Rd10 Mice Research Articles

Caloric restriction induces macrophage reprogramming and improved epithelial ovarian cancer survival

Abstract Calorie restriction (CR) without malnutrition is a dietary intervention where energy intake is reduced by 15 – 40%. The benefits of CR in improving various diseases and extending lifespan has led its investigation in cancer. We have reported 30% CR to limit epithelial ovarian cancer (EOC) progression. Here, we highlight the ability of CR to induce immuno-metabolic reprogramming of tumor associated macrophages and improve EOC survival. Female C57/B6 mice were injected intraperitoneally with 8 × 10 6mouse EOC ID8 (p53 +/+, p53 −/−, or ID8 p53 −/−,BRCA1−/−) cells and fed ad libitum (RD) or subjected to 30% CR. Immune profiling was done by flow cytometry. Metabolic phenotype was determined by XF seahorse analyzer. Macrophage marker were measured by qPCR and western blot. Mice on 30% CR exhibited reduced tumor progression and increased overall survival compared to RD mice in all ID8 models as indicated by median survival; ID8 p53+/+(65 vs 95 days), ID8 p53−/−(50.5 vs 76.5 days), and ID8 p53−/−,BRCA1−/−(44 vs 53 days). Immune profiling showed increase in antitumor associated pro-inflammatory macrophages (F4/80+CD38+) and decrease in protumor anti-inflammatory (F4/80+CD206+) macrophages, increasing the pro / anti-inflammatory ratio. Pro-inflammatory macrophages showed increased intracellular iNOS. The phenotype change in CR mice was accompanied with reprogrammed energy metabolism indicated by increased glycolysis, reflective of pro-inflammatory macrophages. Decrease in anti-inflammatory macrophages resulted in robust anti-tumor T cell response. A 30% CR reprograms macrophage metabolism and promotes their pro-inflammatory anti-tumor phenotype, restores antitumor immunity resulting in reduced EOC growth and improved survival R01 B11209 to RR Henry Ford Cancer institute Post Doctorate Fellowship to HS

Ocular permeability, intraocular biodistribution of lipid nanocapsule formulation intended for retinal drug delivery

Recently, cGMP analogues have been investigated for the treatment of inherited retinal degenerations (IRD) using intravitreal injections. However, higher vitreous elimination rates limit the possibility to treat the retina with small molecule drugs. Here, we investigated the potential of lipid nanocapsules (LNCs) as vehicles to reduce clearance and prolong the delivery of cGMP analogue, CN03 to the retinal photoreceptors. Initially LNCs were investigated for both topical/periocular and intravitreal administration routes. While LNC-mediated drug permeation through the cornea proved to be too low for clinical applications, intravitreal application showed significant promise. Intravitreally administered LNCs containing fluorescent tracer in ex vivo porcine eyes showed complete intravitreal dispersal within 24 h. Ocular bio-distribution on histological sections showed that around 10 % of the LNCs had reached the retina, and 40 % accumulated in the ciliary body. For comparison, we used fluorescently labeled liposomes and these showed a different intraocular distribution with 48 % accumulated in the retina, and almost none were in the ciliary body. LNCs were then tested in retinal explants prepared from wild-type (WT) and rd1 mouse. In WT retina LNCs showed no significant toxic effects up to a concentration of 5 mg/mL. In rd1 retina, the LNC/CN03 formulation protected rd1 photoreceptors with similar efficacy to that of free CN03, demonstrating the usefulness of LNC/CN03 formulation in the treatment of IRD. Overall, our results indicate the suitability of LNCs for intraocular administration and drug delivery to both the retina and the ciliary body.

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.

The photoreceptor protective cGMP-analog Rp-8-Br-PET-cGMPS interacts with cGMP-interactors PKGI, PDE1, PDE6, and PKAI in the degenerating mouse retina.

