Journal: Molecular neurobiology

Article Title: CD38 Coordinates with NF-κB to Promote Cochlear Inflammation in Noise-Induced Hearing Loss: the Protective Effect of Apigenin.

doi: 10.1007/s12035-024-04675-7

Figure Lengend Snippet: Fig. 9 CD38 coordinates with NF-κB to promote cochlear inflammation in NIHL. As the closest immune cell to organ of Corti, activated macrophages can express CD38 and consume NAD in organ of Corti, causing NAD + metabolism dysfunction and ultimately leads to cochlear inflammation. In this process, inflammatory factors such as IL-1, IL-6, and TNF-α pro- moted the activation of NF-κB signaling pathway, forming a positive feedback cycle

Article Snippet: Immunofluorescence The cochlear basilar membrane was prepared with the surface preparation method, antibodies used in this experiment were rabbit anti-NF-κB p65 polyclonal antibody (Abcam, ab32536), rat anti-F4/80 polyclonal antibody (Abcam, ab6640), and mouse anti-CD38 monoclonal antibody (Proteintech, 60,006–1-lg).

Techniques: Activation Assay

Journal: Human Molecular Genetics

Article Title: Mouse models of X-linked juvenile retinoschisis have an early onset phenotype, the severity of which varies with genotype

doi: 10.1093/hmg/ddz122

Figure Lengend Snippet: Localization of RS1 in the developing retina. (A–E) Rs1 KO retinas stained for X-gal at the ages indicated. Red arrows in the insets to (A and B) indicate some of the small number of RGCs that were X-gal positive. After P14, X-gal staining was restricted to the photoreceptors (C–E). (F) WT retina stained for X-gal demonstrates the level of background label in the OPL and inner segments. (G–K) RNAScope® showed Rs1 mRNA in WT mice at the ages indicated. Rs1 was not detected at P1 (G) or P7 (H). At later ages, Rs1 was seen in a small number of cells outside of the ONL at P14 (I) and was restricted to photoreceptors at P21 (J) or adulthood (K). Retinal expression of Rs1 was not detected in the Rs1 KO retina (L).

Article Snippet: Blots were incubated with anti-RS1 antibody (Novus Biologicals USA, 1:4000) for 2 h at room temperature or overnight at 4°C.

Techniques: Staining, Expressing

Journal: Human Molecular Genetics

Article Title: Mouse models of X-linked juvenile retinoschisis have an early onset phenotype, the severity of which varies with genotype

doi: 10.1093/hmg/ddz122

Figure Lengend Snippet: Immunohistochemical localization of RS1 (red) in adult WT (A), KO (B), C59S (C) and R141C (D) retinal cross-sections. In the WT, RS1 is present in all retinal layers (A). This distribution is missing in the KO retina (B) and is restricted to the inner segments of C59S (C) and R141C (D) retina. Non-specific labeling of blood vessels (red arrows) is seen, due to the use of an anti-mouse secondary antibody. Scale bar indicates 100 μm in (A). Western blot analysis of Rs1 in KO (E), C59S (F) and R141C (G) retina. (H) ELISA-based quantification of RS1 demonstrated significant reduction in each mutant. Bars indicate average (±SD) for four retinas.

Article Snippet: Blots were incubated with anti-RS1 antibody (Novus Biologicals USA, 1:4000) for 2 h at room temperature or overnight at 4°C.

Techniques: Immunohistochemical staining, Labeling, Western Blot, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: Human Molecular Genetics

Article Title: Mouse models of X-linked juvenile retinoschisis have an early onset phenotype, the severity of which varies with genotype

doi: 10.1093/hmg/ddz122

Figure Lengend Snippet: Quantitation of retinal parameters used to compare WT and Rs1 mutant retinas. (A) Diagram of method to measure INL + OPL area (top), the thickness of the outer retina (middle) and the thickness of the overall retina (bottom). This image was obtained from the right temporal retina of a 20-week-old KO mouse. (B) Measures plotted as a function of age for each of the mouse lines examined. Data points indicate average (±SEM) for 3–16 mice. Symbols above the data points at each age indicate the results of t-tests comparing Rs1 values to those of WT. * indicates P < 0.05; + indicates P < 0.01; # indicates P < 0.001 and the color indicates which Rs1 model is being compared to WT.

