Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Stable loss of major histocompatibility complex class I (MHC-I) gene DNA methylation (DNAm) in intestinal epithelial organoids (IEOs) derived from patients with Crohn’s disease (CD). (A) (i) Overview of experimental set-up and sample generation. (ii) Representative brightfield images of IEOs. Scale bars: 300 µm. (B) (i) Correlation heat map of comethylated CpG modules identified by weighted gene coexpression network analysis (WGCNA) in terminal ileum (TI) IEOs. Module 17 (ME17) demonstrates hypomethylation and the strongest association with CD diagnosis (R=−0.43, p value<0.001). (ii) Gene set enrichment analysis performed on module 17, showing a significant loss of DNAm in CD organoids compared with healthy controls and UC in TI. (C) DNAm (beta value) of four representative MHC-I related Differetial Methylated Positions (DMPs) showing CD-associated loss of DNAm in TI and sigmoid colon (SC) but not duodenum (DUO) organoids (DUO=54, TI=127 and SC=131). (D) Average DNAm (beta value) of all CpGs located in MHC-I related genes for IEOs split by diagnosis, gut segment and inflammatory status. (E) (i) Correlation of nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) promoter DNAm between early and later passage IEOs from the same individuals including patients diagnosed with CD (blue), UC (yellow) and controls (grey, n=22 patients. (ii) DNAm (beta values) of CpGs located in NLRC5 and TAP1 at high passage (>7) IEOs (cohort 1, n=22). (F) Average MHC-I (i) and NLRC5 (ii) DNAm as well as NLRC5 gene expression (iii) in control patient-derived TI IEOs stimulated with proinflammatory cytokines interferon γ (IFNγ) and tumour necrosis factor α (TNFα) (n=5). (False Discovery Rate (FDR) * < 0.05, FDR **< 0.01, FDR***< 0.001, FDR**** < 0.0001, ns=not significant.)

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, DNA Methylation Assay, Derivative Assay, Biomarker Discovery, Methylation, Binding Assay, Gene Expression, Control

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Loss of major histocompatibility complex class I (MHC-I) DNA methylation (DNAm) correlates with increased gene expression in primary intestinal epithelium of patients with Crohn’s disease (CD). (A) Overview of patient cohort, sample preparation and data generation. (B, C) DNAm and gene expression in purified terminal ileum (TI) (B) and sigmoid colon (SC) (C) epithelium. (i) Average DNAm (beta value) of all and selected MHC-I pathway-related CpGs showing significant, CD-associated loss of DNAm. (ii) Correlation between beta values and corresponding gene expression (R=Spearman’s rank correlation). (D) Nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) promoter DNAm in the IE of healthy, patients with UC and CD at the point of diagnosis and during reassessment. (E) Correlation of NLRC5 promoter DNAm in intestinal epithelial organoids (IEOs) obtained from the same patient at diagnosis and reassessment (Spearman’s rank correlation). (F) NLRC5 promoter DNAm in TI IEOs derived from patients with CD, UC and control (n=3 IEO per condition, two-way analysis of variance (ANOVA) with Turkey’s test for multiple comparisons. ****p<0.0001). (G) NLRC5 mRNA expression in TI IEOs derived from patients with controls, UC and CD at baseline and on interferon γ (IFNγ) treatment (10 ng/mL for 6 hours). Data are normalised to the mean of control lines and shown as mean±SEM (two-way ANOVA with Turkey’s test for multiple comparisons. **P<0.01, *p<0.05, ns=not significant). n=3 IEO lines in each group for three independent experiments.

