Mouse Model Of Retinitis Pigmentosa Research Articles

Early Alterations of RNA Binding Protein (RBP) Homeostasis and ER Stress-Mediated Autophagy Contributes to Progressive Retinal Degeneration in the rd10 Mouse Model of Retinitis Pigmentosa (RP).

The retinal degeneration 10 (rd10) mouse model is widely used to study retinitis pigmentosa (RP) pathomechanisms. It offers a rather unique opportunity to study trans-neuronal degeneration because the cell populations in question are separated anatomically and the mutated Pde6b gene is selectively expressed in rod photoreceptors. We hypothesized that RNA binding protein (RBP) aggregation and abnormal autophagy might serve as early pathogenic events, damaging non-photoreceptor retinal cell types that are not primarily targeted by the Pde6b gene defect. We used a combination of immunohistochemistry (DAB, immunofluorescence), electron microscopy (EM), subcellular fractionation, and Western blot analysis on the retinal preparations obtained from both rd10 and wild-type mice. We found early, robust increases in levels of the protective endoplasmic reticulum (ER) calcium (Ca2+) buffering chaperone Sigma receptor 1 (SigR1) together with other ER-Ca2+ buffering proteins in both photoreceptors and non-photoreceptor neuronal cells before any noticeable photoreceptor degeneration. In line with this, we found markedly altered expression of the autophagy proteins p62 and LC3, together with abnormal ER widening and large autophagic vacuoles as detected by EM. Interestingly, these changes were accompanied by early, prominent cytoplasmic and nuclear aggregation of the key RBPs including pTDP-43 and FET family RBPs and stress granule formation. We conclude that progressive neurodegeneration in the rd10 mouse retina is associated with early disturbances of proteostasis and autophagy, along with abnormal cytoplasmic RBP aggregation.

Protective effect of ZYMT, a traditional Chinese patent medicine in a mouse model of retinitis pigmentosa

Retinitis pigmentosa (RP) is the most common genetic disorder that causes blindness. At present, there exists no remedy for the disease. The aim of the current research was to investigate the protective effect of Zhangyanming Tablets (ZYMT) in a mouse model of RP, and explore the underlying mechanism. Eighty RP mice were randomly divided into two groups. The mice in ZYMT group were administered with ZYMT suspension(0.0378 g/mL), while the mice in model group were given the same volume of distilled water. At day 7 and day 14 after intervention, electroretinogram (ERG), fundus photography, and histological examination were used to assess the retinal function and structure. TUNEL, immunofluorescence and qPCR were used to evaluate cell apoptosis and expressions of Sirt1, Iba1, Bcl-2, Bax and Caspase-3. A significantly shortened latency of ERG waves was observed in ZYMT-treated mice, in comparison to those in the model group (P < 0.05). Histologically, ultrastructure of the retina was better preserved, and the outer nuclear layer (ONL) exhibited marked increase in thickness and cell count in ZYMP group (P < 0.05). The apoptosis rate was decreased markedly in ZYMT group. Immunofluorescence analysis showed that the expressions of Iba1 and Bcl-2 in the retina were increased, Bax and Caspase-3 were decreased after ZYMT intervention, while the qPCR revealed that the expressions of Iba1 and Sirt1 were significantly increased (P < 0.05). This study indicated that ZYMT has protective effect on retinal function and morphology of inherited RP mice in the early stage, possibly mediated via the regulation of antioxidant and anti-/pro-apoptotic factors expressions.

Vision rescue via unconstrained in vivo prime editing in degenerating neural retinas.

Retinitis pigmentosa (RP) is an inherited retinal dystrophy causing progressive and irreversible loss of retinal photoreceptors. Here, we developed a genome-editing tool characterized by the versatility of prime editors (PEs) and unconstrained PAM requirement of a SpCas9 variant (SpRY), referred to as PESpRY. The diseased retinas of Pde6b-associated RP mouse model were transduced via a dual AAV system packaging PESpRY for the in vivo genome editing through a non-NGG PAM (GTG). The progressing cell loss was reversed once the mutation was corrected, leading to substantial rescue of photoreceptors and production of functional PDE6β. The treated mice exhibited significant responses in electroretinogram and displayed good performance in both passive and active avoidance tests. Moreover, they presented an apparent improvement in visual stimuli-driven optomotor responses and efficiently completed visually guided water-maze tasks. Together, our study provides convincing evidence for the prevention of vision loss caused by RP-associated gene mutations via unconstrained in vivo prime editing in the degenerating retinas.

