Stephen E. Moss

Annexin A2 at the interface between F-actin and membranes enriched in phosphatidylinositol 4,5,-bisphosphate.

Vesicle rocketing has been used as a model system for understanding the dynamics of the membrane-associated F-actin cytoskeleton, but in many experimental systems is induced by persistent, non-physiological stimuli. Localised changes in the concentration of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in membranes stimulate the recruitment of actin-remodelling proteins to their sites of action, regulate their activity and favour vesicle rocketing. The calcium and anionic phospholipid-binding protein annexin A2 is necessary for macropinocytic rocketing and has been shown to bind both PI(4,5)P2 and the barbed-ends of F-actin filaments. Here we show that annexin A2 localises to the comet tails which form constitutively in fibroblasts from patients with Lowe Syndrome. These fibroblasts are deficient in OCRL1, a phosphatidylinositol polyphosphate 5-phosphatase with specificity for PI(4,5)P2. We show that upon depletion of annexin A2 from these cells vesicle rocketing is reduced, and that this is also dependent upon PI(4,5)P2 formation. Annexin A2 co-localised with comet-tails induced by pervanadate and hyperosmotic shock in a basophilic cell line, and in an epithelial cell line upon activation of PKC. In vitro annexin A2 promoted comet formation in a bead-rocketing assay and was sufficient to link F-actin filaments to PI(4,5)P2 containing vesicles. These observations are consistent with a role for annexin A2 as an actin nucleator on PI(4,5)P2-enriched membranes.

Regulation of Mitochondrial Morphogenesis by Annexin A6

Mitochondrial homeostasis is critical in meeting cellular energy demands, shaping calcium signals and determining susceptibility to apoptosis. Here we report a role for anxA6 in the regulation of mitochondrial morphogenesis, and show that in cells lacking anxA6 mitochondria are fragmented, respiration is impaired and mitochondrial membrane potential is reduced. In fibroblasts from AnxA6 −/− mice, mitochondrial Ca2+ uptake is reduced and cytosolic Ca2+ transients are elevated. These observations led us to investigate possible interactions between anxA6 and proteins with roles in mitochondrial fusion and fission. We found that anxA6 associates with Drp1 and that mitochondrial fragmentation in AnxA6 −/− fibroblasts was prevented by the Drp1 inhibitor mdivi-1. In normal cells elevation of intracellular Ca2+ disrupted the interaction between anxA6 and Drp1, displacing anxA6 to the plasma membrane and promoting mitochondrial fission. Our results suggest that anxA6 inhibits Drp1 activity, and that Ca2+-binding to anxA6 relieves this inhibition to permit Drp1-mediated mitochondrial fission.

A New Role for Annexin A11 in the Early Secretory Pathway via Stabilizing Sec31A Protein at the Endoplasmic Reticulum Exit Sites (ERES)

Background: Apoptosis-linked gene 2 (ALG-2) is a calcium-dependent adaptor protein that is recruited to the Sec31A-positive ERES. Results: Physical association between annexin A11 and Sec31A is mediated by ALG-2, and annexin A11 is required for stable association of Sec31A at the ERES. Conclusion: Annexin A11 participates in ER-to-Golgi trafficking. Significance: Annexin A11 is the first annexin shown to modulate the early secretory pathway. Exit of cargo molecules from the endoplasmic reticulum (ER) for transport to the Golgi is the initial step in intracellular vesicular trafficking. The coat protein complex II (COPII) machinery is recruited to specialized regions of the ER, called ER exit sites (ERES), where it plays a central role in the early secretory pathway. It has been known for more than two decades that calcium is an essential factor in vesicle trafficking from the ER to Golgi apparatus. However, the role of calcium in the early secretory pathway is complicated and poorly understood. We and others previously identified Sec31A, an outer cage component of COPII, as an interacting protein for the penta-EF-hand calcium-binding protein ALG-2. In this study, we show that another calcium-binding protein, annexin A11 (AnxA11), physically associates with Sec31A by the adaptor function of ALG-2. Depletion of AnxA11 or ALG-2 decreases the population of Sec31A that is stably associated with the ERES and causes scattering of juxtanuclear ERES to the cell periphery. The synchronous ER-to-Golgi transport of transmembrane cargoes is accelerated in AnxA11- or ALG-2-knockdown cells. These findings suggest that AnxA11 maintains architectural and functional features of the ERES by coordinating with ALG-2 to stabilize Sec31A at the ERES.

