Sostdc1 is expressed in all major compartments of developing and adult mammalian eyes.

PURPOSE: This study was conducted in order to study Sostdc1 expression in rat and human developing and adult eyes. METHODS: Using the yeast signal sequence trap screening method, we identified the Sostdc1 cDNA encoding a protein secreted by the adult rat retinal pigment epithelium. We determined by in situ hybridization, RT-PCR, immunohistochemistry, and western blot analysis Sostdc1 gene and protein expression in developing and postnatal rat ocular tissue sections. We also investigated Sostdc1 immunohistolocalization in developing and adult human ocular tissues. RESULTS: We demonstrated a prominent Sostdc1 gene expression in the developing rat central nervous system (CNS) and eyes at early developmental stages from E10.5 days postconception (dpc) to E13 dpc. Specific Sostdc1 immunostaining was also detected in most adult cells of rat ocular tissue sections. We also identified the rat ocular embryonic compartments characterized by a specific Sostdc1 immunohistostaining and specific Pax6, Sox2, Otx2, and Vsx2 immunohistostaining from embryonic stages E10.5 to E13 dpc. Furthermore, we determined the localization of SOSTDC1 immunoreactivity in ocular tissue sections of developing and adult human eyes. Indeed, we detected SOSTDC1 immunostaining in developing and adult human retinal pigment epithelium (RPE) and neural retina (NR) as well as in several developing and adult human ocular compartments, including the walls of choroidal and scleral vessels. Of utmost importance, we observed a strong SOSTDC1 expression in a pathological ocular specimen of type 2 Peters' anomaly complicated by retinal neovascularization as well in the walls ofother pathological extra-ocular vessels.  CONCLUSION: As rat Sostdc1 and human SOSTDC1 are dual antagonists of the Wnt/ß-catenin and BMP signaling pathways, these results underscore the potential crucial roles of these pathways and their antagonists, such as Sostdc1 and SOSTDC1, in developing and adult mammalian normal eyes as well as in syndromic and nonsyndromic congenital eye diseases.

P02-A121†Precut DSAEK stored in an active storage machine: feasibility study.

PURPOSE: The number of endothelial grafts precut by eye banks increases. Their shelf life is limited to a few days. We previously demonstrated the superiority of an active storage machine (ASM) over organ culture (passive) for whole corneas. AIMS: to measure endothelial viability of precut DSAEK after 3 or 10 days of storage in our ASM in a preclinical study. METHODS: Human pairs of corneas were included. The endothelial cell density (ECD in cells/mm2), and central corneal thickness (CCT in µm) were measured to ensure their initial intra pair comparability. After deswelling (CorneaJet, Eurobio) grafts preparation was performed by cutting the anterior stroma with a Moria linear microkeratome and keeping the anterior lamellae attached during storage. After randomization, one cornea was kept in the corneajet bottle (CJ) and the other was inserted into the ASM allowing a renewal or storage medium (CorneaMax, Eurobio) at 2.6 µL/min with 21 mmHg of pressure in the endothelial chamber. Both group of corneas were stored for 3 or 10 days at 31°C. The final viable ECD (vECD) was determined using the triple staining with Hoechst-Ethidium-Calcein-AM by an independent experimenter in a masked fashion. RESULTS: Initial ECDs were comparable: 2595±878 in ASM versus 2654±954 cells/mm2 in CJ for the 3-days period (n=5 pairs) and 2416±712 in ASM versus 2492±764 cells/mm2 in CJ for the 10-period (n=5 pairs). CCTs were also comparable. The anterior lamellae stayed attached in either the ASM or CJ. vECD was significantly higher in ASM than in CJ with respectively 2062±695 cells/mm2 versus 1632±633 cells/mm2 after 3 days either a cell loss of 20.5% and 38.5% respectively (p=0.0062) and 1082±649 versus 935±691 cells/mm2 for the 10-day period either a cell loss of 132% and 164% respectively (p=0.005). Grafts thickness did not differ after 3 days 219±25 µm in ASM versus 182±39 µm (p=0.063) or 10 days respectively 221±58 µm versus 189±48 µm (p=0.06). CONCLUSION: The storage of precut DSAEKs into the ASM allows a better preservation of grafts without use on deswelling storage medium. Nevertheless, the cell loss remains high after 10 days, suggesting a significant cell stress.

