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

2020 ◽  
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.

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

2021 ◽  
Author(s):  
Michael A Robichaux ◽  
Vy Nguyen ◽  
Fung Chan ◽  
Lavanya Kailasam ◽  
John H Wilson ◽  
...  
Keyword(s):  
Cell Death ◽  
Mouse Model ◽  
Retinitis Pigmentosa ◽  
Outer Nuclear Layer ◽  
Retinal Disease ◽  
Tunel Staining ◽  
Protein Accumulation ◽  
Mrna Levels ◽  
Rod Photoreceptor ◽  
Photoreceptor Neurons

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.

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

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.

scholarly journals Minocycline decreases CCR2-positive monocytes in the retina and ameliorates photoreceptor degeneration in a mouse model of retinitis pigmentosa

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0239108
Author(s):  
Ryo Terauchi ◽  
Hideo Kohno ◽  
Sumiko Watanabe ◽  
Saburo Saito ◽  
Akira Watanabe ◽  
...  
Keyword(s):  
Cell Death ◽  
Retinal Degeneration ◽  
Photoreceptor Cell ◽  
Fluorescent Protein ◽  
Outer Nuclear Layer ◽  
Inhibitory Effect ◽  
Subretinal Space ◽  
Photoreceptor Degeneration ◽  
Retinal Inflammation ◽  
Green Fluorescent

Retinal inflammation accelerates photoreceptor cell death caused by retinal degeneration. Minocycline, a semisynthetic broad-spectrum tetracycline antibiotic, has been previously reported to rescue photoreceptor cell death in retinal degeneration. We examined the effect of minocycline on retinal photoreceptor degeneration using c-mer proto-oncogene tyrosine kinase (Mertk)−/−Cx3cr1GFP/+Ccr2RFP/+ mice, which enabled the observation of CX3CR1-green fluorescent protein (GFP)- and CCR2-red fluorescent protein (RFP)-positive macrophages by fluorescence. Retinas of Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice showed photoreceptor degeneration and accumulation of GFP- and RFP-positive macrophages in the outer retina and subretinal space at 6 weeks of age. Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice were intraperitoneally administered minocycline. The number of CCR2-RFP positive cells significantly decreased after minocycline treatment. Furthermore, minocycline administration resulted in partial reversal of the thinning of the outer nuclear layer and decreased the number of apoptotic cells, as assessed by the TUNEL assay, in Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice. In conclusion, we found that minocycline ameliorated photoreceptor cell death in an inherited photoreceptor degeneration model due to Mertk gene deficiency and has an inhibitory effect on CCR2 positive macrophages, which is likely to be a neuroprotective mechanism of minocycline.

scholarly journals Knockout of ccr2 alleviates photoreceptor cell death in a model of retinitis pigmentosa

2012 ◽  
Vol 104 ◽  
pp. 39-47 ◽  
Author(s):  
Congrong Guo ◽  
Atsushi Otani ◽  
Akio Oishi ◽  
Hiroshi Kojima ◽  
Yukiko Makiyama ◽  
...  
Keyword(s):  
Cell Death ◽  
Retinitis Pigmentosa ◽  
Photoreceptor Cell

Apoptosis, retinitis pigmentosa, and degeneration

1994 ◽  
Vol 72 (11-12) ◽  
pp. 489-498 ◽  
Author(s):  
Paul Wong
Keyword(s):  
Cell Death ◽  
Retinitis Pigmentosa ◽  
Retinal Degeneration ◽  
Photoreceptor Cell ◽  
Retinal Cell ◽  
Current Evidence ◽  
Cell Degeneration ◽  
Genetic Lesion ◽  
Common Causes ◽  
Active Cell Death

The mechanism of photoreceptor cell death in different inherited retinal degenerations is not fully understood. Mutations in a number of different genes (such as rhodopsin, the beta subunit of cGMP phosphodiesterase, and peripherin) have been identified as the primary genetic lesion in different forms of human retinitis pigmentosa, one of the most common causes of inherited blindness. In all cases the manifestation of the disorder regardless of the specific primary genetic lesion is similar, resulting in photoreceptor cell degeneration and blindness. A recent hypothesis is that the active photoreceptor cell death, which is characteristic of these genetically distinct disorders, is mediated by a common induction of apoptosis. In the present review, the current evidence for active cell death during retinal cell death in several different rodent models of retinitis pigmentosa and retinal degeneration is examined.Key words: retinal degeneration, apoptosis, retinitis pigmentosa, clusterin, DNA fragmentation.

scholarly journals Dysfunction of Heterotrimeric Kinesin-2 in Rod Photoreceptor Cells and the Role of Opsin Mislocalization in Rapid Cell Death

2010 ◽  
Vol 21 (23) ◽  
pp. 4076-4088 ◽  
Author(s):  
Vanda S. Lopes ◽  
David Jimeno ◽  
Kornnika Khanobdee ◽  
Xiaodan Song ◽  
Bryan Chen ◽  
...  
Keyword(s):  
Cell Death ◽  
Outer Segment ◽  
Photoreceptor Cell ◽  
Photoreceptor Cells ◽  
Cell Loss ◽  
Rod Photoreceptor ◽  
Retinal Degenerations ◽  
Cell Counts ◽  
And Function