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, DNA Methylation Assay, Gene Expression, Sample Prep, Purification, Binding Assay, Biomarker Discovery, Derivative Assay, Control, Expressing

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) acts as transcriptional transactivator of intestinal epithelial cell (IEC) major histocompatibility complex class I (MHC-I) and potentiates the effect of interferon γ (IFNγ). (A) Overview of experimental set-up. (B) Heatmap showing gene expression (RNAseq) of MHC-I pathway genes in terminal ileum (TI) intestinal epithelial organoids (IEOs)± NLRC5 overexpression (dox), and ±exposure to IFNγ (n=4 independent replicates). (C) RNA transcription of HLA-A / -B / -C / -E / -F / -G in response to IFNγ and tumour necrosis factor α (TNFα) in wild type (WT) and NLRC5 OE TI IEOs. (D) Relative expression for MHC-I pathway genes in WT ( NLRC5 +/+ ) and corresponding NLRC5 deficient ( NLRC5 −/ − ) TI IEOs±IFNγ (n=3 replicates. Two-way analysis of variance (ANOVA) with Bonferroni’s test for multiple comparisons, **p<0.01, ***p<0.001, ****p<0.0001). Data are representative of two independent experiments. (E) Immunofluorescence spinning disc microscopy of organoids described in D, ±IFNγ (48 hours). (i) Representative images of untreated (BSA) and treated (IFNγ) WT ( NLRC5 +/+ ) and NLRC5 deficient ( NLRC5 −/ − ) TI IEOs taken by Opera Phoenix. Scale bar=2 mm. (ii) HLA-A,B,C mean intensity quantification of BSA and IFNγ NLRC5 +/+ and NLRC5 −/ − TI IEOs. (n=3 independent replicates. Two-way ANOVA with Bonferroni multiple comparisons test, **p<0.01, ***p<0.001, ****p<0.0001.) (F) Correlation between mRNA gene expression of NLRC5 and (i) HLA-B and (ii) HLA-E, in purified TI and sigmoid colon (SC) epithelium (cohort 2) (Spearman’s rank correlation).

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Binding Assay, Immunopeptidomics, Gene Expression, Over Expression, Expressing, Immunofluorescence, Microscopy, Purification

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Crohn’s disease (CD)-associated increased intestinal epithelial major histocompatibility complex class I (MHC-I) expression affects the stem cell compartment and follows a crypt-villus gradient. (A) Summary of experimental set-up. (B) (i) Schematic representation of intestinal epithelial cell (IEC) subtypes and their location within the small bowel (terminal ileum (TI)) crypt-villus structure (TA—transiently amplifying cells). (ii) Uniform manifold approximation and projection (UMAP) plot demonstrating single IEC transcriptomes present in TI mucosal biopsies obtained from children newly diagnosed with CD and non-IBD controls. (C) Top panel: violin plots showing crypt-villus scores of cells within each identified cell subtype (top left) and total number of cells (top right). Bottom panel: correlation between MHC-I summary score and crypt-villus scores for all IEC transcriptomes. Best fitting correlation is displayed as individual lines for CD (blue), UC (yellow) and non-IBD control samples (grey) (bottom left). Bottom right: box plots of summary MHC-I single-cell transcriptional score split by diagnosis. (D) Summary/average MHC-I score in individual IEC subtypes comparing CD, UC and controls. (E) Nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) expression in TI IEC of patients with CD colocalises with CD8+ T cells. RNA scope of TI biopsies from healthy donors and patients with CD. EPCAM (cyan), NLRC5 (white), TAP1 (yellow), CD8A (red), IFNG (green) and nuclei (DAPI, blue). Proximity of CD8 + T-cells with NLRC5 + EPCAM + cells in the CD biopsy is shown with arrows. Representative images are shown. Scale bar=100 µm and zoom in scale bar=10 µm.

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, Expressing, Control, Biomarker Discovery, Binding Assay, RNAscope

