September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Regulatory mechanisms of the neuroprotective function of αA- and αB-crystallins in retinal neurons and glia
Author Affiliations & Notes
  • Anne Ruebsam
    Departments of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan, United States
  • Mandy Losiewicz
    Departments of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan, United States
  • Jessica Jiang
    Departments of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan, United States
  • Patrice E Fort
    Departments of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan, United States
  • Footnotes
    Commercial Relationships   Anne Ruebsam, None; Mandy Losiewicz, None; Jessica Jiang, None; Patrice Fort, None
  • Footnotes
    Support  research fellowship by the Jackstaedt Foundation, Germany
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 3234. doi:
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    • Get Citation

      Anne Ruebsam, Mandy Losiewicz, Jessica Jiang, Patrice E Fort; Regulatory mechanisms of the neuroprotective function of αA- and αB-crystallins in retinal neurons and glia. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3234.

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

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Abstract

Purpose : αA- and αB-crystallins are potent regulators of stress-induced cell death in multiple retinal diseases and both are highly upregulated in models of type 1 and 2 diabetes. We presume that their initial upregulation serves as a neuroprotective function. In chronic diabetes the protective properties of α-crystallins are altered, coinciding with an increase in cell death. We have previously shown, that upregulation of αA- and αB-crystallin during diabetes is correlated with decreased phosphorylation on Serine 148 and increased phosphorylation on Serine 19, 45 and 59. This study aimed at evaluating the impact of these post-translational modifications on αA- and αB-crystallins protective function of retinal cells under metabolic stress.

Methods : Differentiated R28 cells, a model of retinal neurons, as well as rMC-1, a Müller cell line, were transfected with plasmids encoding either wild-type (WT), phosphomimetic, or non-phosphorylatable mutants of αA- or αB-crystallins, corresponding to the phosphorylation profiles described above. After confirmation of their distinct expression profile, cells were exposed to different metabolic stresses by switching to a medium with varying glucose (5 mM vs 25 mM) or serum concentrations (10% vs No FBS). Cell survival and proliferation were assessed with caspase 3/7 activity assay, DNA fragmentation assay and TUNEL staining. Additionally, functional changes were assessed by anatomical changes and gene and protein expression profiles analysis. Experiments were performed in triplicates in three independent experiments.

Results : Transfection with WT αA and αB-crystallins protects R28 and rMC-1 cells from metabolic stress-induced cell death compared to empty plasmid transfected controls. This protective function was lost when αA- and αB-crystallins were mutated on specific phosphosites shown to be affected by diabetes, and associated with changes of α-crystallins biochemical properties.

Conclusions : Neuronal cells, overexpressing αA- and αB-crystallins are at least transiently less sensitive to metabolic stresses. This effect is abolished by specific alteration of their phosphorylation profile, mimicking what is seen during diabetes and leading to an increased neuronal cell death. We conclude that αA- and αB- crystallins are neuroprotective and the phosphorylation of αA- and αB-crystallins serves as a key regulatory mechanism of their pro-survival function.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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