June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Inhibition of neutral sphingomyelinase enhances GM1 ganglioside expression and transfer of apoptotic cargoes
Author Affiliations & Notes
  • Michael Risner
    Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Marcio Ribeiro
    Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Nolan McGrady
    Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Donald J Zack
    Wilmer Eye Institute, Johns Hopkins Medicine, Baltimore, Maryland, United States
  • David J Calkins
    Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Footnotes
    Commercial Relationships   Michael Risner None; Marcio Ribeiro None; Nolan McGrady None; Donald Zack None; David Calkins None
  • Footnotes
    Support  NIH Grant U24-EY029903, Brightfocus Grant G2022011S
Investigative Ophthalmology & Visual Science June 2023, Vol.64, OD67. doi:
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    • Get Citation

      Michael Risner, Marcio Ribeiro, Nolan McGrady, Donald J Zack, David J Calkins; Inhibition of neutral sphingomyelinase enhances GM1 ganglioside expression and transfer of apoptotic cargoes. Invest. Ophthalmol. Vis. Sci. 2023;64(8):OD67.

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

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Abstract

Purpose : Sphingomyelin is localized within the plasma membrane, regulating membrane permeability and protein trafficking. Metabolism of sphingomyelin impacts intracellular and intercellular signaling. Here, we sought to test the impact of inhibiting degradation of sphingomyelin by neutral sphingomyelinase (nSMase) on cell viability and intercellular signaling in vitro.

Methods : We used H9-BRN3B-tdTomato-Thy1.2 human embryonic stem cells chemically induced toward a retinal ganglion cell (RGC) fate (hRGC). We assayed the impact of inhibiting nSMase with GW4869 (10 μM) on cell viability and intercellular transfer of material using a coculture system. We treated one cohort of hRGCs with GW4869 or DMSO (2.89%) for 24 h, and identfied these cells by labeling with cholera toxin subunit B 488 (CTB, donor cells). After 24 h, we plated a second cohort of naïve hRGCs (receiver cells). The next day, we prepared cells for live and fixed confocal imaging, and we obtained small extracellular vesicles (EV) from the supernatant by differential centrifugation. We assayed small EVs by particle tracking and western blot.

Results : GW4869 reduced nSMase2 expression (p=0.02), decreased the concentration of small EVs (135 to 165 nm, p≦0.01), and increased the concentration of larger EVs (195 to 405 nm, p<0.0001) in the pellet obtained from the hRGC supernatant. We found GW4869 enhanced CTB uptake by donor cells and transfer of CTB to receiver hRGCs (p≦0.008), but also, increased donor cell death (p=0.007). In agreement with these findings, we found GW4869 significantly increased GM1 ganglioside in hRGCs (p<0.0001), and cell death was correlated with increased annexin 5 expression in the EV pellet and phosphatidylserine (PS)-positive particles in the extracellular medium. During live imaging, we noticed hRGC membranes engulf extracellular PS-positive particles, followed by membrane blebbing, and increased PS fluorescence.

Conclusions : Our results indicate metabolism of sphingomyelin influences cell integrity by regulating GM1 ganglioside expression and impacts neighboring cells through intercellular transfer of apoptotic cargoes.

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

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