Rd10 Mice Research Articles

Rhodopsin signaling mediates light-induced photoreceptor cell death in rd10 mice through a transducin-independent mechanism.

Retinitis pigmentosa (RP) is a debilitating blinding disease affecting over 1.5 million people worldwide, but the mechanisms underlying this disease are not well understood. One of the common models used to study RP is the retinal degeneration-10 (rd10) mouse, which has a mutation in Phosphodiesterase-6b (Pde6b) that causes a phenotype mimicking the human disease. In rd10 mice, photoreceptor cell death occurs with exposure to normal light conditions, but as demonstrated in this study, rearing these mice in dark preserves their retinal function. We found that inactivating rhodopsin signaling protected photoreceptors from degeneration suggesting that the pathway activated by this G-protein-coupled receptor is causing light-induced photoreceptor cell death in rd10 mice. However, inhibition of transducin signaling did not prevent the loss of photoreceptors in rd10 mice reared under normal light conditions implying that the degeneration caused by rhodopsin signaling is not mediated through its canonical G-protein transducin. Inexplicably, loss of transducin in rd10 mice also led to photoreceptor cell death in darkness. Furthermore, we found that the rd10 mutation in Pde6b led to a reduction in the assembled PDE6αβγ2 complex, which was corroborated by our data showing mislocalization of the γ subunit. Based on our findings and previous studies, we propose a model where light activates a non-canonical pathway mediated by rhodopsin but independent of transducin that sensitizes cyclic nucleotide gated channels to cGMP and causes photoreceptor cell death. These results generate exciting possibilities for treatment of RP patients without affecting their vision or the canonical phototransduction cascade.

Environmental light intensity determines the temporal progression of retinal degeneration in rd10 mice

AbstractThe rd10 mouse is a model of autosomal recessive retinitis pigmentosa (RP). This animal model carries a mutation in the Pde6b gen that generates a progressive loss of photoreceptors which leads to complete blindness. The aim of this study was to evaluate the effect of environmental light intensity on the degenerative process of the rd10 model. Three rd10 mice groups have been exposed from birth to P30 to different environmental light intensities: low (5 lux), medium (50 lux) and high (300 lux) intensity. Age‐matching C57BL/6J mice were used as controls. At the end of the experimental procedure, retinal function was assessed by electroretinography (ERG) and optomotor test. In addition, OCT images, hematoxylin staining, and immunohistochemistry were performed to analyze the structural integrity of the retinas of the three experimental groups. The rd10 mice housed at 5 lux intensity showed better functional response that the rd10 mice maintained at 50 or 300 lux, exhibiting significantly higher amplitudes in a‐ and b‐waves, as well as higher visual acuity. In the same line, the rd10 mice at 50 lux presented higher responses than the mice at 300 lux. Furthermore, the thickness of the ONL showed differences between the three rd10 groups. As the light intensity is lower, the thickness of the measured ONL is greater. These findings correlated with the morphology of the cones where better‐preserved photoreceptor morphology was found in the dystrophic animals housed at 5 lux. No significant differences were found between normal C57BL/6J animals kept at different light intensities. Environmental light intensity accelerates or delays the degeneration in rd10 mice. These data indicate that light intensity is a key factor to consider in studies carried out in retinal degenerative animal models, determining the temporal progression of the disease.MINECO‐FEDER BFU2015‐67139‐R, RETICS‐FEDER RD16/0008/0016, Prometeo 2016/158, ACIF/2016/055, FPU16/04114.

