Subcellular localization of mutant P23H rhodopsin in an RFP fusion knockin mouse model of retinitis pigmentosa

The P23H mutation in rhodopsin (Rho), the visual pigment protein in rod photoreceptor neurons, is the most common genetic cause of autosomal dominant retinitis pigmentosa (adRP), a retinal disease that causes blindness. Despite multiple studies in animal models, the subcellular details of the fate of misfolded mutant Rho in rod photoreceptors have not been completely defined. We generated a new mouse model of adRP, in which the P23H-Rho mutant allele is fused to Tag-RFP-T (P23HhRhoRFP). In heterozygotes, outer segments formed, and WT rhodopsin was properly localized there, but mutant P23H-Rho protein was specifically mislocalized in the inner segments of rods. Despite this cellular phenotype, the P23HhRhoRFP heterozygous mice exhibited only slowly progressing retinal degeneration; in ERG recordings, scotopic a-wave amplitudes were reduced by 24% and 26% at 30 days and 90 days respectively, and the corresponding scotopic b-waves by 18% and 24%. Outer nuclear layer thickness was still 80% of WT at 90 days, but at 364 days had declined to 40% of WT. Transmission electron microscopy revealed greatly expanded membrane lamellae in the inner segment, and by fluorescence imaging, we determined that the mislocalized P23HhRhoRFP was contained in greatly expanded endoplasmic reticulum (ER) membranes. TUNEL staining revealed a slow pace of cell death involving chromosomal endonucleolytic degradation. Quantification of mRNA for markers of ER stress and the unfolded protein response revealed little or no increases in levels of messages encoding the proteins BiP, CHOP, ATF6, XBP1, PERK, Eif2α and Derlin-1, but a decreased level of total Rhodopsin (mouse + human) mRNA levels. The decline in the rate of cell death after an initial burst suggests that P23HhRhoRFP mutant rods undergo an adaptative process that prolongs survival despite gross P23HhRhoRFP protein accumulation in the ER. Because of its slowly progressing nature, and easy visualization of the mutant protein, the P23H-Rho-RFP mouse may represent a useful tool for the future study of the pathology and treatment of P23H-Rho and adRP.

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Modelling and rescue of RP2 Retinitis Pigmentosa using iPSC Derived Retinal Organoids

10.1101/2020.01.28.923227 ◽

2020 ◽

Cited By ~ 1

Author(s):

Amelia Lane ◽

Katarina Jovanovic ◽

Ciara Shortall ◽

Daniele Ottaviani ◽

Anna Brugulat Panes ◽

...

Keyword(s):

Cell Death ◽

Retinitis Pigmentosa ◽

Photoreceptor Cell ◽

Outer Nuclear Layer ◽

Disease Model ◽

Retinal Disease ◽

Severe Form ◽

Rod Photoreceptor ◽

Knock Out ◽

Induced Pluripotent

SummaryRP2 mutations cause a severe form of X-linked retinitis pigmentosa (XLRP). The mechanism of RP2 associated retinal degeneration in humans is unclear, and animal models of RP2 XLRP do not recapitulate this severe phenotype. Here, we developed gene edited isogenic RP2 knock-out (RP2 KO) induced pluripotent stem cells (iPSC) and RP2 patient derived iPSC to produce 3D retinal organoids as a human retinal disease model. Strikingly, the RP2 KO and RP2 patient derived organoids showed a peak in rod photoreceptor cell death at day 150 (D150) with subsequent thinning of the organoid outer nuclear layer (ONL) by D180 of culture. AAV mediated gene augmentation with human RP2 rescued the degeneration phenotype of the RP2 KO organoids, to prevent ONL thinning and restore rhodopsin expression. Notably, these data show that 3D retinal organoids can be used to model photoreceptor degeneration and test potential therapies to prevent photoreceptor cell death.

