Steroid induced neuroprotection of damaged photoreceptor cells

Retinitis Pigmentosa (RP) is the name given to a group of hereditary diseases causing progressive and degenerative blindness. RP affects over 1 in 4000 individuals, making it the most prevalent inherited retinal disease worldwide, yet currently there is no cure. In 2011, our group released a paper detailing the protective effects of the synthetic progestin ‘Norgestrel’. A common component of the female oral contraceptive pill, Norgestrel was shown to protect against retinal cell death in two distinct mouse models of retinal degeneration: in the Balb/c light damage model and the Pde6brd10 (rd10) model. Little was known of the molecular workings of this compound however and thus this study aimed to elucidate the protective manner in which Norgestrel worked. To this aim, the 661W cone photoreceptor-like cell line and ex vivo retinal explanting was utilised. We found that Norgestrel induces a increase in neuroprotective basic fibroblast growth factor (bFGF) with subsequent downstream actions on the inhibition of glycogen synthase kinase 3β. Progesterone receptor expression was subsequently characterised in the C57 and rd10 retinas and in the 661W cell line. Norgestrel caused nuclear trafficking of progesterone receptor membrane complex one (PGRMC1) in 661W cells and thus Norgestrel was hypothesised to work primarily through the actions of PGRMC1. This trafficking was shown to be responsible for the critical upregulation of bFGF and PGRMC1- Norgestrel binding was proven to cause a neuroprotective bFGF-mediated increase in intracellular calcium. The protective properties of Norgestrel were further studied in the rd10 mouse model of retinitis pigmentosa. Using non-invasive diet supplementation (80mg/kg), we showed that Norgestrel gave significant retinal protection out to postnatal day 40 (P40). Overactive microglia have previously been shown to potentiate photoreceptor cell loss in the degenerating rd10 retina and thus we focussed on Norgestrel-mediated changes in photoreceptor-microglial crosstalk. Norgestrel acted to dampen pro-inflammatory microglial cell reactivity, decreasing chemokine (MCP1, MCP3, MIP-1α, MIP-1β) and subsequent damaging cytokine (TNFα, Il-1β) production. Critically, Norgestrel up-regulated photoreceptor-microglial, fractalkine-CX3CR1 signalling 1000-fold in the P20 rd10 mouse. Known to prevent microglial activation, we hypothesise that Norgestrel acts as a vital anti-inflammatory in the diseased retina, driving fractalkine-CX3CR1 signalling to delay retinal degeneration. This study stands to highlight some of the neuroprotective mechanisms utilised by Norgestrel in the prevention of photoreceptor cell death. We identify for the first time, not only a pro-survival pathway activated directly in photoreceptor cells, but also a Norgestreldriven mediation of an otherwise damaging microglial cell response. All taken, these results form the beginning of a case to bring Norgestrel to clinical trials, as a potential therapeutic for the treatment of RP.

Novel ingredients from brewers' spent grain - bioactivity in cell culture model systems and bioactivity retention in fortified food products