Due to extensive elaboration of the photoreceptor cilium to form the outer segment, axonemal transport (IFT) in photoreceptors is extraordinarily busy, and retinal degeneration is a component of many ciliopathies. Functional loss of heterotrimeric kinesin-2, a major anterograde IFT motor, causes mislocalized opsin, followed by rapid cell death. Here, we have analyzed the nature of protein mislocalization and the requirements for the death of kinesin-2-mutant rod photoreceptors. Quantitative immuno EM showed that opsin accumulates initially within the inner segment, and then in the plasma membrane. The light-activated movement of arrestin to the outer segment is also impaired, but this defect likely results secondarily from binding to mislocalized opsin. Unlike some other retinal degenerations, neither opsin–arrestin complexes nor photoactivation were necessary for cell loss. In contrast, reduced rod opsin expression provided enhanced rod and cone photoreceptor survival and function, as measured by photoreceptor cell counts, apoptosis assays, and ERG analysis. The cell death incurred by loss of kinesin-2 function was almost completely negated by Rho−/−. Our results indicate that mislocalization of opsin is a major cause of photoreceptor cell death from kinesin-2 dysfunction and demonstrate the importance of accumulating mislocalized protein per se, rather than specific signaling properties of opsin, stemming from photoactivation or arrestin binding.

scholarly journals Metabolite therapy guided by liquid biopsy proteomics delays retinal neurodegeneration

2019 ◽  
Author(s):  
Katherine J. Wert ◽  
Gabriel Velez ◽  
Kanchustambham Vijayalakshmi ◽  
Vishnu Shankar ◽  
Jesse D. Sengillo ◽  
...  
Keyword(s):  
Cell Death ◽  
Retinitis Pigmentosa ◽  
Ketogenic Diet ◽  
Proteomic Analysis ◽  
Photoreceptor Cell ◽  
Neuroprotective Effect ◽  
Tca Cycle ◽  
Ionization Mass ◽  
Molecular Pathways ◽  
Aconitic Acid

AbstractNeurodegenerative diseases are debilitating, incurable disorders caused by progressive neuronal cell death. Retinitis pigmentosa (RP) is a blinding neurodegenerative disease that results in retinal photoreceptor cell death and progresses to the loss of the entire neural retinal network. We previously found that proteomic analysis of the adjacent vitreous serves as way to indirectly biopsy the neural retina and identify changes in the retinal proteome. We therefore analyzed protein expression in liquid vitreous biopsies from autosomal recessive retinitis pigmentosa (arRP) patients with PDE6A mutations and arRP mice with Pde6ɑ mutations. Proteomic analysis of retina and vitreous samples identified molecular pathways affected at the onset of photoreceptor cell death. Based on affected molecular pathways, arRP mice were treated with a ketogenic diet or metabolites involved in fatty-acid synthesis, oxidative phosphorylation, and the tricarboxylic acid (TCA) cycle. Dietary supplementation of a single metabolite, ɑ-ketoglutarate, increased docosahexaeonic acid (DHA) levels, provided neuroprotection, and enhanced visual function in arRP mice. A ketogenic diet delayed photoreceptor cell loss, while vitamin B supplementation had a limited effect. Finally, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) revealed restoration of key metabolites that correlated with our proteomic findings: pyrimidine and purine metabolism (uridine, dihydrouridine, and thymidine), glutamine and glutamate (glutamine/glutamate conversion), and succinic and aconitic acid (TCA cycle). This study demonstrates that replenishing TCA cycle metabolites via oral supplementation prolongs vision and provides a neuroprotective effect on the photoreceptor cells and inner retinal network.One Sentence SummaryThe study shows protein and metabolite pathways affected during neurodegeneration and that replenishing metabolites provides a neuroprotective effect on the retina.

scholarly journals Excess Lipin enzyme activity contributes to TOR1A recessive disease and DYT-TOR1A dystonia

Brain ◽  
2020 ◽  
Vol 143 (6) ◽  
pp. 1746-1765 ◽  
Author(s):  
Ana Cascalho ◽  
Joyce Foroozandeh ◽  
Lise Hennebel ◽  
Jef Swerts ◽  
Christine Klein ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Phosphatidic Acid ◽  
Mouse Models ◽  
Disease Model ◽  
Induced Pluripotent Stem Cell ◽  
Motor Dysfunction ◽  
Neurological Dysfunction ◽  
Knock Out ◽  
Induced Pluripotent

Abstract TOR1A/TorsinA mutations cause two incurable diseases: a recessive congenital syndrome that can be lethal, and a dominantly-inherited childhood-onset dystonia (DYT-TOR1A). TorsinA has been linked to phosphatidic acid lipid metabolism in Drosophila melanogaster. Here we evaluate the role of phosphatidic acid phosphatase (PAP) enzymes in TOR1A diseases using induced pluripotent stem cell-derived neurons from patients, and mouse models of recessive Tor1a disease. We find that Lipin PAP enzyme activity is abnormally elevated in human DYT-TOR1A dystonia patient cells and in the brains of four different Tor1a mouse models. Its severity also correlated with the dosage of Tor1a/TOR1A mutation. We assessed the role of excess Lipin activity in the neurological dysfunction of Tor1a disease mouse models by interbreeding these with Lpin1 knock-out mice. Genetic reduction of Lpin1 improved the survival of recessive Tor1a disease-model mice, alongside suppressing neurodegeneration, motor dysfunction, and nuclear membrane pathology. These data establish that TOR1A disease mutations cause abnormal phosphatidic acid metabolism, and suggest that approaches that suppress Lipin PAP enzyme activity could be therapeutically useful for TOR1A diseases.

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