June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Inducible Poly (ADP-Ribose) Polymerase Dependent Cell Death in a Zebrafish Model of Retinitis Pigmentosa
Author Affiliations & Notes
  • Liyun Zhang
    Johns Hopkins University, Baltimore, Maryland, United States
  • Tae-In Kam
    Johns Hopkins University, Baltimore, Maryland, United States
  • Meera T Saxena
    Johns Hopkins University, Baltimore, Maryland, United States
  • Jeff S Mumm
    Johns Hopkins University, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Liyun Zhang, None; Tae-In Kam, None; Meera Saxena, None; Jeff Mumm, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 3078. doi:
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      Liyun Zhang, Tae-In Kam, Meera T Saxena, Jeff S Mumm; Inducible Poly (ADP-Ribose) Polymerase Dependent Cell Death in a Zebrafish Model of Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2021;62(8):3078.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Retinitis pigmentosa (RP) is an inherited retinal degenerative disease characterized by rod cell death followed by cone loss. Recently, non-apoptotic cell death mechanisms have been implicated in RP. In a comprehensive biochemical analysis of ten mammalian RP models, evidence of non-apoptotic cell death, such as activation of Poly (ADP-ribose) Polymerase (PARP), was common across all models while apoptosis was implicated in only one. Previously, our lab generated a nitroreductase (NTR)/metronidazole (Mtz) based transgenic zebrafish model enabling inducible rod cell death to study rod cell regeneration. However, relevance of NTR/Mtz-induced cell death to retinal degeneration was unclear. The purpose of this study was to investigate the cell death mechanism of NTR/Mtz-induced rod photoreceptor degeneration.

Methods : Five day old zebrafish larvae expressing yellow fluorescent protein (YFP) and NTR in rod photoreceptors were pre-treated with cell death pathway inhibitors for four hours followed by Mtz addition to induce rod cell death. After two days, rod cell survival was assessed using an established plate reader assay. Genes encoding key factors for three cell death pathways were knocked down using CRISPR/Cas9 and rod cell survival assessed following NTR/Mtz-induced cell death. Targeted genes included parp1 (parthanatos pathway), receptor (TNFRSF)-interacting serine-threonine kinase 1 like (ripk1l; necroptosis) and caspase 3a/3b (casp3a/3b; apoptosis). qPCR was performed to confirm reduced expression of targeted genes and western blots used to assess PARP activation, i.e., accumulation of poly (ADP-ribose) (PAR) polymers.

Results : Increased rod cell survival was observed in larvae treated with PARP/parthanatos inhibitors and a necroptosis inhibitor, but not an apoptosis inhibitor. Similarly, knock down of parp1 and ripk1l, but not casp3a/3b, improved rod cell survival. Moreover, PAR polymers accumulated in Mtz-treated fish compared to controls, indicating PARP activation, a signature of parthanatos.

Conclusions : Mtz/NTR induced rod cell death involves both the PARP1/parthanatos and ripk1l/necroptosis. As both of these pathways have been implicated in neurodegeneration, including RP, we conclude that the NTR/Mtz system serves as an inducible model of RP providing a useful tool for exploring neuroprotective therapeutics.

This is a 2021 ARVO Annual Meeting abstract.

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