Abstract
Purpose :
TrkB signalling is essential for maturation of photoreceptors during development and promote their survival. Deoxygedunin is naturally occurring tetranortriterpenoid that binds to extracellular domain of TrkB, stimulates its dimerisation and promotes downstream signalling pathways. This study investigated whether deoxygedunin stimulated TrkB receptor intracellular signalling cascade in 661W photoreceptor cell line and evaluated potential mechanism of its action.
Methods :
Mouse retinal photoreceptors 661W (3x105 cell/ml) were cultured in serum-free DMEM media overnight. Cells were treated with deoxygedunin (1µM) and downstream effects on TrkB and downstream intracellular signalling pathways investigated using western blotting. Cells were also subjected to pharmacological treatments with TrkB antagonist Cyclotraxin-B and p75NTR inhibitor TAT PEP-5 (TAT). The band intensities were quantified in the linear range of intensity and data analysed using ImageJ software.
Results :
Our results indicated that TrkB was expressed in 661W cells and was significantly phosphorylated (Tyr 515) upon treatment with deoxygedunin (n=3, 1.5 fold, p<0.05). Deoxygedunin treatment also resulted in significantly elevated pAkt (Ser 473) (p<0.05) and pSTAT3 (Y705) (p<0.01) levels (n=3). TrkB Y515 phosphorylation in deoxygedunin treated cells was significantly decreased when the cells were co-treated with TrkB antagonist cyclotraxin-B (n=3,p <0.05). In contrast, deoxygedunin treatment resulted in TrkB Y515 activation in cells that were also co-treated with p75NTR antagonist TAT and a sustained upregulation of downstream signalling pathways marked by pAkt Ser473 (p<0.05), pErk1/2 (T202/Y204) (p<0.001), pSTAT3 Y705 (p<0.015) was observed.
Conclusions :
Deoxygedunin is a potent TrkB agonist and can activate downstream survival signalling pathways through TrkB activation, independent of any exogenously supplied BDNF in the 661W photoreceptor cells. Deoxygedunin thus imitates BDNF actions and could potentially provide a therapeutic tool for in vivo neuroprotection of photoreceptors in the retina.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.