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Pathogenic STX3 variants affecting the retinal and intestinal transcripts cause an early-onset severe retinal dystrophy in microvillus inclusion disease subjects

Human Genetics ◽

10.1007/s00439-021-02284-1 ◽

2021 ◽

Author(s):

Andreas R. Janecke ◽

Xiaoqin Liu ◽

Rüdiger Adam ◽

Sumanth Punuru ◽

Arne Viestenz ◽

...

Keyword(s):

Early Onset ◽

Single Nucleotide Polymorphism Array ◽

Outer Nuclear Layer ◽

Retinal Dystrophy ◽

Geographic Origin ◽

Retinal Disease ◽

Homozygosity Mapping ◽

Rod Photoreceptor ◽

Loss Of Function ◽

Knockout Mouse Model

AbstractBiallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic—intestinal and retinal—disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.

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New In Vitro Cellular Model for Molecular Studies of Retinitis Pigmentosa

International Journal of Molecular Sciences ◽

10.3390/ijms22126440 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6440

Author(s):

Li Huang ◽

Meltem Kutluer ◽

Elisa Adani ◽

Antonella Comitato ◽

Valeria Marigo

Keyword(s):

Cell Death ◽

Retinitis Pigmentosa ◽

Leucine Zipper ◽

Target Genes ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Rod Photoreceptor ◽

Photoreceptor Degeneration ◽

Molecular Studies

Retinitis pigmentosa (RP) is an inherited form of retinal degeneration characterized by primary rod photoreceptor cell death followed by cone loss. Mutations in several genes linked to the disease cause increased levels of cyclic guanosine monophosphate (cGMP) and calcium ion influxes. The purpose of this project was to develop a new in vitro photoreceptor degeneration model for molecular studies of RP. 661W cells were genetically modified to stably express the neural retina leucine zipper (NRL) transcription factor. One clone (661W-A11) was selected based on the expression of Nrl target genes. 661W-A11 showed a significant increase in expression of rod-specific genes but not of cone-specific genes, compared with 661W cells. Zaprinast was used to inhibit phosphodiesterase 6 (PDE6) activity to mimic photoreceptor degeneration in vitro. The activation of cell death pathways resulting from PDE6 inhibition was confirmed by detection of decreased viability and increased intracellular cGMP and calcium, as well as activation of protein kinase G (PKG) and calpains. In this new in vitro system, we validated the effects of previously published neuroprotective drugs. The 661W-A11 cells may serve as a new model for molecular studies of RP and for high-throughput drug screening.

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Blocking the Necroptosis Pathway Decreases RPE and Photoreceptor Damage Induced by NaIO3

10.1101/387068 ◽

2018 ◽

Cited By ~ 1

Author(s):

Haijiang Lin ◽

Miin Roh ◽

Hidetaka Matsumoto ◽

Alp Atik ◽

Peggy Bouzika ◽

...

Keyword(s):