P01-A120†Pre-dissected dmek stored in an active storage machine: feasibility study.

PURPOSE: The number of endothelial grafts precut by eye banks increases. Their shelf life is limited to a few days. We previously demonstrated the superiority of an active storage machine (ASM) over organ culture (passive) for whole corneas. AIMS: To measure the endothelial viability of pre-dissected DMEK after 3 and 10 days of storage in our ASM in a preclinical study. METHODS: Pairs of human corneas were included. The endothelial cell density (ECD in cells/mm2), thickness and transparency of corneas were measured before graft preparation. Descemet's membrane (DM) was peeled using the no-touch technique leaving the graft attached to the center of the cornea (on approx. 1mm2). After randomization, one cornea was kept in organ culture (OC) and the other in the ASM (21 mmHg, 2.6 µL/min) in the same medium (CorneaMax, Eurobio). The final viable ECD was determined using the triple staining with Hoechst-Ethidium-Calcein-AM. In addition, the expression of CD166 and NCAM (lateral membranes), ZO-1 (apical junctions), Na+/K+ ATPase (endothelial pump function) and COX-IV (mitochondrial content) was studied by immunostaining to characterize endothelial cells after the storage. RESULTS: Initial ECDs were comparable: 2185±232 cells/mm2 in the ASM versus 2276±328 in OC for the 3-day period and 2680±416 cells/mm2 in the ASM versus 2644±420 in OC for the 10-day period. The DMs did not fold back in either BR or OC. The viable ECD did not differ significantly between the ASM and OC for either storage period: 2378±501 (ASM) versus 2342±503 (OC) for the 3-day period (n=8 pairs and p=0.624) and 2482±288 (ASM) versus 2579±315 (OC) for the 10-day period (n=5 pairs and p=0.176). Corneas were more transparent and thinner in the ASM than in OC after 3 days (916±86 versus 1193±136µm, p=0.0001) and 10 days (957±128 versus 1220±105µm, p=0.0625). The functional and structural markers studied were expressed in both groups after 3 and 10 days, some better preserved in the ASM. CONCLUSION: The storage of precut DMEKs is possible in ASM and OC for at least 10 days. Interestingly, a pre-dissected endothelium continues to partially exert its pump function into the ASM. In practice, this could allow the stroma to be used for DALK without further deswelling. In addition to improving the storage of whole grafts, the ASM allows the storage of precut DMEKs for up to 10 days with excellent endothelial survival.

Nanotopography by chromatic confocal microscopy of the endothelium in Fuchs endothelial corneal dystrophy, pseudophakic bullous keratopathy and healthy corneas.

AIM: To investigate the interest of chromatic confocal microscopy (CCM) to characterise guttae in Fuchs endothelial corneal dystrophy (FECD). METHODS: Descemet's membranes (DM) were obtained during endothelial keratoplasty in patients with FECD and pseudophakic bullous keratopathy (PBK). They were compared with healthy samples obtained from body donation to science. Samples were fixed in 0.5% paraformaldehyde and flat mounted. Surface roughness of DMs was quantified using CCM and the AltiMap software that provided the maximum peak (Sp) and valley (Sv) heights, the mean square roughness (Rq) and the asymmetry coefficient (Ssk). RESULTS: The physiological roughness of healthy samples was characterised by an Rq of 0.12±0.05 µm, which was two times rougher than in PBK (Rq=0.06±0.03 µm), but both were still flat with a symmetrical distribution between peaks and valleys (Ssk close to 0, npeaks=nvalleys), smaller than 1 µm. In FECD, the maximum peak height was 5.10±2.40 µm, up to 5.8 and 8.3 times higher than the control and PBK, respectively. The maximum valley depth was half than the peak (2.28±0.89 µm). The surface with guttae was very rough (Rq=0.45±0.14 µm) and the Ssk=1.84± 0.43 µm, greater than 0, confirms an asymmetric surface with high peaks and low valleys (npeaks>nvalleys). Moreover, the CCM provided quantitative parameters allowing to distinguish different types of guttae from different patients. CONCLUSIONS: CCM is an innovative approach to describe and quantify different morphologies of guttae. It could be useful to analyse the different stages of FECD and define subgroups of patients.

