June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Time-dependent changes in ON bipolar cell transcriptomes before and after genetic rescue from rod degeneration
Author Affiliations & Notes
  • Miranda Scalabrino
    Neurobiology, Duke University, Durham, North Carolina, United States
  • Mishek Thapa
    Neurobiology, Duke University, Durham, North Carolina, United States
  • Emily Davis
    Neurobiology, Duke University, Durham, North Carolina, United States
  • Alapakkam P Sampath
    Ophthalmology, University of California Los Angeles David Geffen School of Medicine, Los Angeles, California, United States
  • Jeannie Chen
    Cell & Neurobiology, University of Southern California, Los Angeles, California, United States
  • Greg Field
    Neurobiology, Duke University, Durham, North Carolina, United States
  • Footnotes
    Commercial Relationships   Miranda Scalabrino, None; Mishek Thapa, None; Emily Davis, None; Alapakkam Sampath, None; Jeannie Chen, None; Greg Field, None
  • Footnotes
    Support  NIH R01 EY027193, Holland-Trice Award
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 3146. doi:
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      Miranda Scalabrino, Mishek Thapa, Emily Davis, Alapakkam P Sampath, Jeannie Chen, Greg Field; Time-dependent changes in ON bipolar cell transcriptomes before and after genetic rescue from rod degeneration. Invest. Ophthalmol. Vis. Sci. 2021;62(8):3146.

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

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Abstract

Purpose : Photoreceptor death induces remodeling of bipolar cell (BC) dendrites. This remodeling is accompanied by changes in gene expression, but the gene networks that are involved in this rewiring are poorly understood. Furthermore, gene therapies are being developed to halt and rescue photoreceptors from degeneration. The halting of photoreceptor death at different timepoints in the degeneration process is likely to have distinct consequences on the morphologies and gene expression patterns of postsynaptic BCs. The purpose of this study was to examine how progressive photoreceptor degeneration impacts the transcriptomes of BCs and how these transcriptomes are altered by the genetic rescue of rods from degeneration.

Methods : Mice with a floxed neomycin cassette inserted into the Cngb1 locus (Cngb1neo/neo) were crossed with UBC-cre and Grm6-GFP mice to create a triple transgenic line. These mice express green fluorescent protein (GFP) in BCs and exhibit a slow form of rod degeneration (all rods are lost ~6 months). Also, this line allows for genetic rescue of rods by tamoxifen-induced cre-recombination that triggers the expression of Cngb1 from the endogenous locus. Littermates heterozygous for the neomycin cassette were used as controls. At designated ages, mice were sacrificed, retinas dissociated using papain, and FACS sorted into GFP+ and GFP- cell populations, which yielded relatively pure populations of sorted BCs for RNA extraction and transcriptomic analysis.

Results : We found significant BC gene expression differences between groups that depended on the amount of rod degeneration and the time-point of rod rescue: WT vs degenerating (P30, P90, P210), untreated vs treated, and early treatment (Tx at P30) vs late treatment (Tx at P90). There were fewer differentially expressed genes between late-stage degenerating bipolar cells (P90 untreated) and late treated bipolar cells (Tx at P90, sac at P150), indicating that late therapy is not fully reversing pathology.

Conclusions : These results identify gene networks in postsynaptic BCs that respond to rod degeneration and death. These results also point toward the development of novel therapies to ameliorate blinding conditions and increase the effectiveness of vision restoration.

This is a 2021 ARVO Annual Meeting abstract.

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