Subcellular localization of the FLT3-ITD oncogene plays a significant role in the production of NOX- and p22phox-derived reactive oxygen species in acute myeloid leukemia

Internal tandem duplication of the juxtamembrane domain of FMS-like tyrosine kinase 3 (FLT3-ITD) receptor is the most prevalent FLT3 mutation accounting for 20% of acute myeloid leukemia (AML) patients. FLT3-ITD mutation results in ligand-independent constitutive activation of the receptor at the plasma membrane and ‘impaired trafficking’ of the receptor in compartments of the endomembrane system, such as the endoplasmic reticulum (ER). FLT3-ITD expressing cells have been shown to generate increased levels of reactive oxygen species (ROS), in particular NADPH oxidase (NOX)-generated ROS which act as pro-survival signals. The purpose of this study is to investigate FLT3-ITD production of ROS at the plasma membrane and ER in the FLT3-ITD expressing AML cell line MV4-11. Receptor trafficking inhibitors; Tunicamycin and Brefeldin A induce ER retention of FLT3-ITD, resulting in a decrease in protein expression of NOX4 and its partner protein p22phox, thus demonstrating the critical importance of FLT3-ITD localization for the generation of pro-survival ROS. NOX-generated ROS contribute to total endogenous hydrogen peroxide (H2O2) in AML as quantified by flow cytometry using the cell-permeable H2O2-probe Peroxy Orange 1 (PO1). We found that PI3K/AKT signaling only occurs when FLT3-ITD is expressed at the plasma membrane and is required for the production of NOX-generated ROS. ER retention of FLT3-ITD resulted in NOX4 deglycosylation and p22phox protein degradation

NOX-driven ROS formation in cell transformation of FLT3-ITD positive AML

In different types of myeloid leukemia, increased formation of reactive oxygen species (ROS) has been noted and associated with aspects of cell transformation including the promotion of leukemic cell proliferation and migration, as well as DNA-damage and accumulation of mutations. Work reviewed in this article has shown the involvement of NADPH oxidase (NOX)-derived ROS downstream of oncogenic protein-tyrosine kinases in both processes, and the related pathways have been partially identified. FLT3-ITD, an important oncoprotein in a subset of AML, causes activation of AKT and subsequently stabilization of p22phox, a regulatory subunit for NOX1-4. This process is linked to ROS formation and DNA damage. Moreover, FLT3-ITD signaling through STAT5 enhances expression of NOX4, ROS formation and inactivation of the protein-tyrosine phosphatase DEP-1/PTPRJ, a negative regulator of FLT3 signaling, by reversible oxidation of its catalytic cysteine residue. Genetic inactivation of NOX4 restored DEP-1 activity and attenuated cell transformation by FLT3-ITD in vitro and in vivo. Future work is required to further explore these mechanisms and their causal involvement in leukemic cell transformation, which may result in the identification of novel candidate targets for therapy

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

Norgestrel, a Progesterone Analogue, Promotes Significant Long-Term Neuroprotection of Cone Photoreceptors in a Mouse Model of Retinal Disease

Purpose: Retinitis pigmentosa (RP) refers to a group of inherited blinding retinal diseases, whereby the death of mutated rod photoreceptors is followed closely by the death of cone photoreceptors. Cone cell death can be hugely debilitating as color/daytime vision becomes impaired. Thus, treatments that are effective against cone cell death are urgently needed. Our research has been working toward development of a neuroprotective treatment for RP. We have previously demonstrated significant neuroprotective properties of norgestrel, a progesterone analogue, in the mouse retina. The current study further investigates the potential of norgestrel as a treatment for RP, with a focus on long-term preservation of cone photoreceptors. Methods: Using the well-established rd10 mouse model of RP, we administered a norgestrel-supplemented diet at postnatal day (P)30, following widespread loss of rod photoreceptors and at the outset of cone degeneration. We subsequently assessed cone cell morphology and retinal function at P50, P60, and P80, using immunohistochemistry, electroretinograph recordings, and optomotor testing. Results: While cone cell degeneration was widespread in the untreated rd10 retina, we observed profound preservation of cone photoreceptor morphology in the norgestrel-treated mice for at least 50 days, out to P80. This was demonstrated by up to 28-fold more cone arrestin-positive photoreceptors. This protection transpired to functional preservation at all ages. Conclusions: This work presents norgestrel as an incredibly promising long-term neuroprotective compound for the treatment of RP. Crucially, norgestrel could be used in the mid-late stages of the disease to protect remaining cone cells and help preserve color/daytime vision.Supported by grants from Science Foundation Ireland (SFI 13/IA/1783), Fighting Blindness Ireland (FB13COT), Spanish Ministry of Economy and Competitiveness (MINECO-FEDER-BFU2015-67139-R), Generalitat Valenciana (Prometeo 2016/158), and Carlos III Institute (ISCIII RETICS-FEDER RD16/0008/0016)

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

Effects of Noise on Ecological Invasion Processes: Bacteriophage-mediated Competition in Bacteria

Pathogen-mediated competition, through which an invasive species carrying and transmitting a pathogen can be a superior competitor to a more vulnerable resident species, is one of the principle driving forces influencing biodiversity in nature. Using an experimental system of bacteriophage-mediated competition in bacterial populations and a deterministic model, we have shown in [Joo et al 2005] that the competitive advantage conferred by the phage depends only on the relative phage pathology and is independent of the initial phage concentration and other phage and host parameters such as the infection-causing contact rate, the spontaneous and infection-induced lysis rates, and the phage burst size. Here we investigate the effects of stochastic fluctuations on bacterial invasion facilitated by bacteriophage, and examine the validity of the deterministic approach. We use both numerical and analytical methods of stochastic processes to identify the source of noise and assess its magnitude. We show that the conclusions obtained from the deterministic model are robust against stochastic fluctuations, yet deviations become prominently large when the phage are more pathological to the invading bacterial strain.Comment: 39 pages, 7 figure