In vivo base editing rescues photoreceptors in a mouse model of retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of retinal diseases that cause the progressive death of retinal photoreceptor cells and eventually blindness. Mutations in the β-domain of the phosphodiesterase 6 (Pde6b) gene are the most identified causes of autosomal recessive RP. Clinically, there is no effective treatment so far that can stop the progression of RP and restore the vision. Here, we report a base editing approach in which adeno-associated virus (AAV)-mediated adenine base editor (ABE) delivering to postmitotic photoreceptors was conducted to correct the Pde6b mutation in a retinal degeneration 10 (rd10) mouse model of RP. Subretinal delivery of AAV8-ABE corrected Pde6b mutation with averaging up to 20.79% efficiency at the DNA level and 54.97% efficiency at the cDNA level without bystanders, restored PDE6B expression, preserved photoreceptors, and rescued visual function. RNA-seq revealed the preservation of genes associated with phototransduction and photoreceptor survival. Our data have demonstrated that base editing is a potential gene therapy that could provide durable protection against RP.

Phagocytosis in the retina promotes local insulin production in the eye.

The retina is highly metabolically active, relying on glucose uptake and aerobic glycolysis. Situated in close contact to photoreceptors, a key function of cells in the retinal pigment epithelium (RPE) is phagocytosis of damaged photoreceptor outer segments (POS). Here we identify RPE as a local source of insulin in the eye that is stimulated by POS phagocytosis. We show that Ins2 messenger RNA and insulin protein are produced by RPE cells and that this production correlates with RPE phagocytosis of POS. Genetic deletion of phagocytic receptors ('loss of function') reduces Ins2, whereas increasing the levels of the phagocytic receptor MerTK ('gain of function') increases Ins2 production in male mice. Contrary to pancreas-derived systemic insulin, RPE-derived local insulin is stimulated during starvation, which also increases RPE phagocytosis. Global or RPE-specific Ins2 gene deletion decreases retinal glucose uptake in starved male mice, dysregulates retinal physiology, causes defects in phototransduction and exacerbates photoreceptor loss in a mouse model of retinitis pigmentosa. Collectively, these data identify RPE cells as a phagocytosis-induced local source of insulin in the retina, with the potential to influence retinal physiology and disease.

TLR2 Is Highly Overexpressed in Retinal Myeloid Cells in the rd10 Mouse Model of Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a genetically heterogeneous disease and the predominant cause of hereditary blindness. Irrespective of the causative mutation, traits common to all forms of RP include photoreceptor dysfunction and death, activation of the retinal glial component, and retinal inflammation. Activation of Toll-like receptors (TLRs) in response to tissue damage is associated with inflammatory processes that contribute to neurodegeneration. We show that retinal expression of the genes Tlr1 to Tlr9 is increased in the rd10 mouse model of RP, with Tlr2 showing the greatest increase (36-fold). Flow cytometry analysis of the retinal myeloid population revealed significant increases in numbers of microglia and infiltrating monocytes and macrophages in rd10 retinas. Furthermore, TLR2 expression, which was restricted to myeloid cells, was increased in rd10 retinal microglia. These observations, together with our previous finding of delayed RP progression following Tlr2 deletion, point to TLR2 as a potential therapeutic target for RP.

Pyruvate Kinase 2, an Energy Metabolism Related Enzyme, May Have a Neuroprotective Function in Retinal Degeneration.

Retinitis pigmentosa (RP) is an inherited disorder that results in vision impairment but general and mutation-independent therapeutic strategies are not available. However, it is widely regarded that the cGMP system, including cGMP and its interactor cGMP-dependent protein kinase (PKG), acts as a crucial effector during retinal degeneration. We have previously identified a list of cGMP-PKG-dependent genes in the context of RP, and in this study, we further validated one of these, namely pyruvate kinase 2 (PKM2), and investigated the potential role of PKM2 for the photoreceptors' well-being during RP. With the aid of organotypic retinal explant cultures, we pharmacologically manipulated the PKM2 activities in two different RP mouse models (rd2 and rd10) via the addition of TEPP-46 (a PKM2 activator) and found that activation of PKM2 alleviates the progress of photoreceptor death in the rd10 mouse model. We also noted that the expression of both PKM2 and one of its targets, glucose transporter-1 (Glut1), showed alterations depending on the degeneration state. The observations provide supportive evidence that PKM2 may serve as a novel potential molecular target in RP.

