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Calcium signalling is involved in many processes in mammalian retina, from development to mature functions and neurodegeneration. Although proteins involved in Ca2+ entry in retinal cells have been well studied, less is known about Ca2+-clearance. Among the Ca2+ pumps, plasma membrane Ca2+-ATPases (PMCAs) have been identified as key proteins extruding Ca2+ across the plasma membrane with specific distribution in developing and adult retina. However, the two main isoforms of intracellular Ca2+-ATPases in the central nervous system, the sarco(endo)plasmic reticulum (ER) Ca2+-ATPase 2b (SERCA2b) and the secretory pathway Ca2+-ATPase 1 (SPCA1), which remove cytosolic Ca2+ into intracellular stores, have been less or not at all analysed, respectively. In this study, we described for the first time the SPCA1 localisation in adult mouse retina and we report differential distributions of SERCA2b and SPCA1 transporters within various classes of retinal neurons and distinct subcellular localisations. In addition, we studied the expression and localisation of both Ca2+ pumps in 661W cells, a cone photoreceptor-derived cell line. Since continuous exposure to high light intensity induces photodegeneration, we analysed the effect of LED light exposure on these cells and SERCA2b and SPCA1 distribution. We found that continuous mild LED-light exposure compromised cell survival and produced stress in the ER and Golgi, the Ca2+ stores where the two pumps are localised. These effects were reversed after halting light exposure and washing. This study demonstrates that Ca2+ signalling may be involved in light-induced photoreceptor cell damage and points to previously unrecognised functions of intracellular Ca2+-ATPases in retina physiology. [Display omitted] • Ca2+ pumps SERCA2b and SPCA1 are differentially distributed in adult mouse retina. • SERCA2b and SPCA1 are highly expressed in cone photoreceptors. • Light-induced stress in cone-661W cells affects SERCA2b and SPCA1 distribution. • Organelle integrity can be recovered from light stress after washing and darkness. [ABSTRACT FROM AUTHOR]

Purpose: Vital dyes have become a clinical standard during vitrectomy to visualize anatomical structures. It was the aim of this study to test the effect of two vital dyes (AV 17-M with 5% mannitol and MBB Dual) on different intraocular cells, to see whetherin vitrotest can be used as reliable preclinical testing tool. Methods: Cell morphology was assessed via phase contrast pictures, cell viability via MTS assay and total cell amount via crystal violet staining. ARPE19 and 661W cells were chosen for toxicology testing at different exposure times (60 seconds, 15 minutes and 30 minutes). Vital dyes were completely removed after the staining period. Results: Treatment with AV 17-M changed the morphology and the cell number at every time point investigated on ARPE19 and 661 W cells. ARPE19 cells treated with AV 17-M or MBB Dual displayed only a slight or no decrease in cell viability after the three different exposure times. AV-17 without medium to simulate a possible intraoperative use after fluid-air exchange showed a decrease in viability of 6%, 24% and 14%. A difference in cell density of 21%, 46% and 34% was noted after CV staining for AV 17-M, MBB Dual led to a decrease of 2%, 16% and 3% after 30 minutes compared to BSS. AV 17-M directly applied on 661W decreased viability significantly by 18% after 60 seconds, 33% after 15 minutes and 40% after 30 minutes. Cell density of 661W cells exposed relevant negative effects; after incubation of 60 seconds with AV 17-M, the cell amount was significantly lowered by 41% and MBB Dual by 12%. After 15 minutes, a loss of 48% cell amount was detected with AV 17-M and after 30 min 51%. MBB Dual led to 37% loss after 15 minutes and to 28% loss after 30 minutes. Conclusion: AV 17-M with 5% mannitol has a negative effect on different ocular cells. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

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 μ m) showed this rise to be PGRMC1-dependent, primarily utilizing calcium from extracellular sources, for blockade of L-type calcium channels by verapamil (50 μ m) prevented a Norgestrel-induced calcium influx in stressed cells. Calcium influx was also shown to be bFGF-dependent, for si RNA 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. [ABSTRACT FROM AUTHOR]

The synthetic progesterone Norgestrel has been shown to have proven neuroprotective efficacy in two distinct models of retinitis pigmentosa: the rd10/rd10 (B6. CXBI-Pde6brd10/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 si RNA 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. [ABSTRACT FROM AUTHOR]

Retinitis pigmentosa (RP) is an inherited form of retinal degeneration characterized by primary rod photoreceptor cell death followed by cone loss. Mutations in several genes linked to the disease cause increased levels of cyclic guanosine monophosphate (cGMP) and calcium ion influxes. The purpose of this project was to develop a new in vitro photoreceptor degeneration model for molecular studies of RP. 661W cells were genetically modified to stably express the neural retina leucine zipper (NRL) transcription factor. One clone (661W-A11) was selected based on the expression of Nrl target genes. 661W-A11 showed a significant increase in expression of rod-specific genes but not of cone-specific genes, compared with 661W cells. Zaprinast was used to inhibit phosphodiesterase 6 (PDE6) activity to mimic photoreceptor degeneration in vitro. The activation of cell death pathways resulting from PDE6 inhibition was confirmed by detection of decreased viability and increased intracellular cGMP and calcium, as well as activation of protein kinase G (PKG) and calpains. In this new in vitro system, we validated the effects of previously published neuroprotective drugs. The 661W-A11 cells may serve as a new model for molecular studies of RP and for high-throughput drug screening. [ABSTRACT FROM AUTHOR]

Inherited retinal degenerative diseases (IRDs), which ultimately lead to photoreceptor cell death, are characterized by high genetic heterogeneity. Many IRD-associated genetic defects affect 3′,5′-cyclic guanosine monophosphate (cGMP) levels. cGMP-dependent protein kinases (PKGI and PKGII) have emerged as novel targets, and their inhibition has shown functional protection in IRDs. The development of such novel neuroprotective compounds warrants a better understanding of the pathways downstream of PKGs that lead to photoreceptor degeneration. Here, we used human recombinant PKGs in combination with PKG activity modulators (cGMP, 3′,5′-cyclic adenosine monophosphate (cAMP), PKG activator, and PKG inhibitors) on a multiplex peptide microarray to identify substrates for PKGI and PKGII. In addition, we applied this technology in combination with PKG modulators to monitor kinase activity in a complex cell system, i.e. the retinal cell line 661W, which is used as a model system for IRDs. The high-throughput method allowed quick identification of bona fide substrates for PKGI and PKGII. The response to PKG modulators helped us to identify, in addition to ten known substrates, about 50 novel substrates for PKGI and/or PKGII which are either specific for one enzyme or common to both. Interestingly, both PKGs are able to phosphorylate the regulatory subunit of PKA, whereas only PKGII can phosphorylate the catalytic subunit of PKA. In 661W cells, the results suggest that PKG activators cause minor activation of PKG, but a prominent increase in the activity of cAMP-dependent protein kinase (PKA). However, the literature suggests an important role for PKG in IRDs. This conflicting information could be reconciled by cross-talk between PKG and PKA in the retinal cells. This must be explored further to elucidate the role of PKGs in IRDs. [ABSTRACT FROM AUTHOR]