Abstract
Purpose: :
To investigate the role and regulation of alpha-crystallin proteins in the retinal cells in relation with stress such as diabetic retinopathy. We previously showed that alpha-crystallins are expressed in the retina and over-expressed during diabetes. We also demonstrated that they have neuroprotective functions in retinal neurons but that these functions could be altered by diabetes by a mechanism that remained unclear.
Methods: :
Mice lacking either one, or both, of the alpha-crystallins were used to analyze the regulation in the retina of the remaining crystallins as well as the impact on retinal cell survival in response to diabetes. Protein changes were analyzed using western-blot and immunohistochemistry analysis, respectively, to determine levels of protein expression and cellular localization while cell death was assessed using a Cell Death DNA fragmentation Elisa assay. Alpha-crystallins were isolated from retinal tissues of control and diabetic animal models by immunoprecipitation methods and post-translational modifications were then characterized using mass spectrometry analysis.
Results: :
We demonstrated that the diabetes-induced crystallin upregulation is not a general stress response leading to the induction of all heat-shock proteins. It is rather an adaptive mechanism that becomes negated through different post-translational modifications. Using alpha-crystallin knock-out mice we demonstrated that the absence of alphaA-crystallin led to a larger increase in retinal cell death during dibetes. We also showed that alpha-crystallins are targeted for post-translational modifications in the retina during diabetes. Using biochemical and proteomic method, we identified and studied specific alphaA- and alphaB-crystallin phosphorylation sites.
Conclusions: :
This study demonstrates the crucial but differential neuroprotective function that both alphaA- and alphaB-crystallin play in retinal degenerative diseases such as diabetic retinopathy. This study also points out the specificity of the response in regard to heat shock proteins as well as how this function is altered in these diseases. Understanding alpha-crystallins function and their regulation in these conditions could lead to the development of new therapies.
Keywords: crystallins • diabetic retinopathy • cell survival