Metabolic Analysis of Vitreous/Lens and Retina in Wild Type and Retinal Degeneration Mice.
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ABSTRACT:
SUBMITTER: Murenu E
PROVIDER: S-EPMC7956175 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mutations in the retinitis pigmentosa 1 (RP1) gene are a common cause of autosomal dominant retinitis pigmentosa (adRP), and have also been found to cause autosomal recessive RP (arRP) in a few families. The 33 dominant mutations and 6 recessive RP1 mutations identified to date are all nonsense or frameshift mutations, and almost exclusively (38 out of 39) are located in the 4(th) and final exon of RP1. To better understand the underlying disease mechanisms of and help develop therapeutic strategies for RP1 disease, we performed a series of human genetic and animal studies using gene targeted and transgenic mice. Here we report that a frameshift mutation in the 3(rd) exon of RP1 (c.686delC; p.P229QfsX35) found in a patient with recessive RP1 disease causes RP in the homozygous state, whereas the heterozygous carriers are unaffected, confirming that haploinsufficiency is not the causative mechanism for RP1 disease. We then generated Rp1 knock-in mice with a nonsense Q662X mutation in exon 4, as well as Rp1 transgenic mice carrying a wild-type BAC Rp1 transgene. The Rp1-Q662X allele produces a truncated Rp1 protein, and homozygous Rp1-Q662X mice experience a progressive photoreceptor degeneration characterized disorganization of photoreceptor outer segments. This phenotype could be prevented by expression of a normal amount of Rp1 protein from the BAC transgene without removal of the mutant Rp1-Q662X protein. Over-expression of Rp1 protein in additional BAC Rp1 transgenic lines resulted in retinal degeneration. These findings suggest that the truncated Rp1-Q662X protein does not exert a toxic gain-of-function effect. These results also imply that in principle gene augmentation therapy could be beneficial for both recessive and dominant RP1 patients, but the levels of RP1 protein delivered for therapy will have to be carefully controlled.
| S-EPMC3424119 | biostudies-literature
Project description:Recent success in functional recovery by photoreceptor precursor transplantation in dysfunctional retina has led to an increased interest in using embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC)-derived retinal progenitors to treat retinal degeneration. However, cell-based therapies for end-stage degenerative retinas that have lost the outer nuclear layer (ONL) are still a big challenge. In the present study, by transplanting mouse iPSC-derived retinal tissue (miPSC retina) in the end-stage retinal-degeneration model (rd1), we visualized the direct contact between host bipolar cell terminals and the presynaptic terminal of graft photoreceptors by gene labeling, showed light-responsive behaviors in transplanted rd1 mice, and recorded responses from the host retina with transplants by ex vivo micro-electroretinography and ganglion cell recordings using a multiple-electrode array system. Our data provides a proof of concept for transplanting ESC/iPSC retinas to restore vision in end-stage retinal degeneration.
| S-EPMC5233464 | biostudies-literature
Project description:PurposeThe purpose of this study was to present our hypothesis that aging alters metabolic function in ocular tissues. We tested the hypothesis by measuring metabolism in aged murine tissues alongside retinal responses to light.MethodsScotopic and photopic electroretinogram (ERG) responses in young (3-6 months) and aged (23-26 months) C57Bl/6J mice were recorded. Metabolic flux in retina and eyecup explants was quantified using U-13C-glucose or U-13C-glutamine with gas chromatography-mass spectrometry (GC-MS), O2 consumption rate (OCR) in a perifusion apparatus, and quantifying adenosine triphosphatase (ATP) with a bioluminescence assay.ResultsScotopic and photopic ERG responses were reduced in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected in aged mice. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable.ConclusionsOur examination of metabolism showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The metabolic stability of these tissues ex vivo suggests age-related metabolic alterations may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or structural changes influence ocular metabolism in vivo.
| S-EPMC8606884 | biostudies-literature
Project description:PurposeDetermining the role of DYNC2H1 variants in nonsyndromic inherited retinal disease (IRD).MethodsGenome and exome sequencing were performed for five unrelated cases of IRD with no identified variant. In vitro assays were developed to validate the variants identified (fibroblast assay, induced pluripotent stem cell [iPSC] derived retinal organoids, and a dynein motility assay).ResultsFour novel DYNC2H1 variants (V1, g.103327020_103327021dup; V2, g.103055779A>T; V3, g.103112272C>G; V4, g.103070104A>C) and one previously reported variant (V5, g.103339363T>G) were identified. In proband 1 (V1/V2), V1 was predicted to introduce a premature termination codon (PTC), whereas V2 disrupted the exon 41 splice donor site causing incomplete skipping of exon 41. V1 and V2 impaired dynein-2 motility in vitro and perturbed IFT88 distribution within cilia. V3, homozygous in probands 2-4, is predicted to cause a PTC in a retina-predominant transcript. Analysis of retinal organoids showed that this new transcript expression increased with organoid differentiation. V4, a novel missense variant, was in trans with V5, previously associated with Jeune asphyxiating thoracic dystrophy (JATD).ConclusionThe DYNC2H1 variants discussed herein were either hypomorphic or affecting a retina-predominant transcript and caused nonsyndromic IRD. Dynein variants, specifically DYNC2H1 variants are reported as a cause of non syndromic IRD.
