July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Specific induction of the internalization and degradation of inflammatory Wnt5a in damaged RPE cells by neuroprotection D1 (NPD1).
Author Affiliations & Notes
  • Jorgelina Muriel Calandria
    Neuroscience Center, LSU Health Sciences Center, New Orleans, Louisiana, United States
  • Sayantani Kala-Bhattacharjee
    Neuroscience Center, LSU Health Sciences Center, New Orleans, Louisiana, United States
  • Nicolas G Bazan
    Neuroscience Center, LSU Health Sciences Center, New Orleans, Louisiana, United States
  • Footnotes
    Commercial Relationships   Jorgelina Calandria, None; Sayantani Kala-Bhattacharjee, None; Nicolas Bazan, None
  • Footnotes
    Support  Supported by NIGMS grant P30 GM103340 and NEI grant R01 EY005121, the Eye, Ear, Nose and Throat Foundation.
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 2463. doi:
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      Jorgelina Muriel Calandria, Sayantani Kala-Bhattacharjee, Nicolas G Bazan; Specific induction of the internalization and degradation of inflammatory Wnt5a in damaged RPE cells by neuroprotection D1 (NPD1).. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2463.

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

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Abstract

Purpose : We previously demonstrated that Wnt5a is expressed and secreted by RPE cells undergoing uncompensated oxidative stress (UOS). Wnt5a expression is mediated by NFκB/p65 as part of a positive feedback loop involving FZD5 and ROR2 receptors. At that time, we proposed that Wnt5a is secreted by affected tissue to induce apoptosis in susceptible cells with no effect on healthy tissue. NPD1 blocked the expression of Wnt5a and FZD5 and decreases Wnt5a secretion. We hypothesize now that NPD1 decrease the abundance of soluble Wnt5a by enhancing its internalization and degradation and thus interfering with the inflammatory response in a beneficial manner

Methods : mRNA was measured using SYBR green real-time PCR assay. Exosomes were obtained using two methods: a commercial one-step exosome isolation system and differential ultracentrifugation and precipitation of 100000-rpm supernatant. Wnt5a was detected via western blot or immunocytochemistry of human primary RPE cells undergoing uncompensated oxidative stress (UOS) triggered by 1600 µM H2O2 and 10 ng/ml TNFα, in the presence or absence of 100nM NPD1, 25 μM Pitstop2, 100 μg/ml Box5 (Wnt5a-FZD5 antagonist) and 50 ng/ml recombinant Wnt5a.

Results : Human primary RPE cells secreted Wnt5a in a non-exosome form. Wnt5a and FZD5 co-localized in vesicle-like structures inside human primary RPE cells suggesting that internalization of the ligand-receptor complex occurs. Recombinant Wnt5a addition increased and Box5 and NPD1 reduced the number of vesicles showing colocalization of FZD5 and Wnt5a. Wnt5a-FZD5 internalization utilizes clathrin-mediated endocytosis and its disruption affects the ability of Wnt5a to activate p65/NFkB in human primary RPE cells.

Conclusions : A systemic inflammatory role of Wnt5a is controlled by NPD1 at two main levels: transcriptional and via degradation. While the transcription modulation of Wnt5a is not specific of NPD1, the degradation occurring after internalization responds only to the mentioned lipid messenger, which make its effect inclusive to autocrine and paracrine signals. These events may be relevant to the onset and progression of dry form of age-related macular degeneration.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Proposed mechanism of internalization, recycling and degradation of Wnt5a in RPE cells.

Proposed mechanism of internalization, recycling and degradation of Wnt5a in RPE cells.

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