The inherited eye disease retinitis pigmentosa (RP) causes the loss of photoreceptors by a still unknown cell death mechanism. During this degeneration, cyclic guanosine-3',5'-monophosphate (cGMP) levels become elevated, leading to over-activation of the cGMP-binding protein cGMP-dependent protein kinase (PKG). cGMP analogs selectively modified to have inhibitory actions on PKG have aided in impeding photoreceptor death, and one such cGMP analog is Rp-8-Br-PET-cGMPS. However, cGMP analogs have previously been shown to interact with numerous targets, so to better understand the therapeutic action of Rp-8-Br-PET-cGMPS, it is necessary to elucidate its target-selectivity and hence what potential cellular mechanism(s) it may affect within the photoreceptors. Here, we, therefore, applied affinity chromatography together with mass spectrometry to isolate and identify Rp-8-Br-PET-cGMPS interactors from retinas derived from three different murine RP models (i.e., rd1, rd2, and rd10 mice). Our findings revealed that Rp-8-Br-PET-cGMPS bound seven known cGMP-binding proteins, including PKG1β, PDE1β, PDE1c, PDE6α, and PKA1α. Furthermore, an additional 28 proteins were found to be associated with Rp-8-Br-PET-cGMPS. This latter group included MAPK1/3, which is known to connect with cGMP/PKG in other systems. However, in organotypic retinal cultures, Rp-8-Br-PET-cGMPS had no effect on photoreceptor MAPK1/3 expression or activity. To summarize, Rp-8-Br-PET-cGMPS is more target specific compared to regular cGMP.

CRISPR-mediated optogene expression from a cell-specific endogenous promoter in retinal ON-bipolar cells to restore vision

Retinitis pigmentosa, an inherited form of retinal degeneration, is characterized by a progressive loss of rods and subsequent degeneration of cones, leading to blindness. However, the remaining neural portion of the retina (bipolar and ganglion cells) remains anatomically and functionally intact for an extended time. A possible treatment to restore the light sensitivity of the retina consists of rendering the remaining retinal cells photosensitive using optogenetic tools like, for example, Opto-mGluR6, a light-sensitive mGluR6 receptor. We have previously demonstrated that AAV vector-mediated expression of Opto-mGluR6 in ON-bipolar cells restores visual function in otherwise blind mice. However, classical gene supplementation therapy still suffers from high off-target expression rates and uncontrollable target gene expression levels that may lead to either cytotoxicity or lack of functional restoration. To address these issues and achieve cell-specific and endogenously controlled Opto-mGluR6 expression, we employed the CRISPR/Cas technology—in particular, homology-independent targeted integration (HITI) and microhomology-dependent targeted integration (MITI)—to knock-in the Opto-mGluR6 gene behind the ON-bipolar cell-specific GRM6 promoter. We compared four Cas systems in vitro and show that SpCas9 for HITI and LbCpf1 for MITI are well suited to promoting knock-in. As AAV2-mediated ON-bipolar cell transduction resulted in inefficiency, we evaluated Exo-AAVs as delivery vehicles and found Exo-AAV1 efficient for targeting ON-bipolar cells. We demonstrate that intravitreal injection of Exo-AAV1 carrying vectors that promote MITI significantly improved visual acuity in otherwise blind rd1 mice. We conclude by confirming and providing a qualitative evaluation of the MITI-mediated knock-in in the correct genomic locus.

Inherited Retinal Degeneration: Towards the Development of a Combination Therapy Targeting Histone Deacetylase, Poly (ADP-Ribose) Polymerase, and Calpain

Inherited retinal degeneration (IRD) represents a diverse group of gene mutation-induced blinding diseases. In IRD, the loss of photoreceptors is often connected to excessive activation of histone-deacetylase (HDAC), poly-ADP-ribose-polymerase (PARP), and calpain-type proteases (calpain). Moreover, the inhibition of either HDACs, PARPs, or calpains has previously shown promise in preventing photoreceptor cell death, although the relationship between these enzyme groups remains unclear. To explore this further, organotypic retinal explant cultures derived from wild-type mice and rd1 mice as a model for IRD were treated with different combinations of inhibitors specific for HDAC, PARP, and calpain. The outcomes were assessed using in situ activity assays for HDAC, PARP, and calpain, immunostaining for activated calpain-2, and the TUNEL assay for cell death detection. We confirmed that inhibition of either HDAC, PARP, or calpain reduced rd1 mouse photoreceptor degeneration, with the HDAC inhibitor Vorinostat (SAHA) being most effective. Calpain activity was reduced by inhibition of both HDAC and PARP whereas PARP activity was only reduced by HDAC inhibition. Unexpectedly, combined treatment with either PARP and calpain inhibitors or HDAC and calpain inhibitors did not produce synergistic rescue of photoreceptors. Together, these results indicate that in rd1 photoreceptors, HDAC, PARP, and calpain are part of the same degenerative pathway and are activated in a sequence that begins with HDAC and ends with calpain.