Cell Death ◽

Photoreceptor Cell ◽

Cell Damage ◽

Outer Nuclear Layer ◽

Cell Loss ◽

Tunel Staining ◽

Programmed Necrosis ◽

Cell Death Pathway ◽

Whole Mount

AbstractPurposeSodium iodate (NaIO3) has been extensively used as a retinotoxin to induce RPE cell damage and degeneration of photoreceptorsin vitroandin vivo. RIP-Kinase dependent programmed necrosis is an important redundant cell death pathway involved in photoreceptor cell death. We wanted to determine whether these pathways are actively involved in RPE and photoreceptor cell death after NaIO3 insult.MethodsARPE-19 cells were exposed to different concentrations of NaIO3 in the presence or absence of various concentrations of a RIPK inhibitor (Nec-1) or a pan-caspase inhibitor (Z-VAD), individually or combined. Cell death was determined at different time points by MTT (Sigma-Aldrich), LDH (Promega) and TUNEL (Millipore) assay. C57BL/6 and RIP3−/-mice were treated with a peritoneal injection of NaIO3 and eyes were enucleated at day 3 or 7. TUNEL staining was used to evaluate photoreceptor cell death. Photoreceptor cell loss was evaluated by measuring the thickness of outer nuclear layer (ONL). Microglia in the ONL were quantified in a retinal whole mount with Iba-1 antibody. RPE degeneration was also assessed in a RPE whole mount, with ZO-1 antibody.ResultsNaIO3 resulted in significant cell death of ARPE-19 cells. Treatment with Nec-1 resulted in better protection than treatment with Z-VAD (P<0.01). A synergistic protective effect was observed when co-treating the cells with Nec-1 and Z-VAD. Nec-1 treatment also decreased the ARPE-19 mitochondrial damage caused by NaIO3.In vivoadministration of NaIO3 resulted in significant RPE and photoreceptor destruction with substantial inflammatory cell infiltration. RIP3 knockout animals displayed considerably less RPE and photoreceptor cell loss, as well as drastically less inflammation.ConclusionsProgrammed necrosis is an important cell death pathway mediating NaIO3 RPE and photoreceptor cell toxicity. Blocking the necroptosis pathway may serve as a novel therapeutic strategy for various RPE degenerative diseases.

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Inhibition of MicroRNA 6937 Delays Photoreceptor and Vision Loss in a Mouse Model of Retinitis Pigmentosa

Pharmaceutics ◽

10.3390/pharmaceutics12100913 ◽

2020 ◽

Vol 12 (10) ◽

pp. 913

Author(s):

Ander Anasagasti ◽

Araceli Lara-López ◽

Santiago Milla-Navarro ◽

Leire Escudero-Arrarás ◽

María Rodríguez-Hidalgo ◽

...

Keyword(s):

Mouse Model ◽

Retinitis Pigmentosa ◽

Molecular Mechanisms ◽

Vision Loss ◽

Outer Nuclear Layer ◽

Photoreceptor Cells ◽

Inherited Retinal Dystrophies ◽

Gene Expression Control ◽

Inhibit Gene Expression ◽

Post Transcriptional Regulation

Inherited retinal dystrophies (IRDs) are a group of rare retinal conditions, including retinitis pigmentosa (RP), caused by monogenic mutations in 1 out of more than 250 genes. Despite recent advancements in gene therapy, there is still a lack of an effective treatment for this group of retinal conditions. MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNAs that inhibit gene expression. Control of miRNAs-mediated protein expression has been described as a widely used mechanism for post-transcriptional regulation in many physiological and pathological processes in different organs, including the retina. Our main purpose was to test the hypothesis that modulation of a group of miRNAs can protect photoreceptor cells from death in the rd10 mouse model of retinitis pigmentosa. For this, we incorporated modulators of three miRNAs in adeno-associated viruses (AAVs), which were administered through sub-retinal injections. The results obtained indicate that inhibition of the miR-6937-5p slows down the visual deterioration of rd10 mice, reflected by an increased electroretinogram (ERG) wave response under scotopic conditions and significant preservation of the outer nuclear layer thickness. This work contributes to broadening our knowledge on the molecular mechanisms underlying retinitis pigmentosa and supports the development of novel therapeutic approaches for RP based on miRNA modulation.

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The Role of Caspase-12 in Retinal Bystander Cell Death and Innate Immune Responses against MCMV Retinitis

International Journal of Molecular Sciences ◽

10.3390/ijms22158135 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8135

Author(s):

Xinyan Zhang ◽

Jinxian Xu ◽

Brendan Marshall ◽

Zheng Dong ◽

Sylvia B. Smith ◽

...