Single-Cell Transcriptomic Profiling of Müller Glia in the rd10 Retina.

Müller glia are the principal macroglia of the retina and support retinal neurons both in health and disease. In retinitis pigmentosa (RP), a highly heterogeneous inherited retinal disorder, the most common form of pathology involves primary rod degeneration, followed by secondary cone death. To investigate Müller glia responses to rod degeneration, we performed droplet-based single-cell RNA sequencing in the rd10 mouse model of RP during primary rod degeneration. We confirmed known MG behavior on gliosis, metabolic, and immune functions. Pde6brd10 Müller glia also exhibited an increased expression of histocompatibility complex members, which might arise from a novel immune function of Müller glia in RP. We also describe a possible decrease in glial lipid biogenesis, which might affect degenerating photoreceptors.

Early alterations of RNA binding protein (RBP) homeostasis and ER stress-mediated autophagy trigger progressive retinal degeneration in the rd10 mouse model of retinitis pigmentosa (RP)

Early alterations of RNA binding protein (RBP) homeostasis and ER stress-mediated autophagy trigger progressive retinal degeneration in the rd10 mouse model of retinitis pigmentosa (RP)

Biodistribution of progesterone in the eye after topical ocular administration via drops or inserts

Progesterone (PG) has been shown to have a slowing effect on photoreceptor cell death in mouse models of retinitis pigmentosa when administered orally. The aim of this study was to investigate whether ophthalmically administered progesterone was able to reach neuroretina and thus, the distribution through ocular tissues of different PG formulations was studied. The effect of different initial PG concentration was also investigated. Different formulations with PG in their composition (drops, a corneal/scleral-insert and scleral-inserts) were prepared and assayed. Using whole porcine eyes, the different formulations were topically administered to the ocular surface. Frozen eyes were dissected, the PG in each tissue was extracted in acetonitrile and the amount of PG quantified by UHPLC-MS/MS. Our results show that after topical administration, PG diffuses from the ocular surface and distributes throughout all tissues of the eye. Lower levels of PG were found in sclera, choroid and neuroretina when PG was applied as drops compared to inserts. Our results also show that an increase in the initial PG concentrations applied, resulted in a statistically significant increase in the amounts of PG in aqueous humour, sclera, choroid and neuroretina.

Mesenchymal stem cell-derived extracellular vesicles protect retina in a mouse model of retinitis pigmentosa by anti-inflammation through miR-146a-Nr4a3 axis

BackgroundRetinitis pigmentosa is a rod-cone degenerative disease that induces irreversible vision loss. This study probed the protective capacity of mesenchymal stem cell-derived small EVs (MSC-EVs) on the retinas of rd10 mice and the underlying mechanism.MethodsMSC-EVs were injected into the vitreous of rd10 mice at postnatal day 14 and P21; morphology and function were examined at P28. The mechanism of action was explored by using co-culture of photoreceptor cell line 661 W and microglia cell line BV2.ResultsTreatment with MSC-EVs increased the survival of photoreceptors and preserved their structure. Visual function, as reflected by optomotor and electroretinogram responses, was significantly enhanced in MSC-EVs-treated rd10 mice. Mechanistically, staining for Iba1, GFAP, F4/80, CD68 and CD206 showed that MSC-EVs suppressed the activation of microglial, Müller glial and macrophages. Furthermore, western blotting showed that the treatment inhibited the NF-κB pathway. RNA-seq and qPCR showed that MSC-EVs upregulated anti-inflammatory cytokines while downregulating pro-inflammatory cytokines. MSC-EVs application in vitro decreased the number of TUNEL-positive 661 W cells co-cultured with LPS-stimulated BV2, with similar impact on the cytokine expression as in vivo study. Genetic screening predicted miR-146a to be the downstream target of MSC-EVs, which was detected in MSC-EVs and upregulated in co-cultured 661 W cells and BV2 cells after MSC-EVs treatment. Upregulation of miR-146a by using its mimic decreased the expression of the transcription factor Nr4a3, and its downregulation inhibition promoted Nr4a3 expression in both 661 W and BV2 cells. Nr4a3 was further identified as the target gene of miR-146a by dual-luciferase assay. Furthermore, overexpressing miR-146a significantly decreased the expression of LPS-induced pro-inflammatory cytokines in BV2 cells.ConclusionsMSC-EVs delays retinal degeneration in rd10 mice mainly by its anti-inflammatory effect via the miR-146a-Nr4a3axis. Hence, MSC-EVs may be used in the treatment of neurodegenerative diseases.

