June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Notch inhibition promotes regeneration and immunosuppression supports cone survival in a zebrafish model of inherited retinal dystrophy
Author Affiliations & Notes
  • Brian D Perkins
    Cleveland Clinic Cole Eye Institute, Cleveland, Ohio, United States
  • Ping Song
    Cleveland Clinic Cole Eye Institute, Cleveland, Ohio, United States
  • Joseph Fogerty
    Cleveland Clinic Cole Eye Institute, Cleveland, Ohio, United States
  • Patrick Boyd
    Biological Sciences, University of Notre Dame, Notre Dame, Indiana, United States
  • Sarah Grabinski
    Cleveland Clinic Cole Eye Institute, Cleveland, Ohio, United States
  • Thanh V Hoang
    Neuroscience, Johns Hopkins University, Baltimore, Maryland, United States
  • Seth Blackshaw
    Neuroscience, Johns Hopkins University, Baltimore, Maryland, United States
  • Jeff S Mumm
    Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • David Hyde
    Biological Sciences, University of Notre Dame, Notre Dame, Indiana, United States
  • Footnotes
    Commercial Relationships   Brian Perkins None; Ping Song None; Joseph Fogerty None; Patrick Boyd None; Sarah Grabinski None; Thanh Hoang None; Seth Blackshaw Genentech, Code F (Financial Support), CDI Labs, LLC, Code O (Owner); Jeff Mumm US8431768B2, Code P (Patent); David Hyde None
  • Footnotes
    Support  NIH grants R01-EY017037 and R01-EY030574 to B.D.P., U01-EY027267 to D.R.H. and S.B., a Knights Templar Eye Foundation Career Initiation Grant to J.F., and a Doris and Jules Stein Professorship Award from Research to Prevent Blindness B.D.P. Additional support was provided by an NIH P30 core grant (P30-EY025585), a Foundation Fighting Blindness (FFB) Center Grant, and an Unrestricted Award from Research to Prevent Blindness and the Cleveland Eye Bank Foundation to the Cole Eye Institute. The LRI Genomics Core is supported by an NIH P30 core grant (P30-CA043703).
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1870. doi:
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      Brian D Perkins, Ping Song, Joseph Fogerty, Patrick Boyd, Sarah Grabinski, Thanh V Hoang, Seth Blackshaw, Jeff S Mumm, David Hyde; Notch inhibition promotes regeneration and immunosuppression supports cone survival in a zebrafish model of inherited retinal dystrophy. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1870.

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

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Abstract

Purpose : Müller glia (MG) in several zebrafish models of chronic photoreceptor degeneration fail to re-enter the cell cycle and regenerate lost cells. Our goals were to determine the transcriptional and cellular responses to chronic degeneration and to identify mechanisms that prevent regeneration in cep290 and bbs2 mutant zebrafish.

Methods : RNA-seq, single-cell RNA-seq, and qRT-PCR were used to assess gene expression changes. Immunohistochemistry was performed on retinal cryosections to monitor retinal cell types. Acute injury was generated by exposing animals to high intensity light. Dexamethasone and irf8 mutant zebrafish were used for temporary and chronic immune suppression, respectively. The gamma-secretase inhibitor RO4929097A was used to inhibit Notch signaling. A minimum of 6 animals per group were tested. Data was quantified and analyzed using ANOVA with post hoc comparisons.

Results : Zebrafish cep290 and bbs2 mutants exhibit progressive photoreceptor degeneration beginning at 3 months of age. Degeneration leads to an immune response with accumulation of 4C4+/L-plastin+ cells in the outer nuclear layer (ONL) and subretinal space and the upregulation of inflammatory signaling pathways. Both mutants exhibit increased numbers of PCNA+ cells in the ONL but only a 2-fold increase in the number of proliferating Müller glia. At 1 month of recovery following acute light damage, the density of regenerated photoreceptors in cep290 or bbs2 mutants at all ages remained significantly lower than that of wild-type animals. In irf8;cep290 double mutants, which possessed significantly fewer microglia, there was reduced inflammation and cone degeneration was rescued. Finally, single-cell RNA-sequencing revealed sustained notch3 expression in MG of cep290 mutants and inhibition of Notch signaling induced MG to re-enter the cell cycle in both mutants.

Conclusions : Zebrafish cep290 and bbs2 mutants progressively lose cones beginning by 3 months of age. Regeneration of lost cones occurs only following acute damage, suggesting that the mutants retain the ability to stimulate reprogramming and proliferation of Müller glia. Finally, we show that inhibition of Notch signaling releases that constraint. Our results indicate that therapies to suppress microglia function may prolong photoreceptor survival and that triggering regeneration requires more than inflammation alone.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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