March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Disruption of the P4-ATPase Aminophospholipid Flippase Atp8a2 Gene Suggests a Role for Phosphatidylserine in Photoreceptor Outer Segments
Author Affiliations & Notes
  • Jonathan A. Coleman
    Biochemistry and Molecular Biology,
    University of British Columbia, Vancouver, British Columbia, Canada
  • Hidayat R. Djajadi
    Biochemistry and Molecular Biology,
    University of British Columbia, Vancouver, British Columbia, Canada
  • Laurie L. Molday
    Biochemistry and Molecular Biology,
    University of British Columbia, Vancouver, British Columbia, Canada
  • Robert S. Molday
    Biochemistry and Molecular Biology and Ophthalmology and Visual Sciences,
    University of British Columbia, Vancouver, British Columbia, Canada
  • Footnotes
    Commercial Relationships  Jonathan A. Coleman, None; Hidayat R. Djajadi, None; Laurie L. Molday, None; Robert S. Molday, None
  • Footnotes
    Support  CIHR, NEI, MVRF, and NSERC
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 743. doi:
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      Jonathan A. Coleman, Hidayat R. Djajadi, Laurie L. Molday, Robert S. Molday; Disruption of the P4-ATPase Aminophospholipid Flippase Atp8a2 Gene Suggests a Role for Phosphatidylserine in Photoreceptor Outer Segments. Invest. Ophthalmol. Vis. Sci. 2012;53(14):743.

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

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Abstract

Purpose: : ATP8A2 is a P4-ATPase which transports phosphatidylserine (PS) from the exocytoplasmic to the cytoplasmic side of photoreceptor outer segment (OS) disc membranes to generate and maintain PS asymmetry. The goal of this study was to determine the importance of ATP8A2 in photoreceptors and other cell types by the targeted disruption of the atp8a2 gene in mice.

Methods: : Exons 11 - 13 of atp8a2 were replaced using a targeting construct containing a neomycin cassette in hybrid 129 SvEv and C57BL/6 ES cells. Retina membranes were prepared using the Optiprep method. Lipids were separated by thin layer chromatography and quantified by phosphorus assays. Retinoids were analyzed by HPLC. Antibodies specific to OS proteins were used to detect their expression and localization by western blotting and immunofluorescence microscopy. Outer nuclear layer (ONL) thickness was measured by DAPI labeling using the optic nerve as a reference. Ultrathin sections of glutaraldehyde-fixed resin-embedded retina were analyzed by transmission electron microscopy (TEM).

Results: : Atp8a2 knockout mice are smaller and catatonic compared to wild type littermates. RT-PCR, western blotting, and immunocytochemistry confirmed the absence of ATP8A2 in the retinas of the atp8a2 knockout mice. CDC50A, the β-subunit of ATP8A2, was present at significantly lower levels. Opsin, CNGA1, ABCA4 and other outer segment (OS) proteins were reduced by approximately 50%, but correctly localized to the OS. The OS layer was approximately 50% shorter. OS contained significantly less phosphatidylserine and higher levels of phosphatidylcholine (PC). The number of photoreceptors was reduced by 15% at P23 with no further reduction evident at P50. The ultrastructure of the OS as visualized by EM appeared normal. Spectrophotometry and HPLC measurements revealed that rhodopsin and retinoid levels are 66% lower in the knockout retina.

Conclusions: : Analysis of atp8a2 knockout mice suggests that PS asymmetry and composition plays a role in photoreceptor OS morphogenesis possibly associated with the trafficking of proteins to the OS. PS asymmetry may also play a crucial role in protein trafficking in other neurons.

Keywords: photoreceptors • lipids • ion transporters 
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