Activating an optogenetically engineered retina with a wearable headset in patients with retinitis pigmentosa

AbstractPurposeOptogenetics allows the expression of channelrhodopsins like ChrimsonR (ChR) in the retina of blind patients in order to confer light sensitivity to retinal ganglion cells (RGCs). However, ChR has a low sensitivity to light and is unlikely to be activated by ambient light. To activate ChR and to obtain a functional response from the RGCs, we developed and tested a wearable headset that delivers light stimulation to the human retina at an appropriate irradiance and wavelength.MethodsWe designed and constructed a wearable headset using electronics, optics, mechanics and software engineering. The proper range of light intensity required for the activation of ChR fused with tdTomato (ChRtdT) was tested on explanted retinas of non‐human primates (NHPs) transfected with AAV2.7m8‐ChRtdT. The ocular safety of exposure to 595 nm LED light following intravitreal injection of AAV2.7m8‐ChRdtT was assessed in rd1 mice, an established animal model for retinitis pigmentosa.ResultsActivation of ChRtdT in explanted retinas of NHPs transfected with AAV2.7m8‐ChRtdT showed that a minimum of 1015 photons/cm2/s (0.33 mW/cm2) of amber light was required to get a statistically significant electrophysiological response.In rd1 mice transfected with AAV2.7m8‐ChRtdT, fourteen days after exposure to 595‐nm LED light for 2 hours, no light‐related findings were identified in the retina at the highest intensity tested (nominal 1.7 × 1017 photons/cm2/s).As a result, our wearable headset provides a retinal irradiance up to 4 × 1016 photons/cm2/s for a period leading to a dose matching the one received by the retina of rd1 mouse exposed for 2 hours, a level shown to be safe for the retina.ConclusionOur medical device GS030‐MD was designed to send a light irradiance to the human retina both sufficient to activate ChRtdT and safe for the eye. GS030‐MD is currently being tested in combination with AAV2.7m8‐ChRtdT in the first‐in‐man PIONEER Phase I/II clinical trial.

NAD+/NADH precursor treatment protects against retinal degeneration

AbstractPurposeThe retina is highly metabolically active, suggesting that metabolic dysfunction could underlie many retinal degenerative diseases. Nicotinamide adenine dinucleotide (NAD+) is a coenzyme and a co‐substrate in several cellular energetic metabolic pathways. Retinal NAD+ levels decline with age and during retinal damage or degeneration. Maintaining NAD+ levels may be therapeutic in retinal disease. The purpose of this study was to investigate whether systemic treatment with nicotinamide riboside (NR), a NAD+ precursor, is protective in disparate models of retinal damage or degeneration.MethodsFour mouse models of retinal degeneration were tested: a mouse model of light‐induced retinal degeneration, the IRBP knock‐out mouse, rhodopsin‐Tvrm4 mutant mouse, and the rd10 mouse. Mice were IP injected with various concentrations of NR at various times relative to degeneration onset. Retinal function was assessed by ERG, retinal morphology was assessed by OCT and H & E or TUNEL staining, and visual function was assessed by OMR. Retinal NAD+/NADH levels were enzymatically assayed.ResultsEach retinal degeneration model exhibited significantly suppressed retinal and visual function, a severely disrupted photoreceptor cell layer, and significantly decreased numbers of nuclei and increased accumulation of TUNEL‐labeled cells in the outer nuclear layer. These effects were prevented by various NR treatment regimens. Treatment with NR also resulted in increased levels of NAD+ in retina.ConclusionsThis is the first study to report protective effects of NR treatment in models of retinal degeneration and suggests that maintaining retinal NAD+ via systemic NR treatment should be further explored for clinical relevance.

Rational Engineering and Preclinical Evaluation of Neddylation and SUMOylation Site Modified Adeno-Associated Virus Vectors in Murine Models of Hemophilia B and Leber Congenital Amaurosis.

Synthetic engineering of viral vectors such as adeno-associated virus (AAV) is crucial to overcome host transduction barriers observed during clinical gene therapy. We reasoned that exploring the role of cellular ubiquitin-like modifiers (UBLs) such as Neddylation or SUMOylation during AAV transduction could be beneficial. Using a combination of in silico biochemical and molecular engineering strategies, we have studied the impact of these UBLs during AAV2 infection and further developed Neddylation or SUMOylation site–modified AAV vectors and validated them in multiple disease models in vitro and in vivo. Hepatic gene transfer of two novel vectors developed, K105Q (SUMOylation-site mutant) and K665Q (Neddylation-site mutant), demonstrated a significantly improved human coagulation factor (F) IX expression (up to two-fold) in a murine model of hemophilia B. Furthermore, subretinal gene transfer of AAV2-K105Q vector expressing RPE65 gene demonstrated visual correction in a murine model of a retinal degenerative disease (rd12 mice). These vectors did not have any adverse immunogenic events in vivo. Taken together, we demonstrate that gene delivery vectors specifically engineered at UBLs can improve the therapeutic outcome during AAV-mediated ocular or hepatic gene therapy.

