June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Low level glycogen synthase kinase 3 (GSK3) inhibition broadly reduces extracellular matrix protein production and pathology associated with a juvenile macular dystrophy
Author Affiliations & Notes
  • John Hulleman
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Sophia DiCesare
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Antonio J Ortega
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Gracen Collier
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Steffi Daniel
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Footnotes
    Commercial Relationships   John Hulleman Aldebaran Therapeutics, Code C (Consultant/Contractor); Sophia DiCesare None; Antonio Ortega None; Gracen Collier None; Steffi Daniel None
  • Footnotes
    Support  R01-EY027785, Fichtenbaum Charitable Trust, P30-EY030413, Hirl Endowed Research Fund
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 4435. doi:
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      John Hulleman, Sophia DiCesare, Antonio J Ortega, Gracen Collier, Steffi Daniel; Low level glycogen synthase kinase 3 (GSK3) inhibition broadly reduces extracellular matrix protein production and pathology associated with a juvenile macular dystrophy. Invest. Ophthalmol. Vis. Sci. 2023;64(8):4435.

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

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Abstract

Purpose : An R345W mutation in fibulin-3 (F3, aka EFEMP1) causes Doyne Honeycomb Retinal Dystrophy (DHRD), an autosomal dominant disease which bears similarities to age-related macular degeneration. Using high-throughput screening, we identified compounds that reduced the production of F3 from retinal pigmented epithelial (RPE) cells, serving as a potential therapy for DHRD. Identified hit compounds were enriched in GSK3 inhibitors. One compound, CHIR99021 (CHIR) was selected for in-depth analysis in RPE, fibroblast, and primary cells, followed by testing its effects in vivo in a mouse model of DHRD.

Methods : We endogenously tagged F3 with a luminescent peptide, HiBiT, to easily track the protein in immortalized human RPE cells, mouse fibroblasts, and primary dermal fibroblasts. HiBiT assays were used to quantify the fate of F3 after CHIR treatment (0.1-10 μM, 3-7 d). Unbiased omics techniques (RNAseq, label-free proteomics) were used to assess broad effects of CHIR treatment. CHIR was i.p.-administered to wild-type or R345W+/+ C57BL/6 mice followed by pharmacokinetic (PK) analysis, ERG, histology, and TEM for basal laminar deposit (BLamD) quantification.

Results : CHIR treatment (or genetic GSK3 knockdown) dose-dependently reduced F3 secretion and intracellular levels in all cell lines tested. qPCR demonstrated that this reduction was mediated through reduced F3 transcription. A dose of 1 μM CHIR for 7d was selected to prevent Wnt-based effects. RNAseq analysis ID’d 165 downregulated genes most of which were also confirmed by proteomics. GO analysis highlighted a significant downregulation of ECM genes and those related to sub-RPE deposit formation (e.g., F3, COL4A, AMTN, FN1, C3, etc). Moreover, CHIR reversed elevated MMP2 activity levels in R345W RPE cells. CHIR (25 mg/kg i.p.) readily entered the mouse retina in PK studies (up to 1.6 μM), and 1 mo CHIR (5 d/wk) had no effect on scotopic ERG or gross ocular histology. However, BLamD formation was reduced in CHIR-treated R345W+/+ mice (8.2% FOV positive for BLamD vs. 12.1% in controls).

Conclusions : Our data suggest that low-level GSK3 inhibition transcriptionally downregulates the production of multiple ECM genes involved in sub-RPE deposit formation, including F3, and may be an attractive target for prevention of sub-RPE deposits, including BLamDs.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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