Determining the localization of Tubulin Polymerization Promoting Protein TPPP/p25 in the mice and human retina: effect of zinc supplementation

Abstract

Purpose : Tubulin Polymerization Promoting Protein (TPPP/p25) plays an important role stabilizing microtubule structures and is able to bind zinc through a zinc finger motif that stabilizes the protein. Therefore, in zinc rich tissues like the retina, if it is present, TPPP/p25 might play an important role in tubulin polymerization and acetylation and/or microtubule bundling. In this study we investigated TPPP/p25 localization in mice and human retina and the effect of zinc supplementation on protein levels.

Methods : Human eyes were obtained from UCL Institute of Ophthalmology and Boston University with Institutional Ethics Committee approval. Eyes from control and Zn supplemented mice were obtained from George Mason University. We used specific mono- and polyclonal antibodies that were visualized by confocal and electron microscopy to detect TPPP/p25, GABA and Glycine immunoreactivity. Quantitative Western blot was used to determine protein concentration.

Results : TPPP/p25 immunoreactivity was localized to the inner plexiform layer (IPL; S1, S3 and S5 sub-laminas) and some but not all cell bodies of amacrine cells (AC) in both mice and human retina. Labelled cell bodies were associated with interplexyform projections and cells that were identified as displaced AC. TPPP/p25 positive cells were co-labelled with GABA and/or Glycine antibodies. Immunogold labeling localized TPPP/p25 to boutons with few synaptic vesicles, cell nuclei and cytoplasm. Quantitative Western blot of human retinal extracts showed that the level of TPPP/p25 was 0.05-0.13 ng/µg protein, comparable to that of brain tissues. Interestingly, TPPP/p25 levels showed a central to peripheral retinal gradient. The levels of TPPP/p25 immunoreactivity significantly increased by oral zinc supplementation but only in central retinal regions in mice eyes.

Conclusions : These data suggest that TPPP/p25 has a very specific localization to the IPL and some but not all AC. Therefore, TPPP/p25 might play a role in the organization and reorganization of the synaptic architecture and visual integration. The increased immunoreactivity suggests that changes in zinc levels can directly affect level of TPPP/p25 in the IPL and as such affects the synaptic connectivity mainly on the central retina.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.