June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Sphingolipid biosynthetic inhibitor, SPT-I, prevents oxidative-stress-mediated cell death in mouse photoreceptor-derived 661W cells
Author Affiliations & Notes
  • Faiza Tahia
    Pharmaceutical Sciences, The University of Tennessee Health Science Center, Memphis, Tennessee, United States
    Ophthalmology, The University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Sandip Basu
    Ophthalmology, The University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Koushik Mondal
    Ophthalmology, The University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Harry Kochat
    Pharmaceutical Sciences, The University of Tennessee Health Science Center, Memphis, Tennessee, United States
    Plough Center for Sterile Drug Delivery Solutions, The University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Kennard Brown
    Plough Center for Sterile Drug Delivery Solutions, The University of Tennessee Health Science Center, Memphis, Tennessee, United States
    Executive Vice Chancellor and Chief Operations Officer, The University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Nawajes Mandal
    Ophthalmology, The University of Tennessee Health Science Center, Memphis, Tennessee, United States
    Pharmaceutical Sciences, Anatomy and Neurobiology, The University of Tennessee Health Science Center, Memphis, Tennessee, United States
  • Footnotes
    Commercial Relationships   Faiza Tahia None; Sandip Basu None; Koushik Mondal None; Harry Kochat None; Kennard Brown None; Nawajes Mandal None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 1610. doi:
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      Faiza Tahia, Sandip Basu, Koushik Mondal, Harry Kochat, Kennard Brown, Nawajes Mandal; Sphingolipid biosynthetic inhibitor, SPT-I, prevents oxidative-stress-mediated cell death in mouse photoreceptor-derived 661W cells. Invest. Ophthalmol. Vis. Sci. 2023;64(8):1610.

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

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Abstract

Purpose : Photoreceptor cell death culminates into vision loss in many forms of heterogeneous retinal degenerative diseases (RD) like Retinitis Pigmentosa and Age-related Macular Degeneration. A complex inheritance pattern and environmental factors like white light exposure, oxidative damage, and inflammatory stresses are some of the common pathogenic contributors to photoreceptor cell death. There is no effective treatment available in these various forms of RD, and recent studies have recognized bioactive sphingolipid, ceramide, as a second messenger activating the process of photoreceptor cell death. We hypothesize that SPT-I, the small molecule inhibitor of the first enzyme, Serine Palmitoyl Transferase (SPT), for de novo ceramide biosynthesis, can inhibit photoreceptor cell death by preventing ceramide generation.

Methods : 661W cells were cultured and subjected to different doses of H2O2 (100-750 uM) for 3 hours to provide oxidative stress. 300 uM H2O2 was chosen to provide an optimum level of stress, against which different doses of SPT-I (5-50 uM) were tested to see its cytoprotective effect. Cell viability was determined by measuring the released lactate dehydrogenase in the cell culture medium. The involvement of oxidative stress, apoptosis, and sphingolipid biosynthesis pathway was determined by analyzing RNA and protein expression from the treated cells. The effect of SPT-I on ceramide biosynthesis was tested by treating the cells with different doses of SPT-I (10-50 uM) and measuring the ceramide levels by mass spectrometry.

Results : Treatment with 300 uM H2O2 for 3 hours in 661W cells caused 20% cell death. Co-treatment with SPT-I significantly protected the cells from H2O2 stress in a dose-dependent manner. Gene expression analysis reveal significantly induced expression of multiple genes, particularly heme-oxygenase 1 (Ho1), compared to the control, and co-treatment with SPT-I, significantly reduced the expression of these genes back to normal. The sphingolipid analysis suggested SPT-I can cause a significant reduction of ceramide synthesis in a dose-dependent manner in 661W cells.

Conclusions : We observed significant protection of 661W cells by SPT-I against H2O2-induced cell death. Our results suggest ceramide biosynthesis can be targeted with a novel drug candidate to reduce photoreceptor cell death in various human retinal degenerative diseases.

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

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