Fractalkine-CX3CR1 signaling is critical for progesterone-mediated neuroprotection in the retina

Retinitis pigmentosa (RP) encompasses a group of retinal diseases resulting in photoreceptor loss and blindness. We have previously shown in the rd10 mouse model of RP, that rd10 microglia drive degeneration of viable neurons. Norgestrel, a progesterone analogue, primes viable neurons against potential microglial damage. In the current study we wished to investigate this neuroprotective effect further. We were particularly interested in the role of fractalkine-CX3CR1 signaling, previously shown to mediate photoreceptor-microglia crosstalk and promote survival in the rd10 retina. Norgestrel upregulates fractalkine-CX3CR1 signaling in the rd10 retina, coinciding with photoreceptor survival. We show that Norgestrel-treated photoreceptor-like cells, 661Ws, and C57 explants modulate rd10 microglial activity in co-culture, resulting in increased photoreceptor survival. Assessment of Norgestrel’s neuroprotective effects when fractalkine was knocked-down in 661 W cells and release of fractalkine was reduced in rd10 explants confirms a crucial role for fractalkine-CX3CR1 signaling in Norgestrel-mediated neuroprotection. To further understand the role of fractalkine in neuroprotection, we assessed the release of 40 cytokines in fractalkine-treated rd10 microglia and explants. In both cases, treatment with fractalkine reduced a variety of pro-inflammatory cytokines. These findings further our understanding of Norgestrel’s neuroprotective properties, capable of modulating harmful microglial activity indirectly through photoreceptors, leading to increased neuroprotection

Pro-survival redox signalling in progesterone-mediated retinal neuroprotection

Retinitis pigmentosa (RP) is a group of hereditary retinal diseases, characterised by photoreceptor cell loss. Despite a substantial understanding of the mechanisms leading to cell death, an effective therapeutic strategy is sought. Our laboratory has previously demonstrated the neuroprotective properties of Norgestrel, a progesterone analogue, in the degenerating retina, mediated in part by the neurotrophic factor basic fibroblast growth factor (bFGF). In other retinal studies, we have also presented a pro-survival role for reactive oxygen species (ROS), downstream of bFGF. Thus, we hypothesized that Norgestrel utilises bFGF-driven ROS production to promote photoreceptor survival. Using the 661W photoreceptor-like cell line, we now show that Norgestrel, working through progesterone receptor membrane complex 1 (PGRMC1); generates an early burst of pro-survival bFGF-induced ROS. Using the rd10 mouse model of RP, we confirm that Norgestrel induces a similar early pro-survival increase in retinal ROS. Norgestrel-driven protection in the rd10 retina was attenuated in the presence of antioxidants. This study therefore presents an essential role for ROS signalling in Norgestrel-mediated neuroprotection in vitro and demonstrates that Norgestrel employs a similar pro-survival mechanism in the degenerating retina

Progesterone analogue protects stressed photoreceptors via bFGF-mediated calcium influx.

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

The activity of cAMP-Phosphodiesterase 4D7 (PDE4D7) is regulated by protein kinase A-dependent phosphorylation within its unique N-terminus

The cyclic AMP phosphodiesterases type 4 (PDE4s) are expressed in a cell specific manner, with intracellular targeting directed by unique N-terminal anchor domains. All long form PDE4s are phosphorylated and activated by PKA phosphorylation within their upstream conserved region 1 (UCR1). Here, we identify and characterise a novel PKA site (serine 42) within the N-terminal region of PDE4D7, an isoform whose activity is known to be important in prostate cancer progression and ischemic stroke. In contrast to the UCR1 site, PKA phosphorylation of the PDE4D7 N-terminus appears to occur constitutively and inhibits PDE4 activity to allow cAMP signalling under basal conditions

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

The synthetic progestin norgestrel modulates Nrf2 signaling and acts as an antioxidant in a model of retinal degeneration

Retinitis pigmentosa (RP) is one of the most common retinal degenerative conditions affecting people worldwide, and is currently incurable. It is characterized by the progressive loss of photoreceptors, in which the death of rod cells leads to the secondary death of cone cells; the cause of eventual blindness. As rod cells die, retinal-oxygen metabolism becomes perturbed, leading to increased levels of reactive oxygen species (ROS) and thus oxidative stress; a key factor in the secondary death of cones. In this study, norgestrel, an FDA-approved synthetic analog of progesterone, was found to be a powerful neuroprotective antioxidant, preventing light-induced ROS in photoreceptor cells, and subsequent cell death. Norgestrel also prevented light-induced photoreceptor morphological changes that were associated with ROS production, and that are characteristic of RP. Further investigation showed that norgestrel acts via post-translational modulation of the major antioxidant transcription factor Nrf2; bringing about its phosphorylation, subsequent nuclear translocation, and increased levels of its effector protein superoxide dismutase 2 (SOD2). In summary, these results demonstrate significant protection of photoreceptor cells from oxidative stress, and underscore the potential of norgestrel as a therapeutic option for RP

