Knockout of PARG110 confers resistance to cGMP-induced toxicity in mammalian photoreceptors.

Hereditary retinal degeneration (RD) relates to a heterogeneous group of blinding human diseases in which the light sensitive neurons of the retina, the photoreceptors, die. RD is currently untreatable and the underlying cellular mechanisms remain poorly understood. However, the activity of the enzyme poly-ADP-ribose polymerase-1 (PARP1) and excessive generation of poly-ADP-ribose (PAR) polymers in photoreceptor nuclei have been shown to be causally involved in RD. The activity of PARP1 is to a large extent governed by its functional antagonist, poly-ADP-glycohydrolase (PARG), which thus also may have a role in RD. To investigate this, we analyzed PARG expression in the retina of wild-type (wt) mice and in the rd1 mouse model for human RD, and detected increased PARG protein in a subset of degenerating rd1 photoreceptors. Knockout (KO) animals lacking the 110 kDa nuclear PARG isoform were furthermore analyzed, and their retinal morphology and function were indistinguishable from wild-type animals. Organotypic wt retinal explants can be experimentally treated to induce rd1-like photoreceptor death, but PARG110 KO retinal explants were unexpectedly highly resistant to such treatment. The resistance was associated with decreased PAR accumulation and low PARP activity, indicating that PARG110 may positively regulate PARP1, an event that therefore is absent in PARG110 KO tissue. Our study demonstrates a causal involvement of PARG110 in the process of photoreceptor degeneration. Contrasting its anticipated role as a functional antagonist, absence of PARG110 correlated with low PARP activity, suggesting that PARG110 and PARP1 act in a positive feedback loop, which is especially active under pathologic conditions. This in turn highlights both PARG110 and PARP1 as potential targets for neuroprotective treatments for RD

Retinitis pigmentosa: rapid neurodegeneration is governed by slow cell death mechanisms

For most neurodegenerative diseases the precise duration of an individual cell's death is unknown, which is an obstacle when counteractive measures are being considered. To address this, we used the rd1 mouse model for retinal neurodegeneration, characterized by phosphodiesterase-6 (PDE6) dysfunction and photoreceptor death triggered by high cyclic guanosinemono-phosphate (cGMP) levels. Using cellular data on cGMP accumulation, cell death, and survival, we created mathematical models to simulate the temporal development of the degeneration. We validated model predictions using organotypic retinal explant cultures derived from wild-type animals and exposed to the selective PDE6 inhibitor zaprinast. Together, photoreceptor data and modeling for the first time delineated three major cell death phases in a complex neuronal tissue: (1) initiation, taking up to 36 h, (2) execution, lasting another 40 h, and finally (3) clearance, lasting about 7 h. Surprisingly, photoreceptor neurodegeneration was noticeably slower than necrosis or apoptosis, suggesting a different mechanism of death for these neurons. Cell Death and Disease (2013) 4, e488; doi: 10.1038/cddis.2013.12; published online 7 February 201

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

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

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

Sphingolipids as critical players in retinal physiology and pathology

Sphingolipids have emerged as bioactive lipids involved in the regulation of many physiological and pathological processes. In the retina, they have been established toparticipate in numerousprocesses, suchas neuronal survival and death, proliferation and migration of neuronal and vascular cells, inflammation, and neovascularization. Dysregulation of sphingolipids is therefore crucial in the onset and progression of retinal diseases. This review examines the involvement of sphingolipids in retinal physiology and diseases. Ceramide (Cer) has emerged as a common mediator of inflammation and death of neuronal and retinal pigment epithelium cells in animal models of retinopathies such as glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa. Sphingosine- 1-phosphate (S1P) has opposite roles, preventing photoreceptor and ganglion cell degeneration but also promoting inflammation, fibrosis, and neovascularization in AMD, glaucoma, and pro-fibrotic disorders. Alterations in Cer, S1P, and ceramide 1- phosphate may also contribute to uveitis. Notably, use of inhibitors that either prevent Cer increase or modulate S1P signaling, such as Myriocin, desipramine, and Fingolimod (FTY720), preserves neuronal viability and retinal function. These findings underscore the relevance of alterations in the sphingolipid metabolic network in the etiology of multiple retinopathies and highlight the potential of modulating their metabolism for the design of novel therapeutic approaches.Fil: Simon, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Basu, Sandip K.. University of Tennessee; Estados UnidosFil: Qaladize, Bano. University of Tennessee; Estados UnidosFil: Grambergs, Richards. University of Tennessee; Estados UnidosFil: Rotstein, Nora Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Mandal, Nawajes .A.. University of Tennessee; Estados Unido

Aag DNA Glycosylase Promotes Alkylation-Induced Tissue Damage Mediated by Parp1

Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag−/− mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.National Institutes of Health (U.S.) (NIH grant R01-CA075576)National Institutes of Health (U.S.) (NIH grant R01-CA055042)National Institutes of Health (U.S.) (NIH grant R01-CA149261)National Institutes of Health (U.S.) (NIH grant P30-ES00002)National Institutes of Health (U.S.) (NIH grant P30-ES02109)National Center for Research Resources (U.S.) (grant number M01RR-01066)National Center for Research Resources (U.S.) (grant number UL1 RR025758, Harvard Clinical and Translational Science Center

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

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