Functional food ingredients, with scientifically proven and validated bioactive effects, present an effective means of inferring physiological health benefits to consumers to reduce the risk of certain diseases. The search for novel bioactive compounds for incorporation into functional foods is particularly active, with brewers’ spent grain (BSG, a brewing industry co-product) representing a unique source of potentially bioactive compounds. The DNA protective, antioxidant and immunomodulatory effects of phenolic extracts from both pale (P1 - P4) and black (B1 – B4) BSG were examined. Black BSG extracts significantly (P < 0.05) protected against DNA damage induced by hydrogen peroxide (H2O2) and extracts with the highest total phenolic content (TPC) protected against 3-morpholinosydnonimine hydrochloride (SIN-1)-induced oxidative DNA damage, measured by the comet assay. Cellular antioxidant activity assays were used to measured antioxidant potential in the U937 cell line. Extracts P1 – P3 and B2 - B4 demonstrated significant (P < 0.05) antioxidant activity, measured by the superoxide dismutase (SOD) activity, catalase (CAT) activity and gluatathione (GSH) content assays. Phenolic extracts P2 and P3 from pale BSG possess anti-inflammatory activity measured in concanavalin-A (conA) stimulated Jurkat T cells by an enzyme-linked immunosorbent assay (ELISA); significantly (P < 0.05) reducing production of interleukin-2 (IL-2), interleukin-4 (IL-4, P2 only), interleukin-10 (IL-10) and interferon-γ (IFN-γ). Black BSG phenolic extracts did not exhibit anti-inflammatory effects in vitro. Hydroxycinnamic acids (HA) have previously been shown to be the phenolic acids present at highest concentration in BSG; therefore the HA profile of the phenolic extracts used in this research, the original barley (before brewing) and whole BSG was characterised and quantified using high performance liquid chromatography (HPLC). The concentration of HA present in the samples was in the order of ferulic acid (FA) > p-coumaric acid (p-CA) derivatives > FA derivatives > p-CA > caffeic acid (CA) > CA derivatives. Results suggested that brewing and roasting decreased the HA content. Protein hydrolysates from BSG were also screened for their antioxidant and anti-inflammatory potential. A total of 34 BSG protein samples were tested. Initial analyses of samples A – J found the protein samples did not exert DNA protective effects (except hydrolysate H) or antioxidant effects by the comet and SOD assays, respectively. Samples D, E, F and J selectively reduced IFN-γ production (P < 0.05) in Jurkat T cells, measured using enzyme linked immunosorbent assay (ELISA). Further testing of hydrolysates K – W, including fractionated hydrolysates with molecular weight < 3, < 5 and > 5 kDa, found that higher molecular weight (> 5 kDa) and unfractionated hydrolysates demonstrate greatest anti-inflammatory effects, while fractionated hydrolysates were also shown to have antioxidant activity, by the SOD activity assay. A commercially available yogurt drink (Actimel) and snack-bar and chocolate-drink formulations were fortified with the most bioactive phenolic and protein samples – P2, B2, W, W < 3 kDa, W < 5 kDa, W > 5 kDa. All fortified foods were subjected to a simulated gastrointestinal in vitro digestion procedure and bioactivity retention in the digestates was determined using the comet and ELISA assays. Yogurt fortified with B2 digestate significantly (P < 0.05) protected against H2O2-induced DNA damage in Caco-2 cells. Greatest immunomodulatory activity was demonstrated by the snack-bar formulation, significantly (P < 0.05) reducing IFN-γ production in con-A stimulated Jurkat T cells. Hydrolysate W significantly (P < 0.05) increased the IFN-γ reducing capacity of the snack-bar. Addition of fractionated hydrolysate W < 3 kDa and W < 5 kDa to yogurt also reduced IL-2 production to a greater extent than the unfortified yogurt (P < 0.05).

Molecular genetics of the imprinted gene - SPROUTY3 (SPRY3)

Sprouty proteins are key regulators of cell growth and branching morphogenesis during development. Human SPRY3 which maps to the pseudoautosomal region 2, undergoes random X-inactivation in females and preferential Y-inactivation in males, behaving as though genetically X-linked. Spry3 is widely expressed in neuronal tissues, being found at high levels in the cerebellum and particularly in the Purkinje cells which, notably, are deficient in the autistic brain. Spry3 is also highly expressed in other ganglia in adults including retinal ganglion cells, dorsal root ganglion and superior cervical ganglion. SPRY3 enhancer can drive SPRY3 expression in the lung airway, which is consistent with a role in branching morphogenesis and the function of the original Drosophila Spry gene, which is critical for lung morphogenesis, providing a possible explanation for an observed anatomic abnormality in the autistic lung airway. In the human and mouse, the SPRY3 core promoter contains an AG-rich repeat and we found evidence of coexpression, promoter binding and regulation of SPRY3 expression by transcription factors EGR1, ZNF263 and PAX6. Spry3 over-expression in mouse superior cervical ganglion cells inhibits axon branching and Spry3 knockdown in those cells increases axon branching, consistent with known functions of other Sprouty proteins. Novel SPRY3 upstream transcripts that I characterised originate from three start sites in the X-linked F8A3 – TMLHE gene region, which is recently implicated in autism causation. Arising from these findings, I propose that the lung airway abnormality and low levels of blood carnitine found in autism suggest that deregulation of SPRY3 may underpin a subset of autism cases.

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.