Targeting retinoic acid receptor alpha-corepressor interaction activates chaperone-mediated autophagy and protects against retinal degeneration

Chaperone-mediated autophagy activity, essential in the cellular defense against proteotoxicity, declines with age, and preventing this decline in experimental genetic models has proven beneficial. Here, we have identified the mechanism of action of selective chaperone-mediated autophagy activators previously developed by our group and have leveraged that information to generate orally bioavailable chaperone-mediated autophagy activators with favorable brain exposure. Chaperone-mediated autophagy activating molecules stabilize the interaction between retinoic acid receptor alpha - a known endogenous inhibitor of chaperone-mediated autophagy - and its co-repressor, nuclear receptor corepressor 1, resulting in changes of a discrete subset of the retinoic acid receptor alpha transcriptional program that leads to selective chaperone-mediated autophagy activation. Chaperone-mediated autophagy activators molecules activate this pathway in vivo and ameliorate retinal degeneration in a retinitis pigmentosa mouse model. Our findings reveal a mechanism for pharmacological targeting of chaperone-mediated autophagy activation and suggest a therapeutic strategy for retinal degeneration.

Possible Role of Insulin-Degrading Enzyme in the Physiopathology of Retinitis Pigmentosa.

Insulin-degrading enzyme (IDE) was named after its role as a proteolytic enzyme of insulin. However, recent findings suggest that IDE is a widely expressed, multitask protein, with both proteolytic and non-proteolytic functions. Here, we characterize the expression of IDE in the mammalian retina in both physiological and pathological conditions. We found that IDE was enriched in cone inner segments. IDE levels were downregulated in the dystrophic retina of several mouse models of retinitis pigmentosa carrying distinct mutations. In rd10 mice, a commonly studied mouse model of retinitis pigmentosa, treatment with an IDE activator (a synthetic peptide analog of preimplantation factor) delayed loss of visual function and preserved photoreceptor cells. Together, these results point to potential novel roles for IDE in retinal physiology and disease, further extending the list of diverse functions attributed to this enzyme.

Subcellular localization of mutant P23H rhodopsin in an RFP fusion knock-in mouse model of retinitis pigmentosa.

ABSTRACTThe P23H mutation in rhodopsin (Rho), the rod visual pigment, is the most common allele associated with autosomal-dominant retinitis pigmentosa (adRP). The fate of misfolded mutant Rho in rod photoreceptors has yet to be elucidated. We generated a new mouse model, in which the P23H-Rho mutant allele is fused to the fluorescent protein Tag-RFP-T (P23HhRhoRFP). In heterozygotes, outer segments formed, and wild-type (WT) rhodopsin was properly localized, but mutant P23H-Rho protein was mislocalized in the inner segments. Heterozygotes exhibited slowly progressing retinal degeneration. Mislocalized P23HhRhoRFP was contained in greatly expanded endoplasmic reticulum (ER) membranes. Quantification of mRNA for markers of ER stress and the unfolded protein response revealed little or no increases. mRNA levels for both the mutant human rhodopsin allele and the WT mouse rhodopsin were reduced, but protein levels revealed selective degradation of the mutant protein. These results suggest that the mutant rods undergo an adaptative process that prolongs survival despite unfolded protein accumulation in the ER. The P23H-Rho-RFP mouse may represent a useful tool for the future study of the pathology and treatment of P23H-Rho and adRP. This article has an associated First Person interview with the first author of the paper.

First person - Michael Robichaux.