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Intestinal epithelial cells (IECs) present antigen via major histocompatibility complex class I (MHC-I) and activate CD8 + T cells in vitro with nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) acting as key modulator of mucosal inflammation in vivo. (A) Overview of experimental set-up. (B) Quantification of H2K b -SIINFEKL and pan-H2K b flow cytometry on live EpCAM + cells in murine intestinal epithelial organoids (IEOs) stimulated with or without interferon γ (IFNγ) (48 hours) and pulsed with or without OVA257–264 peptide (SIINFEKL) peptide. Data are representative of two independent experiments run in triplicates. GMFI, geometric mean fluorescence intensity; AU, arbitrary units. P values were calculated by two-way analysis of variance (ANOVA) with Bonferroni test for multiple comparisons (**p<0.01, ****p<0.0001). (C) Overview of experimental design. (D) Quantitative PCR gene expression of Ifng for coculture experiment in murine IEOs±SIINFEKL peptide pulse and cocultured with SIINFEKL-activated OTI T-cells. Data are presented as fold change over unstimulated OTI cells minus murine IEOs, normalised to Cd8a . P values were calculated using two-way ANOVA with Bonferroni’s multiple comparisons test (***p<0.001, ns=not significant). (E) Body weight changes over time during and after a 6-day course of 2% dextran sulphate sodium (DSS) exposure. (n=8 and n=5 Nlrc5fl/fl and Nlrc5-/- mice, respectively. P values calculated by multiple t-tests with Holm-Šídák correction for multiple comparisons.) (F) Quantification of H2K b surface expression on EpCAM+ cell populations within the lamina propria extractions of DSS-treated mice. All panels: data are representative of two independent experiments (**p<0.01). (G) Colon weight per unit length and mesenteric lymph node (MLN) weight and spleen weight of Nlrc5 wild type and knockout mice, on day 14 after initiation of 6-day course of 2% DSS (**p<0.01).

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, In Vitro, Binding Assay, In Vivo, Flow Cytometry, Fluorescence, Real-time Polymerase Chain Reaction, Gene Expression, Expressing, Knock-Out

Journal: bioRxiv

Article Title: Age-related loss of brain demyelinating and remyelinating potential is overcome by microglia renewal

doi: 10.64898/2026.06.15.732390

Figure Lengend Snippet: Ai-iii) Comparison of RNA sequencing signatures between young and aged healthy naïve brains (CPZ0 aged vs CPZ0 young). Ai) Volcano Plot displaying differentially expressed genes (DEGs) in aged vs young naïve (CPZ0) mice. Aii) Top regulated statistically significant GO terms for biological processes between aged and young naïve (CPZ0) mice. DotPlots showing z-score for normalized gene counts between young and aged naïve (CPZ0) mice for, Aiii) DAM and, Aiv) microglia activation statistically significant DEGs. Bi-iii) Comparison of brain RNA sequencing signatures between young and aged healthy naïve (CPZ0) and demyelination (CPZ4) brains. DotPlots showing z-score for normalized gene counts between young and aged mice for both naïve (CPZ0) and demyelination (CPZ4) timepoints, for Bi) myelin, Bii) DAM, Biii) microglia activation and Biv) cell senescence genes. C) Meta-analysis of datasets from our previously published single cell RNA-Seq of cortex from young mice at CPZ0 (naïve) and CPZ3 (demyelination onset) with published replicative senescence signatures. From the microglia signature at CPZ3 (1700 DEGs), 9 showed overlap with 64 genes reported by Hernandez-Segura et al., and Hu et al. , , and 12 of 119 in the SenMayo cellular senescence gene set reported by Saul et al., . Di-v) Comparison of RNA sequencing signatures between young and aged demyelination brains (CPZ4 aged vs CPZ4 young). Di) Volcano Plot displaying differentially expressed genes (DEGs) between young and aged demyelination (CPZ4) groups . Dii) Top regulated statistically significant GO terms for biological processes between aged and young CPZ4 mice. DotPlots showing z-score for normalized gene counts between young and aged CPZ4 mice for, Diii) DAM, Div) microglia activation and, Dv) phagocytosis statistically significant DEGs. Ei) Spatial distribution of PLP mRNA+ oligodendrocytes by RNAscope in coronal brain sections through corpus callosum (cc) and hippocampus (hi) from young and aged mice at CPZ0 (naïve) and CPZ4 (demyelination) (upper panels), and higher power insets (lower panels) Eii) Quantification of PLP mRNA+ oligodendrocytes/mm3 by RNAscope in the CC of young and aged mice in CPZ0, CPZ4. (n = 5-6 mice/group. ∗p-adjusted ≤ 0.05, ∗∗ p-adjusted ≤ 0.001, and ∗∗∗ p-adjusted ≤ 0.0001).