Loss of Müller cell glutamine synthetase immunoreactivity is associated with neuronal changes in late-stage retinal degeneration.

A hallmark of photoreceptor degenerations is progressive, aberrant remodeling of the surviving retinal neurons and glia following photoreceptor loss. The exact relationship between neurons and glia remodeling in this late stage of retinal degeneration, however, is unclear. This study assessed this by examining Müller cell dysfunction via glutamine synthetase immunoreactivity and its spatial association with retinal neuron subpopulations through various cell markers. Aged Rd1 mice retinae (P150 - P536, n = minimum 5 per age) and control heterozygous rd1 mice retinae (P536, n = 5) were isolated, fixed and cryosectioned. Fluorescent immunolabeling of glutamine synthetase was performed and retinal areas quantified as having low glutamine synthetase immunoreactivity if proportion of labeled pixels in an area was less than two standard deviations of the mean of the total retina. Other Müller cell markers such as Sox9 and Glial fibrillary acidic protein along with neuronal cell markers Calbindin, Calretinin, recoverin, Protein kinase C-α, Glutamic acid decarboxylase 67, and Islet-1 were then quantified within areas of low and normal synthetase immunoreactivity. Glutamine synthetase immunoreactivity was lost as a function of age in the rd1 mouse retina (P150 - P536). Immunoreactivity of other Müller cell markers, however, were unaffected suggesting Müller cells were still present in these low glutamine synthetase immunoreactive regions. Glutamine synthetase immunoreactivity loss affected specific neuronal populations: Type 2, Type 8 cone, and rod bipolar cells, as well as AII amacrine cells based on reduced recoverin, protein kinase Ca and parvalbumin immunoreactivity, respectively. The number of cell nuclei within regions of low glutamine synthetase immunoreactivity was also reduced suggesting possible neuronal loss rather than reduced cell marker immunoreactivity. These findings further support a strong interplay between glia-neuronal alterations in late-stage degeneration and highlight a need for future studies and consideration in intervention development.

Rhodopsin-positive cell production by intravitreal injection of small molecule compounds in mouse models of retinal degeneration.

We aimed to verify whether the intravitreal injection of small molecule compounds alone can create photoreceptor cells in mouse models of retinal degeneration. Primary cultured mouse Müller cells were stimulated in vitro with combinations of candidate compounds and the rhodopsin expression was measured on day 7 using polymerase chain reaction and immunostaining. We used 6-week-old N-methyl-N-nitrosourea-treated and 4-week-old rd10 mice as representative in vivo models of retinal degeneration. The optimal combination of compounds selected via in vitro screening was injected into the vitreous and the changes in rhodopsin expression were investigated on day 7 using polymerase chain reaction and immunostaining. The origin of rhodopsin-positive cells was also analyzed via lineage tracing and the recovery of retinal function was assessed using electroretinography. The in vitro mRNA expression of rhodopsin in Müller cells increased 30-fold, and 25% of the Müller cells expressed rhodopsin protein 7 days after stimulation with a combination of 4 compounds: transforming growth factor-β inhibitor, bone morphogenetic protein inhibitor, glycogen synthase kinase 3 inhibitor, and γ-secretase inhibitor. The in vivo rhodopsin mRNA expression and the number of rhodopsin-positive cells in the outer retina were significantly increased on day 7 after the intravitreal injection of these 4 compounds in both N-methyl-N-nitrosourea-treated and rd10 mice. Lineage tracing in td-Tomato mice treated with N-methyl-N-nitrosourea suggested that the rhodopsin-positive cells originated from endogenous Müller cells, accompanied with the recovery of the rhodopsin-derived scotopic function. It was suggested that rhodopsin-positive cells generated by compound stimulation contributes to the recovery of retinal function impaired by degeneration.

