Deborah M. Wallace

Rab11-FIP4 interacts with Rab11 in a GTP-dependent manner and its overexpression condenses the Rab11 positive compartment in HeLa cells

We have recently identified Rab11-FIP4 as the sixth member of the Rab11-FIP family of Rab11 interacting proteins. Here, we demonstrate that Rab11-FIP4 interacts with Rab11 in a GTP-dependent manner and that its C-terminal region allows the protein to self-interact and interact with pp75/Rip11, Rab11-FIP2, and Rab11-FIP3. However, Rab11-FIP4 does not appear to interact directly with Rab coupling protein (RCP). We investigated the subcellular localisation of Rab11-FIP4 in HeLa cells and show that it colocalises extensively with transferrin and with Rab11. Furthermore, when overexpressed, it causes a condensation of the Rab11 compartment in the perinuclear region. We demonstrate that the carboxy-terminal region of Rab11-FIP4 (Rab11-FIP4(C-ter)) is necessary and sufficient for its endosomal membrane association. Expression of Rab11-FIP4(C-ter) causes a dispersal of the Rab11 compartment towards the cell periphery and does not inhibit transferrin recycling in HeLa cells. It is likely that Rab11-FIP4 serves as a Rab11 effector in a Rab11 mediated function other than transferrin recycling.

Redox survival signalling in retina-derived 661W cells

Reactive oxygen species have been implicated in processes involving cellular damage and subsequent cell death, especially in organs such as the eye that are constantly exposed to excitatory signals. However, recent studies have shown that oxidant species can also act as intracellular signalling molecules promoting cell survival, but little is known about this mechanism in the retina. The present study demonstrates for the first time that hydrogen peroxide (H2O2) is generated rapidly and acts as a pro-survival signal in response to a variety of apoptotic stimuli in retina-derived 661W cells and in the retinal ganglion cell line RGC-5. Focussing on 661Ws and serum deprivation, we systematically investigated pro-survival and pro-death pathways and discovered that the rapid and transient burst of H2O2 activates the AKT survival pathway. Activation of the apoptotic machinery takes place following the decline of H2O2 to basal levels. To substantiate this proposed pro-survival role of H2O2, we inhibited the oxidant burst, which exacerbated cell death. Conversely, maintenance of the oxidant signal using exogenous H2O2 enhanced cell survival. Overall, the results presented in this study provide evidence for a novel role of H2O2 in mediating survival of retinal cells in response to apoptotic stimuli.

A Novel Antioxidant Function for the Tumor-Suppressor Gene p53 in the Retinal Ganglion Cell

PURPOSE Recent evidence has suggested that the tumor-suppressor gene p53 has a role in regulating antioxidant response in cancer cells. This study was conducted to determine whether p53 regulates redox enzymes in a neuronal context in RGCs and whether this regulation contributes to an increased survival signal. METHODS The expression of p53, and its putative responsive antioxidant enzymes sestrin 2, catalase, Cu/ZnSOD, and MnSOD were evaluated in the developing rat retina by immunohistochemistry and Western blot. Small interfering (si)RNA to p53 was used in an RGC cell line, RGC-5, and downstream effects on antioxidants observed by Western blot. Transcription factor-analysis software was used to identify p53 binding sites on the catalase promoter, and chromatin immunoprecipitation (ChIP) assays on whole retina to demonstrate in vivo binding. The effect of p53 deficiency on basal reactive oxygen species levels (ROS) within the RGC and on susceptibility to oxidative-signaling-induced apoptosis was measured by flow cytometry. RESULTS Developmental expression patterns of p53 and catalase mirrored each other. p53 knockdown resulted in a significant decrease in catalase. p53-binding sites were identified on the rat catalase promoter and confirmed in vivo. p53 knockdown resulted in a corresponding increase in basal cellular ROS levels and increased susceptibility to oxidative-signaling-induced cell death. CONCLUSIONS The results suggest a novel regulating influence of p53 on catalase in the retina--more specifically in the RGC--and an influence of p53 on the susceptibility of the cell to oxidative-signaling-induced apoptosis, which could implicate p53 as a potential neuroprotectant for the RGC.

The role of matricellular proteins in glaucoma

Glaucoma is an optic neuropathy affecting approximately 60million people worldwide and is the second most common cause of irreversible blindness. Elevated intraocular pressure (IOP) is the main risk factor for developing glaucoma and is caused by impaired aqueous humor drainage through the trabecular meshwork (TM) and Schlemms canal (SC). In primary open angle glaucoma (POAG), this elevation in IOP in turn leads to deformation at the optic nerve head (ONH) specifically at the lamina cribrosa (LC) region where there is also a deposition of extracellular matrix (ECM) molecules such as collagen and fibronectin. Matricellular proteins are non-structural secreted glycoproteins that help cells communicate with their surrounding ECM. This family of proteins includes connective tissue growth factor (CTGF), also known as CCN2, thrombospondins (TSPs), secreted protein acidic and rich in cysteine (SPARC), periostin, osteonectin, and Tenascin-C and -X and other ECM proteins. All members appear to play a role in fibrosis and increased ECM deposition. Most are widely expressed in tissues particularly in the TM and ONH and deficiency of TSP1 and SPARC have been shown to lower IOP in mouse models of glaucoma through enhanced outflow facility. The role of these proteins in glaucoma is emerging as some have an association with the pathophysiology of the TM and LC regions and might therefore be potential targets for therapeutic intervention in glaucoma.