Article Snippet: RNAscope with the PLP mRNA probe was performed according to the Advanced Cell Diagnostics RNAscope Multiplex Fluorescent ReagentKit user manual.

Techniques: Comparison, RNA Sequencing, Activation Assay, Single Cell, RNAscope

Journal: bioRxiv

Article Title: FUS post-transcriptional splicing is autoregulated via RNA condensation with therapeutic potential for ALS-FUS

doi: 10.1101/2025.02.01.633781

Figure Lengend Snippet: a,b YTHDC1 depletion or m6A downregulation leads to a loss of FUSint6&7-RNA condensate integrity. Representative images (general plane) and quantification of condensates (a) as well as high-resolution images (b) are shown. 107 and 65 cells (4 or 5 FoV) were analysed for scrambled and YTHDC1 siRNA, respectively, from a representative experiment. **p<0.01, Mann-Whitney U test. HeLa cells were used in a and SH-SY5Y cells were used in b . Representative images are shown. STM2457-treated cells are also shown. In b , PNN was used as a speckle marker. Scale bar, 20 μm in a and 5 μm in b . c YTHDC1 depletion does not affect FUSint6&7-RNA level but downregulates FUS mRNA. qRT-PCR analysis with FUS intron 6- and 7-specific primers was performed in HeLa cells. N=5-7, *p<0.05, Mann-Whitney U test. d High-confidence DRACH motif in FUS intron 7 shown in a structural context, as predicted by SRAMP. e,f FUS introns 6 and 7 are extensively methylated. m6A-Atlas 2.0 database was used for mapping methylation sites (e) and calculating the m6A mark density (f). Also see Supplementary Table S3. g,h Pharmacological inhibition of a m6A writer METTL3 affects FUSint6&7-RNA condensate integrity without changing levels of this RNA. Representative images and quantification of the condensate number in STM2457-treated cells (g) and qRT-PCR analysis of RNA level (h) are shown. In g , cells were treated for 16 h, and >200 cells (5 FoV) were analysed per condition from a representative experiment, **p<0.01, Mann-Whitney U test. Scale bar, 20 μm. In h , qRT-PCR analysis was done using FUS intron 6- and -7 specific primers. N=4. i Pharmacological inhibition of a m6A eraser FTO promotes FUSint6&7-RNA condensate assembly. Representative images and quantification of the condensate number in FB23-2 treated cells are shown. Cells were treated with the compound for either 4 or 16 h. 79, 106 and 117 cells (5 FoV) were analysed for DMSO, 4-h FB23-2 and 16-h FB23-2 treatments, respectively, from a representative experiment. *p<0.05, Kruskal-Wallis with Dunn’s test. Scale bar, 20 μm. j FUS intron 6 RNAscope-ISH reveals partial cytoplasmic redistribution of FUSint6&7-RNA in STM2457-treated cells. Arrows indicate cytoplasmic accumulations of this RNA; nucleus is circled in the insets. Scale bar, 20 μm. k FUSΔNLS lines have preserved or enhanced ability to form FUSint6&7-RNA condensates. Representative images and quantification are shown. 234, 239, 234, 141 and 62 cells were analysed for WT, ΔNLS4, ΔNLS7, ΔNLS10 and ΔNLS11 lines, respectively (from 4-9 FoV). *p<0.05, **p<0.01, Kruskal-Wallis with Dunn’s test. l MeRIP demonstrates increased FUSint6&7-RNA methylation in FUSΔNLS lines. Total RNA purified from ΔNLS7 (homozygous) and ΔNLS10 (heterozygous) lines was subjected to pulldown using m6A antibody-coated beads and used for qRT-PCR analysis with FUSint6-specific primers. Methylated FUSint6&7-RNA level was normalised to GAPDH mRNA (extensively methylated transcript), then to total FUSint6&7-RNA level in the respective cell line, and finally, to no-antibody beads control. N=4, *p<0.05, Kruskal-Wallis with Dunn’s test. m Enhanced association of FUSint6&7-RNA with YTHDC1 in FUSΔNLS lines. RIP was performed using Flag-Trap agarose in YTHDC1-Flag expressing WT and FUSΔNLS cells, followed by qRT-PCR analysis with FUSint6-specific primers. Venus-Flag was used as a control (“vector”). FUSint6&7-RNA level was normalised to GAPDH and then to total FUSint6&7-RNA level in the respective cell line. Results for ΔNLS4, ΔNLS7 and ΔNLS10 lines were combined. N=2. *p<0.05, Mann-Whitney U test (WT vs . ΔNLS).

