CX3CL1 Action on Microglia Protects from Diet-Induced Obesity by Restoring POMC Neuronal Excitability and Melanocortin System Activity Impaired by High-Fat Diet Feeding.

Both hypothalamic microglial inflammation and melanocortin pathway dysfunction contribute to diet-induced obesity (DIO) pathogenesis. Previous studies involving models of altered microglial signaling demonstrate altered DIO susceptibility with corresponding POMC neuron cytological changes, suggesting a link between microglia and the melanocortin system. We addressed this hypothesis using the specific microglial silencing molecule, CX3CL1 (fractalkine), to determine whether reducing hypothalamic microglial activation can restore POMC/melanocortin signaling to protect against DIO. We performed metabolic analyses in high fat diet (HFD)-fed mice with targeted viral overexpression of CX3CL1 in the hypothalamus. Electrophysiologic recording in hypothalamic slices from POMC-MAPT-GFP mice was used to determine the effects of HFD feeding and microglial silencing via minocycline or CX3CL1 on GFP-labeled POMC neurons. Finally, mice with hypothalamic overexpression of CX3CL1 received central treatment with the melanocortin receptor antagonist SHU9119 to determine whether melanocortin signaling is required for the metabolic benefits of CX3CL1. Hypothalamic overexpression of CX3CL1 increased leptin sensitivity and POMC gene expression, while reducing weight gain in animals fed an HFD. In electrophysiological recordings from hypothalamic slice preparations, HFD feeding was associated with reduced POMC neuron excitability and increased amplitude of inhibitory postsynaptic currents. Microglial silencing using minocycline or CX3CL1 treatment reversed these HFD-induced changes in POMC neuron electrophysiologic properties. Correspondingly, blockade of melanocortin receptor signaling in vivo prevented both the acute and chronic reduction in food intake and body weight mediated by CX3CL1. Our results show that suppressing microglial activation during HFD feeding reduces DIO susceptibility via a mechanism involving increased POMC neuron excitability and melanocortin signaling.

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.

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.

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.

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 24 h. Specific PGRMC1 inhibition by AG205 (1 µm) showed this rise to be PGRMC1-dependent, primarily utilizing 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 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.

Alterations to retinal architecture prior to photoreceptor loss in a mouse model of retinitis pigmentosa.

Mouse models of retinitis pigmentosa (RP) are essential tools in the pursuit to understand fully what cell types and processes underlie the degeneration observed in RP. Knowledge of these processes is required if we are to develop successful therapies to treat this currently incurable disease. We have used the rd10 mouse model of RP to study retinal morphology prior to photoreceptor loss, using immunohistochemistry and confocal microscopy on cryosections, since little is known about how the mutation affects the retina during this period. We report novel findings that the mutation in the rd10 mouse results in retinal abnormalities earlier than was previously thought. Defects in rod and cone outer segments, bipolar cells, amacrine cells and photoreceptor synapses were apparent in the retina during early stages of postnatal retinal development and prior to the loss of photoreceptors. Additionally, we observed a dramatic response of glial cells during this period. Microglia responded as early as postnatal day (P) 5; ?13 days before any photoreceptor loss is detected with Müller glia and astrocytes exhibiting changes from P10 and P15 respectively. Overall, these findings present pathological aspects to the postnatal development of the rd10 retina, contributing significantly to our understanding of disease onset and progression in the rd10 mouse and provide a valuable resource for the study of retinal dystrophies.

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 3ß signalling.

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 3ß (GSK3ß) through phosphorylation at serine 9. The effects of Norgestrel on GSK3ß were dependent on protein kinase A (PKA) pathway activation. Specific inhibition of both the PKA and GSK3ß 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 GSK3ß.