The Circular RNome of Developmental Retina in Mice

Circular RNAs (circRNAs) represent a class of noncoding RNAs with a wide expression pattern, and they constitute an important layer of the genome regulatory network. To date, the expression pattern and regulatory potency of circRNAs in the retina, a key part of the central nervous system, are not yet well understood. In this study, RNAs from five stages (E18.5, P1, P7, P14, and P30) of mouse retinal development were sequenced. A total of 9,029 circRNAs were identified. Most circRNAs were expressed in different stages with a specific signature, and their expression patterns were different from those of their host linear transcripts. Some circRNAs could act as sponges for several retinal microRNAs (miRNAs). Furthermore, circTulp4 could function as a competitive endogenous RNA (ceRNA) to regulate target genes. Remarkably, silencing circTulp4 in vivo led to mice having a thin outer nuclear layer (ONL) and defective retinal function. In addition, we found that circRNAs were dysregulated at a much earlier time point than that of disease onset in a retinal degeneration model (rd8 mice). In summary, we provide the first circRNA expression atlas during retinal development and highlight a key biological role for circRNAs in retinal development and degeneration.

Role of Translational Attenuation in Inherited Retinal Degeneration.

PurposeWe reported previously that retinas of mice with inherited retinal degeneration make less protein than retinas of normal mice. Despite recent studies suggesting that diminished protein synthesis rates may contribute to neurologic disorders, a direct link between protein synthesis rates and the progression of neurodegeneration has not been established. Moreover, it remains unclear whether reduced protein synthesis could be involved in retinal pathogenesis. Dysregulation of AKT/mTOR signaling has been reported in the retina during retinal degeneration, but to what extent this signaling contributes to translational attenuation in these mice remains uncertain.MethodsC57BL/6J and rd16 mice were subcutaneously injected with anisomycin to chronically inhibit protein synthesis rates. An AAV2 construct encoding constitutively active 4ebp1 was subretinally delivered in wildtype animals to lower protein synthesis rates. 4ebp1/2 were knocked out in rd16 mice.ResultsAnisomycin treatment lowered retinal translation rates, accelerated retinal degeneration in rd16 mice, and initiated cell death in the retinas of C57BL/6J mice. AAV-mediated transfer of constitutively active 4ebp1-4A into the subretinal space of wildtype animals inhibited protein synthesis, and led to reduced electroretinography amplitudes and fewer ONL nuclei. Finally, we report that restoring protein synthesis rates by knocking out 4ebp1/2 was associated with an approximately 2-fold increase in rhodopsin levels and a delay in retinal degeneration in rd16 mice.ConclusionsOur study indicates that protein synthesis inhibition is likely not a cell defense mechanism in the retina by which deteriorating photoreceptors survive, but may be harmful to degenerating retinas, and that restoring protein synthesis may have therapeutic potential in delaying the progression of retinal degeneration.

Investigations Into Bioenergetic Neuroprotection of Cone Photoreceptors: Relevance to Retinitis Pigmentosa.