Shotgun proteomics identification of proteins expressed in the Descemet's membrane of patients with Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is a slowly evolving, bilateral disease of the corneal endothelium, characterized by an abnormal accumulation of extracellular matrix (ECM) in the basement membrane (Descemet's membrane, DM). This results in the formation of small round excrescences, called guttae, and a progressive disappearance of endothelial cells. In the intermediate stage, the numerous guttae create significant optical aberrations, and in the late stage, the loss of endothelial function leads to permanent corneal edema. The molecular components of guttae have not been fully elucidated. In the current study, we conducted shotgun proteomics of the DMs, including guttae, obtained from patients with FECD and revealed that 32 proteins were expressed only in the FECD-DMs but not in the DMs of control subjects. Subsequent enrichment analyses identified associations with multiple ECM-related pathways. Immunostaining of flat-mounted DMs confirmed that 4 of the top 5 identified proteins (hemoglobin α, SRPX2, tenascin-C, and hemoglobin γδεß) were expressed in FECD-DMs but not in non-FECD-DMs. Fibrinogen α was strongly expressed in FECD-DMs, but weakly expressed in non-FECD-DMs. We also demonstrated that matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) can display the in situ spatial distribution of biomolecules expressed in the DM, including the guttae.

CCR2/CCL2-mediated inflammation protects photoreceptor cells from amyloid-ß-induced apoptosis.

Age-related macular degeneration is characterized by the formation of drusen containing amyloid-ß (Aß) and the degeneration of photoreceptors. To explore the largely unknown role of Aß in the retina, we investigated the effects on photoreceptors of the oligomeric form of Aß(1-42). Subretinal injection of the Aß peptide induced misplaced expression of recoverin and synaptophysin in the photoreceptors, oxidative stress in their inner and outer segments, and finally apoptosis. Aß did not induce cell death in purified photoreceptor cell cultures, but did so in retinal cell cultures, thereby suggesting that the cellular environment plays a role in Aß-induced photoreceptor apoptosis. Subretinal injection of Aß was followed by activation and migration of microglial cells and then by photoreceptor apoptosis. Microglial cells phagocytosed rhodopsin-containing debris and Aß in the subretinal space. Quantitative RT-PCR allowed us to identify a specific gene expression profile associated with the Aß-induced progression of retinal degeneration and consistent with oxidative stress, inflammation, and an apoptotic program. The gene most highly upregulated in Aß-injected retinas was that for the chemokine CCL2, and its absence or that of its cognate receptor CCR2 greatly reduced migration of activated microglial cells to the site of retinal injury and profoundly worsened photoreceptor degeneration and disorganization of the retinal pigment epithelium in Aß-injected retinas. Our study pinpoints the roles of Aß and of CCL2/CCR2 axis-dependent inflammation in photoreceptor apoptosis.

APP involvement in retinogenesis of mice.

Very few studies have examined expression and function of amyloid precursor protein (APP) in the retina. We showed that APP mRNA and protein are expressed according to the different waves of retinal differentiation. Depletion of App led to an absence of amacrine cells, a 50% increase in the number of horizontal cells and alteration of the synapses. The retinas of adult APP(-/-) mice showed only half as many glycinergic amacrine cells as wild-type retinas. We identified Ptf1a, which plays a role in controlling both amacrine and horizontal cell fates, as a downstream effector of APP. The observation of a similar phenotype in sorLA knockout mice, a major regulator of APP processing, suggests that regulation of APP functions via sorLA controls the determination of amacrine and horizontal cell fate. These findings provide novel insights that indicate that APP plays an important role in retinal differentiation.

[New lighting technology and our eyes]. / Les nouveaux éclairages et nos yeux.

The retina is the neurosensitive layer of the eye. In this tissue, photoreceptors convert light into nerve signals to be relayed to the brain. Despite retinal specialization in the treatment of light, excessive exposure can cause retinal damage, called retinal phototoxicity. In recent years, lighting devices rich in wavelengths of high energy (blue light) appeared, raising new concerns about retinal protection against light damage. We focus here on light-induced ocular diseases and the possible influence on visual health of new lighting technologies.

