T-type voltage-gated channels, Na+/Ca2+-exchanger, and calpain-2 promote photoreceptor cell death in inherited retinal degeneration.

Inherited retinal degenerations (IRDs) are a group of untreatable and commonly blinding diseases characterized by progressive photoreceptor loss. IRD pathology has been linked to an excessive activation of cyclic nucleotide-gated channels (CNGC) leading to Na+- and Ca2+-influx, subsequent activation of voltage-gated Ca2+-channels (VGCC), and further Ca2+ influx. However, a connection between excessive Ca2+ influx and photoreceptor loss has yet to be proven.Here, we used whole-retina and single-cell RNA-sequencing to compare gene expression between the rd1 mouse model for IRD and wild-type (wt) mice. Differentially expressed genes indicated links to several Ca2+-signalling related pathways. To explore these, rd1 and wt organotypic retinal explant cultures were treated with the intracellular Ca2+-chelator BAPTA-AM or inhibitors of different Ca2+-permeable channels, including CNGC, L-type VGCC, T-type VGCC, Ca2+-release-activated channel (CRAC), and Na+/Ca2+ exchanger (NCX). Moreover, we employed the novel compound NA-184 to selectively inhibit the Ca2+-dependent protease calpain-2. Effects on the retinal activity of poly(ADP-ribose) polymerase (PARP), sirtuin-type histone-deacetylase, calpains, as well as on activation of calpain-1, and - 2 were monitored, cell death was assessed via the TUNEL assay.While rd1 photoreceptor cell death was reduced by BAPTA-AM, Ca2+-channel blockers had divergent effects: While inhibition of T-type VGCC and NCX promoted survival, blocking CNGCs and CRACs did not. The treatment-related activity patterns of calpains and PARPs corresponded to the extent of cell death. Remarkably, sirtuin activity and calpain-1 activation were linked to photoreceptor protection, while calpain-2 activity was related to degeneration. In support of this finding, the calpain-2 inhibitor NA-184 protected rd1 photoreceptors.These results suggest that Ca2+ overload in rd1 photoreceptors may be triggered by T-type VGCCs and NCX. High Ca2+-levels likely suppress protective activity of calpain-1 and promote retinal degeneration via activation of calpain-2. Overall, our study details the complexity of Ca2+-signalling in photoreceptors and emphasizes the importance of targeting degenerative processes specifically to achieve a therapeutic benefit for IRDs. Video Abstract.

HDAC inhibition delays photoreceptor loss in Pde6b mutant mice of retinitis pigmentosa: insights from scRNA-seq and CUT&Tag.

Purpose: This research aimed to ascertain the neuroprotective effect of histone deacetylase (HDAC) inhibition on retinal photoreceptors in Pde6brd1 mice, a model of retinitis pigmentosa (RP). Methods: Single-cell RNA-sequencing (scRNA-seq) explored HDAC and poly (ADP-ribose) polymerase (PARP)-related gene expression in both Pde6b-mutant rd1 and wild-type (WT) mice. The CUT&Tag method was employed to examine the functions of HDAC in rd1 mice. Organotypic retinal explant cultures from WT and rd1 mice were exposed to the HDAC inhibitor SAHA (suberoylanilide hydroxamic acid) postnatally, from day 5 to day 11. The terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay was applied to quantify the percentage of photoreceptor loss in the outer nuclear layer (ONL). HDAC activity was confirmed to be inhibited by SAHA through an HDAC activity assay. Moreover, the study evaluated PARP activity, a key driver of the initial response to DNA damage during photoreceptor degeneration, following HDAC inhibition. Results: The scRNA-seq revealed that diverse roles of HDAC and PARP isoforms in photoreceptor cell death. HDAC-related genes appeared to regulate cell death and primary immunodeficiency. Alterations in HDAC activity were consistent with the TUNEL-positive cells in the ONL at different time points. Notably, SAHA significantly postponed photoreceptor loss and decreased HDAC and PARP activity, thereby implicating both in the same degenerative pathway. Conclusions: This study highlights that the interaction between HDAC inhibition and PARP can delay photoreceptor cell death, proposing a promising therapeutic approach for RP.

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.

Inhibition of the MAPK/c-Jun-EGR1 Pathway Decreases Photoreceptor Cell Death in the rd1 Mouse Model for Inherited Retinal Degeneration.

Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies that typically results in photoreceptor cell death and vision loss. Here, we explored the effect of early growth response-1 (EGR1) expression on photoreceptor cell death in Pde6brd1 (rd1) mice and its mechanism of action. To this end, single-cell RNA-seq (scRNA-seq) was used to identify differentially expressed genes in rd1 and congenic wild-type (WT) mice. Chromatin immunoprecipitation (ChIP), the dual-luciferase reporter gene assay, and western blotting were used to verify the relationship between EGR1 and poly (ADP-ribose) polymerase-1 (PARP1). Immunofluorescence staining was used to assess PARP1 expression after silencing or overexpression of EGR1. Photoreceptor cell death was assessed using the TUNEL assay following silencing/overexpression of EGR1 or administration of MAPK/c-Jun pathway inhibitors tanzisertib and PD98059. Our results showed differential expression of ERG1 in rd1 and WT mice via scRNA-seq analysis. The ChIP assay demonstrated EGR1 binding to the PARP1 promoter region. The dual-luciferase reporter gene assay and western blotting results revealed that EGR1 upregulated PARP1 expression. Additionally, the TUNEL assay showed that silencing EGR1 effectively reduced photoreceptor cell death. Similarly, the addition of tanzisertib and PD98059 reduced the expression of c-Jun and EGR1 and decreased photoreceptor cell death. Our study revealed that inhibition of the MAPK/c-Jun pathway reduced the expression of EGR1 and PARP1 and prevented photoreceptor cell death. These results highlight the importance of EGR1 for photoreceptor cell death and identify a new avenue for therapeutic interventions in RP.

Single-Cell Transcriptomic Profiling in Inherited Retinal Degeneration Reveals Distinct Metabolic Pathways in Rod and Cone Photoreceptors.

The cellular mechanisms underlying hereditary photoreceptor degeneration are still poorly understood. The aim of this study was to systematically map the transcriptional changes that occur in the degenerating mouse retina at the single cell level. To this end, we employed single-cell RNA-sequencing (scRNA-seq) and retinal degeneration-1 (rd1) mice to profile the impact of the disease mutation on the diverse retinal cell types during early post-natal development. The transcriptome data allowed to annotate 43,979 individual cells grouped into 20 distinct clusters. We further characterized cluster-specific metabolic and biological changes in individual cell types. Our results highlight Ca2+-signaling as relevant to hereditary photoreceptor degeneration. Although metabolic reprogramming in retina, known as the 'Warburg effect', has been documented, further metabolic changes were noticed in rd1 mice. Such metabolic changes in rd1 mutation was likely regulated through mitogen-activated protein kinase (MAPK) pathway. By combining single-cell transcriptomes and immunofluorescence staining, our study revealed cell type-specific changes in gene expression, as well as interplay between Ca2+-induced cell death and metabolic pathways.

Organotypic Retinal Explant Cultures from Macaque Monkey.

Hereditary retinal degeneration (RD) is characterized by progressive photoreceptor cell death. Overactivation of the cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) pathway in photoreceptor cells causes photoreceptor cell death, especially in models harboring phosphodiesterase 6b (PDE6b) mutations. Previous studies on RD have used mainly murine models such as rd1 or rd10 mice. Given the genetic and physiological differences between mice and humans, it is important to understand to which extent the retinas of primates and rodents are comparable. Macaques share a high level of genetic similarity with humans. Therefore, wild-type macaques (aged 1-3 years) were selected for the in vitro culture of retinal explants that included the retina-retinal pigment epithelium (RPE)-choroid complex. These explants were treated with different concentrations of the PDE6 inhibitor zaprinast to induce the cGMP-PKG signaling pathway and simulate RD pathogenesis. cGMP accumulation and cell death in primate retinal explants were subsequently verified using immunofluorescence and the TUNEL assay. The primate retinal model established in this study may serve for relevant and effective studies into the mechanisms of cGMP-PKG-dependent RD, as well as for the development of future treatment approaches.

Inherited Retinal Degeneration: PARP-Dependent Activation of Calpain Requires CNG Channel Activity.

Inherited retinal degenerations (IRDs) are a group of blinding diseases, typically involving a progressive loss of photoreceptors. The IRD pathology is often based on an accumulation of cGMP in photoreceptors and associated with the excessive activation of calpain and poly (ADP-ribose) polymerase (PARP). Inhibitors of calpain or PARP have shown promise in preventing photoreceptor cell death, yet the relationship between these enzymes remains unclear. To explore this further, organotypic retinal explant cultures derived from wild-type and IRD-mutant mice were treated with inhibitors specific for calpain, PARP, and voltage-gated Ca2+ channels (VGCCs). The outcomes were assessed using in situ activity assays for calpain and PARP and immunostaining for activated calpain-2, poly (ADP-ribose), and cGMP, as well as the TUNEL assay for cell death detection. The IRD models included the Pde6b-mutant rd1 mouse and rd1*Cngb1-/- double-mutant mice, which lack the beta subunit of the rod cyclic nucleotide-gated (CNG) channel and are partially protected from rd1 degeneration. We confirmed that an inhibition of either calpain or PARP reduces photoreceptor cell death in rd1 retina. However, while the activity of calpain was decreased by the inhibition of PARP, calpain inhibition did not alter the PARP activity. A combination treatment with calpain and PARP inhibitors did not synergistically reduce cell death. In the slow degeneration of rd1*Cngb1-/- double mutant, VGCC inhibition delayed photoreceptor cell death, while PARP inhibition did not. Our results indicate that PARP acts upstream of calpain and that both are part of the same degenerative pathway in Pde6b-dependent photoreceptor degeneration. While PARP activation may be associated with CNG channel activity, calpain activation is linked to VGCC opening. Overall, our data highlights PARP as a target for therapeutic interventions in IRD-type diseases.