Recent studies suggest cone degeneration in retinitis pigmentosa (RP) may result from intracellular energy depletion. We tested the hypothesis that cones die when depleted of energy by examining the effect of two bioenergetic, nutraceutical agents on cone survival. The study had three specific aims: firstly, we, studied the neuroprotective efficacies of glucose and creatine in an in vitro model of RP. Next, we utilized a well-characterized mouse model of RP to examine whether surviving cones, devoid of their inner segments, continue to express genes vital for glucose, and creatine utilization. Finally, we analyzed the neuroprotective properties of glucose and creatine on cone photoreceptors in a mouse model of RP. Two different bioenergy-based therapies were tested in rd1 mice: repeated local delivery of glucose and systemic creatine. Optomotor responses were tested and cone density was quantified on retinal wholemounts. The results showed that glucose supplementation increased survival of cones in culture subjected to mitochondrial stress or oxidative insult. Despite losing their inner segments, surviving cones in the rd1 retina continued to express the various glycolytic enzymes. Following a single subconjunctival injection, the mean vitreous glucose concentration was significantly elevated at 1 and 8 h, but not at 16 h after injection; however, daily subconjunctival injection of glucose neither enhanced spatial visual performance nor slowed cone cell degeneration in rd1 mice relative to isotonic saline. Creatine dose-dependently increased survival of cones in culture subjected to mitochondrial dysfunction, but not to oxidative stress. Despite the loss of their mitochondrial-rich inner segments, cone somas and axonal terminals in the rd1 retina were strongly positive for both the mitochondrial and cytosolic forms of creatine kinase at each time point examined. Creatine-fed rd1 mice displayed enhanced optomotor responses compared to mice fed normal chow. Moreover, cone density was significantly greater in creatine-treated mice compared to controls. The overall results of this study provide tentative support for the hypothesis that creatine supplementation may delay secondary degeneration of cones in individuals with RP.

Longitudinal OCT and OCTA monitoring reveals accelerated regression of hyaloid vessels in retinal degeneration 10 (rd10) mice

The hyaloid vascular system (HVS) is known to have an important role in eye development. However, physiological mechanisms of HVS regression and their correlation with developmental eye disorders remain unclear due to technical limitations of conventional ending point examination with fixed tissues. Here, we report comparative optical coherence tomography (OCT) and OCT angiography (OCTA) monitoring of HVS regression in wild-type and retinal degeneration 10 (rd10) mice. Longitudinal OCTA monitoring revealed accelerated regression of hyaloid vessels correlated with retinal degeneration in rd10. Quantitative OCT measurement disclosed significant distortions of both retinal thickness and the vitreous chamber in rd10 compared to WT mice. These OCT/OCTA observations confirmed the close relationship between HVS physiology and retinal neurovascular development. The distorted HVS regression might result from retinal hyperoxia or dopamine abnormality due to retinal remodeling in rd10 retina. By providing a noninvasive imaging platform for longitudinal monitoring of HVS regression, further OCT/OCTA study may lead to in-depth understanding of the physiological mechanisms of HVS regression in normal and diseased eyes, which is not only important for advanced study of the nature of the visual system but also may provide insights into the development of better treatment protocols of congenital eye disorders.

(Z)-7,4'-Dimethoxy-6-hydroxy-aurone-4-O-β-glucopyranoside mitigates retinal degeneration in Rd10 mouse model through inhibiting oxidative stress and inflammatory responses

Purpose: As an inherited retinal dystrophy characterised by progressive degeneration of photoreceptor cells, retinitis pigmentosa (RP) leads to partial or total blindness eventually. Possible causes of the photoreceptor cell death are oxidative stress and inflammatory responses. (Z)-7,4'-Dimethoxy-6-hydroxy-aurone-4-O-β-glucopyranoside (DHAG) is a novel compound with potent antioxidant properties. The aim of this study was to investigate whether DHAG could mitigate photoreceptor cell degeneration in an established mouse model of RP.Materials and method: Rd10 mice were treated with DHAG daily by gavage from postnatal day 12 (P12) to P33. Retinal morphology was evaluated by haematoxylin and eosin staining. Apoptosis-positive cells were detected by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. Oxidative stress markers and inflammatory factors were measured by enzyme-linked immunosorbent assay (ELISA). Real-time polymerase chain reaction, immunostaining and western blot were applied to measure the gene and protein change to explore the underlying mechanisms.Results: Results showed that DHAG significantly preserved the retinal morphology, reducing photoreceptor cell apoptosis, decreasing oxidative stress and inhibiting inflammatory responses in Rd10 mice. The mechanism might be related to inhibit the activation of P38 pathway.Conclusions: This study showed the beneficial effects of DHAG, a compound possessing antioxidant properties, and provided scientific rationale to develop DHAG as a potential agent to treat RP.

