anti-GLUA2 immunolabeling, green) per climbing fiber presynaptic bouton (anti-VGLUT2 immunolabeling, red) and their intensity were quantified in cerebellar sections of juvenile Susd4 -/- KO mice and Susd4 +/+ WT littermates. Cumulative plot for the mean GLUA2 intensity per VGLUT2 bouton shows no significant change between WT and KO. The distribution of the VGLUT2 boutons according to the number of associated GLUA2 clusters is significantly different between WT and KO. Mean ± s.e.m. (WT n= 5 and KO n= 5 mice; Intensity: Kolmogorov-Smirnov test, P=0.5009; Distribution: Chi-square contingency test, **** P<0.0001). Scale bars: 30 µm (top) and 15 µm (bottom). (B) Activity-dependent changes in surface localization of GLUA2 was studied in cerebellar acute slices from Susd4 KO mice and control Susd4 WT littermates using a chemical LTD protocol (cLTD; K-Glu: K + 50mM and glutamate 10µM for 5 minutes followed by 30 minutes of recovery). Surface biotinylation of GLUA2 subunits was performed followed by affinity-purification of biotinylated GLUA2 subunits and anti-GLUA2 immunoblot analysis. The fraction of biotinylated GLUA2 was obtained by measuring the levels of biotinylated GLUA2 in affinity-purified samples and total GLUA2 normalized to beta-actin in input samples for each condition. The ratios between the fraction of biotinylated GLUA2 after cLTD and control conditions are represented. Mean ± s.e.m. (n=8 independent experiments; Two-tailed Student’s one sample t-test was performed on the ratios with a null hypothesis of 1, P WT = 0.0212 and P KO = 0.0538). (C) Activity-dependent degradation of GLUA2 was assessed in cerebellar acute slices from Susd4 KO and control mice after induction of chemical LTD (cLTD; K-Glu: K + 50mM and glutamate 10µM for 5 minutes followed by 30 minutes of recovery). This degradation was absent when slices were incubated with 100µg/mL leupeptin and with 50µM MG132 (to inhibit lysosomal and proteasome degradation, respectively), or when slices were obtained from Susd4 KO mice. Band intensities of GLUA2 were normalized to β-ACTIN. The ratios between levels with cLTD induction (K-Glu) and without cLTD induction (CTL) are represented. See also Figure S8 . Mean ± s.e.m. (n=8 independent experiments; Two-tailed Student’s one sample t-test was performed on the ratios with a null hypothesis of 1, P WT = 0.0107, P WT+Leu/MG132 = 0.3755, P KO = 0.3176 and P KO+Leu/MG132 = 0.2338). (D) Purkinje cells from primary cerebellar cultures of L7-Cre mice were transduced at 3 days in vitro (DIV3) with a virus driving expression of HA-tagged SUSD4 (AAV2-hSYN-DIO-HA-SUSD4-2A-eGFP) and immunolabeled at DIV17 in non-permeabilizing conditions to localize surface SUSD4 (anti-HA, red) and surface GLUA2 subunits (anti-GluA2, blue). Direct green fluorescent protein is shown (GFP, green). Examples of spines containing either SUSD4 alone, GLUA2 alone or both are shown using red, blue and yellow arrowheads, respectively (maximum projection of a 1.8 µm z-stack). Scale bar: 5 µm.

Article Snippet: AAV2 particles were generated using a hSYN-DIO-HA-SUSD4-2A-eGFP-WPRE construct (Vector biolabs, Malvern, USA) and injected stereotaxically in cerebella of adult mice expressing the CRE recombinase in cerebellar Purkinje cells (PCs) using the L7Cre mice.

Techniques: Immunolabeling, Activity Assay, Affinity Purification, Western Blot, Two Tailed Test, Incubation, In Vitro, Expressing

Journal: eLife

Article Title: Therapeutic deep brain stimulation disrupts movement-related subthalamic nucleus activity in parkinsonian mice

doi: 10.7554/eLife.75253

Figure Lengend Snippet:

Article Snippet: WT mice were injected with a retrograde virus encoding Cre recombinase (either CAV-Cre [Montpellier, 100 nL injected undiluted, undiluted titer 1.0 × 10 13 /mL; https://plateau-igmm.pvm.cnrs.fr/?vector=cav-cre ] or AAV2 retro -Cre-mCherry [Addgene/UPenn Vector Core RRID Addgene_55632, 100 nL injected undiluted, undiluted titer 7.8 × 10 13 /mL]) in the STN (–1.8 AP, –1.65 ML, 4.5 DV) and Cre-dependent GCaMP6s (500 nL injected diluted 1:8 in normal saline) in the primary motor cortex (M1, +2 AP, –1.56 ML, 1 DV) and implanted with a fiber-optic ferrule above M1 (0.8 DV).

Techniques: Saline, Plasmid Preparation, Software