Journal: Frontiers in Cellular Neuroscience

Article Title: The Endocannabinoid/Cannabinoid Receptor 2 System Protects Against Cisplatin-Induced Hearing Loss

doi: 10.3389/fncel.2018.00271

Figure Lengend Snippet: Distribution of the GFP-tagged CB2R knock-in mice. (A) The genetic background of the knock-in mice is shown where the GFP reporter gene was inserted within exon 3 of CB2R and the expression of GFP was driven by internal ribosomal entry (IRE) promoter. Cochleae isolated from these GFP-CB2R co-expressing knock-in mice were fixed in 4% paraformaldehyde followed by decalcification and then sectioned. (B) The representative mid-modiolar section shows endogenous GFP fluorescence (green) in different anatomical regions of the cochlea including organ of Corti (OC), stria vascularis (SV), spiral ligament (SL), and spiral ganglion (SG) neuron. (C) Immunolabeling of GFP-tagged CB2R (green) along with hair cell marker, Myo VIIa (red), show colocalization of CB2R in both, the OHCs and IHCs. (D) Co-labeling of these cochleae with Tuj1 (red), a neuronal marker, show the distribution of GFP-CB2R (green) in spiral ganglion neurons. (E) Whole mount sections of cochleae from GFP-CB2R co-expressing knock-in mice show co-localization of GFP-CB2R (green) and myosin VIIa (red) in the OHCs and IHCs of OC. (F) GFP-CB2R whole mounts sections were also co-stained with neuronal marker, Tuj1 (red), and pre-synaptic ribbon marker, CtBP2 (magenta), showing pre-synaptic localization of CB2R. Cell nuclei (blue) are stained with Hoechst stain in (B,E,F) . Studies reported here were repeated in at least three different animals and similar results were obtained.

Article Snippet: Two different myosin VIIa antibodies were used for different purposes: Mouse IgG1 anti-myosin-VIIa antibody was obtained from Developmental Studies Hybridoma Bank (#138-1), while rabbit anti-myosin-VIIa was purchased from Proteus Biosciences (#25-6790).

Techniques: Knock-In, Expressing, Isolation, Fluorescence, Immunolabeling, Marker, Labeling, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: The Endocannabinoid/Cannabinoid Receptor 2 System Protects Against Cisplatin-Induced Hearing Loss

doi: 10.3389/fncel.2018.00271

Figure Lengend Snippet: Distribution of the CB2R in the rat cochlea. (A) Immunolabeling of mid-modiolar cochlear sections with CB2R antibody shows labeling of cells in the organ of Corti (green). Labeling was observed in the outer hair cells (OHC), inner hair cells (IHC), inner pillar cell (IPC), and outer pillar cell (OPC). (B) Co-labeling of the organ of Corti with antibodies for CB2R (green) and myosin VIIa (red) showed co-localization of these proteins in the OHC and IHC. (C) Staining of CB2R was also observed in the intermediate and basal cells of the SV and type II fibrocytes in SL. CB2R (green) showed some overlap with Na + /K + -ATPase α1 subunit (red) in the SV. (D) CB2R was localized to the plasma membranes of SG. Approximately 60% of cells in this section were stained with both CB2R (green) and Hoechst (blue). (E) Co-labeling of CB2R and myosin VIIa in OHCs and IHCs in whole mount preparations of the organ of Corti shows that distribution of CB2R (green) is more widespread than that of myosin VIIa (red). Hoechst was used as a nuclear stain (blue) in (B–E) . Studies reported here were repeated in at least three different animals and similar results were obtained.

Article Snippet: Two different myosin VIIa antibodies were used for different purposes: Mouse IgG1 anti-myosin-VIIa antibody was obtained from Developmental Studies Hybridoma Bank (#138-1), while rabbit anti-myosin-VIIa was purchased from Proteus Biosciences (#25-6790).