Activation of Sigma 1 Receptor Extends Survival of Cones and Improves Visual Acuity in a Murine Model of Retinitis Pigmentosa.

PurposeRetinitis pigmentosa (RP), a retinal photoreceptor degeneration, typically affects rod function and subsequently cones. Activation of sigma 1 receptor (Sig1R) has been shown to preserve cone function through 6 weeks in the rd10 mouse model of RP, when mice were treated systemically with the Sig1R ligand (+)-pentazocine (PTZ). This study determined the extent to which cone function is preserved in rd10 mice when Sig1R is activated.MethodsRd10 mice were administered (+)-PTZ (alternate days beginning at postnatal day [P]14) over a period of 180 days. Mouse visual function and structure were measured in vivo using optokinetic tracking response, scotopic and photopic electroretinography plus photopic assessment using “natural” noise stimuli, and optical coherence tomography (OCT). Immunofluorescent methods were used to detect cones in retinal cryosections.ResultsVisual acuity was maintained in rd10(+)-PTZ-treated mice through P56, whereas rd10 nontreated mice showed marked decline by P28. Cone responses were detected in (+)-PTZ-treated mice through P60, which were more robust when tested with natural noise stimuli; cone responses were minimal in nontreated rd10 mice. OCT revealed significantly thicker retinas in (+)-PTZ-treated rd10 mice through P60 compared to nontreated mice. Cones were detected by immunofluorescence in (+)-PTZ-treated rd10 retinas through P120.ConclusionsThe extent to which cone rescue could be sustained in (+)-PTZ-treated rd10 mice was evaluated comprehensively, showing that activation of Sig1R is associated with prolonged visual acuity, extended detection of cone function, and detection of cones in retinal histologic sections. The data reflect promising long-term neuroprotection when Sig1R is activated.

Cytoglobin deficiency potentiates Crb1-mediated retinal degeneration in rd8 mice

PurposeThe purpose of this study is to determine the effect of Cytoglobin (Cygb) deficiency on Crb1-related retinopathy. The Crb1 cell polarity complex is required for photoreceptor function and survival. Crb1-related retinopathies encompass a broad range of phenotypes which are not completely explained by the variability of Crb1 mutations. Genes thought to modify Crb1 function are therefore important targets of research. The biological function of Cygb involves oxygen delivery, scavenging of reactive oxygen species, and nitric oxide metabolism. However, the relationship of Cygb to diseases involving the Crb1 cell polarity complex is unknown. MethodsCygb knockout mice homozygous for the rd8 mutation (Cygb−/-rd8/rd8) were screened for ocular abnormalities and imaged using optical coherence tomography and fundus photography. Electroretinography was performed, as was histology and immunohistochemistry. Quantitative PCR was used to determine the effect of Cygb deficiency on transcription of Crb1 related cell polarity genes. ResultsCygb−/-rd8/rd8 mice develop an abnormal retina with severe lamination abnormalities. The retina undergoes progressive degeneration with the ventral retina more severely affected than the dorsal retina. Cygb expression is in neurons of the retinal ganglion cell layer and inner nuclear layer. Immunohistochemical studies suggest that cell death predominates in the photoreceptors. Electroretinography amplitudes show reduced a- and b-waves, consistent with photoreceptor disease. Cygb deficient retinas had only modest transcriptional perturbations of Crb1-related cell polarity genes. Cygb−/- mice without the rd8 mutation did not exhibit obvious retinal abnormalities. ConclusionsCygb is necessary for retinal lamination, maintenance of cell polarity, and photoreceptor survival in rd8 mice. These results are consistent with Cygb as a disease modifying gene in Crb1-related retinopathy. Further studies are necessary to investigate the role of Cygb in the human retina.

CRISPR-Cas9-mediated therapeutic editing of Rpe65 ameliorates the disease phenotypes in a mouse model of Leber congenital amaurosis.