TITLE: Les nouveaux éclairages et nos yeux. ABSTRACT: Dans la rétine, couche neurosensorielle de l'œil, les photorécepteurs transforment le signal lumineux en influx nerveux interprétable par le cerveau. Malgré sa spécialisation dans le traitement des signaux lumineux, la rétine peut subir des dommages, à la suite d'une exposition excessive à la lumière ; on parle alors de phototoxicité rétinienne. Ces dernières années, l'apparition de dispositifs d'éclairage riches en longueurs d'onde de forte énergie (ce que l'on nomme lumière bleue), remet le problème de la phototoxicité rétinienne à l'ordre du jour. Nous discutons des pathologies oculaires induites par la lumière et de la possible influence des nouvelles technologies d'éclairage sur notre santé visuelle.

Mechanisms of FH Protection Against Neovascular AMD.

A common allele (402H) of the complement factor H (FH) gene is the major risk factor for age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. Development and progression of AMD involves vascular and inflammatory components partly by deregulation of the alternative pathway of the complement system (AP). The loss of central vision results from atrophy and/or from abnormal neovascularization arising from the choroid. The functional link between FH, the main inhibitor of AP, and choroidal neovascularization (CNV) in AMD remains unclear. In a murine model of CNV used as a model for neovascular AMD (nAMD), intraocular human recombinant FH (recFH) reduced CNV as efficiently as currently used anti-VEGF (vascular endothelial growth factor) antibody, decreasing deposition of C3 cleavage fragments, membrane attack complex (MAC), and microglia/macrophage recruitment markers in the CNV lesion site. In sharp contrast, recFH carrying the H402 risk variant had no effect on CNV indicating a causal link to disease etiology. Only the recFH NTal region (recFH1-7), containing the CCPs1-4 C3-convertase inhibition domains and the CCP7 binding domain, exerted all differential biological effects. The CTal region (recFH7-20) containing the CCP7 and CCPs19-20 binding domains was antiangiogenic but did not reduce the microglia/macrophage recruitment. The antiangiogenic effect of both recFH1-20 and recFH-CCP7-20 resulted from thrombospondin-1 (TSP-1) upregulation independently of the C3 cleavage fragments generation. This study provides insight on the mechanistic role of FH in nAMD and invites to reconsider its therapeutic potential.

Prematurely senescent ARPE-19 cells display features of age-related macular degeneration.

The etiology of age-related macular degeneration (AMD), the leading cause of blindness in the developed world, remains poorly understood, but may be related to cumulative oxidative stress. The prime target of the disease is the retinal pigmented epithelium (RPE). To study the molecular mechanisms underlying RPE degeneration, we investigated whether repetitive oxidative stress induced premature senescence in RPE cells from the human ARPE-19 cell line. After exposure to 8 mM tert-butylhydroperoxide (tert-BHP) for 1 h daily for 5 days, the cells showed four well-known senescence biomarkers: hypertrophy, senescence-associated beta-galactosidase activity, growth arrest, and cell cycle arrest in G1. A specific low-density array followed by qRT-PCR validation allowed us to identify 36 senescence-associated genes differentially expressed in the prematurely senescent cells. Functional analysis demonstrated that premature senescence induced amyloid beta secretion, resistance to acute stress by tert-BHP and amyloid beta, and defects in adhesion and transepithelial permeability. Coculture assays with choroidal endothelial cells showed the proangiogenic properties of the senescent RPE cells. These results demonstrate that chronic oxidative stress induces premature senescence in RPE cells that modifies the transcriptome and substantially alters cell processes involved in the pathophysiology of AMD. Oxidative stress-induced premature senescence may represent an in vitro model for screening therapeutics against AMD and other retinal degeneration disorders.

17-AAG and 17-DMAG-induced inhibition of cell proliferation through B-Raf downregulation in WT B-Raf-expressing uveal melanoma cell lines.

