June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Ceruloplasmin gene transfer into the retina causes increased retinal iron levels and retinal degeneration in ceruloplasmin/hephaestin-deficient mice
Author Affiliations & Notes
  • Rupak Bhuyan
    Sheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Delu Song
    Sheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Joshua L. Dunaief
    Sheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Rupak Bhuyan, None; Delu Song, None; Joshua Dunaief, None
  • Footnotes
    Support  NIH Grant EY015240, NIH Grant KL2TR001879
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 232. doi:
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      Rupak Bhuyan, Delu Song, Joshua L. Dunaief; Ceruloplasmin gene transfer into the retina causes increased retinal iron levels and retinal degeneration in ceruloplasmin/hephaestin-deficient mice. Invest. Ophthalmol. Vis. Sci. 2017;58(8):232.

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

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Abstract

Purpose : A mouse model systemically deficient in the ferroxidases ceruloplasmin (Cp) and hephaestin (Heph) exhibits retinal iron overload similar to that seen in humans with aceruloplasminemia. We tested the hypothesis that retinal ceruloplasmin production in this mouse model can protect against retinal iron accumulation.

Methods : 8-wk-old wild-type (WT) and Cp/Heph double knock-out (DKO) C57BL/6J mice received intravitreal injections of adeno-associated virus (AAV) containing human Cp cDNA into each OS (AAV-Cp). Control OD eyes were injected with GFP (AAV-GFP) for early DKO trials and null vector (AAV-null) for later DKO and WT trials. Fundoscopy was performed 1 wk post injection for WT and 7 mos post injection for DKO. Enucleation occurred immediately after imaging for flat mount preparation. Cp and transferrin receptor (Tfrc) mRNAs were quantified by qPCR to ensure transduction and to measure iron levels in neurosensory retina (NSR) and retinal pigment epithelium (RPE). Two-tailed student’s t-test and one-way ANOVA were used for statistical analyses.

Results : Imaging of AAV-Cp DKO mice at 9 mos of age revealed widespread hypopigmentation with increased lipofuscin autofluorescence (Fig 1,2). qPCR showed decreased Tfrc mRNA in AAV-Cp injected eyes from WT mice in the NSR, indicating increased iron levels. There was no difference in the RPE (n=8). Subgroup analysis showed no difference in Tfrc mRNA between AAV-Cp WT mice with high versus low Cp expression.

Conclusions : Instead of rescuing the DKO phenotype, intravitreal AAV-Cp injections worsened retinal degeneration as seen in fundus photographs and flat mounts. This degree of degeneration asymmetry between the experimental and control eyes has never been observed in uninjected DKO eyes (n>50). Moreover, lower Tfrc mRNA levels in the NSR of AAV-Cp WT mice suggest that the injections paradoxically caused retinal iron overload. The lack of differences in Tfrc mRNA levels in WT RPE suggests that intravitreal Cp delivery did not cause iron overload in the RPE in the short term. These results suggest that Cp in the neural retina may promote iron influx across the blood-retinal barrier.

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

 

Micron III fundoscopic images show extensive hypopigmentation (A) and lipofuscin autofluorescence (B) in an AAV-Cp DKO eye vs control.

Micron III fundoscopic images show extensive hypopigmentation (A) and lipofuscin autofluorescence (B) in an AAV-Cp DKO eye vs control.

 

Flat mount image shows greater lipofuscin autofluorescence in an AAV-Cp DKO eye vs control.

Flat mount image shows greater lipofuscin autofluorescence in an AAV-Cp DKO eye vs control.

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