Integrative RNA-seq and ATAC-seq analyses of phosphodiesterase 6 mutation-induced retinitis pigmentosa.

PURPOSE: Inhibition of poly-ADP-ribose polymerase 1 (PARP1) could relieve phosphodiesterase 6 mutation-induced retinitis pigmentosa (RP). However, the mechanism related to PARP1 overexpression in the RP has not been clarified. We attempted to explore the potential mechanism related to PARP1 regulating RP. METHODS: ATAC-seq and RNA-seq were performed for retina tissues of C3H and rd1 mice. The differentially expressed genes (DEGs) were identified, followed by the construction of PARP1-DEG co-expression and protein-protein interaction (PPI) networks. Gene ontology-biological process and pathway enrichment of DEGs were performed by clusterProfiler software. The overlapped genes that might play regulatory roles in PARP1 expression were mined by integrated analysis of RNA-seq and ATAC-seq data. RESULTS: A total of 1061 DEGs were identified between C3H and rd1 group. Co-expression network was constructed with 313 PARP1-gene co-expression pairs. The down-regulated DEGs were closely related to visual perception and light stimulus-related biological process, while the up-regulated DEGs were significantly enriched in phototransduction and PPAR signaling pathway. PPI network was constructed with 202 nodes and 375 edges, which was clustered into 3 modules. Module 1 genes were closely related to detection of light stimulus, visual perception related biological process and phototransduction pathway (involved with Gnat1/Guca1b/Gnat2/Sag/Pde6g). By integrated analysis of the RNA-seq and ATAC-seq, the overlapped up-regulated genes were Asxl3 and Nyap2, while the down-regulated genes were Tmem136 and Susd3. CONCLUSION: Gnat1 may play a key role in RP development by interacting with PARP1. Susd3 may play a regulatory role in PARP1 expression and affect RP formation.

Expression of glucose transporter-2 in murine retina: Evidence for glucose transport from horizontal cells to photoreceptor synapses.

The retina has the highest relative energy consumption of any tissue, depending on a steady supply of glucose from the bloodstream. Glucose uptake is mediated by specific transporters whose regulation and expression are critical for the pathogenesis of many diseases, including diabetes and diabetic retinopathy. Here, we used immunofluorescence to show that glucose transporter-2 (GLUT2) is expressed in horizontal cells of the mouse neuroretina in proximity to inner retinal capillaries. To study the function of GLUT2 in the murine retina, we used organotypic retinal explants, cultivated under entirely controlled, serum-free conditions and exposed them to streptozotocin, a cytotoxic drug transported exclusively by GLUT2. Contrary to our expectations, streptozotocin did not measurably affect horizontal cell viability, while it ablated rod and cone photoreceptors in a concentration-dependent manner. Staining for poly-ADP-ribose (PAR) indicated that the detrimental effect of streptozotocin on photoreceptors may be associated with DNA damage. The negative effect of streptozotocin on the viability of rod photoreceptors was counteracted by co-administration of either the inhibitor of connexin-formed hemi-channels meclofenamic acid or the blocker of clathrin-mediated endocytosis dynasore. Remarkably, cone photoreceptors were not protected from streptozotocin-induced degeneration by neither of the two drugs. Overall, these data suggest the existence of a GLUT2-dependent glucose transport shuttle, from horizontal cells into photoreceptor synapses. Moreover, our study points at different glucose uptake mechanisms in rod and cone photoreceptors.

RNA Biological Characteristics at the Peak of Cell Death in Different Hereditary Retinal Degeneration Mutants.