PURPOSE: The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) has been shown to have promising results in antitumor activity through the degradation of the activated V600E mutant of B-Raf (V600E B-Raf) in cutaneous melanoma cell lines. It has different effects, however, on the wild-type form of B-Raf (WT B-Raf), according to the WT B-Raf activation levels in the tumor cells. Uveal melanoma cells express WT B-Raf and only rarely express V600E B-Raf. This study was conducted to investigate the effects of HSP90 inhibition on uveal melanoma cell lines. METHODS: Human uveal melanoma cell lines were treated with the HSP90 inhibitors 17-AAG and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG). Cell proliferation was assessed by MTT staining, and apoptosis was quantified by flow cytometry. Analysis of the expression of HSP90 and activation of the MEK/ERK downstream signaling of B-Raf was performed by Western blot. Effects of the downregulation of the HSP90 cochaperone, cdc37, on cell proliferation and activation of MEK/ERK was investigated by siRNA strategy. RESULTS: The inhibition of HSP90 downregulated B-Raf, decreased cell proliferation, and reduced activation of MEK/ERK in uveal melanoma cell lines expressing WT B-Raf. HSP90 inhibition also reduced the expression of Akt, but the inhibition of Akt had no effect on cell proliferation, ruling out a role of Akt in the 17-AAG-induced inhibition of cell proliferation. The downregulation of cdc37 did not affect MEK/ERK signaling and cell proliferation, demonstrating that the cochaperone was not required for HSP90-controlled stability of B-Raf. c-Kit was also downregulated after HSP90 inhibition. The combination of 17-DMAG with imatinib mesylate, the inhibitor of c-kit, had synergistic inhibitory effects on cell proliferation in WT B-Raf uveal melanoma cell lines. CONCLUSIONS: These results suggest that targeting HSP90 in tandem with c-Kit inhibition may be a promising therapeutic approach to uveal melanoma.

AßPP-induced UPR Transcriptomic Signature of Glial Cells to Oxidative Stress as an Adaptive Mechanism to Preserve Cell Function and Survival.

BACKGROUND: Alzheimer's disease (AD) and age-related macular degeneration (AMD) present similarities, particularly with respect to oxidative stress, including production of 4-Hydroxy-2- nonenal (HNE). AMD has been named the AD in the eye. The Müller cells (MC) function as a principal glia of the retina and maintain water/potassium, glutamate homeostasis and redox status. Any MC dysfunction results in retinal neurodegeneration. OBJECTIVES: We investigated the effects of HNE in human MC. RESULTS: HNE induced an increase of the reactive oxygen species associated with mitochondrial dysfunction and apoptosis. HNE induced endoplasmic reticulum (ER) stress (upregulation of GRP78/Bip, and the proapoptotic factor, CHOP). HNE also impaired expression of genes controlling potassium homeostasis (KCNJ10), glutamate detoxification (GS), and the visual cycle (RLBP1). MC adaptive response to HNE included upregulation of amyloid-ß protein precursor (AßPP). To determine the role of AßPP, we overexpressed AßPP in MC. Overexpression of AßPP induced strong antioxidant and anti-ER stress (PERK downregulation and GADD34 upregulation) responses accompanied by activation of the prosurvival branch of the unfolded protein response. It was also associated with upregulation of major genes involved in MC-controlled retinal homeostasis (KCNJ10, GS, and RLBP1) and protection against HNE-induced apoptosis. Therefore, AßPP is an ER and oxidative stress responsive molecule, and is able to stimulate the transcription of major genes involved in MC functions impaired by HNE. CONCLUSION: Our study suggests that targeting oxidative and ER stress might be a potential therapeutic strategy against glia impairment in AMD and AD, in light of the common features between the two pathologies.

Sustained versus transient ERK1/2 signaling underlies the anti- and proapoptotic effects of oxidative stress in human RPE cells.