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping early-career researchers promote themselves alongside their papers. Michael Robichaux is first author on ‘ Subcellular localization of mutant P23H rhodopsin in an RFP fusion knock-in mouse model of retinitis pigmentosa’, published in DMM. Michael conducted the research described in this article while a postdoctoral fellow in Ted Wensel's lab at Baylor College of Medicine, Houston, TX, USA. He is now an assistant professor at West Virginia University, Morgantown, WV, USA, investigating the intricate subcellular processes in photoreceptor neurons that enable vision.

Enhanced cGMP Interactor Rap Guanine Exchange Factor 4 (EPAC2) Expression and Activity in Degenerating Photoreceptors: A Neuroprotective Response?

The disease retinitis pigmentosa (RP) leads to photoreceptor degeneration by a yet undefined mechanism(s). In several RP mouse models (i.e., rd mice), a high cyclic GMP (cGMP) level within photoreceptors is detected, suggesting that cGMP plays a role in degeneration. The rap guanine exchange factor 4 (EPAC2) is activated by cyclic AMP (cAMP) and is an accepted cGMP-interacting protein. It is unclear whether and how cGMP interacts with EPAC2 in degenerating photoreceptors; we therefore investigated EPAC2 expression and interactions with cGMP and cAMP in retinas of the rd1 and rd10 models for retinal degeneration. EPAC2 expression in the photoreceptor layer increased significantly during rd1 and rd10 degeneration, and an increase in EPAC2 interactions with cGMP but not cAMP in the rd1 was also seen via a proximity ligation assay on histological sections. Retinal explant cultures revealed that pharmacological inhibition of the EPAC2 activity reduced the photoreceptor layer thickness in the rd10 retina, suggesting that EPAC2 inhibition promotes degeneration. Taken together, our results support the hypothesis that high degeneration-related cGMP leads to increased EPAC2 and cGMP interactions, inhibiting EPAC2. By inference, EPAC2 could have neuroprotective capacities that may be exploited in the future.

Structural abnormalities of retinal pigment epithelial cells in a light-inducible, rhodopsin mutant mouse.

Retinal pigment epithelium (RPE) is a specialized pigmented monolayer dedicated to retinal support and protection. Given the fact that photoreceptor outer segments are the primary energy resource of RPE metabolism, it follows that, when photoreceptor function is compromised, RPE cells are impaired and vice versa. In retinitis pigmentosa (RP), genetic mutations lead to a massive degeneration of photoreceptors but only few studies have addressed systematically the consequences of rod and cone death on RPE cells, which, among others, undergo an abnormal organization of tight junctions (TJs) and a compromised barrier function. The biological mechanisms driving these barrier reorganizations are largely unknown. Studies aimed at addressing general and mutation-independent changes of the RPE in RP are relevant to reveal new pathogenic mechanisms of this heterogeneous family of diseases and prospectively develop effective therapeutic strategies. Here, we take advantage of a mouse model of RP in which retinal degeneration is spatially restricted to investigate a possible involvement of inflammatory responses in RPE remodeling. By immunostaining for Zona Occludens-1 (ZO-1), a structural and functional marker of TJs with pleiotropic functions, we found a partial rescue of TJs organization following local restoration of retinal organization, revealing that TJs structure can recover. Since lack of ZO-1 from TJs can alter cell density, we counted RPE cells without finding any differences between degenerated and controls animals, indicating preservation of RPE cells. However, we found an increased number of immune cells adhering to the RPE apical surface and a spatial correlation with areas of abnormal ZO-1 distribution. This suggests that inflammatory processes following photoreceptor degeneration can be responsible for TJs alterations during RP progression and deserve further investigation.

Single-cell RNA sequencing of the retina in a model of retinitis pigmentosa reveals early responses to degeneration in rods and cones