Article Snippet: Blots were incubated with anti-RS1 antibody (Novus Biologicals USA, 1:4000) for 2 h at room temperature or overnight at 4°C.

Techniques: Quantitation Assay, Mutagenesis

Journal: Human Molecular Genetics

Article Title: Mouse models of X-linked juvenile retinoschisis have an early onset phenotype, the severity of which varies with genotype

doi: 10.1093/hmg/ddz122

Figure Lengend Snippet: Dark-adapted electroretinography. Representative dark-adapted ERGs obtained from mice aged P15 (A), P24 (B) or 10 weeks (C). Note that responses of Rs1 mutant mice are reduced at all ages and display the characteristic negative ERG waveform. Scale bars indicate 500 μV and 100 ms. Luminance-response functions for the major ERG components compare data from mice aged P15 (D), P24 (E) or 10 weeks (F). Data points indicate the average (±SEM) for 3–16 mice.

Article Snippet: Blots were incubated with anti-RS1 antibody (Novus Biologicals USA, 1:4000) for 2 h at room temperature or overnight at 4°C.

Techniques: Mutagenesis

Journal: Human Molecular Genetics

Article Title: Mouse models of X-linked juvenile retinoschisis have an early onset phenotype, the severity of which varies with genotype

doi: 10.1093/hmg/ddz122

Figure Lengend Snippet: Light-adapted electroretinography. Representative light-adapted cone ERGs obtained from mice aged P15 (A), P24 (B) or 10 weeks (C). Note that responses of Rs1 mutant mice are reduced at all ages. Scale bars indicate 100 μV and 100 ms. Luminance-response functions for the cone ERG from mice aged P15 (D), P24 (E) or 10 weeks (F). Data points indicate the average (±SEM) for 3–16 mice.

Article Snippet: Blots were incubated with anti-RS1 antibody (Novus Biologicals USA, 1:4000) for 2 h at room temperature or overnight at 4°C.

Techniques: Mutagenesis

Journal: Human Molecular Genetics

Article Title: Mouse models of X-linked juvenile retinoschisis have an early onset phenotype, the severity of which varies with genotype

doi: 10.1093/hmg/ddz122

Figure Lengend Snippet: Age-related changes in ERG amplitude parameters. RmP3 (A) and A (B) parameters obtained by fitting the Lamb & Pugh equation to the leading edge of the dark-adapted ERG a-wave. Rmax (C) and K (D) parameters obtained by plotting the Naka–Rushton equation to the initial limb of the dark-adapted b-wave response function. Rmax (E) and K (F) parameters obtained by plotting the Naka–Rushton equation to the light-adapted b-wave response function. Data points indicate the average (±) SEM for 3–16 mice. Symbols above the data points at each age indicate the results of t-tests comparing Rs1 values to those of WT. * indicates P < 0.05; + indicates P < 0.01; # indicates P < 0.001 and the color indicates which Rs1 model is being compared to WT. Filled arrows indicate where values for one Rs1 mutant are significantly different from both of the other mutants; the color of the arrow identifies that mutant.

Article Snippet: Blots were incubated with anti-RS1 antibody (Novus Biologicals USA, 1:4000) for 2 h at room temperature or overnight at 4°C.

Techniques: Mutagenesis

Journal: Human Molecular Genetics

Article Title: Mouse models of X-linked juvenile retinoschisis have an early onset phenotype, the severity of which varies with genotype

doi: 10.1093/hmg/ddz122

Figure Lengend Snippet: Altered patterns of spontaneous activity in Rs1 mutant mice. (A, C) Confocal reconstructions of representative RGCs following electrophysiological assessment showing distinction between ON and OFF RGC types. Raster plots and peristimulus time histograms show spontaneous activity in ON (B) and OFF (D) RGCs that were recorded in cell-attached mode at P19–P21 in WT and age-matched Rs1 mutant male mice. (E) Representative traces from a WT and three KO RGCs illustrate a range of activity patterns encountered in the mutants. (F) Quantification of spontaneous activity of ON and OFF RGCs in WT and Rs1 mutant mice. Numbers above each graph bar indicate P-values of one-way ANOVA pairwise multiple comparison (Tukey test) of each mutant to the WT RGCs. Each group contains n = 6–8 mice with 2–4 RGC of each class per animal. Error bars indicate ±SEM, AP/s—action potentials per second.