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

Retinitis pigmentosa (RP) is a degenerative disease leading to photoreceptor cell loss. Mouse models of RP, such as the rd10 mouse (B6.CXBl-Pde6brd10/J), have enhanced our understanding of the disease, allowing for development of potential therapeutics. In 2011, our group first demonstrated that the synthetic progesterone analogue 'Norgestrel' is neuroprotective in two mouse models of retinal degeneration, including the rd10 mouse. We have since elucidated several mechanisms by which Norgestrel protects stressed photoreceptors, such as upregulating growth factors. This study consequently aimed to further characterize Norgestrel's neuroprotective effects. Specifically, we sought to investigate the role that microglia might play; for microglial-derived inflammation has been shown to potentiate neurodegeneration. Dams of post-natal day (P) 10 rd10 pups were given a Norgestrel-supplemented diet (80mg/kg). Upon weaning, pups remained on Norgestrel. Tissue was harvested from P15-P50 rd10 mice on control or Norgestrel-supplemented diet. Norgestrel-diet administration provided significant retinal protection out to P40 in rd10 mice. Alterations in microglial activity coincided with significant protection, implicating microglial changes in Norgestrel-induced neuroprotection. Utilizing primary cultures of retinal microglia and 661W photoreceptor-like cells, we show that rd10 microglia drive neuronal cell death. We reveal a novel role of Norgestrel, acting directly on microglia to reduce pro-inflammatory activation and prevent neuronal cell death. Norgestrel effectively suppresses cytokine, chemokine and danger-associated molecular pattern molecule (DAMP) expression in the rd10 retina. Remarkably, Norgestrel upregulates fractalkine-CX3CR1 signaling 1 000-fold at the RNA level, in the rd10 mouse. Fractalkine-CX3CR1 signaling has been shown to protect neurons by regulating retinal microglial activation and migration. Ultimately, these results present Norgestrel as a promising treatment for RP, with dual actions as a neuroprotective and anti-inflammatory agent in the retina

Norgestrel upregulates fractalkine-CX3CR1 signaling in the rd10 retina.

<p><b>(A)</b> 661W cells were serum-starved and treated with 20 μM Norgestrel (Norgestrel) or the equivalent DMSO control (Control) over 6 and 24h. RT-qPCR analysis detected a significant increase in mRNA levels of fractalkine in serum-starved cells treated with Norgestrel. N = 6 biological replicates, n = 3 technical replicates per sample. <b>(B)</b> Norgestrel-supplementation in rd10 mice induced a significant increase in fractalkine and CX3CR1 mRNA expression in P20-P30 retinas compared to control. Control column represents age-matched rd10 mice on control diet. N = 3 mice per group, n = 3 technical replicates per mouse. <b>(C)</b> Western blot for fractalkine at P20 and P25 confirmed an increase in full-length fractalkine (100kDa; red box) at both time-points with Norgestrel treatment. Total protein stain shows similar loading between samples. Blots are representative of 6 biological replicates per group. <b>(D)</b> Fluorescent microscopic images of fractalkine (green) in the central retina from P15-40 of control and Norgestrel-fed rd10 mice. Fractalkine staining is evident in the outer nuclear layer (ONL) of the retina in Norgestrel-supplemented mice. Hoechst (blue) staining reveals the cell layers in the retina. Scale bar 50μm. N = 3 mice per group, n = 3 sections per mouse. <b>(E)</b> 661W cells were cultured in the absence of serum (serum-starved 661W) over 3 h. 661W cells were then placed in culture with microglia that had been treated with (Microglia Fract.) or without (Microglia) 1.2μg/ml recombinant full-length fractalkine over 24h. Co-cultures were incubated for a further 24h. Apoptosis of 661W cells was detected by TUNEL of DNA strand breaks in cell nuclei. Co-cultures with fractalkine-treated microglia had less TUNEL-positive 661W cells than equivalent vehicle control. Scale bar 30μm. <b>(F)</b> Quantification of TUNEL-positive 661W cells cultured in the absence of serum and following co-incubation with primary rd10 microglial cells pre-treated with recombinant full-length fractalkine or vehicle. N = 9 mice for primary culture, n = 5 technical replicates per group. Results are presented as mean ± SEM (t-test, *<i>p<</i>0.05, ***<i>p<</i>0.005, ****<i>p<</i>0.001).</p

Norgestrel dampens pro-inflammatory microglial activity <i>in vivo</i> and decreases DAMP release from photoreceptors.

<p><b>(A)</b> Confocal microscopic images of microglia (Iba1; red) and activated microglia (CD68; green) in the central retina from P15-P25 control and Norgestrel-fed rd10 mice. Less microglia including CD68+ microglia were observed at P15 and P20 in Norgestrel-fed mice. Hoechst (blue) staining reveals the cell layers in the retina. Scale bar 50μm. N = 3 mice per group, n = 8 sections per mouse. <b>(B & C)</b> Quantification of microglial number and CD68-positivity confirmed (B) significantly less microglia at P15 and P20 and (C) significantly less CD68-positivity at P20 with Norgestrel-supplementation. <b>(D)</b> RT-qPCR analysis of mRNA encoding (i) pro-inflammatory (M1) and (ii) & (iii) anti-inflammatory (M2) microglial markers in whole retina from P20-P30 control and Norgestrel-fed rd10 mice. Norgestrel significantly reduced pro-inflammatory and increased anti-inflammatory markers in the retina. Control column represents age-matched rd10 mice on control diet. N = 3 mice per group, n = 3 technical replicates per mouse. <b>(E)</b> RT-qPCR analysis of mRNA encoding danger associated molecular patterns (DAMP), (i) high motility box group 1 (HMGB1) and (ii) interleukin-1α (IL-1α) in whole retina from P20-P30 control and Norgestrel-fed rd10 mice. Norgestrel significantly reduced DAMP expression at P20 and P25 in Norgestrel-fed mice. Control column represents age-matched rd10 mice on control diet. N = 3 mice per group, n = 3 technical replicates per mouse. Results are presented as mean ± SEM (t-test, *p<0.05, ***p<0.005, ****p<0.0001).</p