July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Identification of tau protein kinases in mixed rat retinal cell cultures
Author Affiliations & Notes
  • Teresa Mammone
    Ophthalmic Research Laboratories, University of Adelaide, Adelaide, South Australia, Australia
  • Glyn Chidlow
    Ophthalmic Research Laboratories, University of Adelaide, Adelaide, South Australia, Australia
  • Robert James Casson
    Ophthalmic Research Laboratories, University of Adelaide, Adelaide, South Australia, Australia
  • John P M Wood
    Ophthalmic Research Laboratories, University of Adelaide, Adelaide, South Australia, Australia
  • Footnotes
    Commercial Relationships   Teresa Mammone, None; Glyn Chidlow, None; Robert Casson, None; John Wood, None
  • Footnotes
    Support  BrightFocus Foundation (Grant G2013135)
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 5997. doi:https://doi.org/
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    • Get Citation

      Teresa Mammone, Glyn Chidlow, Robert James Casson, John P M Wood; Identification of tau protein kinases in mixed rat retinal cell cultures. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5997. doi: https://doi.org/.

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

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Abstract

Purpose : Tau is a microtubule associated protein. Abnormal tau phosphorylation can impair neuronal function. We aimed to understand more about the relationship between tau phosphorylation and retinal pathology by identifying the protein kinases involved in this process

Methods : An in vitro model of tau phosphorylation was established using a mixed rat retinal cell culture. Tau phosphorylation was examined under basal conditions and after elevation with the protein phosphatase inhibitor, calyculin A (CA). The optimal CA regimen to detect tau phosphorylation was determined by Western immunoblot and immunocytochemistry. A range of protein kinase inhibitors were tested to see whether they blocked CA-induced tau phosphorylation; LiCl (glycogen synthase kinase), calphostin C (protein kinase C), SP600125 (SAPK), FR180204 (P42/44 MAPK) and roscovitine (Cdk5). Concurrent toxicity to cells was also examined

Results : Tau phosphorylation was seen under basal conditions in retinal cell cultures. CA markedly stimulated tau phosphorylation, particularly at residue T181. Optimal phosphorylation was determined at a concentration of 5nM CA, with a 1 hour treatment. This treatment provided no obvious adverse effects to glia or neurons. The effect of selected protein kinase inhibitors were then tested. LiCl, calphostin C, SP600125 and FR180204 all showed no effect on baseline phosphorylation compared to control. A modest but significant reduction of 10-20% in tau phosphorylation was observed by each inhibitor. However, roscovitine demonstrated both an approximate 40% reduction in basal phosphorylation of tau at residue T181 and a particularly marked reduction of 80% phosphorylation in the presence of CA. In no case did treatment with any protein kinase inhibitor have any apparent detrimental effect to retinal cells

Conclusions : These results demonstrate that CA was effective in elevating tau phosphorylation at various epitopes to a detectable degree, creating a model system with which to identify tau kinases in retinal cells. It can thus be considered as an in vitro model of tau phosphorylation for potentially studying tauopathies. The results also suggest that several different protein kinases can contribute to phosphorylation of Tau at epitope T181, but, in particular, Cdk5. Inhibitors of these kinases may potentially provide a clinical avenue for treatment of retinal diseases where tau phosphorylation is known to occur, such as glaucoma

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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