Progesterone analogue protects stressed photoreceptors via bFGF-mediated calcium influx.
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Accepted Version
Date
2016-10-20
Authors
Wyse-Jackson, Alice C.
Roche, Sarah L.
Ruiz-Lopez, Ana M.
Moloney, Jennifer N.
Byrne, Ashleigh M.
Cotter, Thomas G.
Journal Title
Journal ISSN
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Publisher
Wiley
Published Version
Abstract
Retinitis pigmentosa (RP) is a degenerative retinal disease leading to photoreceptor cell loss. In 2011, our group identified the synthetic progesterone ‘Norgestrel’ as a potential treatment for RP. Subsequent research showed Norgestrel to work through progesterone receptor membrane component 1 (PGRMC1) activation and upregulation of neuroprotective basic fibroblast growth factor (bFGF). Using trophic factor deprivation of 661W photoreceptor-like cells, we aimed to further elucidate the mechanism leading to Norgestrel-induced neuroprotection. In the present manuscript, we show by flow cytometry and live-cell immunofluorescence that Norgestrel induces an increase in cytosolic calcium in both healthy and stressed 661Ws over 24h. Specific PGRMC1 inhibition by AG205 (1 μM) showed this rise to be PGRMC1-dependent, primarily utilising calcium from extracellular sources, for blockade of L-type calcium channels by verapamil (50 μM) prevented a Norgestrel-induced calcium influx in stressed cells. Calcium influx was also shown to be bFGF-dependent, for siRNA knock down of bFGF prevented Norgestrel-PGRMC1 induced changes in cytosolic calcium. Notably, we demonstrate PGRMC1-activation is necessary for Norgestrel-induced bFGF upregulation. We propose that Norgestrel protects through the following pathway: binding to and activating PGRMC1 expressed on the surface of photoreceptor cells, PGRMC1 activation drives bFGF upregulation and subsequent calcium influx. Importantly, raised intracellular calcium is critical to Norgestrel's protective efficacy, for extracellular calcium chelation by EGTA abrogates the protective effects of Norgestrel on stressed 661W cells in vitro.
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Keywords
Norgestrel , 661W , PGRMC1 , Neuroprotection , Retina
Citation
Wyse-Jackson, A. C., Roche, S. L., Ruiz-Lopez, A. M., Moloney, J. N., Byrne, A. M. and Cotter, T. G. (2016) 'Progesterone analogue protects stressed photoreceptors via bFGF-mediated calcium influx', European Journal of Neuroscience, 44(12), pp. 3067-3079. doi: 10.1111/ejn.13445
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Copyright
This is the peer reviewed version of the following article: Wyse-Jackson, A. C., Roche, S. L., Ruiz-Lopez, A. M., Moloney, J. N., Byrne, A. M. and Cotter, T. G. (2016) 'Progesterone analogue protects stressed photoreceptors via bFGF-mediated calcium influx', European Journal of Neuroscience, 44(12), pp. 3067-3079, doi: 10.1111/ejn.13445, which has been published in final form at https://doi.org/10.1111/ejn.13445. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.