Article Snippet: For dot blot, 500 ng of total RNA was UV-crosslinked to Hybond-N+ membrane (Merck), which was subsequently probed with an m6A-specific antibody (mouse monoclonal, Proteintech, 68055-1-Ig) as described for western blot.

Techniques: MANN-WHITNEY, Marker, Quantitative RT-PCR, Methylation, Inhibition, RNAscope, Purification, Control, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: FUS post-transcriptional splicing is autoregulated via RNA condensation with therapeutic potential for ALS-FUS

doi: 10.1101/2025.02.01.633781

Figure Lengend Snippet: a Ectopically expressed GFP-tagged FUS is not enriched in the endogenous FUSint6&7-RNA condensates. FUS intron 6 Stellaris probe was used in HeLa cells. Representative image is shown. Note FUS accumulation in paraspeckles (arrows). Scale bar, 5 µm. b FUS overexpression does not affect the FUSint6&7-RNA condensate size. Representative images and quantification for HeLa cells are shown. NT, non-transfected (cells analysed in the same FoV). Condensate size was measured in 15 FoV per condition. n.s., non-significant. Scale bar, 10 µm. c m6A limits FUS condensation in vitro . Recombinant human his-tagged FUS and synthetic Cy5-labelled RNA oligonucleotides, fully methylated or unmodified, were used. Representative images of FUS condensates and quantification of average condensate size and fusion events are shown. 5-8 FoV were analysed per condition from a representative experiment. *p<0.05, ***p<0.001, Kruskal-Wallis with Dunn’s test. d FUS-RNA complex formation is diminished by methylation, as analysed by EMSA. Recombinant FUS protein and RNA oligonucleotides as in c were used. Recombinant YTHDF1 protein was used in parallel as a positive control. Complex formation in the indicated area (blue line) was quantified by densitometry (5.0 and 7.5 μM data-points combined). N=3, *p<0.05, Mann-Whitney U test. e Confocal nanoscanning assay (CONA) demonstrates reduced binding of FUS to methylated RNA, as compared to its unmodified counterpart. Cy5-labelled RNA oligonucleotides as above and lysates of cells expressing GFP-tagged FUS were used. Representative images of beads and quantification of GFP ring fluorescence intensity (normalised by Cy5 fluorescence – RNA coating) are shown. 80-100 beads were analysed per condition using an automated pipeline. ****p<0.0001, Kruskal-Wallis with Dunn’s test. f AlphaFold3 modelling confirms the repelling properties of m6A in FUS binding to RNA. FUS RRM is in blue and RNA is in orange (unmodified) or green (methylated). Interacting interfaces of FUS protein and the DRACH motif-containing RNA are boxed. Amino acid sequence interacting with the UUGG motif in the oligonucleotide is also indicated.

Article Snippet: For dot blot, 500 ng of total RNA was UV-crosslinked to Hybond-N+ membrane (Merck), which was subsequently probed with an m6A-specific antibody (mouse monoclonal, Proteintech, 68055-1-Ig) as described for western blot.

Techniques: Over Expression, Transfection, In Vitro, Recombinant, Methylation, Positive Control, MANN-WHITNEY, Binding Assay, Expressing, Fluorescence, Sequencing

Journal: Developmental cell

Article Title: Specialized endothelial tip cells guide neuroretina vascularization and blood-retina-barrier formation

doi: 10.1016/j.devcel.2021.06.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Total RNA was purified from mBECs using RNeasy Plus Mini Kit (Qiagen, #74134), and reverse transcribed to cDNA using iScript cDNA Synthesis Kit (Bio-Rad, #170–8891).