Anti-Connective Tissue Growth Factor Antibody Treatment Reduces Extracellular Matrix Production in Trabecular Meshwork and Lamina Cribrosa Cells

PURPOSE We have previously demonstrated elevated levels of connective tissue growth factor (CTGF/CCN2) in the aqueous humor (AqH) of pseudoexfoliation glaucoma (PXFG) patients when compared with cataract controls. Furthermore, there is a significant trabecular meshwork (TM) and lamina cribrosa (LC) fibrotic phenotype associated with glaucoma, possibly driven by CTGF. The purpose of this study was to investigate the potential of anti-CTGF immunotherapy in glaucoma. METHODS Primary TM and LC cells were cultured from human donors with (GTM/GLC) and without (NTM/NLC) primary open angle glaucoma (POAG). Aqueous humor samples from PXFG, POAG, and control cataract patients were applied to N/GTM and N/GLC cells in the presence or absence of a therapeutic, humanized monoclonal anti-CTGF antibody FG-3019 (10 μg/mL). Hydrogen peroxide (H2O2) was also used as a stimulus. Expression of fibrotic genes (fibronectin-1, fibrillin-1, CTGF, collagen type I α1, and α-smooth muscle actin) was assessed by q-PCR. Protein expression of collagen 1A1 and α-smooth muscle actin was examined in N/G TM cells by SDS-PAGE. The modulatory effect of FG-3019 (10 μg/mL) and IgG (10 μg/mL) were also assessed. RESULTS Treatment of cells with AqH from PXFG and POAG patients and H2O2 induced a significant (P < 0.05) increase in expression of profibrotic genes, which was significantly reduced by pretreatment with FG-3019 (P < 0.05). FG-3019 also reduced expression of α-smooth muscle actin and collagen 1A1 protein expression in N/GTM cells. CONCLUSIONS FG-3019 is effective in blocking extracellular matrix production in TM and LC cells, thus supporting a role for the use of anti-CTGF immunotherapy in the treatment of glaucoma.

Histone deacetylase activity in conjunction with E2F‐1 and p53 regulates Apaf‐1 expression in 661W cells and the retina

Apaf‐1 and the cysteine proteases known as caspases are genes central to the intrinsic apoptotic pathway in the retina. Previously, we have shown that histone deacetylase (HDAC) activity regulates Apaf‐1 expression in the retina. In this study, we unravel the detailed molecular mechanism of HDAC‐mediated regulation of Apaf‐1 initially by use of a cell line (661W), which expresses some cone‐specific genes and then by means of an ex vivo retinal explant system. Inhibition of HDAC activity by trichostatin A (TSA) up‐regulates Apaf‐1 expression, which precedes the induction of apoptosis. Furthermore, by a bioinformatics approach, we identify E2F‐1 and p53 binding sites on the mouse Apaf‐1 promoter and show by chromatin immunoprecipitation assays that these sites are occupied in vitro and that treatment with TSA results in increased binding of E2F‐1 and p53 to the Apaf‐1 promoter. By performing siRNA to these transcription factors, we illustrate that they govern Apaf‐1 expression levels in vitro. Finally, in a retinal explant system, we show that similar to our 661W results, E2F‐1 and p53 are up‐regulated after inhibition of HDAC activity in the retina. This correlates with our previous observation in the explant system that Apaf‐1 expression increases significantly and leads to an induction of apoptosis after inhibition of HDAC activity. Overall, we propose a role for HDAC activity, E2F‐1, and p53 in the regulation of Apaf‐1 expression in 661W cells; initial data also indicate a regulatory role in the retina.

Rosiglitazone acts as a neuroprotectant in retinal cells via up-regulation of sestrin-1 and SOD-2.

Rosiglitazone is a member of the thiazolidinedione family of synthetic peroxisome proliferator‐activated receptor (PPAR) agonists. It is a selective ligand of the PPARγ subtype and functions by regulating the transcription of insulin‐responsive genes. A screen of FDA‐approved compounds identified rosiglitazone as a novel anti‐apoptotic agent in retinal cells both in vitro and in vivo, functioning as a neuroprotectant in response to oxidative and calcium stress. We have found that the likely mechanism of action is via increased protein expression of the antioxidant enzymes superoxide dismutase 2 (SOD‐2) and sestrin‐1, boosting antioxidant defences. Transcription of both genes appears to be mediated by PPARγ as their up‐regulation is reversed by the PPARγ antagonist GW9662 and proliferator hormone response elements were found in the putative promoter regions of mouse SOD‐2 and sestrin‐1. However, further investigation revealed that p53 expression was also induced in response to rosiglitazone and chromatin immunoprecipitation assays confirm that it is a bona fide target of PPARγ. Furthermore, inhibition of p53 partially blocks the observed increase in SOD‐2 and sestrin‐1 expression indicating that p53 expression is upstream of both antioxidants. We conclude that rosiglitazone may increase cell survival in retinal diseases and potentially other neuronal diseases in which oxidative stress is a key factor.