Lowe Syndrome Protein OCRL1 Supports Maturation of Polarized Epithelial Cells

Mutations in the inositol polyphosphate 5-phosphatase OCRL1 cause Lowe Syndrome, leading to cataracts, mental retardation and renal failure. We noted that cell types affected in Lowe Syndrome are highly polarized, and therefore we studied OCRL1 in epithelial cells as they mature from isolated individual cells into polarized sheets and cysts with extensive communication between neighbouring cells. We show that a proportion of OCRL1 targets intercellular junctions at the early stages of their formation, co-localizing both with adherens junctional components and with tight junctional components. Correlating with this distribution, OCRL1 forms complexes with junctional components α-catenin and zonula occludens (ZO)-1/2/3. Depletion of OCRL1 in epithelial cells growing as a sheet inhibits maturation; cells remain flat, fail to polarize apical markers and also show reduced proliferation. The effect on shape is reverted by re-expressed OCRL1 and requires the 5′-phosphatase domain, indicating that down-regulation of 5-phosphorylated inositides is necessary for epithelial development. The effect of OCRL1 in epithelial maturation is seen more strongly in 3-dimensional cultures, where epithelial cells lacking OCRL1 not only fail to form a central lumen, but also do not have the correct intracellular distribution of ZO-1, suggesting that OCRL1 functions early in the maturation of intercellular junctions when cells grow as cysts. A role of OCRL1 in junctions of polarized cells may explain the pattern of organs affected in Lowe Syndrome.

Retinal changes precede visual dysfunction in the complement factor H knockout mouse.

We previously reported that aged mice lacking complement factor H (CFH) exhibit visual defects and structural changes in the retina. However, it is not known whether this phenotype is age-related or is the consequence of disturbed development. To address this question we investigated the effect of Cfh gene deletion on the retinal phenotype of young and mid-age mice. Cfh −/− mouse eyes exhibited thickening of the retina and reduced nuclear density, but relatively normal scotopic and photopic electroretinograms. At 12 months there was evidence of subtle astroglial activation in the Cfh −/− eyes, and significant elevation of the complement regulator, decay-accelerating factor (DAF) in Müller cells. In the retinal pigment epithelium (RPE) of young control and Cfh −/− animals mitochondria and melanosomes were oriented basally and apically respectively, whereas the apical positioning of melanosomes was significantly perturbed in the mid-age Cfh −/− RPE. We conclude that deletion of Cfh in the mouse leads to defects in the retina that precede any marked loss of visual function, but which become progressively more marked as the animals age. These observations are consistent with a lifelong role for CFH in retinal homeostasis.

Immortalized human fetal retinal cells retain progenitor characteristics and represent a potential source for the treatment of retinal degenerative disease.

Human fetal retinal cells have been widely advocated for the development of cellular replacement therapies in patients with retinal dystrophies and age-related macular degeneration. A major limitation, however, is the lack of an abundant and renewable source of cells to meet therapeutic demand, although theoretically this may be addressed through the use of immortalized retinal progenitor cell lines. Here, we have used the temperature-sensitive tsA58 simian virus SV40 T antigen to conditionally immortalize human retinal progenitor cells isolated from retinal tissue at 10–12 weeks of gestation. We show that immortalized human fetal retinal cells retain their progenitor cell properties over many passages, and are comparable with nonimmortalized human fetal retinal cultures from the same gestational period with regard to expression of certain retinal genes. To evaluate the capacity of these cells to integrate into the diseased retina and to screen for potential tumorigenicity, cells were grafted into neonatal hooded Lister rats and RCS dystrophic rats. Both cell lines exhibited scarce integration into the host retina and failed to express markers of mature differentiated retinal cells. Moreover, although immortalized cells showed a greater propensity to survive, the cell lines demonstrated poor long-term survival. All grafts were infiltrated with host macrophage/microglial cells throughout their duration of survival. This study demonstrates that immortalized human fetal retinal progenitor cells retain their progenitor characteristics and may therefore have therapeutic potential in strategies that demand a renewable and consistent supply of donor cells for the treatment of degenerative retinal diseases.

In vivo imaging of retinal cell apoptosis following acute light exposure

PURPOSE Outer nuclear apoptosis following acute light exposure has previously only been shown histologically. This study investigated whether in vivo detection with DARC (detection of apoptosing retinal cells) technology could identify cells undergoing apoptosis. METHODS Acute blue light damage (lambda=405 nm; 3.2 mW/cm(2)) was applied to eyes of dark Agouti rats over 2 h. In vivo retinal imaging using confocal scanning laser ophthalmoscopy was performed before and directly after light exposure as well as after 24 h of dark adaptation. Development of retinal cell apoptosis was then assessed using intravitreal fluorescent-labeled annexin-5 with DARC technology in vivo. RESULTS Directly after light exposure, no pathological retinal changes were observed by in vivo imaging. However, retinal flattening and the development of apoptosis within the irradiated retina occurred 1 day later and following dark adaptation. Confocal live scanning through the exposed retina revealed hyperfluorescent apoptotic cells at the level of the outer retina. Histological analysis confirmed the occurrence of photoreceptor cell death and the development of cellular damage at the outer retina. DISCUSSION This study confirms acute light-induced outer nuclear apoptosis using in vivo DARC technology. This may open new and promising ways to assess programmed cell death of the photoreceptor cells, which - until now - was possible only with postmortem analysis.