PURPOSE: Oxidative stress is thought to contribute to the pathogenesis of age-related macular degeneration (AMD), which involves retinal pigmented epithelial (RPE) cell death. However, signaling pathways involved in the oxidative-stress-induced RPE cell death are poorly understood. This study was conducted to investigate the involvement of the MAP kinase pathways during the induction of RPE cell death by oxidative stress. METHODS: ARPE-19 cells were exposed to the oxidant tert-butyl hydroperoxide (t-BHP). Cell viability was assessed by cell counting and MTT-staining, and apoptosis was quantified by TUNEL and flow cytometry. Activation of JNK1/3, p38 alphabeta MAPKs and ERK1/2 and their potential targets was detected by Western blot analysis and immunochemistry with specific anti-phospho protein antibodies. Specific pharmacologic inhibitors directed against the MAPKs were used to analyze the signaling involved in cell death of RPE cells exposed to t-BHP. RESULTS: Exposure of RPE cells to t-BHP, associated with increase in reactive oxygen species and intracellular glutathione depletion, induced time- and concentration-dependent apoptosis, which was associated with the accumulation of inactive ERK1/2 in cell nuclei and a transient and weak ERK1/2 activation. This activation was accompanied by a deactivation of P90(RSK), the major target of ERK1/2 and consequently by the delayed activation of its transcription factor CREB. MEK1/2 inhibition completely suppressed the transient activation of ERK1/2 and completely blocked apoptosis, demonstrating the role of the MEK-ERK module in mediating oxidative-stress-induced RPE cell death. In contrast, neither JNKs nor p38 alphabeta MAPKs were involved in mediating t-BHP-induced apoptotic signaling in RPE cells. CONCLUSIONS: The results suggest that inhibiting the MEK-ERK module may allow the development of selective methods for treating oxidative-stress-induced RPE degeneration, such as AMD.

Amyloidosis in Retinal Neurodegenerative Diseases.

As a part of the central nervous system, the retina may reflect both physiological processes and abnormalities related to pathologies that affect the brain. Amyloidosis due to the accumulation of amyloid-beta (Aß) was initially regarded as a specific and exclusive characteristic of neurodegenerative alterations seen in the brain of Alzheimer's disease (AD) patients. More recently, it was discovered that amyloidosis-related alterations, similar to those seen in the brain of Alzheimer's patients, also occur in the retina. Remarkably, these alterations were identified not only in primary retinal pathologies, such as age-related macular degeneration (AMD) and glaucoma, but also in the retinas of Alzheimer's patients. In this review, we first briefly discuss the biogenesis of Aß, a peptide involved in amyloidosis. We then discuss some pathological aspects (synaptic dysfunction, mitochondrial failure, glial activation, and vascular abnormalities) related to the neurotoxic effects of Aß. We finally highlight common features shared by AD, AMD, and glaucoma in the context of Aß amyloidosis and further discuss why the retina, due to the transparency of the eye, can be considered as a "window" to the brain.

Amyloid ß Peptide Induces Apoptosis Through P2X7 Cell Death Receptor in Retinal Cells: Modulation by Marine Omega-3 Fatty Acid DHA and EPA.

Retinal Müller glial cells have already been implicated in age-related macular degeneration (AMD). AMD is characterized by accumulation of toxic amyloid-ß peptide (Aß); the question we raise is as follows: is P2X7 receptor, known to play an important role in several degenerative diseases, involved in Aß toxicity on Müller cells? Retinal Müller glial cells were incubated with Aß for 48 h. Cell viability was assessed using the alamarBlue assay and cytotoxicity using the lactate dehydrogenase (LDH) release assay. P2X7 receptor expression was highlighted by immunolabeling observed on confocal microscopy and its activation was evaluated by YO-PRO-1 assay. Hoechst 33342 was used to evaluate chromatin condensation, and caspases 8 and 3 activation was assessed using AMC assays. Lipid formulation rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) used in Age-Related Eye Disease Study 2 was incubated on cells for 15 min prior to Aß incubation. For the first time, we showed that Aß induced caspase-independent apoptosis through P2X7 receptor activation on our retinal model. DHA and EPA are polyunsaturated fatty acids recommended in food supplement to prevent AMD. We therefore modulated Aß cytotoxicity using a lipid formulation rich in DHA and EPA to have a better understanding of the results observed in clinical studies. We showed that fish oil rich in EPA and DHA, in combination with a potent P2X7 receptor antagonist, represents an efficient modulator of Aß toxicity and that P2X7 could be an interesting therapeutic target to prevent AMD.

Amyloid Precursor-Like Protein 2 deletion-induced retinal synaptopathy related to congenital stationary night blindness: structural, functional and molecular characteristics.