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.

Electrically-evoked responses for retinal prostheses are differentially altered depending on ganglion cell types in outer retinal neurodegeneration caused by Crb1 gene mutation.

Microelectronic prostheses for artificial vision stimulate neurons surviving outer retinal neurodegeneration such as retinitis pigmentosa (RP). Yet, the quality of prosthetic vision substantially varies across subjects, maybe due to different levels of retinal degeneration and/or distinct genotypes. Although the RP genotypes are remarkably diverse, prosthetic studies have primarily used retinal degeneration (rd) 1 and 10 mice, which both have Pde6b gene mutation. Here, we report the electric responses arising in retinal ganglion cells (RGCs) of the rd8 mouse model which has Crb1 mutation. We first investigated age-dependent histological changes of wild-type (wt), rd8, and rd10 mice retinas by H&E staining. Then, we used cell-attached patch clamping to record spiking responses of ON, OFF and direction selective (DS) types of RGCs to a 4-ms-long electric pulse. The electric responses of rd8 RGCs were analyzed in comparison with those of wt RGCs in terms of individual RGC spiking patterns, populational characteristics, and spiking consistency across trials. In the histological examination, the rd8 mice showed partial retinal foldings, but the outer nuclear layer thicknesses remained comparable to those of the wt mice, indicating the early-stage of RP. Although spiking patterns of each RGC type seemed similar to those of the wt retinas, correlation levels between electric vs. light response features were different across the two mouse models. For example, in comparisons between light vs. electric response magnitudes, ON/OFF RGCs of the rd8 mice showed the same/opposite correlation polarity with those of wt mice, respectively. Also, the electric response spike counts of DS RGCs in the rd8 retinas showed a positive correlation with their direction selectivity indices (r = 0.40), while those of the wt retinas were negatively correlated (r = -0.90). Lastly, the spiking timing consistencies of late responses were largely decreased in both ON and OFF RGCs in the rd8 than the wt retinas, whereas no significant difference was found across DS RGCs of the two models. Our results indicate the electric response features are altered depending on RGC types even from the early-stage RP caused by Crb1 mutation. Given the various degeneration patterns depending on mutation genes, our study suggests the importance of both genotype- and RGC type-dependent analyses for retinal prosthetic research.

Dual-AAV split prime editor corrects the mutation and phenotype in mice with inherited retinal degeneration

The prime editor (PE) can edit genomes with almost any intended changes, including all 12 possible types of base substitutions, small insertions and deletions, and their combinations, without the requirement for double strand breaks or exogenous donor templates. PE demonstrates the possibility of correcting a variety of disease-causing mutations and might expand the therapeutic application of gene editing. In this study, PE was optimized based on a dual-adeno-associated virus (AAV) split-intein system in vitro by screening different split sites and split inteins. We found that splitting PE before amino acid 1105(Ser) of SpCas9 with Rma intein resulted in the highest on-target editing. The orientations of pegRNA and nicking sgRNA in the AAV vector were further optimized. To test the in vivo performance of the optimized dual-AAV split-PE3, it was delivered by subretinal injection in rd12 mice with inherited retinal disease Leber congenital amaurosis. The prime editors corrected the pathogenic mutation with up to 16% efficiency in a precise way, with no detectable off-target edits, restored RPE65 expression, rescued retinal and visual function, and preserved photoceptors. Our findings establish a framework for the preclinical development of PE and motivate further testing of PE for the treatment of inherited retinal diseases caused by various mutations.

Photoreceptor laminin drives differentiation of human pluripotent stem cells to photoreceptor progenitors that partially restore retina function