Article Snippet: Blots were incubated with anti-RS1 antibody (Novus Biologicals USA, 1:4000) for 2 h at room temperature or overnight at 4°C.

Techniques: Activity Assay, Mutagenesis, Comparison

Journal: Human Molecular Genetics

Article Title: Mouse models of X-linked juvenile retinoschisis have an early onset phenotype, the severity of which varies with genotype

doi: 10.1093/hmg/ddz122

Figure Lengend Snippet: Morphological changes of cones and sprouting of cone BCs. (A) Vertical views reconstructed from confocal z-stack images of the retina whole-mounts. Cone arrestin (green) was concentrated in the OS of WT and was redistributed through the entire cone cell body in Rs1 mutants. (B) Horizontal confocal sections from (A) demonstrate shortened cone OSs and malformation of cone axon terminals. Cone BCs labeled for synaptotagmin II (magenta) exhibited sprouting dendrites in the ONL. (C) Cone arrestin was redistributed in Rs1 mutants as shown by the averaged fluorescent intensity profile. (D) A vertical view of a 250 μm2 z-stack through the ONL rotated 90° and collapsed onto a single plane. Blue line marks the ONL position reached by 90% of sprouts. ONL was defined between the OPL (0%) and IS (100%). (E) Distribution of sprouts in the OPL from (D). Area under the curve represents total length of sprouts measured as a percentage of the ONL. (F) In all Rs1 mutants, cone BC sprouts reached far into the IPL. The numbers represent depth of the ONL reached by 90% sprouts, as shown by the blue line in (D). (G) KO mice had greater total length of cone BC sprouts than C59S or R141C mice. The numbers indicate the area under curve in (E), marked by red vertical dashed lines. Bars indicate average (±SD) for 5–6 mice. Red P-values indicate significant difference from WT. Scale bar in (A) indicates 10 μm. OS: outer segment; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer and GCL: ganglion cell layer.

Article Snippet: Blots were incubated with anti-RS1 antibody (Novus Biologicals USA, 1:4000) for 2 h at room temperature or overnight at 4°C.

Techniques: Labeling

Journal: Human Molecular Genetics

Article Title: Mouse models of X-linked juvenile retinoschisis have an early onset phenotype, the severity of which varies with genotype

doi: 10.1093/hmg/ddz122

Figure Lengend Snippet: Aberrant activity impairs responses to light in Rs1 mutant mice. (A, B) Series of spiking responses were obtained to a 200 μm spot of light with increasing positive contrast in ON (A) and negative contrast in OFF (B) RGCs. Red steps below activity traces indicate stimulus timing. (C) Average spiking activity during light stimulus and the response amplitude, adjusted to spontaneous activity (D) in P19–P21 WT and Rs1 mutant male mice. (E) In the recordings from RGCs, elevated spontaneous activity reduces SNR (see Materials and Methods for computation) of retinal output, diminishing discrimination of responses to visual stimulus. Numbers above each graph bar represent P-values of one-way ANOVA pairwise multiple comparison (Tukey test) to the WT RGCs. Each group contains n = 6–8 mice with 2–4 RGC of each class per animal. Error bars indicate ±SEM, AP/s—action potentials per second. (F) Whole-cell recording of excitatory and inhibitory currents underlying spontaneous and light-evoked activities in RGCs in WT and Rs1 mutant mice.

Article Snippet: Blots were incubated with anti-RS1 antibody (Novus Biologicals USA, 1:4000) for 2 h at room temperature or overnight at 4°C.

