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Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo

BACKGROUND: Cell-to-cell communication is vital for tissues to respond, adapt, and thrive in the prevailing milieu. Several mechanisms mediate intercellular signaling, including tunneling nanotubes, gap junctions, and extracellular vesicles (EV). Depending on local and systemic conditions, EVs may c...

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Autores principales: Risner, Michael L., Ribeiro, Marcio, McGrady, Nolan R., Kagitapalli, Bhanu S., Chamling, Xitiz, Zack, Donald J., Calkins, David J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10614343/
https://www.ncbi.nlm.nih.gov/pubmed/37904133
http://dx.doi.org/10.1186/s12964-023-01291-1
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author Risner, Michael L.
Ribeiro, Marcio
McGrady, Nolan R.
Kagitapalli, Bhanu S.
Chamling, Xitiz
Zack, Donald J.
Calkins, David J.
author_facet Risner, Michael L.
Ribeiro, Marcio
McGrady, Nolan R.
Kagitapalli, Bhanu S.
Chamling, Xitiz
Zack, Donald J.
Calkins, David J.
author_sort Risner, Michael L.
collection PubMed
description BACKGROUND: Cell-to-cell communication is vital for tissues to respond, adapt, and thrive in the prevailing milieu. Several mechanisms mediate intercellular signaling, including tunneling nanotubes, gap junctions, and extracellular vesicles (EV). Depending on local and systemic conditions, EVs may contain cargoes that promote survival, neuroprotection, or pathology. Our understanding of pathologic intercellular signaling has been bolstered by disease models using neurons derived from human pluripotent stems cells (hPSC). METHODS: Here, we used hPSC-derived retinal ganglion cells (hRGC) and the mouse visual system to investigate the influence of modulating EV generation on intercellular trafficking and cell survival. We probed the impact of EV modulation on cell survival by decreasing the catabolism of sphingomyelin into ceramide through inhibition of neutral sphingomyelinase (nSMase), using GW4869. We assayed for cell survival in vitro by probing for annexin A5, phosphatidylserine, viable mitochondria, and mitochondrial reactive oxygen species. In vivo, we performed intraocular injections of GW4869 and measured RGC and superior colliculus neuron density and RGC anterograde axon transport. RESULTS: Following twenty-four hours of dosing hRGCs with GW4869, we found that inhibition of nSMase decreased ceramide and enhanced GM1 ganglioside accumulation. This inhibition also reduced the density of small EVs, increased the density of large EVs, and enriched the pro-apoptotic protein, annexin A5. Reducing nSMase activity increased hRGC apoptosis initiation due to enhanced density and uptake of apoptotic particles, as identified by the annexin A5 binding phospholipid, phosphatidylserine. We assayed intercellular trafficking of mitochondria by developing a coculture system of GW4869-treated and naïve hRGCs. In treated cells, inhibition of nSMase reduced the number of viable mitochondria, while driving mitochondrial reactive oxygen species not only in treated, but also in naive hRGCs added in coculture. In mice, 20 days following a single intravitreal injection of GW4869, we found a significant loss of RGCs and their axonal recipient neurons in the superior colliculus. This followed a more dramatic reduction in anterograde RGC axon transport to the colliculus. CONCLUSION: Overall, our data suggest that perturbing the physiologic catabolism of sphingomyelin by inhibiting nSMase reorganizes plasma membrane associated sphingolipids, alters the profile of neuron-generated EVs, and promotes neurodegeneration in vitro and in vivo by shifting the balance of pro-survival versus -degenerative EVs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12964-023-01291-1.
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spelling pubmed-106143432023-10-31 Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo Risner, Michael L. Ribeiro, Marcio McGrady, Nolan R. Kagitapalli, Bhanu S. Chamling, Xitiz Zack, Donald J. Calkins, David J. Cell Commun Signal Research BACKGROUND: Cell-to-cell communication is vital for tissues to respond, adapt, and thrive in the prevailing milieu. Several mechanisms mediate intercellular signaling, including tunneling nanotubes, gap junctions, and extracellular vesicles (EV). Depending on local and systemic conditions, EVs may contain cargoes that promote survival, neuroprotection, or pathology. Our understanding of pathologic intercellular signaling has been bolstered by disease models using neurons derived from human pluripotent stems cells (hPSC). METHODS: Here, we used hPSC-derived retinal ganglion cells (hRGC) and the mouse visual system to investigate the influence of modulating EV generation on intercellular trafficking and cell survival. We probed the impact of EV modulation on cell survival by decreasing the catabolism of sphingomyelin into ceramide through inhibition of neutral sphingomyelinase (nSMase), using GW4869. We assayed for cell survival in vitro by probing for annexin A5, phosphatidylserine, viable mitochondria, and mitochondrial reactive oxygen species. In vivo, we performed intraocular injections of GW4869 and measured RGC and superior colliculus neuron density and RGC anterograde axon transport. RESULTS: Following twenty-four hours of dosing hRGCs with GW4869, we found that inhibition of nSMase decreased ceramide and enhanced GM1 ganglioside accumulation. This inhibition also reduced the density of small EVs, increased the density of large EVs, and enriched the pro-apoptotic protein, annexin A5. Reducing nSMase activity increased hRGC apoptosis initiation due to enhanced density and uptake of apoptotic particles, as identified by the annexin A5 binding phospholipid, phosphatidylserine. We assayed intercellular trafficking of mitochondria by developing a coculture system of GW4869-treated and naïve hRGCs. In treated cells, inhibition of nSMase reduced the number of viable mitochondria, while driving mitochondrial reactive oxygen species not only in treated, but also in naive hRGCs added in coculture. In mice, 20 days following a single intravitreal injection of GW4869, we found a significant loss of RGCs and their axonal recipient neurons in the superior colliculus. This followed a more dramatic reduction in anterograde RGC axon transport to the colliculus. CONCLUSION: Overall, our data suggest that perturbing the physiologic catabolism of sphingomyelin by inhibiting nSMase reorganizes plasma membrane associated sphingolipids, alters the profile of neuron-generated EVs, and promotes neurodegeneration in vitro and in vivo by shifting the balance of pro-survival versus -degenerative EVs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12964-023-01291-1. BioMed Central 2023-10-30 /pmc/articles/PMC10614343/ /pubmed/37904133 http://dx.doi.org/10.1186/s12964-023-01291-1 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Risner, Michael L.
Ribeiro, Marcio
McGrady, Nolan R.
Kagitapalli, Bhanu S.
Chamling, Xitiz
Zack, Donald J.
Calkins, David J.
Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo
title Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo
title_full Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo
title_fullStr Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo
title_full_unstemmed Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo
title_short Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo
title_sort neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10614343/
https://www.ncbi.nlm.nih.gov/pubmed/37904133
http://dx.doi.org/10.1186/s12964-023-01291-1
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