BackgroundIn inherited retinal disorders such as retinitis pigmentosa (RP), rod photoreceptor-specific mutations cause primary rod degeneration that is followed by secondary cone death and loss of high-acuity vision. Mechanistic studies of retinal degeneration are challenging because of retinal heterogeneity. Moreover, the detection of early cone responses to rod death is especially difficult due to the paucity of cones in the retina. To resolve heterogeneity in the degenerating retina and investigate events in both types of photoreceptors during primary rod degeneration, we utilized droplet-based single-cell RNA sequencing in an RP mouse model, rd10.ResultsUsing trajectory analysis, we defined two consecutive phases of rod degeneration at P21, characterized by the early transient upregulation of Egr1 and the later induction of Cebpd. EGR1 was the transcription factor most significantly associated with the promoters of differentially regulated genes in Egr1-positive rods in silico. Silencing Egr1 affected the expression levels of two of these genes in vitro. Degenerating rods exhibited changes associated with metabolism, neuroprotection, and modifications to synapses and microtubules. Egr1 was also the most strongly upregulated transcript in cones. Its upregulation in cones accompanied potential early respiratory dysfunction and changes in signaling pathways. The expression pattern of EGR1 in the retina was dynamic during degeneration, with a transient increase of EGR1 immunoreactivity in both rods and cones during the early stages of their degenerative processes.ConclusionOur results identify early and late changes in degenerating rd10 rod photoreceptors and reveal early responses to rod degeneration in cones not expressing the disease-causing mutation, pointing to mechanisms relevant for secondary cone degeneration. In addition, our data implicate EGR1 as a potential key regulator of early degenerative events in rods and cones, providing a potential broad target for modulating photoreceptor degeneration.

Human primitive mesenchymal stem cell-derived retinal progenitor cells improved neuroprotection, neurogenesis, and vision in rd12 mouse model of retinitis pigmentosa

BackgroundCurrently, there is no treatment for retinal degenerative diseases (RDD) such as retinitis pigmentosa (RP). Stem cell-based therapies could provide promising opportunities to repair the damaged retina and restore vision. Thus far, primarily adult mesenchymal stem cells (MSCs) have been investigated in preclinical and clinical studies, and the results have not been convincing. We applied a new approach in which primitive (p) MSC-derived retinal progenitor cells (RPCs) were examined to treat retinal degeneration in an rd12 mouse model of RP.MethodsWell-characterized pMSCs and RPCs labeled with PKH26 were intravitreally injected into rd12 mice. The vision and retinal function of transplanted animals were analyzed using electroretinography. Animals were killed 4 and 8 weeks after cell transplantation for histological, immunological, molecular, and transcriptomic analyses of the retina.ResultsTransplanted RPCs significantly improved vision and retinal thickness as well as function in rd12 mice. pMSCs and RPCs homed to distinct retinal layers. pMSCs homed to the retinal pigment epithelium, and RPCs migrated to the neural layers of the retina, where they improved the thickness of the respective layers and expressed cell-specific markers. RPCs induced anti-inflammatory and neuroprotective responses as well as upregulated the expression of genes involved in neurogenesis. The transcriptomic analysis showed that RPCs promoted neurogenesis and functional recovery of the retina through inhibition of BMP and activation of JAK/STAT and MAPK signaling pathways.ConclusionsOur study demonstrated that RPCs countered inflammation, provided retinal protection, and promoted neurogenesis resulting in improved retinal structure and physiological function in rd12 mice.

Insulin receptor activation by proinsulin preserves synapses and vision in retinitis pigmentosa

Synaptic loss, neuronal death, and circuit remodeling are common features of central nervous system neurodegenerative disorders. Retinitis pigmentosa (RP), the leading cause of inherited blindness, is a group of retinal dystrophies characterized by photoreceptor dysfunction and death. The insulin receptor, a key controller of metabolism, also regulates neuronal survival and synaptic formation, maintenance, and activity. Indeed, deficient insulin receptor signaling has been implicated in several brain neurodegenerative pathologies. We present evidence linking impaired insulin receptor signaling with RP. We describe a selective decrease in the levels of the insulin receptor and its downstream effector phospho-S6 in retinal horizontal cell terminals in the rd10 mouse model of RP, as well as aberrant synapses between rod photoreceptors and the postsynaptic terminals of horizontal and bipolar cells. A gene therapy strategy to induce sustained proinsulin, the insulin precursor, production restored retinal insulin receptor signaling, by increasing S6 phosphorylation, without peripheral metabolic consequences. Moreover, proinsulin preserved photoreceptor synaptic connectivity and prolonged visual function in electroretinogram and optomotor tests. These findings point to a disease-modifying role of insulin receptor and support the therapeutic potential of proinsulin in retinitis pigmentosa.