Abstract
Purpose: :
Ongoing proteomics studies have identified many retinal proteins phosphorylated following experimental elevation of IOP. This study focused on 14-3-3 proteins identified through proteomic analysis.
Methods: :
IOP elevation was induced in rats by hypertonic saline injections into episcleral veins. Complementary proteomic approaches, including 2D-PAGE-based and gel-free approaches, were utilized to identify phosphorylated proteins during glaucomatous neurodegeneration. Specific protein complexes were eluted using co-immunoprecipitation and recombinant protein-based affinity pull-down for subsequent mass spectrometric analysis. For further validation of the proteomic findings, western blot analysis and immunohistochemistry were performed. To determine functional importance and phosphorylation dependence, in vivo treatment experiments were performed using D-JNKI1, a c-jun amino(N)-terminal kinase (JNK) inhibitor; and FK506, a protein phosphatase inhibitor.
Results: :
Findings of mass spectrometry, western blot analysis, and tissue immunolabeling revealed the presence of different 14-3-3 isotopes in RGCs and their up-regulation and phosphorylation during glaucomatous neurodegeneration. Consecutive experiments through proteomic analysis of eluted 14-3-3-containing protein complexes identified various interacting proteins, which included calmodulin and a pro-apoptotic member of the Bcl-2 family, Bad. A series of experiments supported that 14-3-3 binding to phospho-Bad keeps this pro-apoptotic protein sequestered in the cytoplasm. However, this association was disrupted in ocular hypertensive eyes in correlation with Bad dephosphorylation and 14-3-3 phosphorylation, thereby leading to mitochondrial translocation of Bad for apoptotic function. Inhibition of JNK activity and inhibition of protein phosphatase activity complementarily secured the 14-3-3-scaffold of Bad in the cytoplasm and preserved optic nerve axons in ocular hypertensive eyes.
Conclusions: :
These findings identify that an important protein family associated with checkpoint control pathways, 14-3-3, is involved in cellular signaling during glaucomatous neurodegeneration in a phosphorylation-dependent manner. The 14-3-3 appears to constitute an important regulatory pathway of cell death signaling, which controls the subcellular localization and function of many important proteins, including Bad.
Keywords: ganglion cells • mitochondria • neuroprotection