Olaparib significantly delays photoreceptor loss in a model for hereditary retinal degeneration

The enzyme poly-ADP-ribose-polymerase (PARP) mediates DNA-repair and rearrangements of the nuclear chromatin. Generally, PARP activity is thought to promote cell survival and in recent years a number of PARP inhibitors have been clinically developed for cancer treatment. Paradoxically, PARP activity is also connected to many diseases including the untreatable blinding disease Retinitis Pigmentosa (RP), where PARP activity appears to drive the pathogenesis of photoreceptor loss. We tested the efficacy of three different PARP inhibitors to prevent photoreceptor loss in the rd1 mouse model for RP. In retinal explant cultures in vitro, olaparib had strong and long-lasting photoreceptor neuroprotective capacities. We demonstrated target engagement by showing that olaparib reduced photoreceptor accumulation of poly-ADP-ribosylated proteins. Remarkably, olaparib also reduced accumulation of cyclic-guanosine-monophosphate (cGMP), a characteristic marker for photoreceptor degeneration. Moreover, intravitreal injection of olaparib in rd1 animals diminished PARP activity and increased photoreceptor survival, confirming in vivo neuroprotection. This study affirms the role of PARP in inherited retinal degeneration and for the first time shows that a clinically approved PARP inhibitor can prevent photoreceptor degeneration in an RP model. The wealth of human clinical data available for olaparib highlights its strong potential for a rapid clinical translation into a novel RP treatment.Peer reviewe

Identification of a Common Non-Apoptotic Cell Death Mechanism in Hereditary Retinal Degeneration

Cell death in neurodegenerative diseases is often thought to be governed by apoptosis; however, an increasing body of evidence suggests the involvement of alternative cell death mechanisms in neuronal degeneration. We studied retinal neurodegeneration using 10 different animal models, covering all major groups of hereditary human blindness (rd1, rd2, rd10, Cngb1 KO, Rho KO, S334ter, P23H, Cnga3 KO, cpfl1, Rpe65 KO), by investigating metabolic processes relevant for different forms of cell death. We show that apoptosis plays only a minor role in the inherited forms of retinal neurodegeneration studied, where instead, a non-apoptotic degenerative mechanism common to all mutants is of major importance. Hallmark features of this pathway are activation of histone deacetylase, poly-ADP-ribose-polymerase, and calpain, as well as accumulation of cyclic guanosine monophosphate and poly-ADP-ribose. Our work thus demonstrates the prevalence of alternative cell death mechanisms in inherited retinal degeneration and provides a rational basis for the design of mutation-independent treatments

Calpain and PARP Activation during Photoreceptor Cell Death in P23H and S334ter Rhodopsin Mutant Rats

Retinitis pigmentosa (RP) is a heterogeneous group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness. Many human cases are caused by mutations in the rhodopsin gene. An important question regarding RP pathology is whether different genetic defects trigger the same or different cell death mechanisms. To answer this question, we analysed photoreceptor degeneration in P23H and S334ter transgenic rats carrying rhodopsin mutations that affect protein folding and sorting respectively. We found strong activation of calpain and poly(ADP-ribose) polymerase (PARP) in both mutants, concomitant with calpastatin down-regulation, increased oxidative DNA damage and accumulation of PAR polymers. These parameters were strictly correlated with the temporal progression of photoreceptor degeneration, mirroring earlier findings in the phosphodiesterase-6 mutant rd1 mouse, and suggesting execution of non-apoptotic cell death mechanisms. Interestingly, activation of caspases-3 and -9 and cytochrome c leakage—key events in apoptotic cell death—were observed only in the S334ter mutant, which also showed increased expression of PARP-1. The identification of the same metabolic markers triggered by different mutations in two different species suggests the existence of common cell death mechanisms, which is a major consideration for any mutation independent treatment

