June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Analysis of Secretory Lipidomics and Proteomics of Late-Onset Retinal Degeneration iPSC-derived RPE
Author Affiliations & Notes
  • Kiyoharu J Miyagishima
    NEI, NIH, Bethesda, Maryland, United States
  • Ruchi Sharma
    NEI, NIH, Bethesda, Maryland, United States
  • Katharina Clore-Gronenborn
    NEI, NIH, Bethesda, Maryland, United States
  • Zoya Qureshy
    NEI, NIH, Bethesda, Maryland, United States
  • Bokkyoo Jun
    Neuroscience Center of Excellence, Louisiana State University Health, New Orleans, Louisiana, United States
  • William C Gordon
    Neuroscience Center of Excellence, Louisiana State University Health, New Orleans, Louisiana, United States
  • Nathan Hotaling
    NEI, NIH, Bethesda, Maryland, United States
  • Congxiao Zhang
    NEI, NIH, Bethesda, Maryland, United States
  • Catherine A Cukras
    NEI, NIH, Bethesda, Maryland, United States
  • Paul A Sieving
    NEI, NIH, Bethesda, Maryland, United States
  • Nicolas Guillermo Bazan
    Neuroscience Center of Excellence, Louisiana State University Health, New Orleans, Louisiana, United States
  • Sheldon S Miller
    NEI, NIH, Bethesda, Maryland, United States
  • Kapil Bharti
    NEI, NIH, Bethesda, Maryland, United States
  • Footnotes
    Commercial Relationships   Kiyoharu Miyagishima, None; Ruchi Sharma, None; Katharina Clore-Gronenborn, None; Zoya Qureshy, None; Bokkyoo Jun, None; William Gordon, None; Nathan Hotaling, None; Congxiao Zhang, None; Catherine Cukras, None; Paul Sieving, None; Nicolas Bazan, None; Sheldon Miller, None; Kapil Bharti, None
  • Footnotes
    Support  Supported by NEI IRP, NEI, EY005121, NIGMS ,GM103340 and the Eye, Ear, Nose and Throat Foundation (NGB)
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3019. doi:
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      Kiyoharu J Miyagishima, Ruchi Sharma, Katharina Clore-Gronenborn, Zoya Qureshy, Bokkyoo Jun, William C Gordon, Nathan Hotaling, Congxiao Zhang, Catherine A Cukras, Paul A Sieving, Nicolas Guillermo Bazan, Sheldon S Miller, Kapil Bharti; Analysis of Secretory Lipidomics and Proteomics of Late-Onset Retinal Degeneration iPSC-derived RPE. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3019.

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

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Abstract

Purpose : Late-Onset Retinal Degeneration (L-ORD) is a rare autosomal dominant disorder caused by a single S163R amino acid substitution in a secreted protein known as CTRP5. The disease phenotype (delayed dark adaptation, sub-RPE deposits) is reminiscent of more prevalent retinal degenerations, thus providing valuable insight into the shared mechanisms underlying their pathogenesis.

Methods : Fibroblasts from two L-ORD affected and two unaffected siblings were reprogrammed into induced pluripotent stem cells (iPSCs) using sendai viral vectors containing the four Yamanaka factors. iPSCs were fully characterized via immunocytochemistry, karyotyping, and spontaneous differentiation of embryoid bodies into all three germ layers. iPSCs were then differentiated into RPE using a developmentally guided protocol. Cell shapemetric analysis was performed using custom software on iPSC-derived RPE stained with ZO-1. Apical media was analyzed for protein and lipid composition.

Results : Although iPSC-derived RPE from patients and unaffected siblings have similar hexagonality (score, side ratio, area ratio), L-ORD patient iPSC-derived RPE tended be more variable in size and on average possessed greater cell areas. Mass Spect analysis revealed elevated sequence coverage of glutathione, cystatin C, keratins, vimentin, actin, and tubulin secreted by patient iPSC-RPE. Lipidomic analysis of patient iPSC-RPE showed higher secretion levels of very long chain- polyunsaturated fatty acids (VLC-PUFAs) corresponding with higher DHA secretion into the apical media compared to unaffected siblings.

Conclusions : The more variable and larger RPE cell sizes in L-ORD patient iPSC-RPE are indicative of cytoskeletal rearrangement perhaps resulting from enhanced uncompensated oxidative stress. In agreement, proteomic analysis of the apical media revealed proteins identified as biomarkers for oxidative stress. Lastly, lipidomic analysis revealed an over-abundance of DHA and VLC-PUFAs in L-ORD patient iPSC-RPE suggesting that elevated levels of DHA may reflect an attempt to set in motion protective instructive signals from DHA and when overproduced may contribute to lipid peroxidation and susceptibility of these cells to oxidative stress-mediated damage.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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