Blindness caused by advanced stages of inherited retinal diseases and age-related macular degeneration are characterized by photoreceptor loss. Cell therapy involving replacement with functional photoreceptor-like cells generated from human pluripotent stem cells holds great promise. Here, we generated a human recombinant retina-specific laminin isoform, LN523, and demonstrated the role in promoting the differentiation of human embryonic stem cells into photoreceptor progenitors. This chemically defined and xenogen-free method enables reproducible production of photoreceptor progenitors within 32days. We observed that the transplantation into rd10 mice were able to protect the host photoreceptor outer nuclear layer (ONL) up to 2weeks post transplantation as measured by full-field electroretinogram. At 4weeks post transplantation, the engrafted cells were found to survive, mature, and associate with the host's rod bipolar cells. Visual behavioral assessment using the water maze swimming test demonstrated visual improvement in the cell-transplanted rodents. At 20weeks post transplantation, the maturing engrafted cells were able to replace the loss of host ONL by extensive association with host bipolar cells and synapses. Post-transplanted rabbit model also provided congruent evidence for synaptic connectivity with the degenerated host retina. The results may pave the way for the development of stem cell-based therapeutics for retina degeneration.

Functional integrity of visual coding following advanced photoreceptor degeneration

Photoreceptor degeneration sufficient to produce severe visual loss often spares the inner retina. This raises hope for vision restoration treatments using optogenetics or electrical stimulation, which generate a replacement light input signal in surviving neurons. The success of these approaches is dependent on the capacity of surviving circuits of the visual system to generate and propagate an appropriate visual code in the face of neuroanatomical remodeling. To determine whether retinally degenerate animals possess this capacity, we generated a transgenic mouse model expressing the optogenetic actuator ReaChR in ON bipolar cells (second-order neurons in the visual projection). After crossing this with the rd1 model of photoreceptor degeneration, we compared ReaChR-derived responses with photoreceptor-driven responses in wild-type (WT) mice at the level of retinal ganglion cells and the visual thalamus. The ReaChR-driven responses in rd1 animals showed low photosensitivity, but in other respects generated a visual code that was very similar to the WT. ReaChR rd1 responses had high trial-to-trial reproducibility and showed sensitivity normalization to code contrast across background intensities. At the single unit level, ReaChR-derived responses exhibited broadly similar variations in response polarity, contrast sensitivity, and temporal frequency tuning as the WT. Units from the WT and ReaChR rd1 mice clustered together when subjected to unsupervised community detection based on stimulus-response properties. Our data reveal an impressive ability for surviving circuitry to recreate a rich visual code following advanced retinal degeneration and are promising for regenerative medicine in the central nervous system.

Lycium barbarum Polysaccharides Regulating miR-181/Bcl-2 Decreased Autophagy of Retinal Pigment Epithelium with Oxidative Stress.

Disturbed structure and dysfunction of the retinal pigment epithelium (RPE) lead to degenerative diseases of the retina. Excessive accumulation of reactive oxygen species (ROS) in the RPE is thought to play an important role in RPE dysfunction and degeneration. Autophagy is a generally low-activity degradation process of cellular components that increases significantly when high levels of oxidative stress are present. Agents with antioxidant properties may decrease autophagy and provide protection against RPE dysfunction and damage caused by ROS. Lycium barbarum polysaccharide (LBP) has been widely studied as an antioxidant and cell-protective agent. Therefore, we designed this study to investigate the effects of LBP, which inhibits miR-181, on autophagy in retinal pigment epithelium (RPE) with oxidative stress in vitro and in vivo. In the current study, we found that the highly expressed miR-181 downregulated the expression of Bcl-2 in hydrogen peroxide- (H2O2-) induced ARPE-19 cells, resulting in an increase in ROS, apoptosis, and autophagy flux. LBP inhibited the expression of miR-181, decreased the levels of ROS, apoptosis, and autophagy flux, and increased cell viability in H2O2-induced ARPE-19 cells, suggesting that LBP provides protection against oxidative damage in ARPE-19 cells. We also found that LBP decreased RPE atrophy and autophagy flux in rd10 mice. Taken together, the results showed that LBP has a protective effect for RPE under oxidative stress by inhibiting miR-181 and affecting the Bcl-2/Beclin1 autophagy signaling pathway.

Assessment of Murine Retinal Acuity Ex Vivo Using Multielectrode Array Recordings.