Techniques: Recombinant, Saline, Fluorescence, Plasmid Preparation, Modification, Western Blot, cDNA Synthesis, SYBR Green Assay, Imaging, RNAscope, Multiplex Assay, Gene Expression, Software, Laser-Scanning Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration

doi: 10.1073/pnas.1802519115

Figure Lengend Snippet: Photoreceptors are preserved in RPE-Abca4-Tg/Abca4 −/− vs. Abca4 −/− mice. ( A ) Representative retina images from 1-y-old albino mice acquired by light microscopy. (Scale bars, 20 μm.) ( B ) Total numbers of photoreceptor nuclei were counted per 100-μm 2 cell area. Note the increased number of cells in the ONL of RPE-Abca4-Tg/Abca4 −/− mice compared with Abca4 −/− mice indicating partial rescue of photoreceptor degeneration. Data are presented as mean ± SD; n = 5–9 mice per group; RPE-Abca4-Tg/Abca4 −/− vs. Abca4 −/− , * P = 0.0319; Abca4 −/− vs. BALB/c, ** P < 0.0001; and RPE-Abca4-Tg/Abca4 −/− vs. BALB/c, P = 0.0061.

Article Snippet: Retina sections from pigmented mice were bleached for 30 s using the Melanin Bleach Kit (Polysciences); bleaching was quenched with 50 mM ammonium chloride for 25 min, and sections were washed, and blocked with 1% BSA and 5% goat serum before probing with rabbit polyclonal anti-ABCA4 primary antibody (1:100; Abcam).

Techniques: Light Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration

doi: 10.1073/pnas.1802519115

Figure Lengend Snippet: ABCA4 colocalizes with endolysosomal markers. ( A ) Representative merged confocal images of retina/RPE sections from 2-mo-old wild-type BALB/c ( Upper ) and albino Abca4 −/− ( Lower ) mice reacted with antibodies to ABCA4 (red) and LAMP1 (green). Note that ABCA4 and LAMP1 colocalize in wild-type RPE but not in the OS. LAMP1, but not ABCA4, immunoreactivity is also present in the Abca4 −/− RPE cells. ( B ) Representative merged confocal images of retina sections from 5-mo-old wild-type (129/Sv) ( Top ), Abca4 −/− ( Middle ), and Mertk −/− ( Bottom ) mice immunostained with ABCA4 (red) and Rab5 (green) antibodies. Colocalization of ABCA4 and Rab5 is observed in both 129/Sv and Mertk −/− RPE cells as indicated by the orange signal. In the RPE of Abca4 −/− retina section only Rab5 immunoreactivity is seen. White arrows indicate retinal detachment, and the white asterisk indicates the absence of OS in the Mertk −/− retina due to photoreceptor degeneration. ( C ) Representative confocal images of fixed hfRPE cells labeled with ABCA4 (red) ( Top ) or endosomal CAV1 (green) ( Middle ) antibodies. ( Bottom ) Merged confocal images of ABCA4 and CAV1. Note the colocalization of ABCA4 and CAV1. The green labeling of the filter in the CAV1 panel is due to nitrocellulose autofluorescence. Nuclei are stained with DAPI (blue). For murine RPE sections, n = 3 mice per group. For hfRPE cells, each experiment was repeated three times with three different donor cell lines. (Magnification: C , 60×.) (Scale bars, 10 μm.)

Article Snippet: Retina sections from pigmented mice were bleached for 30 s using the Melanin Bleach Kit (Polysciences); bleaching was quenched with 50 mM ammonium chloride for 25 min, and sections were washed, and blocked with 1% BSA and 5% goat serum before probing with rabbit polyclonal anti-ABCA4 primary antibody (1:100; Abcam).