| S-EPMC7708302 | biostudies-literature
Project description:The neurosensory retina is an outgrowth of the Central Nervous System (CNS), and the eye is considered "a window to the brain." Reelin glycoprotein is directly involved in neurodevelopment, in synaptic plasticity, learning and memory. Consequently, abnormal Reelin signaling has been associated with brain neurodegeneration but its contributing role in ocular degeneration is still poorly explored. To this aim, experimental procedures were assayed on vitreous or retinas obtained from Reeler mice (knockout for Reelin protein) at different postnatal days (p) p14, p21 and p28. At p28, a significant increase in the expression of Amyloid Precursor Protein (APP) and its amyloidogenic peptide (Aβ1-42 along with truncated tau fragment (i.e., NH2htau)- three pathological hallmarks of Alzheimer's disease (AD)-were found in Reeler mice when compared to their age-matched wild-type controls. Likewise, several inflammatory mediators, such as Interleukins, or crucial biomarkers of oxidative stress were also found to be upregulated in Reeler mice by using different techniques such as ELLA assay, microchip array or real-time PCR. Taken together, these findings suggest that a dysfunctional Reelin signaling enables the expression of key pathological features which are classically associated with AD neurodegenerative processes. Thus, this work suggests that Reeler mouse might be a suitable animal model to study not only the pathophysiology of developmental processes but also several neurodegenerative diseases, such as AD and Age-related Macular Degeneration (AMD), characterized by accumulation of APP and/or Aβ1-42, NH2htau and inflammatory markers.
| S-EPMC9708893 | biostudies-literature
Project description:BackgroundCeroid lipofuscinosis type 8 belongs to a heterogenous group of vision and life-threatening neurodegenerative diseases, neuronal ceroid lipofuscinosis (NCL). Effective therapy is limited to a single drug for treatment of ceroid lipofuscinosis type 2, necessitating animal disease models to facilitate further therapeutic development. Murine models are advantageous for therapeutic development due to easy genetic manipulation and rapid breeding, however appropriate genetic models need to be identified and characterized before being used for therapy testing. To date, murine models of ocular disease associated with ceroid lipofuscinosis type 8 have only been characterized in motor neuron degeneration mice.MethodsCln8-/- mice were produced by CRISPR/Cas9 genome editing through the International Mouse Phenotyping Consortium. Ophthalmic examination, optical coherence tomography, electroretinography, and ocular histology was performed on Cln8-/- mice and controls at 16 weeks of age. Quantification of all retinal layers, retinal pigmented epithelium, and the choriocapillaris was performed using images acquired with ocular coherence tomography and planimetry of histologic sections. Necropsy was performed to investigate concurrent systemic abnormalities. Clinical correlation with human patients with CLN8-associated retinopathy is provided.ResultsRetinal degeneration characterized by retinal pigment epithelium mottling, scattered drusen, and retinal vascular attenuation was noted in all Cln8-/- mice. Loss of inner and outer photoreceptor segment demarcation was noted on optical coherence tomography, with significant thinning of the whole retina (P=1e-9), outer nuclear layer (P=1e-9), and combined photoreceptor segments (P=1e-9). A global reduction in scotopic and photopic electroretinographic waveforms was noted in all Cln8-/- mice. Slight thickening of the inner plexiform layer (P=0.02) and inner nuclear layer (P=0.004), with significant thinning of the whole retina (P=0.03), outer nuclear layer (P=0.01), and outer photoreceptor segments (P=0.001) was appreciated on histologic sections. Scattered lipid vacuoles were noted in splenic red pulp of all Cln8-/- mice, though no gross systemic abnormalities were detected on necropsy. Retinal findings are consistent with those seen in patients with ceroid lipofuscinosis type 8.ConclusionsThis study provides detailed clinical characterization of retinopathy in adult Cln8-/- mice. Findings suggest that Cln8-/- mice may provide a useful murine model for development of novel therapeutics needed for treating ocular disease in patients with ceroid lipofuscinosis type 8.
| S-EPMC8421982 | biostudies-literature
Project description:In normal mice, the lentiviral vector (LV) is very efficient to target the RPE cells, but transduces retinal neurons well only during development. In the present study, the tropism of LV has been investigated in the degenerating retina of mice, knowing that the retina structure changes during degeneration. We postulated that the viral transduction would be increased by the alteration of the outer limiting membrane (OLM). Two different LV pseudotypes were tested using the VSVG and the Mokola envelopes, as well as two animal models of retinal degeneration: light-damaged Balb-C and Rhodopsin knockout (Rho-/-) mice. After light damage, the OLM is altered and no significant increase of the number of transduced photoreceptors can be obtained with a LV-VSVG-Rhop-GFP vector. In the Rho-/- mice, an alteration of the OLM was also observed, but the possibility of transducing photoreceptors was decreased, probably by ongoing gliosis. The use of a ubiquitous promoter allows better photoreceptor transduction, suggesting that photoreceptor-specific promoter activity changes during late stages of photoreceptor degeneration. However, the number of targeted photoreceptors remains low. In contrast, LV pseudotyped with the Mokola envelope allows a wide dispersion of the vector into the retina (corresponding to the injection bleb) with preferential targeting of Müller cells, a situation which does not occur in the wild-type retina. Mokola-pseudotyped lentiviral vectors may serve to engineer these glial cells to deliver secreted therapeutic factors to a diseased area of the retina.
