Efficacy of PARP inhibition in Pde6a mutant mouse models for retinitis pigmentosa depends on the quality and composition of individual human mutations.

Retinitis pigmentosa (RP), an inherited blinding disease, is caused by a variety of different mutations that affect retinal photoreceptor function and survival. So far there is neither effective treatment nor cure. We have previously shown that poly(ADP-ribose)polymerase (PARP) acts as a common and critical denominator of cell death in photoreceptors, qualifying it as a potential target for future therapeutic intervention. A significant fraction of RP-causing mutations affect the genes for the rod photoreceptor phosphodiesterase 6A (PDE6A) subunit, but it is not known whether they all engage the same death pathway. Analysing three homozygous point mutations (Pde6a R562W, D670G, and V685M) and one compound heterozygous Pde6a (V685M/R562W) mutation in mouse models that match human RP patients, we demonstrate excessive activation of PARP, which correlated in time with the progression of photoreceptor degeneration. The causal involvement of PARP activity in the neurodegenerative process was confirmed in organotypic retinal explant cultures treated with the PARP-selective inhibitor PJ34, using different treatment time-points and durations. Remarkably, the neuroprotective efficacy of PARP inhibition correlated inversely with the strength of the genetically induced insult, with the D670G mutant showing the best treatment effects. Our results highlight PARP as a target for neuroprotective interventions in RP caused by PDE6A mutations and are a first attempt towards personalized, genotype-matched therapy development for RP. In addition, for each of the different mutant situations, our work identifies windows of opportunity for an optimal treatment regimen for further in vivo experimentation and possibly clinical studies.

DNA methylation and differential gene regulation in photoreceptor cell death.

Retinitis pigmentosa (RP) defines a group of inherited degenerative retinal diseases causing progressive loss of photoreceptors. To this day, RP is still untreatable and rational treatment development will require a thorough understanding of the underlying cell death mechanisms. Methylation of the DNA base cytosine by DNA methyltransferases (DNMTs) is an important epigenetic factor regulating gene expression, cell differentiation, cell death, and survival. Previous studies suggested an involvement of epigenetic mechanisms in RP, and in this study, increased cytosine methylation was detected in dying photoreceptors in the rd1, rd2, P23H, and S334ter rodent models for RP. Ultrastructural analysis of photoreceptor nuclear morphology in the rd1 mouse model for RP revealed a severely altered chromatin structure during retinal degeneration that coincided with an increased expression of the DNMT isozyme DNMT3a. To identify disease-specific differentially methylated DNA regions (DMRs) on a genomic level, we immunoprecipitated methylated DNA fragments and subsequently analyzed them with a targeted microarray. Genome-wide comparison of DMRs between rd1 and wild-type retina revealed hypermethylation of genes involved in cell death and survival as well as cell morphology and nervous system development. When correlating DMRs with gene expression data, we found that hypermethylation occurred alongside transcriptional repression. Consistently, motif analysis showed that binding sites of several important transcription factors for retinal physiology were hypermethylated in the mutant model, which also correlated with transcriptional silencing of their respective target genes. Finally, inhibition of DNMTs in rd1 organotypic retinal explants using decitabine resulted in a substantial reduction of photoreceptor cell death, suggesting inhibition of DNA methylation as a potential novel treatment in RP.

Novel in situ activity assays for the quantitative molecular analysis of neurodegenerative processes in the retina.

The mechanisms of neuronal cell death are still only poorly understood, which has hindered the advancement of therapies for many currently untreatable neurodegenerative diseases. This calls for the development of new methods which reveal critical molecular mechanisms of the celldeath machinery with both high sensitivity and cellular resolution. Using animal models for hereditary neurodegeneration in the retina, we have developed or adapted different biochemical assays to determine the enzymatic activities of calpain, poly-ADP-ribose-polymerase (PARP), and histone deacetylase (HDAC) directly and in situ. Additionally, the enzymatic activity of cGMP-dependent protein kinase (PKG) was assessed indirectly using in situ immunohistological techniques to detect PKG-activity-dependent products. Combining these assays with in situ cell death markers revealed close temporospatial correlations, suggesting causal connections between the PKG, HDAC, PARP and calpain activities and neuronal cell death. Using different pharmacological and genetic manipulations, causality could indeed be demonstrated. Surprisingly, the often dramatic rises in metabolic activities didnot match by corresponding increases in expression, highlighting the importance of analyses of protein activities at the cellular level. The above mentioned studies identified a number of metabolic processes previously unknownto be involved in inherited retinal degeneration. Comparing different animal retinal degeneration models uncovered striking similarities in enzymatic activities, suggesting a generality of the destructive pathways. Taken together, these findings provided a number of novel targets for neuroprotection and as such opened up new perspectives for the therapy of hereditary neurodegeneration in the retina and possibly other parts of the central nervous system.

How long does a photoreceptor cell take to die? Implications for the causative cell death mechanisms.

The duration of cell death may allow deducing the underlying degenerative mechanism. To find out how long a photoreceptor takes to die, we used the rd1 mouse model for retinal neurodegeneration, which is characterized by phosphodiesterase-6 (PDE6) dysfunction and photoreceptor death triggered by high cGMP levels. Based on cellular data on the progression of cGMP accumulation, cell death, and survival, we created a mathematical model to simulate the temporal development of the degeneration and the clearance of dead cells. Both cellular data and modelling suggested that at the level of the individual cell, the degenerative process was rather slow, taking around 80 h to complete. Organotypic retinal explant cultures derived from wild-type animals and exposed to the selective PDE6 inhibitor zaprinast, confirmed the surprisingly long duration of an individual photoreceptor cell's death. We briefly discuss the possibility to link different cell death stages and their temporal progression to specific enzymatic activities known to be causally connected to cell death. This in turn opens up new perspectives for the treatment of inherited retinal degeneration, both in terms of therapeutic targets and temporal windows-of-opportunity.

Significant photoreceptor rescue by treatment with a combination of antioxidants in an animal model for retinal degeneration.

The purpose of this study was to investigate the presence of oxidative DNA damage in the photoreceptors of the rd1 mouse, an animal model for retinitis pigmentosa, and to determine if antioxidants could delay the progress of photoreceptor cell death. Retinas of rd1 mice and congenic wild type controls were examined for DNA oxidation and fragmentation. To study the rescue effect of antioxidants on retinal degeneration, rd1 retinas were studied in vitro and in vivo using lutein, zeaxanthin, alpha lipoic acid and reduced l-glutathione. For the in vitro studies, antioxidants were added to the culture medium. For the in vivo studies, postnatal day (PN3) pups of rd1 mice were fed antioxidants either individually or in combination and control rd1 animals received vehicle alone. Histological evaluation was performed using hematoxylin/eosin and avidin staining, as well as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Many of the rd1 rod photoreceptors at PN11 displayed oxidative DNA damage and TUNEL positive reaction which co-localized in a subset of rod photoreceptors. Avidin-labeled rod photoreceptors were more abundant than the TUNEL positive photoreceptors of the rd1 mouse, indicating that oxidative DNA damage precedes fragmentation. The number of TUNEL positive and avidin positive cells was considerably decreased upon treatment with the combination of the antioxidants. Rescue of rd1 photoreceptors was significant at PN18 and PN17, respectively, in the in vitro and in vivo studies. In conclusion individual antioxidants had no significant rescue effect but the combination slowed down the rd1 rod photoreceptor degeneration, indicating an additive or synergistic effect.