May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Anti–Enolase Antibodies Block Enolase Function and Induce Apoptosis of Retinal Cells
Author Affiliations & Notes
  • G. Adamus
    Neurological Sciences Inst, Oregon Health Sciences Univ, Beaverton, OR
  • A. Magrys
    Neurological Sciences Inst, Oregon Health Sciences Univ, Beaverton, OR
  • G. Ren
    Neurological Sciences Inst, Oregon Health Sciences Univ, Beaverton, OR
  • Footnotes
    Commercial Relationships  G. Adamus, None; A. Magrys, None; G. Ren, None.
  • Footnotes
    Support  NIH Grant EY13053
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 3478. doi:
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      G. Adamus, A. Magrys, G. Ren; Anti–Enolase Antibodies Block Enolase Function and Induce Apoptosis of Retinal Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3478.

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

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Abstract

Abstract: : Purpose: Autoantibodies against α–enolase, a glycolytic enzyme, are often associated with visual loss in patients with paraneoplastic and autoimmune retinopathy, but their role in pathogenesis is not fully understood. Our objective was to determine whether anti–enolase antibodies are responsible for blocking the function of enolase in retinal cells, which in effect would activate the apoptotic death of retinal cells. Methods: We generated a monoclonal antibody against retinal α–enolase Enol–1, which we used in subsequent experiments. Anti–recoverin antibody Rec–1 was used as a positive control. Enolase activity was measured spectrophotometrically in E1A.NR3 retinal cells (developed by Dr. Gail Seigel) treated and untreated with Enol–1 antibody using the enolase substrate 2–phosphoglycerate. The apoptotic protein Bax was evaluated by immunocytochemistry. The mitochondrial potential of antibody–treated and –untreated retinal cells was examined with the fluorescent cationic dye JC–1. Caspase 3 activation was determined by the application of caspase 3 fluorescent substrate DEVD (Magic Red) to antibody–treated and –untreated retinal cells. Results: Treatment of retinal cells with the anti–enolase antibody Enol–1 significantly blocked the activity of the enolase enzyme (p=0.0012). In the presence of cytochalasin B, an inhibitor of endocytosis and antibody uptake, enolase activity was not affected, suggesting that after rapid cellular entry, the antibody interacts with the enolase protein. The Enol–1 antibody induced the translocation of Bax to the cytoplasm and its aggregation in the mitochondria, events that usually correlate with the initiation of apoptosis. Antibody–treated, but not –untreated, retinal cells showed changes in mitochondrial potential, as determined by the application of JC–1 dye. Using the fluorescent substrate DEVD to detect caspase 3 activation, caspase 3 was detected 24 hours after the treatment of cells with Enol–1. Conclusions: We demonstrated that anti–enolase antibody is a potent inducer of mitochondrial injury and the caspase–3–dependent apoptotic pathway. Autoantibodies that are directed against this glycolytic enzyme by the blockage of the enolase function may be implicated in retinopathy pathogenesis in terms of the dysregulation of glucose metabolism in retinal cells.

Keywords: autoimmune disease • CAR • apoptosis/cell death 
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