Leber congenital amaurosis (LCA), one of the leading causes of childhood-onset blindness, is caused by autosomal recessive mutations in several genes including RPE65. In this study, we performed CRISPR-Cas9-mediated therapeutic correction of a disease-associated nonsense mutation in Rpe65 in rd12 mice, a model of human LCA. Subretinal injection of adeno-associated virus carrying CRISPR-Cas9 and donor DNA resulted in >1% homology-directed repair and ~1.6% deletion of the pathogenic stop codon in Rpe65 in retinal pigment epithelial tissues of rd12 mice. The a- and b-waves of electroretinograms were recovered to levels up to 21.2 ± 4.1% and 39.8 ± 3.2% of their wild-type mice counterparts upon bright stimuli after dark adaptation 7 months after injection. There was no definite evidence of histologic perturbation or tumorigenesis during 7 months of observation. Collectively, we present the first therapeutic correction of an Rpe65 nonsense mutation using CRISPR-Cas9, providing new insight for developing therapeutics for LCA.

Gene Editing Preserves Visual Functions in a Mouse Model of Retinal Degeneration.

Inherited retinal dystrophies (IRDs) are a large and heterogeneous group of degenerative diseases caused by mutations in various genes. Given the favorable anatomical and immunological characteristics of the eye, gene therapy holds great potential for their treatment. Our goal is to validate the preservation of visual functions by viral-free homology directed repair (HDR) in an autosomal recessive loss of function mutation. We used a tailored gene editing system based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to prevent retinal photoreceptor death in the retinal degeneration 10 (Rd10) mouse model of retinitis pigmentosa. We tested the gene editing tool in vitro and then used in vivo subretinal electroporation to deliver it to one of the retinas of mouse pups at different stages of photoreceptor differentiation. Three months after gene editing, the treated eye exhibited a higher visual acuity compared to the untreated eye. Moreover, we observed preservation of light-evoked responses both in explanted retinas and in the visual cortex of treated animals. Our study validates a CRISPR/Cas9-based therapy as a valuable new approach for the treatment of retinitis pigmentosa caused by autosomal recessive loss-of-function point mutations.

Spatio-temporal characterization of S- and M/L-cone degeneration in the Rd1 mouse model of retinitis pigmentosa

BackgroundThe Pde6brd1 (Rd1) mouse is widely used as a murine model for human retinitis pigmentosa. Understanding the spatio-temporal patterns of cone degeneration is important for evaluating potential treatments. In the present study we performed a systematic characterization of the spatio-temporal patterns of S- and M/L-opsin+ cone outer segment and cell body degeneration in Rd1 mice, described the distribution and proportion of dual cones in Rd1 retinas, and examined the kinetics of microglial activation during the period of cone degeneration.ResultsOuter segments of S- and M/L-cones degenerated far more rapidly than their somas. Loss of both S- and M/L-opsin+ outer segments was fundamentally complete by P21 in the central retina, and 90% complete by P45 in the peripheral retina. In comparison, degeneration of S- and M/L-opsin+ cell bodies proceeded at a slower rate. There was a marked hemispheric asymmetry in the rate of S-opsin+ and M/L-opsin+ cell body degeneration. M/L-opsin+ cones were more resilient to degeneration in the superior retina, whilst S-opsin+ cones were relatively preserved in the inferior retina. In addition, cone outer segment and cell body degeneration occurred far more rapidly in the central than the peripheral retina. At P14, the superior retina comprised a minority of genuine S-cones with a much greater complement of genuine M/L-opsin cones and dual cones, whilst the other three retinal quadrants had broadly similar numbers of genuine S-cones, genuine M/L-cones and dual cones. At P60, approximately 50% of surviving cones in the superior, nasal and temporal quadrants were dual cones. In contrast, the inferior peripheral retina at P60 contained almost exclusively genuine S-cones with a tiny minority of dual cones. Microglial number and activity were stimulated during rod breakdown, remained relatively high during cone outer segment degeneration and loss of cone somas in the central retina, and decreased thereafter in the period coincident with slow degeneration of cone cell bodies in the peripheral retina.ConclusionThe results of the present study provide valuable insights into cone degeneration in the Rd1 mouse, substantiating and extending conclusions drawn from earlier studies.