Techniques: Activity Assay, Mutagenesis, Comparison

Journal: Human Molecular Genetics

Article Title: Mouse models of X-linked juvenile retinoschisis have an early onset phenotype, the severity of which varies with genotype

doi: 10.1093/hmg/ddz122

Figure Lengend Snippet: Morphological changes of rod BCs and HCs. (A) Vertical views reconstructed from confocal z-stacks of retinal whole mounts reveal sprouting of rod BCs (magenta) and HCs (green). (B) Horizontal confocal planes from (A). Rod BC and HC processes extend together into the ONL. Rod BCs and HCs had malformed and displaced somas. In Rs1 mutants, astrocytes (blue) had dense processes and Müller cells (blue) expressed high levels of GFAP through the entire cell. (C–F) Quantification of rod BC and HC processes revealed prominent sprouting in all Rs1 mutants, with the greatest amount seen in the KO. Bar indicates average (±SD) of 5–6 mice. Red P-values indicate significant difference from WT. Scale bar in (A) indicates 10 μm. OS: outer segment; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer and PKC: protein kinase C.

Article Snippet: Blots were incubated with anti-RS1 antibody (Novus Biologicals USA, 1:4000) for 2 h at room temperature or overnight at 4°C.

Techniques:

Journal: Human Molecular Genetics

Article Title: Mouse models of X-linked juvenile retinoschisis have an early onset phenotype, the severity of which varies with genotype

doi: 10.1093/hmg/ddz122

Figure Lengend Snippet: Primer/probe sets for TAQMAN ® assays

Article Snippet: Blots were incubated with anti-RS1 antibody (Novus Biologicals USA, 1:4000) for 2 h at room temperature or overnight at 4°C.

Techniques: Sequencing

Journal: Biochemistry and Biophysics Reports

Article Title: Intravitreal injection of new adeno-associated viral vector: Enhancing retinoschisin 1 gene transduction in a mouse model of X-linked retinoschisis

doi: 10.1016/j.bbrep.2024.101646

Figure Lengend Snippet: Improvement of retinal function and structure in Rs1-KO mouse model after 3 months of intravitreal injection of AAV-DJ(K137R)-Rs1 and AAV-DJ-Rs1 vector. ERG, OCT, HE, and IHC were used for evaluation. (A): Representative ERG waveforms. (B): Representative OCT images. (C): HE staining. (D): Immunohistochemistry staining. The red arrow indicates the boundary of the cavity. The untreated eye has large cavities spanning layers proximal to the photoreceptor nuclear layer (ONL). Scale bar, 500um in B, 50 μm in C and D. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The sections were stained using RS1 Polyclonal antibody (Proteintech) and a secondary antibody conjugated to CoraLite®488-Conjugated AffiniPure Goat Anti-Rabbit IgG(H + L) (Proteintech).

Techniques: Injection, Plasmid Preparation, Staining, Immunohistochemistry

Journal: Biochemistry and Biophysics Reports

Article Title: Intravitreal injection of new adeno-associated viral vector: Enhancing retinoschisin 1 gene transduction in a mouse model of X-linked retinoschisis

doi: 10.1016/j.bbrep.2024.101646

Figure Lengend Snippet: Statistical analysis of ERG and OCT data after 3 months of treatment with AAV-DJ (K137R)-Rs1. The ratio of the treated eye to the untreated eye (T/UT) is plotted for the b-wave/a-wave amplitude and opl-inl thickness. (A): The scatter plots show the b-wave/a-wave amplitude results from individual mice at each vector. (B): The bar graphs show the averages and SEM of b-wave/a-wave amplitude for each vector. (C): The scatter plots show the opl-inl thickness results from individual mice at each vector. (D): The bar graphs show the averages and SEM of opl-inl thickness for each vector. Asterisks indicate significance of treatment effect compared with vehicle (****p < 0.0001, **p < 0.01, *p < 0.05). NS indicates ‘not significant’.

Article Snippet: The sections were stained using RS1 Polyclonal antibody (Proteintech) and a secondary antibody conjugated to CoraLite®488-Conjugated AffiniPure Goat Anti-Rabbit IgG(H + L) (Proteintech).

Techniques: Plasmid Preparation