Protein Macroarray Profiling of Serum Autoantibodies in Pseudoexfoliation Glaucoma

PURPOSE Complex repertoires of IgG autoantibodies have been detected against ocular antigens in patients with glaucoma. The goal was to identify and characterize the IgG autoantibody repertoires in sera of patients with pseudoexfoliation glaucoma (PXFG) with protein macroarrays. METHODS Serum samples of 21 patients with PXFG and 19 age- and sex-matched control subjects were profiled on high-density colony protein macroarrays expressing His-tagged recombinant human proteins derived from a human fetal brain cDNA library. Statistically prevalent expression clones in the PXFG group were sequenced. mRNA expression of identified antigens was examined in the rat ganglion cell line RGC-5 and in human brain and optic nerve cDNA. The IgG immunoreactivity of the sera of 20 control and 26 PXFG patients to purified C6orf129 was analyzed in a reverse enzyme-linked immunosorbent assay. RESULTS An increased prevalence was detected among the PXFG patients of serum antibodies to seven proteins: C6orf129; stathmin-like 4; transmembrane protein 9 domain family, member B; fibroblast growth factor receptor 3; cleft lip and palate transmembrane protein 1; EH-domain-containing protein 1; and eukaryotic translation elongation factor 2. All antigens were expressed in the RGC-5 cells and in cDNA from human brain and optic nerve, with the exception of stathmin-like 4, which was not expressed in the RGC-5 cells. The patients with PXFG had increased anti-C6orf129 IgG immunoreactivity compared with that in the control subjects (P < 0.05). CONCLUSIONS Screening high-density protein arrays identifies unique antibody profiles that may discriminate between patients with and without PXFG. Characterization of the autoantibody repertoire may provide new insights into the pathophysiology of PXFG.

IGF-I maintains calpastatin expression and attenuates apoptosis in several models of photoreceptor cell death.

Retinitis pigmentosa is a heterogeneous group of inherited retinal dystrophies in which the loss of photoreceptor cells via apoptosis leads to blindness. In this study we have experimentally mimicked this condition by treating 661W cells and wild‐type mouse retinal explants with a Ca2+ ionophore. Ca2+ overload induced apoptosis, which was correlated with calpain‐2 activation, loss of calpastatin, its endogenous inhibitor, as well as the loss of its transcriptional activator, phospho‐cAMP response element binding (CREB). All are similar changes to those observed in the rd1 mouse model of retinitis pigmentosa. Insulin like‐growth factor‐I (IGF‐I) attenuated this Ca2+‐induced apoptosis, as well as decreased the activation of calpain‐2 and maintained calpastatin levels through the activation of the Akt‐CREB pathway. Similarly, IGF‐I decreased photoreceptor apoptosis in rd1 mouse retinal explants in parallel with reduced activation of calpain‐2 and increased levels of calpastatin and activation of phospho‐CREB. In conclusion, IGF‐I seems to protect neural cells following a physiopathological or an experimental increase in intracellular Ca2+, an observation that may have therapeutic consequences in neurodegenerative diseases such as retinitis pigmentosa.

Therapeutic potential of AAV-mediated MMP-3 secretion from corneal endothelium in treating glaucoma

Abstract Intraocular pressure (IOP) is maintained as a result of the balance between production of aqueous humour (AH) by the ciliary processes and hydrodynamic resistance to its outflow through the conventional outflow pathway comprising the trabecular meshwork (TM) and Schlemm’s canal (SC). Elevated IOP, which can be caused by increased resistance to AH outflow, is a major risk factor for open-angle glaucoma. Matrix metalloproteinases (MMPs) contribute to conventional aqueous outflow homeostasis in their capacity to remodel extracellular matrices, which has a direct impact on aqueous outflow resistance and IOP. We observed decreased MMP-3 activity in human glaucomatous AH compared to age-matched normotensive control AH. Treatment with glaucomatous AH resulted in significantly increased transendothelial resistance of SC endothelial and TM cell monolayers and reduced monolayer permeability when compared to control AH, or supplemented treatment with exogenous MMP-3. Intracameral inoculation of AAV-2/9 containing a CMV-driven MMP-3 gene (AAV-MMP-3) into wild type mice resulted in efficient transduction of corneal endothelium and an increase in aqueous concentration and activity of MMP-3. Most importantly, AAV-mediated expression of MMP-3 increased outflow facility and decreased IOP, and controlled expression using an inducible promoter activated by topical administration of doxycycline achieved the same effect. Ultrastructural analysis of MMP-3 treated matrices by transmission electron microscopy revealed remodelling and degradation of core extracellular matrix components. These results indicate that periodic induction, via use of an eye drop, of AAV-mediated secretion of MMP-3 into AH could have therapeutic potential for those cases of glaucoma that are sub-optimally responsive to conventional pressure-reducing medications.