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Abubakar Siddiq Mangani, Chitra Joseph, Mehdi Mirzaei, Vivek Kumar Gupta, Stuart L Graham; Deoxygedunin activates autophagic mechanisms in retinal cells through its modulatory effects on TrkB and p75NTR. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5992.
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© ARVO (1962-2015); The Authors (2016-present)
Autophagy maintains cellular homeostasis by clearing misfolded proteins and helps prevent neurodegeneration. It is involved in retinal physiology and protects the cells against stress with defective autophagy implicated in retinal neurodegeneration. A natural compound, deoxygedunin (DG) has been shown to be a potent BDNF mimetic with neurotrophic activity. We investigated mechanisms underlying DG actions and determined its modulatory effects on autophagy pathways in culture in photoreceptor cells and mice retinas.
661W photoreceptor cells (3x105) were subjected to pharmacological treatments with DG and TrkB/ p75NTR antagonists- Cyclotraxin-B (CTX-B) and TAT PEP-5 (TAT) respectively. Alterations in autophagic flux were investigated by treating cells with (V)-ATPase inhibitor, Bafilomycin A1. Autophagic changes were probed in mice retinas ex vivo by incubating fresh retinas with various pharmacological modulators in culture media. Autophagy markers were assessed by WB of sample lysates and immunocytochemistry analysis using specific antibodies (n=3 each).
DG treatment resulted in elevated autophagy response demonstrated by increased pmTOR S2448 (p<0.05) and Beclin 1 (p<0.04) expression. Selectively blocking p75NTR and TrkB using TAT/ CTX-B resulted in decreased pmTOR s2448 and Beclin 1 levels and the loss was more profound upon TrkB inhibition. DG treatment further resulted in upregulation of ATG12 conjugate system consisting of Atg12(p<0.03), Atg5(p<0.05), Atg 16L1 (p<0.02) and LC3 A/B (p<0.05). TrkB and p75NTR inhibition resulted in reduced levels of these molecules although CTX-B treatment resulted in greater downregulation. DG treatment and blocking TrkB and p75NTR in mice retinas resulted in similar modulation of these autophagic networks. Bafilomycin A1 treatment of 661W cells (p<0.01) and retina (p<0.05) induced LC3 A/B upregulation, suggesting blocking of autophagosome fusion process with lysosomes, which was significantly rescued by DG.
DG positively regulates the autophagic mechanisms in photoreceptor cells and retinal tissue. Autophagy activation is mediated more robustly through TrkB but also via p75NTR as demonstrated by pharmacological blocking experiments. This study highlights the potential of DG as a retinal neuroprotective drug due to its effects on autophagy activation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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