Visual acuity, measured by resolution of optotypes on a standard eye chart, is a critical clinical test for function of the visual system in humans. Behavioral tests in animals can be used to estimate visual acuity. However, such tests may be limited in the study of mutants or after synthetic vision restoration techniques. Because the total response of the retina to a visual scene is encoded in spiking patterns of retinal ganglion cells, it should be possible to estimate visual acuity in vitro from the retina by analyzing retinal ganglion cell output in response to test stimuli. We created a method, EyeCandy, that combines a visual stimulus-generating engine with analysis of multielectrode array retinal recordings via a machine learning approach to measure murine retinal acuity in vitro. Visual stimuli included static checkerboards, drifting gratings, and letter optotypes. In retinas from wild-type C57Bl/6 mice, retinal acuity measurement for a drifting grating was 0.4 cycles per degree. In contrast, retinas from adult rd1 mice with outer retinal degeneration showed no detectable acuity. A comparison of acuities among different regions of the retina revealed substantial variation, with the inferior-nasal quadrant having highest RA. Letter classification accuracy of a projected Early Treatment Diabetic Retinopathy eye chart reached 99% accuracy for logMAR 3.0 letters. EyeCandy measured a restored RA of 0.05 and 0.08 cycles per degree for static and dynamic stimuli respectively from the retina of the rd1 mouse treated with the azobenzene photoswitch BENAQ. Machine learning may be used to estimate retinal acuity. The use of ex vivo retinal acuity measurement may allow determination of effects of mutations, drugs, injury, or other manipulations on retinal visual function.

Repopulated retinal microglia promote Müller glia reprogramming and preserve visual function in retinal degenerative mice.

Rationale: Müller glia (MG) play a key role in maintaining homeostasis of the retinal microenvironment. In zebrafish, MG reprogram into retinal progenitors and repair the injured retina, while this MG regenerative capability is suppressed in mammals. It has been revealed that microglia in zebrafish contribute to MG reprogramming, whereas those in mammals are over-activated during retinal injury or degeneration, causing chronic inflammation, acceleration of photoreceptor apoptosis, and gliosis of MG. Therefore, how to modulate the phenotype of microglia to enhance MG reprogramming rather than gliosis is critical. Methods: PLX3397, a colony-stimulating factor 1 receptor inhibitor, was applied to deplete microglia in the retinas of retinal degeneration 10 (rd10) mice, and withdrawal of PLX3397 was used to induce the repopulated microglia (Rep-MiG). The protective roles of the Rep-MiG on the degenerative retina were assessed using a light/dark transition test, and scotopic electroretinogram recordings. Immunofluorescence, western blot, transcriptomic sequencing, and bioinformatics analysis were performed to investigate the effects and mechanisms of microglia on MG reprogramming. Results: Following PLX3397 withdrawal, Rep-MiG replenished the entire retina with a ramified morphology and significantly improved the retinal outer nuclear layer structure, the electroretinography response, and the visual behavior of rd10 mice. Coincidentally, MG were activated, de-differentiated, and showed properties of retina progenitors in a spatial correlation with Rep-MiG. Morphological and transcriptomic analyses revealed Rep-MiG significantly enhanced protease inhibitor activity and suppressed extracellular matrix (ECM) levels during retinal degeneration. Conclusions: It suggested that Rep-MiG with the homeostasis characteristic stimulated the progenitor cell-like properties of MG, probably through regulating ECM remodeling, which protected photoreceptors and improved visual function of rd10 mice. It might be a potential protocol to reprogram MG and delay mammal retinal degeneration.

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.

Measuring the Release of Lactate from Wild-Type and rd1 Mouse Retina.

The retina has the highest energy consumption of any tissue in the human body. Remarkably, to satisfy its energy demand, the retina appears to rely mostly on aerobic glycolysis, which results in the production and release of large amounts of lactate. In the present study, we compared two different methods to assess lactate release from in vitro organotypic retinal explants cultured under entirely controlled, serum-free conditions. We used a standard lactate assay kit and 1H-nuclear magnetic resonance(NMR) spectroscopy-based analysis. We found that during the culturing of retinal explants derived from wild-type mice, lactate was released in large amounts and that the two different methods agreed well with each other. When comparing wild-type retina with degenerating rd1 mouse retina, we found the latter to release significantly higher amounts of lactate. Hence, degenerating retina may have an even higher energy demand and metabolic rate compared to healthy retina. We conclude that the use of lactate measurement can be a reliable and simple readout to evaluate ongoing retinal metabolism.