Improved Charge Pump Design and Ex Vivo Experimental Validation of CMOS 256-Pixel Photovoltaic-Powered Subretinal Prosthetic Chip.

An improved design of CMOS 256-pixel photovoltaic-powered implantable chip for subretinal prostheses is presented. In the proposed subretinal chip, a high-efficiency fully-integrated 4× charge pump is designed and integrated with on-chip photovoltaic (PV) cells and a 256-pixel array with active pixel sensors (APS) for image light sensing, biphasic constant current stimulators, and electrodes. Thus the PV voltage generated by infrared (IR) light can be boosted to above 1V so that the charge injection is increased. The proposed chip adopts the 32-phase divisional power supply scheme (DPSS) to reduce the required supply current and thus the required area of the PV cells. The proposed chip is designed and fabricated in 180-nm CMOS image sensor (CIS) technology and post-processed with biocompatible IrOx electrodes and silicone packaging. From the electrical measurement results, the measured stimulation frequency is 28.3 Hz under the equivalent electrode impedance load. The measured maximum output stimulation current is 7.1 μA and the amount of injected charges per pixel is 7.36 nC under image light intensity of 3200 lux and IR light intensity of 100 mW/cm2. The function of the proposed chip has been further validated successfully with the ex vivo experimental results by recording the electrophysiological responses of retinal ganglion cells (RGCs) of retinas from retinal degeneration (rd1) mice with a multi-electrode array (MEA). The measured average threshold injected charge is about 3.97 nC which is consistent with that obtained from the patch clamp recording on retinas from wild type (C57BL/6) mice with a single electrode pair.

Visual responses in the dorsal lateral geniculate nucleus at early stages of retinal degeneration in rd1 PDE6β mice.

Inherited retinal degenerations encompass a wide range of diseases that result in the death of rod and cone photoreceptors, eventually leading to irreversible blindness. Low vision survives at early stages of degeneration, at which point it could rely on residual populations of rod/cone photoreceptors as well as the inner retinal photoreceptor, melanopsin. To date, the impact of partial retinal degeneration on visual responses in the primary visual thalamus (dorsal lateral geniculate nucleus, dLGN) remains unknown, as does their relative reliance on surviving rod and cone photoreceptors vs. melanopsin. To answer these questions, we recorded visually evoked responses in the dLGN of anesthetized rd1 mice using in vivo electrophysiology at an age (3-5 wk) at which cones are partially degenerate and rods are absent. We found that excitatory (ON) responses to light had lower amplitude and longer latency in rd1 mice compared with age-matched visually intact controls; however, contrast sensitivity and spatial receptive field size were largely unaffected at this early stage of degeneration. Responses were retained when those wavelengths to which melanopsin is most sensitive were depleted, indicating that they were driven primarily by surviving cones. Inhibitory responses appeared absent in the rd1 thalamus, as did light-evoked gamma oscillations in firing. This description of fundamental features of the dLGN visual response at this intermediate stage of retinal degeneration provides a context for emerging attempts to restore vision by introducing ectopic photoreception to the degenerate retina.NEW & NOTEWORTHY This study provides new therapeutically relevant insights to visual responses in the dorsal lateral geniculate nucleus during progressive retinal degeneration. Using in vivo electrophysiology, we demonstrate that visual responses have lower amplitude and longer latency during degeneration, but contrast sensitivity and spatial receptive fields remain unaffected. Such visual responses are driven predominantly by surviving cones rather than melanopsin photoreceptors. The functional integrity of this visual pathway is encouraging for emerging attempts at visual restoration.

Drug repurposing studies of PARP inhibitors as a new therapy for inherited retinal degeneration.