Exposure to the complement C5b-9 complex sensitizes 661W photoreceptor cells to both apoptosis and necroptosis

The loss of photoreceptors is the defining characteristic of many retinal degenerative diseases, but the mechanisms that regulate photoreceptor cell death are not fully understood. Here we have used the 661W cone photoreceptor cell line to ask whether exposure to the terminal complement complex C5b-9 induces cell death and/or modulates the sensitivity of these cells to other cellular stressors. 661W cone photoreceptors were exposed to complete normal human serum following antibody blockade of CD59. Apoptosis induction was assessed morphologically, by flow cytometry, and on western blotting by probing for cleaved PARP and activated caspase-3. Necroptosis was assessed by flow cytometry and Sirtuin 2 inhibition using 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furyl]-N-5-quinolinylacrylamide (AGK2). The sensitivity of 661W cells to ionomycin, staurosporine, peroxide and chelerythrine was also investigated, with or without prior formation of C5b-9. 661W cells underwent apoptotic cell death following exposure to C5b-9, as judged by poly(ADP-ribose) polymerase 1 cleavage and activation of caspase-3. We also observed apoptotic cell death in response to staurosporine, but 661W cells were resistant to both ionomycin and peroxide. Interestingly, C5b-9 significantly increased 661W sensitivity to staurosporine-induced apoptosis and necroptosis. These studies show that low levels of C5b-9 on 661W cells can induce apoptosis, and that C5b-9 specifically sensitizes 661W cells to certain apoptotic and necroptotic pathways. Our observations provide new insight into the potential role of the complement system in photoreceptor loss, with implications for the molecular aetiology of retinal disease.

In-vivo-Imaging retinaler Zellapoptose nach akuter Lichtexposition

PURPOSE Outer nuclear apoptosis following acute light exposure has previously only been shown histologically. This study investigated whether in vivo detection with DARC (detection of apoptosing retinal cells) technology could identify cells undergoing apoptosis. METHODS Acute blue light damage (lambda=405 nm; 3.2 mW/cm(2)) was applied to eyes of dark Agouti rats over 2 h. In vivo retinal imaging using confocal scanning laser ophthalmoscopy was performed before and directly after light exposure as well as after 24 h of dark adaptation. Development of retinal cell apoptosis was then assessed using intravitreal fluorescent-labeled annexin-5 with DARC technology in vivo. RESULTS Directly after light exposure, no pathological retinal changes were observed by in vivo imaging. However, retinal flattening and the development of apoptosis within the irradiated retina occurred 1 day later and following dark adaptation. Confocal live scanning through the exposed retina revealed hyperfluorescent apoptotic cells at the level of the outer retina. Histological analysis confirmed the occurrence of photoreceptor cell death and the development of cellular damage at the outer retina. DISCUSSION This study confirms acute light-induced outer nuclear apoptosis using in vivo DARC technology. This may open new and promising ways to assess programmed cell death of the photoreceptor cells, which - until now - was possible only with postmortem analysis.

Regulation of retinal pigment epithelial cell phenotype by Annexin A8

The retinoic acid derivative fenretinide (FR) is capable of transdifferentiating cultured retinal pigment epithelial (RPE) cells towards a neuronal-like phenotype, but the underlying mechanisms are not understood. To identify genes involved in this process we performed a microarray analysis of RPE cells pre- and post-FR treatment, and observed a marked down-regulation of AnnexinA8 (AnxA8) in transdifferentiated cells. To determine whether AnxA8 plays a role in maintaining RPE cell phenotype we directly manipulated AnxA8 expression in cultured and primary RPE cells using siRNA-mediated gene suppression, and over-expression of AnxA8-GFP in conjunction with exposure to FR. Treatment of RPE cells with AnxA8 siRNA recapitulated exposure to FR, with cell cycle arrest, neuronal transdifferentiation, and concomitant up-regulation of the neuronal markers calretinin and calbindin, as assessed by real-time PCR and immunofluorescence. In contrast, AnxA8 transient over-expression in ARPE-19 cells prevented FR-induced differentiation. Ectopic expression of AnxA8 in AnxA8-depleted cells led to decreased neuronal marker staining, and normal cell growth as judged by phosphohistone H3 staining, cell counting and cleaved caspase-3 levels. These data show that down-regulation of AnxA8 is both necessary and sufficient for neuronal transdifferentiation of RPE cells and reveal an essential role for AnxA8 as a key regulator of RPE phenotype.