Purchase this article with an account.
Chitra Joseph, Karthik Kamath, Filip Veljanoski, Yunqi Wu, Abubakar Siddiq Mangani, Mehdi Mirzaei, Vivek Kumar Gupta, Stuart L Graham; BDNF mediated non-canonical regulation of transcription factor and cytoskeletal proteins in retinal photoreceptor cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6049.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The neuroprotective role of brain derived neurotrophic factor (BDNF) has been shown to augment the survival of photoreceptors and other neuronal cells under stress conditions, however, a detailed molecular network of intracellular targets affected by BDNF still remain elusive. We performed a comprehensive phosphoproteome analysis of BDNF treated 661W photoreceptor cells aiming to identify cellular pathways that are directly modulated in response to BDNF.
661W cells were cultured in high glucose DMEM medium,37°C. Following overnight serum starvation, cells were treated with 50ng/ml BDNF for two hours and mechanically lysed. Extracted proteins from treated and control samples (n:3) were subsequently reduced (DTT), alkylated (iodoacetamide), quantified and digested with trypsin followed by phosphopeptide enrichment using titanium dioxide. Phosphopeptides were analyzed using a quadrupole-orbitrap mass spectrometer (Q-Exactive, Thermo, USA) succeeded by identification and quantification using Proteome Discoverer-2.2 (Thermo). After screening for high confident phospho-peptides (FDR<1%), peptide abundance was compared using t-tests. Phosphosites with fold-change threshold ±1.5 and p-value <0.05 were considered as differentially abundant in expression.
The label-free quantitative proteomics analysis identified a total of 5964 non-redundant phosphosites in BDNF treated and control cells amongst which 252 were differentially expressed. Two distinct clusters comprising 134 upregulated and 118 downregulated phosphosites with BDNF treatment were identified. Apart from activation of known BDNF targets PI3K and Ras, our data revealed elevated levels of transcription factors JunB, Taf6, Gcfc2, Etv6, Tfe3, Fosl2 and cytoskeletal proteins Cttn, Ablim1, Zyx and Nudc(≥2 fold). Further, significant downregulation of Gtf2f1,Tcf4,Zfp423 transcription factors, and microtubule associated proteins (MAP)- Mapt, Map2, Map1b and Map4 (≤0.5 fold)was observed.
This study mapped the phosphoproteome profile of retinal photoreceptor cells in response to BDNF and identified several hitherto unknown targets. The novel changes in transcription factors and cytoskeletal proteins reveal the role of BDNF in modifying non-canonical signaling cascades in photoreceptors. Modulation of MAP proteins is of special interest due to the involvement of these proteins in neuronal diseases.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
This PDF is available to Subscribers Only