The enzyme poly-ADP-ribose-polymerase (PARP) has important roles for many forms of DNA repair and it also participates in transcription, chromatin remodeling and cell death signaling. Currently, some PARP inhibitors are approved for cancer therapy, by means of canceling DNA repair processes and cell division. Drug repurposing is a new and attractive aspect of therapy development that could offer low-cost and accelerated establishment of new treatment options. Excessive PARP activity is also involved in neurodegenerative diseases including the currently untreatable and blinding retinitis pigmentosa group of inherited retinal photoreceptor degenerations. Hence, repurposing of known PARP inhibitors for patients with non-oncological diseases might provide a facilitated route for a novel retinitis pigmentosa therapy. Here, we demonstrate and compare the efficacy of two different PARP inhibitors, BMN-673 and 3-aminobenzamide, by using a well-established retinitis pigmentosa model, the rd1 mouse. Moreover, the mechanistic aspects of the PARP inhibitor-induced protection were also investigated in the present study. Our results showed that rd1 rod photoreceptor cell death was decreased by about 25-40% together with the application of these two PARP inhibitors. The wealth of human clinical data available for BMN-673 highlights a strong potential for a rapid clinical translation into novel retinitis pigmentosa treatments. Remarkably, we have found that the efficacy of 3 aminobenzamide was able to decrease PARylation at the nanomolar level. Our data also provide a link between PARP activity with the Wnt/β-catenin pathway and the major intracellular antioxidant concentrations behind the PARP-dependent retinal degeneration. In addition, molecular modeling studies were integrated with experimental studies for better understanding of the role of PARP1 inhibitors in retinal degeneration.

Sex-related differences in the progressive retinal degeneration of the rd10 mouse

The retinal degeneration 10 (rd10) mouse is a model of autosomal recessive retinitis pigmentosa (RP), a disease that causes blindness through the progressive loss of photoreceptors. This study shows evidence of sex-related differences in RP onset and progression in rd10 retinas. The disease onset was considerably earlier in the female rd10 mice than in the male rd10 mice, as evidenced by a loss of PDE6β proteins and rod-dominated electroretinogram (ERG) responses at an early age. Single photopic flash and flicker ERG responses and immunolabeling of opsin molecules were analyzed in both genders to assess the sex differences in the degeneration of cones in the RP retinas. The averaged amplitudes of cone-mediated ERG responses obtained from the females were significantly smaller than the amplitudes of the responses from the age-matched males in the late stages of the RP, suggesting that cones might degenerate faster in the female retinas as the disease progressed. The rapid degeneration of cones caused a more substantial decrease in the ERG responses derived from the On-pathway than the Off-pathway in the females. In addition, the male rd10 mice had heavier body weights than their female counterparts aged between postnatal (P)18 and P50 days. In summary, female rd10 mice were more susceptible to retinal degeneration, suggesting that the female sex might be a risk factor for RP. The results have important implications for future studies exploring potential sex-related differences in RP development and progression in the clinic.

Activation of the TRAAK two-pore domain potassium channels in rd1 mice protects photoreceptor cells from apoptosis.

To investigate the expression of TWIK-related arachidonic acid-stimulated K+ channel (TRAAK) in retinal degeneration mice (rd1) and further evaluate how TRAAK affect photoreceptor cell apoptosis. The rd1 mice were distributed into blank (no treatment), control (1.4% DMSO, intraperitoneal injection) and riluzole groups (4 mg/kg·d, intraperitoneal injection) from postnatal 7d to 10, 14 and 18d; C57 group (no treatment), as age-matched wild-type control. The thickness of the outer nuclear layer (ONL) of retina was detected by paraffin section hematoxylin and eosin staining. The expression of TRAAK and the apoptosis of the ONL cells were detected by immunostaining, Western blotting, and real-time polymerase chain reaction. The channel agonist riluzole activated TRAAK and delayed the apoptosis of photoreceptor cells in ONL layer of rd1 mice. Both at mRNA and protein levels, after riluzole treatment, TRAAK expression was significantly upregulated, when compared with the control and blank group. Then we detected a series of apoptosis related mRNA and protein. The anti-apoptotic factor Bcl-2 downregulated and the pro-apoptotic factors Bax and cleaved-caspase-3 upregulated significantly. Riluzole elevates the expression of TRAAK and inhibits the development of apoptosis. Activation of TRAAK may have some potential effects to put off photoreceptor apoptosis.