The synthetic progesterone Norgestrel is neuroprotective in stressed photoreceptor‐like cells and retinal explants, mediating its effects via basic fibroblast growth factor, protein kinase A and glycogen synthase kinase 3β signalling

Abstract

The synthetic progesterone Norgestrel has been shown to have proven neuroprotective efficacy in two distinct models of retinitis pigmentosa: the rd10/rd10 (B6.CXBI-Pde6b(rd10)/J) mouse model and the Balb/c light-damage model. However, the cellular mechanism underlying this neuroprotection is still largely unknown. Therefore, this study aimed to examine the downstream signalling pathways associated with Norgestrel both in vitro and ex vivo. In this work, we identify the potential of Norgestrel to rescue stressed 661W photoreceptor-like cells and ex vivo retinal explants from cell death over 24 h. Norgestel is thought to work through an upregulation of neuroprotective basic fibroblast growth factor (bFGF). Analysis of 661W cells in vitro by real-time polymerase chain reaction (rt-PCR), enzyme-linked immunosorbent assay (ELISA) and Western blotting revealed an upregulation of bFGF in response to Norgestrel over 6 h. Specific siRNA knockdown of bFGF abrogated the protective properties of Norgestrel on damaged photoreceptors, thus highlighting the crucial importance of bFGF in Norgestrel-mediated protection. Furthermore, Norgestrel initiated a bFGF-dependent inactivation of glycogen synthase kinase 3β (GSK3β) through phosphorylation at serine 9. The effects of Norgestrel on GSK3β were dependent on protein kinase A (PKA) pathway activation. Specific inhibition of both the PKA and GSK3β pathways prevented Norgestrel-mediated neuroprotection of stressed photoreceptor cells in vitro. Involvement of the PKA pathway following Norgestrel treatment was also confirmed ex vivo. Therefore, these results indicate that the protective efficacy of Norgestrel is, at least in part, due to the bFGF-mediated activation of the PKA pathway, with subsequent inactivation of GSK3β.

Progesterone Attenuates Microglial-Driven Retinal Degeneration and Stimulates Protective Fractalkine-CX3CR1 Signaling

“…Previously shown to protect through an upregulation of growth factors [12, 13], this study highlights a novel role for Norgestrel, in acting directly upon microglial cells to dampen damaging inflammatory processes. This is an important finding as recent literature has shown that microglial cells may potentiate retinal disease [18, 19, 21, 44, 63, 64].…”

“…Due to the difficulty in isolating pure photoreceptor primary cultures and in order to assess the interaction between microglia and photoreceptors, we utilized the cone photoreceptor-like 661W cell line. Our group has previously used 661Ws as an in vitro model to study how photoreceptors respond to stressful stimuli and to understand how Norgestrel works in providing neuroprotection [11–13]. …”

“…The previous experiment was replicated using serum-starved 661W cells, a previously established method for inducing 661W cellular stress [11, 13]. This allowed us to investigate the response of microglia to damaged photoreceptors, in an attempt to mimic photoreceptor stress present in the rd10 mouse retina.…”

“…Published studies have since shown that Norgestrel, working through progesterone receptors [11], significantly increases production of basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) in the retina [10, 12]. These growth factors likely act directly on photoreceptors to provide neuroprotection, through an upregulation of pro-survival and downregulation of apoptotic pathways [13]. …”

Microglial‐induced Müller cell gliosis is attenuated by progesterone in a mouse model of retinitis pigmentosa

“…Norgestrel is a progesterone analogue that acts through progesterone receptors to elicit its downstream effects (Ruiz Lopez et al, ; Wyse‐Jackson et al, ; Wyse Jackson & Cotter, ; Wyse Jackson, Roche, Byrne, Ruiz‐Lopez, & Cotter, ). The presence of several progesterone receptors in the retina has been established (Wyse Jackson et al, ).…”