Die Rolle von PARP1 in cGMP induzierter Photorezeptordegeneration

Retinitis Pigmentosa (RP) is an inherited eye disease which causes progressive photoreceptor degeneration and consequently blindness in humans. So far, there has been no found cure or reliable treatment for RP. The retinal degeneration 1 (rd1)mouse model is a well-studied model of human autosomal recessive RP. In 2007, it was found that over-activation of poly(ADP-ribose) polymerases (PARP) contributed to photoreceptor degeneration in the rd1 mouse. However, it was still unclear which PARP family member exactly was involved in photoreceptor degeneration. Therefore, the aim of this thesis was to investigate the role of PARP1, the most prominently expressed and extensively studied member of the PARP family, in photoreceptor degeneration in the rd1 retina. Electroretinography, optic coherence tomography (OCT), scanning laser ophthalmoscopy (SLO), and histology identified no significant differences between PARP1 knock-out (KO) and wild-type (wt) animals in terms of retinal function, thickness, and structure. In addition to PARP, histone deacetylase (HDAC) activity was shown to be involved in rd1 photoreceptor degeneration. Double-label immunohistochemistry showed poly(ADP-ribose) (PAR) accumulation - i.e. the product of PARP activity - in photoreceptor nuclei that were devoid of acetylated proteins, indicating a causal link between PARP and HDAC activity. We emulated the rd1 situation in organotypic retinal cultures derived from PARP1 KO and wt animals using zaprinast, a selective inhibitor of PDE6. Accumulation of cyclic guanosine monophosphate (cGMP) in zaprinast treated PARP1 KO retina was significantly reduced compared to zaprinast treated wt. At the same time, PAR accumulation and cell death were also significantly reduced in zaprinast treated PARP1 KO when compared with wt. In conclusion, PARP1 appears to have an important role in the progression of photoreceptor cell death. In rd1 photoreceptors, PARylation was linked to cell death as well as to deacetylation and HDAC activity, while PARP1 KO retina showed increased resistance to pharmacologically induced photoreceptor degeneration. Because of the involvement of PARP in photoreceptor degeneration and since retinal function and morphology in PARP1 KO retina appeared normal, PARP1 promises to be an interesting target for future therapy development.Retinitis Pigmentosa (RP) ist eine erbliche Augenkrankheit, die progressive Degeneration von Photorezeptorzellen und damit Blindheit beim Menschen verursacht. Bisher gibt es keine Heilung oder zuverlässige Behandlung für RP. Das Retinadegeneration-1 (rd1) Mausmodell ist ein gut untersuchtes Modell der menschlichen autosomal-rezessiven RP. Im Jahr 2007 wurde festgestellt, dass die Überaktivierung von Poly(ADP-Ribose) Polymerase (PARP) zur Photorezeptordegeneration in der rd1 Maus beiträgt Allerdings war unklar, welche Isoform der PARP-Enzymfamilie an der Photorezeptordegeneration beteiligt ist. Daher war es das Hauptziel dieser Arbeit, die genaue Rolle von PARP1 in der Photorezeptordegeneration der rd1 Retina zu untersuchen. Elektroretinographie (ERG), optische Kohärenztomographie (OCT), Scanning Laser Ophthalmoskopie (SLO) und Histologie zeigten keine signifikanten Unterschiede zwischen PARP1 Knock-out (KO) und Wildtyp (wt) Tieren, im Hinblick auf die retinale Funktion, die Schichtdicke oder die retinale Struktur. Zusätzlich zu PARP konnte gezeigt werden, dass HDAC-Aktivität in der rd1 Photorezeptordegeneration involviert war. Immunohistochemische Doppel-Markierung zeigte eine Akkumulation von PAR – d.h. dem Produkt von PARP Aktivität – in Photorezeptorkernen die negativ für acetylierte Proteine waren. Dies weis auf einen ursächlichen Zusammenhang zwischen PARP- und HDAC-Aktivität hin. Anhand von organotypischen Retina-Kulturen die aus PARP1 KO und wt Tieren gewonnen wurden, haben wir die rd1 Situation durch Verwendung des selektiven PDE6-Inhibitors Zaprinast emuliert. Die Akkumulation von cGMP in der Zaprinast-behandelten PARP1 KO Retina war deutlich reduziert im Vergleich zur Zaprinast-behandelten wt Retina. Außerdem waren die PAR Akkumulation und der Zelltod ebenfalls signifikant reduziert in der PARP1 KO Situation, verglichen mit wt. Zusammenfassend scheint es, dass PARP1 eine wichtige Rolle beim Voranschreiten des Photorezeptor-Zelltods hat. In rd1 Photorezeptoren war PAR Akkumulation ursächlich sowohl mit Proteindeacetylierung und HDAC Aktivität, als auch mit dem Zelltod verbunden. Dahingegen zeigte PARP1 KO Retina eine erhöhte Resistenz zur pharmakologisch induzierten Photorezeptordegeneration. Wegen der offensichtlichen Beteiligung von PARP an der Photorezeptordegeneration und weil die retinale Funktion und Morphologie in PARP1 KO Retina normal erscheinen, könnte PARP1 ein interessantes Target für die Entwicklung von zukünftigen Therapien sein

Release of Retinal Extracellular Vesicles in a Model of Retinitis Pigmentosa

Extracellular vesicles (EVs) are membranous structures released by cells, including those of the retinal pigment epithelium (RPE) and photoreceptors. The cargo of EVs includes genetic material and proteins, making these vesicles essential in cell communication. Among the genetic materials, we find a large number of microRNAs (miRNAs), small chains of noncoding RNA. In the case of EVs from the retina, changes have also been observed in the number and cargo of EVs.Our group confirmed that damaged RPE cells in vitro release a greater number of EVs with a higher pro-angiogenic factor (VEGFR-1 and VEGFR-2) than control non-damaged cells, thus increasing neovascularization in endothelial cell cultures. This indicates that something similar could happen in patients suffering from some types of retinal degeneration that occur with angiogenesis, such as wet AMD or RD.Here, we investigated the role of EVs in photoreceptor degeneration, and we report for the first time on CD9 and CD81, closely related tetraspanins, in wild-type and rd1 retinae. Our study demonstrates the involvement of EVs in the process of inherited photoreceptor degeneration in a PDE6 mutationMedicin