Techniques: Labeling, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration

doi: 10.1073/pnas.1802519115

Figure Lengend Snippet: Proposed function of ABCA4 in the endolysosomal membranes of RPE. ( A ) Normal RPE cell. 11cRAL released during proteolysis of rhodopsin within a phagolysosome condenses with PE on the luminal surface to form 11- cis - N -retinylidene-phosphatidylethanolamine (11c- N -ret-PE), which undergoes isomerization to form a mixture of all- trans (at) and 11c- N- ret-PEs. Both N- ret-PE isomers are flipped by ABCA4 to the cytoplasmic surface, where hydrolysis of N- ret-PE is driven by mass action through binding of 11cRAL by cellular retinaldehyde-binding protein (CRALBP) or the reduction of atRAL to atROL by retinol dehydrogenase type 11 (RDH11). The atROL is processed by the RPE visual cycle through esterification by lecithin retinol acyltransferase (LRAT) to yield an all- trans -retinyl ester such as all- trans -retinyl palmitate (atRP), isomerization by RPE65 to yield 11- cis -retinol (11cROL), and oxidation by retinol dehydrogenase type 5 (RDH5) to yield 11cRAL, which binds to CRALBP. 11cRAL leaves the RPE cell to regenerate visual pigments in the adjacent photoreceptor OS. ( B ) Abca4 −/− mutant RPE cell. The lack of ABCA4 in RPE endolysosomes of Abca4 −/− mice or STGD1 patients causes delayed clearance of retinaldehydes and hence higher concentrations of both free retinaldehydes and N- ret-PE. This leads to secondary condensation of atRAL or 11cRAL with N- ret-PE to form bisretinoids.

Article Snippet: Retina sections from pigmented mice were bleached for 30 s using the Melanin Bleach Kit (Polysciences); bleaching was quenched with 50 mM ammonium chloride for 25 min, and sections were washed, and blocked with 1% BSA and 5% goat serum before probing with rabbit polyclonal anti-ABCA4 primary antibody (1:100; Abcam).

Techniques: Binding Assay, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration

doi: 10.1073/pnas.1802519115

Figure Lengend Snippet: ( A – C ) Abca4 mRNA and protein is expressed in RPE cells. In situ hybridization using the RNAscope assay with an Abca4 -specific probe on human cadaveric ocular sections ( A ), mouse retina sections ( B ), and hfRPE cells in culture ( C ). Note the intense chromogenic reactivity (red punctate staining, indicated by the black arrows) for Abca4 mRNA in outer nuclear layer (ONL) and inner segments (IS) of the photoreceptor cells and in RPE cells of human ( A ) and wild-type BALB/c sections ( B , Left ). This reactivity is absent in Abca4 −/− tissue ( B , Right ). Red punctate staining (white arrows) corresponding to ABCA4 mRNA is also observed in hfRPE cultured cells ( C ). CC, choriocapillaris; INL, inner nuclear layer. (Scale bars, 20 μm.) ( D ) ABCA4 immunohistochemistry (red fluorescence) on retina sections from pigmented wild-type (129/Sv), Mertk −/− , and Abca4 −/− mice. Note that ABCA4 immunoreactivity is seen in the RPE and OS of 129/Sv mice and in the RPE but not in the OS (indicated by white asterisk) of Mertk −/− mice but is not seen in the retina section from an Abca4 −/− mouse. The white arrows indicate retinal detachment. Cell nuclei are stained with DAPI (blue). (Scale bars, 10 μm.) ( E ) Representative immunoblots for ABCA4 protein using neural retina and RPE/eyecup homogenates loaded as a fraction of one mouse eye per lane, as indicated. The RNAscope assay ( A – C ) was done with two human cadaveric eyes, three cultured hfRPE cells of different donor eyes, and n = 3 mice (5-mo-old) of each genotype; Immunohistochemistry experiments ( D ) were repeated three times with n = 3 5-mo-old mice per group. The immunoblotting experiment ( E ) was done in duplicates varying the fraction of the homogenate corresponding to one mouse eye ( n = 4 mice for each experiment).

Article Snippet: Retina sections from pigmented mice were bleached for 30 s using the Melanin Bleach Kit (Polysciences); bleaching was quenched with 50 mM ammonium chloride for 25 min, and sections were washed, and blocked with 1% BSA and 5% goat serum before probing with rabbit polyclonal anti-ABCA4 primary antibody (1:100; Abcam).

Techniques: In Situ Hybridization, RNAscope, Staining, Cell Culture, Immunohistochemistry, Fluorescence, Western Blot