BACKGROUND: Amyloid precursor protein knockout mice (APP-KO) have impaired differentiation of amacrine and horizontal cells. APP is part of a gene family and its paralogue amyloid precursor-like protein 2 (APLP2) has both shared as well as distinct expression patterns to APP, including in the retina. Given the impact of APP in the retina we investigated how APLP2 expression affected the retina using APLP2 knockout mice (APLP2-KO). RESULTS: Using histology, morphometric analysis with noninvasive imaging technique and electron microscopy, we showed that APLP2-KO retina displayed abnormal formation of the outer synaptic layer, accompanied with greatly impaired photoreceptor ribbon synapses in adults. Moreover, APLP2-KO displayed a significant decease in ON-bipolar, rod bipolar and type 2 OFF-cone bipolar cells (36, 21 and 63 %, respectively). Reduction of the number of bipolar cells was accompanied with disrupted dendrites, reduced expression of metabotropic glutamate receptor 6 at the dendritic tips and alteration of axon terminals in the OFF laminae of the inner plexiform layer. In contrast, the APP-KO photoreceptor ribbon synapses and bipolar cells were intact. The APLP2-KO retina displayed numerous phenotypic similarities with the congenital stationary night blindness, a non-progressive retinal degeneration disease characterized by the loss of night vision. The pathological phenotypes in the APLP2-KO mouse correlated to altered transcription of genes involved in pre- and postsynatic structure/function, including CACNA1F, GRM6, TRMP1 and Gα0, and a normal scotopic a-wave electroretinogram amplitude, markedly reduced scotopic electroretinogram b-wave and modestly reduced photopic cone response. This confirmed the impaired function of the photoreceptor ribbon synapses and retinal bipolar cells, as is also observed in congenital stationary night blindness. Since congenital stationary night blindness present at birth, we extended our analysis to retinal differentiation and showed impaired differentiation of different bipolar cell subtypes and an altered temporal sequence of development from OFF to ON laminae in the inner plexiform layer. This was associated with the altered expression patterns of bipolar cell generation and differentiation factors, including MATH3, CHX10, VSX1 and OTX2. CONCLUSIONS: These findings demonstrate that APLP2 couples retina development and synaptic genes and present the first evidence that APLP2 expression may be linked to synaptic disease.

cAMP inhibits the proliferation of retinal pigmented epithelial cells through the inhibition of ERK1/2 in a PKA-independent manner.

Retinal pigmented epithelial (RPE) cell integrity is critical to the maintenance of retina functions and RPE cells do not proliferate in adults. The activation of RPE results in cell proliferation which may be associated with proliferative retinopathy and choroidal melanoma. Mitogen-activated protein kinase (MAPK) is believed to be a key participant in the response to mitogenic stimuli. We therefore investigated the involvement of the extracellular signal-regulated protein kinase (ERK) 1 and 2 during the induction of RPE cell proliferation. After foetal calf serum (FCS) stimulation activation of the Ras/Raf/ERK signalling pathway was detected by Western blotting and immunochemistry, with specific anti-phosphosignalling protein antibodies. Pharmacological and antisense (AS) oligonucleotide (ODN) strategies were used to analyse the signalling involved in FCS-induced RPE cell proliferation. Activation of the small G protein Ras and, to a lesser extent of Raf-1, the kinase directly downstream from Ras, was necessary to FCS-induced cell proliferation. MEK1/2 and ERK1/2 were activated during cell proliferation. Inhibition of MEK1/2 with UO 126 completely abolished ERK1/2 activation and reduced cell proliferation by 33-43%. ERK1/2 depletion by an AS ODN approach reduced cell proliferation by 27-33%, confirming the role of ERK1/2 in the FCS stimulation of RPE cells. We also investigated the role of PKA/cAMP, one of the major inhibitory pathways of ERK1/2. PKA blockade did not modify ERK1/2 activation or cell proliferation. In contrast, agents that increased cAMP concentration, abolished RPE proliferation, and MEK/ERK activation. Moreover, inhibition of the cAMP-activated small G protein Rap1, partially reversed the inhibitory effects of cAMP on cell proliferation and MEK/ERK activation. The requirement for Ras and ERK1/2, the lack of ERK1/2 regulation by PKA and the cAMP/Rap1 counter-regulatory pathway for ERK-mediated cell proliferation suggest complex regulation of signalling in RPE cells. These data may have important implications for the development of more selective models for retinal anti-proliferative therapies.

Opposite long-term regulation of c-Myc and p27Kip1 through overactivation of Raf-1 and the MEK/ERK module in proliferating human choroidal melanoma cells.