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Project description:MicroRNA expression in the mouse eye.MicroRNAs (miRNAs) are key regulators of biological processes. To define miRNA function in the eye, it is essential to determine a high-resolution profile of their spatial and temporal distribution. In this report, we present the first comprehensive survey of miRNA expression in ocular tissues, using both microarray and RNA in situ hybridization (ISH) procedures. We initially determined the expression profiles of miRNAs in the retina, lens, cornea and retinal pigment epithelium of the adult mouse eye by microarray. Each tissue exhibited notably distinct miRNA enrichment patterns and cluster analysis identified groups of miRNAs that showed predominant expression in specific ocular tissues or combinations of them. Next, we performed RNA ISH for over 220 miRNAs, including those showing the highest expression levels by microarray, and generated a high-resolution expression atlas of miRNAs in the developing and adult wild-type mouse eye, which is accessible in the form of a publicly available web database. We found that 122 miRNAs displayed restricted expression domains in the eye at different developmental stages, with the majority of them expressed in one or more cell layers of the neural retina . This analysis revealed miRNAs with differential expression in ocular tissues and provided a detailed atlas of their tissue-specific distribution during development of the murine eye. The combination of the two approaches offers a valuable resource to decipher the contributions of specific miRNAs and miRNA clusters to the development of distinct ocular structures. microRNA profiling of ocular tissues from mouse. In particular we analysed the cornea, lens, Retina Pigment Epithelium (RPE) and retina and compared them against RNA extracted from the entire eye. The purpose of this experiment was to understand which microRNAs are present nd/or show differential expression in the various structures of the eye (cornea, lens, RPE, retina). The samples numbered 1 & 2 (i.e. CORNEA1, CORNEA2 etc ) are biological replicates, prepared from tissues dissecyed from different groups of wild-type animals. RNA extracted from the entire eye (EYE) served as the unique reference sample. For each tissue to be analysed we performed the following hybridizations: - 2 slides for lens (LENS1, LENS2) vs entire eye (EYE) - 2 slides for RPE (RPE1, RPE2) vs entire eye (EYE) - 2 slides for retina (RETINA1, RETINA2) vs entire eye (EYE) - 2 slides for cornea (CORNEA1, CORNEA2) vs entire eye (EYE) - 1 slide for entire eye (EYE) vs entire eye (EYE)
2010-11-22 | E-GEOD-22882 | biostudies-arrayexpress
Project description:We have created a Myrf knockdown (Myrf +/−) mutant mouse strain and investigated the molecular phenotype of reduced MYRF expression in the retina using RNA-sequencing. As an initial finding, photoreceptor defects with impaired functions of spatial vision and retinal electrophysiology were found in Myrf +/− mice, indicating an important role of MYRF in retinal development.It was revealed through RNA-seq that the expression of phototransduction and estrogen signaling pathways reduced in Myrf +/− mice. Thus, alterations of phototransduction and estrogen signaling pathways seem to play important roles in connecting Myrf deficiency with retinal photoreceptor defects.
2020-09-12 | GSE157806 | GEO
Project description:Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world. The retinal pigment epithelium (RPE) is a critical site of pathology in AMD and αB crystallin expression is increased in RPE and associated drusen in AMD. The purpose of this study was to investigate the role of αB crystallin in sodium iodate (NaIO3)-induced retinal degeneration, a model of AMD in which the primary site of pathology is the RPE. Dose dependent effects of intravenous NaIO3 (20-70 mg/kg) on development of retinal degeneration (fundus photography) and RPE and retinal neuronal loss (histology) were determined in wild type and αB crystallin knockout mice. Absence of αB crystallin augmented retinal degeneration in low dose (20 mg/kg) NaIO3-treated mice and increased retinal cell apoptosis which was mainly localized to the RPE layer. Generation of reactive oxygen species (ROS) was observed with NaIO3 in mouse and human RPE which increased further after αB crystallin knockout or siRNA knockdown, respectively. NaIO3 upregulated AKT phosphorylation and peroxisome proliferator-activator receptor-γ (PPARγ) which was suppressed after αB crystallin siRNA knockdown. Further, PPARγ ligand inhibited NaIO3-induced ROS generation. Our data suggest that αB crystallin plays a critical role in protection of NaIO3-induced oxidative stress and retinal degeneration in part through upregulation of AKT phosphorylation and PPARγ expression.
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