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Chitra Joseph, Vivek Kumar Gupta, Stuart L Graham; BDNF effects on retina derived 661W cells indicate distinct photoreceptor intracellular signalling pathways. Invest. Ophthalmol. Vis. Sci. 2018;59(9):976.
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© ARVO (1962-2015); The Authors (2016-present)
Brain derived neurotrophic factor (BDNF) is a major neurotrophin that acts primarily via its high affinity receptor TrkB phosphorylation and mediates the activation of several downstream pathways involved in synapsis regulation, neurogenesis and neuroprotection in the central nervous system. BDNF overexpression and exogenous treatment with BDNF have been shown to augment the survival of photoreceptors. It is debated whether photoreceptor cells are sensory neurons. This study aimed to evaluate the differential effects of BDNF on the downstream intracellular signalling pathways in the 661W photoreceptor cells and compare the findings with SHSY5Y sensory neurons.
661W cone photoreceptor cells were treated with BDNF (50ng/ml) for different time intervals 5 mins, 15mins, 1 hr and 6 hrs. For comparison, SHSY5Y cells were similarly treated with BDNF after differentiation to sensory neurons with 5µm retinoic acid for 4 days. The cells were harvested, lysates prepared and subjected to immunoblot analysis using specific antibodies (Bio-Rad ChemiDoc). The band intensities were quantified in the linear range of detection and data analysed (Image J).
BDNF treatment of 661W cells resulted in substantial phosphorylation of Akt (pSer473) (40±5 fold; p<0.03) and increased pSer9 GSK3β reactivity (p<0.004) within 5 mins. In contrast, significant phosphorylation increase in pAkt (p<0.05) and pGSK3β (2.2±0.17; p<0.002) was observed in differentiated SHSY5Y cells only after 6 hrs. The pattern was reversed with JAK1 (Y1034/1035) phosphorylation, showing 70±5 fold phosphorylation increase in 5 mins in differentiated SHSY5Y cells (p<0.03) while 661W cells demonstrated increased JAK1 phosphorylation at 6hr time interval (p<0.05). 661W cells exhibited immediate STAT3 (Y705) phosphorylation (25±5 fold; p<0.05) as also differentiated SHSY5Y cells (2.6±0.6 fold; p<0.02).
This study provides insights into the unique BDNF downstream signalling pathways in 661W photoreceptor cells highlighting their similarities and differences with the SHSY5Y sensory neurons. Greater understanding of the BDNF actions on photoreceptors in vivo will help understand the photoreceptor biology and help in the specific targeting of BDNF signalling pathway to enhance neuroprotection.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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