Keyword(s):

Cell Death ◽

Caspase 3 ◽

P53 Protein ◽

Plaque Assay ◽

Tunel Staining ◽

Mrna Levels ◽

Bystander Cell ◽

Caspase 12 ◽

Infected Cells ◽

Bystander Cell Death

(1) Background: caspase-12 is activated during cytomegalovirus retinitis, although its role is presently unclear. (2) Methods: caspase-12−/− (KO) or caspase-12+/+ (WT) mice were immunosup eyes were analyzed by plaque assay, TUNEL assay, immunohistochemical staining, western blotting, and real-time PCR. (3) Results: increased retinitis and a more extensive virus spread were detected in the retina of infected eyes of KO mice compared to WT mice at day 14 p.i. Compared to MCMV injected WT eyes, mRNA levels of interferons α, β and γ were significantly reduced in the neural retina of MCMV-infected KO eyes at day 14 p.i. Although similar numbers of MCMV infected cells, similar virus titers and similar numbers of TUNEL-staining cells were detected in injected eyes of both KO and WT mice at days 7 and 10 p.i., significantly lower amounts of cleaved caspase-3 and p53 protein were detected in infected eyes of KO mice at both time points. (4) Conclusions: caspase-12 contributes to caspase-3-dependent and independent retinal bystander cell death during MCMV retinitis and may also play an important role in innate immunity against virus infection of the retina.

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Zebrafish as a Model to Evaluate a CRISPR/Cas9-Based Exon Excision Approach as a Future Treatment Option for EYS-Associated Retinitis Pigmentosa

International Journal of Molecular Sciences ◽

10.3390/ijms22179154 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9154

Author(s):

Renske Schellens ◽

Erik de Vrieze ◽

Pam Graave ◽

Sanne Broekman ◽

Kerstin Nagel-Wolfrum ◽

...

Keyword(s):

Retinitis Pigmentosa ◽

Single Molecule ◽

Therapeutic Potential ◽

Outer Nuclear Layer ◽

Exon Skipping ◽

Retinal Disease ◽

Loss Of Function ◽

Zebrafish Model ◽

Reading Frame ◽

Future Treatment

Retinitis pigmentosa (RP) is an inherited retinal disease (IRD) with an overall prevalence of 1 in 4000 individuals. Mutations in EYS (Eyes shut homolog) are among the most frequent causes of non-syndromic autosomal recessively inherited RP and act via a loss-of-function mechanism. In light of the recent successes for other IRDs, we investigated the therapeutic potential of exon skipping for EYS-associated RP. CRISPR/Cas9 was employed to generate zebrafish from which the region encompassing the orthologous exons 37-41 of human EYS (eys exons 40-44) was excised from the genome. The excision of these exons was predicted to maintain the open reading frame and to result in the removal of exactly one Laminin G and two EGF domains. Although the eysΔexon40-44 transcript was found at levels comparable to wild-type eys, and no unwanted off-target modifications were identified within the eys coding sequence after single-molecule sequencing, EysΔexon40-44 protein expression could not be detected. Visual motor response experiments revealed that eysΔexon40-44 larvae were visually impaired and histological analysis revealed a progressive degeneration of the retinal outer nuclear layer in these zebrafish. Altogether, the data obtained in our zebrafish model currently provide no indications for the skipping of EYS exons 37-41 as an effective future treatment strategy for EYS-associated RP.

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Retinitis Pigmentosa Is Associated With Shifts in The Gut Microbiome

10.21203/rs.3.rs-107256/v1 ◽

2020 ◽

Author(s):

Oksana Kutsyr ◽

Lucía Maestre-Carballa ◽

Mónica Lluesma-Gomez ◽

Manuel Martinez-Garcia ◽

Nicolás Cuenca ◽

...

Keyword(s):