Techniques: Immunolabeling, Labeling, Staining, Clinical Proteomics

Journal: Frontiers in Cellular Neuroscience

Article Title: The Endocannabinoid/Cannabinoid Receptor 2 System Protects Against Cisplatin-Induced Hearing Loss

doi: 10.3389/fncel.2018.00271

Figure Lengend Snippet: Trans -tympanic administration of JWH015 prevents cisplatin-induced hearing loss. (A) Baseline ABR thresholds were recorded in Wistar rats, which were then pre-treated with either trans -tympanic AM630 (2.5 nmoles) for 30 min followed by trans -tympanic JWH015 (2.5 nmoles) or individually with trans -tympanic AM630 or JWH015 for 24 h. Cisplatin (11 mg/kg) was then administered (i.p.) after day 1 and post-ABR was performed after 72 h. Pre-treatment with JWH015 diminished cisplatin-induce ABR threshold shift in all the three frequency regions. The protective action of JWH015 was completely blocked by AM630. Trans -tympanic administration of AM630 alone significantly elevated by ABR thresholds. Data (threshold shift) is plotted as mean ± SEM ( n = 12). (B) The animals were sacrificed at the end point and cochleae were collected, fixed by 4% PFA in PBS followed by decalcification for 3 weeks in 100 mM EDTA. The base of the cochlea were dissected out and stained with hair cell marker, myosin VIIa, to determine the hair cell loss. Representative images are shown. Scale bar is 20 μm. (C) The bar graph represents the average number of hair cells per 100 μm area shown in (B) ( n ≥ 3). Pre-treatment with JWH015 reduced cisplatin-induced outer hair cell loss. Administration of AM630 produced no hair cell loss. ∗ p < 0.05 vs. vehicle; ∗∗ p < 0.05 vs. cisplatin; # p < 0.05 vs. JWH015 + cisplatin; and ∗∗∗ p < 0.05 vs. JWH015 (one-way ANOVA).

Article Snippet: Two different myosin VIIa antibodies were used for different purposes: Mouse IgG1 anti-myosin-VIIa antibody was obtained from Developmental Studies Hybridoma Bank (#138-1), while rabbit anti-myosin-VIIa was purchased from Proteus Biosciences (#25-6790).

Techniques: Staining, Marker, Produced

Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: A . Schematic diagram showing the genetic structure of the integrated EIAV vectors used in this study . EIAV-CMV-MYO7A (UshStat) is based on a non-replicating non-human recombinant lentiviral vector based on the non-pathogenic wild type equine infectious anaemia virus (EIAV). The wild-type EIAV virus has 6 distinct genetic units, however, the majority of these EIAV sequences have been removed to produce a minimal vector system that contains less than 10% of the original viral genome and does not contain any viral promoters or enhancers and there are no coding regions for accessory proteins in either the EIAV genome or in the packaging system. SIN LTR: Self inactivating long term repeat. Neo: Neomycin open reading frame (ORF). CMV: Cytomegalovirus promoter (constitutive). RK: Rhodopsin kinase promoter (photoreceptor specific). eGFP: enhanced green fluorescent protein ORF. MYO7A: Myosin VIIa ORF. WPRE: Woodchuk hepatitis virus post-transcriptional regulatory element. B . Expression analysis of myosin VIIa in 4 weeks mouse eye and HeLa cells transfected with the EIAV-CMV-Null (Null) or UshStat constructs. β-actin was used as loading control. RPE: retinal pigment epithelium. NR: neuroretina. IP/Null: immunoprecipitates of HeLa cells transfected with the null vector. IP/UshStat: immunoprecipitates of HeLa cells transfected with the myosin VIIa vector. IB MYO7A: immunoblot with the mouse anti-myosin VIIa. Molecular weight markers are denoted to the left. C–D : Immunocytochemistry studies of HeLa cells transduced with the null ( C ) or the UshStat vector ( D ) and immunostained for myosin VIIa (red). DAPI was used to counter stain the nucleus. Scale bar: 15 μm.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Recombinant, Plasmid Preparation, Virus, Expressing, Transfection, Construct, Control, Western Blot, Molecular Weight, Immunocytochemistry, Transduction, Staining

Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: Shaker1 mouse retinas were co-transduced by a subretinal injection of UshStat and EIAV-CMV-GFP or EIAV-CMV-Null and EIAV-RK-GFP (the GFP vector was used to identify the transduced region of the retina). After 4 weeks the animals were dark-adapted overnight, then light-adapted for 10 minutes under 200 lux illumination. Retinas were double immunostained with antibodies against GFP (green) and α-transducin (red). Panel I : Low magnification image of a retina transduce with UshStat and GFP, showing that the gradient in α-transducin translocation (left to right) parallels GFP expression (indicative of wild type myosin VIIa). Scale bar: 40 μm. Panel II : A–C : Representative examples of an EIAV transduced region of the retina (determine by the presence of GFP), presenting dual immunostaining for GFP and α-transducin ( C ). In the presence of wild type myosin VIIa, α-transducin is translocated to the inner segment (IS). D–F : An untransduced region of the same retina as in A–C does not show α-transducin translocation to the IS upon illumination. G–I : Retinas co-transduced with EIAV-Null-vector and EIAV-RK-GFP (photoreceptor cell-specific promoter) as a control for the effects of subretinal lentiviral transduction on α-transducin translocation. The region of the retina shown was transduced, evidenced by GFP expression in the photoreceptors ( H ) however there was no translocation of α-transducin to the IS ( G and I ). The qualitative results represented in each of the panels are representative images for at least three replicate experiments. RPE = Retinal Pigment Epithelium; OS = Outer Segments; IS = Inner Segments; ONL = Outer Nuclear Layer; OPL = Outer Plexiform Layer. Scale bar: 25 μm. Arrowheads in D and G indicate translocation of α-transducin in individual photoreceptors. Asterisks in I denote reactivity of the secondary anti-mouse antibody with circulation mouse IgGs present in the blood vessels.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Injection, Plasmid Preparation, Translocation Assay, Expressing, Immunostaining, Transduction, Control

Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: A–B : Show myosin VIIa immunostaining of shaker1 retinas transduced with the EIAV-CMV-Null vector ( A ) or UshStat ( B ). Myosin VIIa expression can be detected in the RPE, OS and IS regions. C–D : monkey retinas transduced with UshStat ( D ) showed high levels of human myosin VIIa in the RPE and moderate expression in outer and inner segments of the photoreceptor cells. Asterisk in C denotes weak immunostaining of the endogenous myosin VIIa in the RPE layer. Note that the anti-myosin VIIa antibody was titrated so as to only detect the exogenous overexpressed virus-derived wild type myosin VIIa ( B, D ) and not the endogenous protein ( A, C ). Scale bars: 25 μm.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Immunostaining, Transduction, Plasmid Preparation, Expressing, Virus, Derivative Assay

Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: Retinas were co-transduced with EIAV-CMV-Null vector and EIAV-RK-GFP ( A–C ) or UshStat and EIAV-CMV-GFP ( D–F, J ). After 4 weeks, mice were exposed to 6 days continuous light at 2000 lux illumination. Retinas were harvested and dual immunostained for myosin VIIa (red) and GFP (green) ( A–F, J ). Immunostaining conditions were chosen as to only detect the exogenous overexpressed myosin VIIa. G–H : Eosin and hematoxylin histochemical staining of shaker1 mouse retinas transduced with either the EIAV-CMV-Null vector ( G ) or UshStat ( H ). I : Eosin and hematoxylin staining of wild type (untransduced) retina for comparison of relative light dependent degeneration of the ONL that is typically observed (double headed arrow). J : Low magnification image of an UshStat+GFP co-transduced retina, showing the area used for the studies (bracket) relative to the area of injection (syringe). This area corresponds to the juxtaposed ∼0.2 mm from the site of injection. K : Schematic representation of the area of injection represented by red boxes, where right box area corresponds to the injection in the right eye and left box area injection in the left eye. The inferior retina was always used for the injections and ONL counting. Scale bars A–F : 25 μm, G–I : 50 μm. J : 90 μm. Labels are as in . Asterisks in C , F and J denote reactivity of the secondary anti-mouse antibody with circulation mouse IgGs present in the blood vessels.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Transduction, Plasmid Preparation, Immunostaining, Staining, Comparison, Injection