Although there is no current evidence for ras gene mutation in choroidal melanoma, there is an increasing body of evidence indicating that deregulated intracellular signalling pathways are involved in choroidal melanoma pathogenesis. The various components of the linear Raf/MEK/ERK signalling pathway have been implicated in various tumours. We therefore investigated the role of Raf-1 and the MEK/ERK module in the proliferation of human normal choroidal melanocytes (NCM) and cells from the ocular choroidal melanoma (OCM-1) cell line. OCM-1 cells proliferated four times faster than NCM. High basal activation of the MEK/ERK module was observed in unstimulated OCM-1 cells, whereas rapid and persistent activation was detected after serum stimulation, throughout the 24-h period of culture. In contrast, the activation of MEK/ERK was barely detectable in unstimulated NCM and occurred late (6 h) after the stimulation of cell proliferation. Inhibition of Raf-1 and MEK1/2 activation by pharmacological approaches and of the production of Raf-1 and ERK1/2 by antisense oligonucleotide approaches demonstrated that Raf-1 and the MEK/ERK module controlled proliferation in OCM-1 cells, but not in NCM. OCM-1 cells produced very low levels of p27Kip1, whereas NCM produced constant, high levels of p27Kip1. The inhibition of Raf-1 or MEK1/2 induced a large increase in p27Kip1 in OCM-1 cells, associated with an arrest of cell proliferation. Levels of c-Myc production were high and constant in OCM-1 cells and low in NCM, in contrast to what was observed for p27Kip1. The inhibition of both Raf-1 and MEK1/2 induced a decrease in c-Myc production and downregulated c-Myc activity by preventing c-Myc phosphorylation in OCM-1 cells. We conclude that Raf-1 and the MEK/ERK module control the production of both p27Kip1 and c-Myc, and the activation of c-Myc for OCM-1 cell proliferation.

VP22 light controlled delivery of oligonucleotides to ocular cells in vitro and in vivo.

PURPOSE: To study VP22 light controlled delivery of antisense oligonucleotide (ODN) to ocular cells in vitro and in vivo. METHODS: The C-terminal half of VP22 was expressed in Escherichia coli, purified and mixed with 20 mer phosphorothioate oligonucleotides (ODNs) to form light sensitive complex particles (vectosomes). Uptake of vectosomes and light induced redistribution of ODNs in human choroid melanoma cells (OCM-1) and in human retinal pigment epithelial cells (ARPE-19) were studied by confocal and electron microscopy. The effect of vectosomes formed with an antisense ODN corresponding to the 3'-untranslated region of the human c-raf kinase gene on the viability and the proliferation of OCM-1 cells was assessed before and after illumination. Cells incubated with vectosomes formed with a mismatched ODN, a free antisense ODN or a free mismatched ODN served as controls. White light transscleral illumination was carried out 24 h after the intravitreal injection of vectosomes in rat eyes. The distribution of fluorescent vectosomes and free fluorescent ODN was evaluated on cryosections by fluorescence microscopy before, and 1 h after illumination. RESULTS: Overnight incubation of human OCM-1 and ARPE-19 cells with vectosomes lead to intracellular internalization of the vectosomes. When not illuminated, internalized vectosomes remained stable within the cell cytoplasm. Disruption of vectosomes and release of the complexed ODN was induced by illumination of the cultures with a cold white light or a laser beam. In vitro, up to 60% inhibition of OCM-1 cell proliferation was observed in illuminated cultures incubated with vectosomes formed with antisense c-raf ODN. No inhibitory effect on the OCM-1 cell proliferation was observed in the absence of illumination or when the cells are incubated with a free antisense c-raf ODN and illuminated. In vivo, 24 h after intravitreal injection, vectosomes were observed within the various retinal layers accumulating in the cytoplasm of RPE cells. Transscleral illumination of the injected eyes with a cold white light induced disruption of the vectosomes and a preferential localization of the "released" ODNs within the cell nuclei of the ganglion cell layer, the inner nuclear layer and the RPE cells. CONCLUSIONS: In vitro, VP22 light controlled delivery of ODNs to ocular cells nuclei was feasible using white light or laser illumination. In vivo, a single intravitreal injection of vectosomes, followed by transscleral illumination allowed for the delivery of free ODNs to retinal and RPE cells.