Retinitis Pigmentosa ◽

Gut Microbiome ◽

Outer Nuclear Layer ◽

Functional Decline ◽

Retinal Disease ◽

Rrna Gene ◽

Sequencing Data ◽

Cell Reactivity ◽

Alpha And Beta Diversity ◽

Potential Biomarker

Abstract Background: The gut microbiome is known to influence the pathogenesis and progression of neurodegenerative diseases. However, there has been relatively little focus upon the implications of the gut microbiome in retinal diseases such as retinitis pigmentosa (RP) that leads to photoreceptor degeneration and is the main worldwide cause of complete blindness in adulthood. Here, we investigated changes in gut microbiome composition linked to RP, by assessing both retinal degeneration and gut microbiome in the rd10 mouse model of RP as compared to control C57BL/6J mice.Results: In rd10 mice, retinal responsiveness to flashlight stimuli was deteriorated with respect to observed in age-matched control mice, with decreased amplitudes in the a- and b-wave responses of the electroretinogram and concomitant reduction of the visual acuity. This functional decline in dystrophic animals was accompanied by photoreceptor loss, evidenced by decreased outer nuclear layer thickness, and morphologic anomalies in photoreceptor cells. Changes in retinal neurons were paralleled to induction of reactive gliosis in retina, with increased microglial cell numbers and higher Müller cell reactivity in rd10 mice. Furthermore, 16S rRNA gene amplicon sequencing data showed a microbial gut dysbiosis with differences in alpha and beta diversity at the genera, species and amplicon sequence variants (ASV) levels between dystrophic and control mice. A taxonomic partitioning of ASV was observed since unique ASVs present in only one group had a cumulative relative microbial abundance between 17.6% (rd10 mice) and 26.7% (C57BL/6J mice). Remarkably, four fairly common ASV in healthy gut microbiome belonging to -Rikenella spp., Muribaculaceace spp., Prevotellaceae UCG-001 spp., and Bacilli spp.- were absent in the gut microbiome of retinal disease mice, while Bacteroides caecimuris was significantly enriched in mice with retinitis pigmentosa. Conclusions: Our results indicate that retinal degenerative changes in retinitis pigmentosa are linked to relevant gut microbiome changes. The findings suggest that microbiome shifting could be considered as potential biomarker and therapeutic target for retinal degenerative diseases.

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Lipopolysaccharide downregulates macrophage-derived IL-22 to modulate alcohol-induced hepatocyte cell death

AJP Cell Physiology ◽

10.1152/ajpcell.00005.2017 ◽

2017 ◽

Vol 313 (3) ◽

pp. C305-C313 ◽

Cited By ~ 10

Author(s):

Yaming Liu ◽

Vikas K. Verma ◽

Harmeet Malhi ◽

Greg J. Gores ◽

Patrick S. Kamath ◽

...

Keyword(s):

Cell Death ◽

Heavy Drinking ◽

Conditioned Media ◽

Tunel Staining ◽

Mrna Levels ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Interleukin 22 ◽

Cytokine Analysis ◽

Lps Activation

Interleukin-22 (IL-22) is a Th17 cell hepatoprotective cytokine that is undergoing clinical trials to treat patients with alcoholic hepatitis (AH). Lipopolysaccharide (LPS) activation of macrophage is implicated in hepatocyte cell death and pathogenesis of AH. The role of IL-22 production from macrophage, its regulation by LPS, and effects on alcohol-induced hepatocyte cell death are unexplored and were examined in this study. Low levels of IL-22 mRNA/protein were detected in macrophage but were significantly upregulated by 6.5-fold in response to the tissue reparative cytokine IL-10. Conversely, LPS significantly decreased IL-22 mRNA levels in a temporal and concentration-dependent manner with a maximum reduction of 5-fold. LPS downregulation of IL-22 mRNA levels was rescued in the presence of a pharmacological inhibitor of c-Jun NH2-terminal kinase (JNK) and by JNK knockdown. Next, we explored whether macrophage-derived IL-22 regulated ethanol-induced hepatocyte death. Conditioned media from IL-10-stimulated macrophages attenuated ethanol-induced hepatocyte caspase-3/7 activity, and apoptosis as assessed by fluorometric assay and TdT-mediated dUTP nick-end labeling (TUNEL) staining, respectively. This effect was diminished in conditioned media from macrophages with IL-22 knockdown. Cytokine analysis in sera samples of patients with AH revealed that IL-22 levels were significantly elevated compared with healthy controls and heavy-drinking controls, implying a state of IL-22 resistance in human AH. Macrophage-derived IL-22 protects hepatocytes from ethanol-induced cell death. IL-22 downregulation is a new regulatory target of LPS in the pathogenesis of AH.

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