Purpose: The present work investigated changes in the gene expression, molecular mechanisms, and pathogenesis of inherited retinal degeneration (RD) in three different disease models, to identify predictive biomarkers for their varied phenotypes and to provide a better scientific basis for their diagnosis, treatment, and prevention. Methods: Differentially expressed genes (DEGs) between retinal tissue from RD mouse models obtained during the photoreceptor cell death peak period (Pde6b rd1 at post-natal (PN) day 13, Pde6b rd10 at PN23, Prph rd2 at PN29) and retinal tissue from C3H wild-type mice were identified using Illumina high-throughput RNA-sequencing. Co-expression gene modules were identified using a combination of GO and KEGG enrichment analyses and gene co-expression network analysis. CircRNA-miRNA-mRNA network interactions were studied by genome-wide circRNA screening. Results: Pde6b rd1 , Pde6b rd10 , and Prph rd2 mice had 1,926, 3,096, and 375 DEGs, respectively. Genes related to ion channels, stress, inflammatory processes, tumor necrosis factor (TNF) production, and microglial cell activation were up-regulated, while genes related to endoplasmic reticulum regulation, metabolism, and homeostasis were down-regulated. Differential expression of transcription factors and non-coding RNAs generally implicated in other human diseases was detected (e.g., glaucoma, diabetic retinopathy, and inherited retinal degeneration). CircRNA-miRNA-mRNA network analysis indicated that these factors may be involved in photoreceptor cell death. Moreover, excessive cGMP accumulation causes photoreceptor cell death, and cGMP-related genes were generally affected by different pathogenic gene mutations. Conclusion: We screened genes and pathways related to photoreceptor cell death. Additionally, up-stream regulatory factors, such as transcription factors and non-coding RNA and their interaction networks were analyzed. Furthermore, RNAs involved in RD were functionally annotated. Overall, this study lays a foundation for future studies on photoreceptor cell death mechanisms.

miR-200-3p suppresses cell proliferation and reduces apoptosis in diabetic retinopathy via blocking the TGF-ß2/Smad pathway.

Increasing evidence has shown that microRNAs (miRNAs) play an important role in the pathogenesis of diabetic retinopathy (DR). However, the role and mechanism of miRNA in regulating high glucose (HG)-induced ARPE-19 cell injury are still not well understood. The present study aimed to investigate the effects of miR-200a-3p on DR progression and reveal the underlying mechanisms of their effects. In the present study, we observed that miR-200a-3p was significantly decreased, while transforming growth factor-ß2 (TGF-ß2) expression was up-regulated in ARPE-19 cells treated with HG and retina tissues of DR rats. Subsequently, overexpression of miR-200a-3p significantly promoted cell proliferation, reduced apoptosis, as well as inhibited the levels of inflammatory cytokines secreted, matrix metalloprotease 2/9 (MMP2/9), and vascular endothelial growth factor (VEGF) in HG-injured ARPE-19 cells. Moreover, miR-200a-3p was proved to target TGF-ß2 mRNA by binding to its 3' untranslated region (3'UTR) using a luciferase reporter assay. Mechanistically, overexpression of miR-200a-3p reduced HG-induced ARPE-19 cell injury and reduced inflammatory cytokines secreted, as well as down-regulated the expression of VEGF via inactivation of the TGF-ß2/Smad pathway in vitro. In vivo experiments, up-regulation of miR-200a-3p ameliorated retinal neovascularization and inflammation of DR rats. In conclusion, our findings demonstrated that miR-200a-3p-elevated prevented DR progression by blocking the TGF-ß2/Smad pathway, providing a new therapeutic biomarker for DR treatment in the clinic.

Retinitis pigmentosa: impact of different Pde6a point mutations on the disease phenotype.

Mutations in the PDE6A gene can cause rod photoreceptors degeneration and the blinding disease retinitis pigmentosa (RP). While a number of pathogenic PDE6A mutations have been described, little is known about their impact on compound heterozygous situations and potential interactions of different disease-causing alleles. Here, we used a novel mouse model for the Pde6a R562W mutation in combination with an existing line carrying the V685M mutation to generate compound heterozygous Pde6a V685M/R562W animals, exactly homologous to a case of human RP. We compared the progression of photoreceptor degeneration in these compound heterozygous mice with the homozygous V685M and R562W mutants, and additionally with the D670G line that is known for a relatively mild phenotype. We investigated PDE6A expression, cyclic guanosine mono-phosphate accumulation, calpain and caspase activity, in vivo retinal function and morphology, as well as photoreceptor cell death and survival. This analysis confirms the severity of different Pde6a mutations and indicates that compound heterozygous mutants behave like intermediates of the respective homozygous situations. Specifically, the severity of the four different Pde6a situations may be categorized by the pace of photoreceptor degeneration: V685M (fastest) > V685M/R562W > R562W > D670G (slowest). While calpain activity was strongly increased in all four mutants, caspase activity was not. This points to the execution of non-apoptotic cell death and may lead to the identification of new targets for therapeutic interventions. For individual RP patients, our study may help to predict time-courses for Pde6a-related retinal degeneration and thereby facilitate the definition of a window-of-opportunity for clinical interventions.