Yuki Morizane

Receptor interacting protein kinases mediate retinal detachment-induced photoreceptor necrosis and compensate for inhibition of apoptosis

Apoptosis has been shown to be a significant form of cell loss in many diseases. Detachment of photoreceptors from the retinal pigment epithelium, as seen in various retinal disorders, causes photoreceptor loss and subsequent vision decline. Although caspase-dependent apoptotic pathways are activated after retinal detachment, caspase inhibition by the pan-caspase inhibitor Z-VAD fails to prevent photoreceptor death; thus, we investigated other pathways leading to cell loss. Here, we show that receptor interacting protein (RIP) kinase-mediated necrosis is a significant mode of photoreceptor cell loss in an experimental model of retinal detachment and when caspases are inhibited, RIP-mediated necrosis becomes the predominant form of death. RIP3 expression, a key activator of RIP1 kinase, increased more than 10-fold after retinal detachment. Morphological assessment of detached retinas treated with Z-VAD showed decreased apoptosis but significantly increased necrotic photoreceptor death. RIP1 kinase inhibitor necrostatin-1 or Rip3 deficiency substantially prevented those necrotic changes and reduced oxidative stress and mitochondrial release of apoptosis-inducing factor. Thus, RIP kinase-mediated programmed necrosis is a redundant mechanism of photoreceptor death in addition to apoptosis, and simultaneous inhibition of RIP kinases and caspases is essential for effective neuroprotection and may be a novel therapeutic strategy for treatment of retinal disorders.

Programmed necrosis, not apoptosis, is a key mediator of cell loss and DAMP-mediated inflammation in dsRNA-induced retinal degeneration

There is no known treatment for the dry form of an age-related macular degeneration (AMD). Cell death and inflammation are important biological processes thought to have central role in AMD. Here we show that receptor-interacting protein (RIP) kinase mediates necrosis and enhances inflammation in a mouse model of retinal degeneration induced by dsRNA, a component of drusen in AMD. In contrast to photoreceptor-induced apoptosis, subretinal injection of the dsRNA analog poly(I : C) caused necrosis of the retinal pigment epithelium (RPE), as well as macrophage infiltration into the outer retinas. In Rip3−/− mice, both necrosis and inflammation were prevented, providing substantial protection against poly(I : C)-induced retinal degeneration. Moreover, after poly(I : C) injection, Rip3−/− mice displayed decreased levels of pro-inflammatory cytokines (such as TNF-α and IL-6) in the retina, and attenuated intravitreal release of high-mobility group box-1 (HMGB1), a major damage-associated molecular pattern (DAMP). In vitro, poly(I : C)-induced necrosis were inhibited in Rip3-deficient RPE cells, which in turn suppressed HMGB1 release and dampened TNF-α and IL-6 induction evoked by necrotic supernatants. On the other hand, Rip3 deficiency did not modulate directly TNF-α and IL-6 production after poly(I : C) stimulation in RPE cells or macrophages. Therefore, programmed necrosis is crucial in dsRNA-induced retinal degeneration and may promote inflammation by regulating the release of intracellular DAMPs, suggesting novel therapeutic targets for diseases such as AMD.

AMP-activated Protein Kinase Suppresses Matrix Metalloproteinase-9 Expression in Mouse Embryonic Fibroblasts

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tissue remodeling under both physiological and pathological conditions. Although MMP-9 expression is low in most cells and is tightly controlled, the mechanism of its regulation is poorly understood. We utilized mouse embryonic fibroblasts (MEFs) that were nullizygous for the catalytic α subunit of AMP-activated protein kinase (AMPK), which is a key regulator of energy homeostasis, to identify AMPK as a suppressor of MMP-9 expression. Total AMPKα deletion significantly elevated MMP-9 expression compared with wild-type (WT) MEFs, whereas single knock-out of the isoforms AMPKα1 and AMPKα2 caused minimal change in the level of MMP-9 expression. The suppressive role of AMPK on MMP-9 expression was mediated through both its activity and presence. The AMPK activators 5-amino-4-imidazole carboxamide riboside and A769662 suppressed MMP-9 expression in WT MEFs, and AMPK inhibition by the overexpression of dominant negative (DN) AMPKα elevated MMP-9 expression. However, in AMPKα−/− MEFs transduced with DN AMPKα, MMP-9 expression was suppressed. AMPKα−/− MEFs showed increased phosphorylation of IκBα, expression of IκBα mRNA, nuclear localization of nuclear factor-κB (NF-κB), and DNA-binding activity of NF-κB compared with WT. Consistently, selective NF-κB inhibitors BMS345541 and SM7368 decreased MMP-9 expression in AMPKα−/− MEFs. Overall, our results suggest that both AMPKα isoforms suppress MMP-9 expression and that both the activity and presence of AMPKα contribute to its function as a regulator of MMP-9 expression by inhibiting the NF-κB pathway.

Heat Shock Protein 70 (HSP70) Is Critical for the Photoreceptor Stress Response after Retinal Detachment via Modulating Anti-Apoptotic Akt Kinase

Photoreceptor apoptosis is a major cause of vision loss in many ocular diseases. Significant progress has been made to elucidate the molecular pathways involved in this process, yet little is known about proteins counteracting these apoptotic pathways. It is established that heat shock proteins (HSPs) function as molecular helper proteins (chaperones) by preventing protein aggregation and facilitating refolding of dysfunctional proteins, critical to the survival of all organisms. Here, we investigated the role of HSP70 on photoreceptor survival after experimental retinal detachment (RD) in mice and rats. We found that HSP70 was up-regulated after RD and associated with phosphorylated Akt, thereby preventing its dephosphorylation and further activation of cell death pathways. Administration of quercetin, which inhibits HSP70 and suppresses Akt phosphorylation significantly increased photoreceptor apoptosis. Similarly, RD-induced photoreceptor apoptosis was augmented in mice carrying hypomorphic mutations of the genes encoding HSP70. On the other hand, administration of geranylgeranylacetone, which induces an increase in HSP70 significantly decreased photoreceptor apoptosis after RD through prolonged activation of Akt pathway. Thus, HSP70 may be a favorable potential target to increase photoreceptor cell survival after RD.

Retinoblastoma cells are inhibited by aminoimidazole carboxamide ribonucleotide (AICAR) partially through activation of AMP-dependent kinase

5‐Aminoimidazole‐4‐carboxamide‐1‐β‐4‐ribofuranoside (AICAR), an analog of AMP, is widely used as an activator of AMP‐kinase (AMPK), a protein that regulates the responses of the cell to energy change. We studied the effects of AICAR on the growth of retinoblastoma cell lines (Y79, WERI, and RB143). AICAR inhibited Rb cell growth, induced apoptosis and S‐phase cell cycle arrest, and led to activation of AMPK. These effects were abolished by treatment with dypiridamole, an inhibitor that blocks entrance of AICAR into cells. Treatment with the adenosine kinase inhibitor 5‐iodotubericidin to inhibit the conversion of AICAR to ZMP (the direct activator of AMPK) reversed most of the growth‐inhibiting effects of AICAR, indicating that some of the antiproliferative effects of AICAR are mediated through AMPK activation. In addition, AICAR treatment was associated with inhibition of the mammalian target of rapamycin pathway, decreased phosphorylation of ribosomal protein‐S6 and 4E‐BP1, down‐regulation of cyclins A and E, and decreased expression of p21. Our results indicate that AICAR‐induced activation of AMPK inhibits retinoblastoma cell growth. This is one of the first descriptions of a nonchemotherapeutic drug with low toxicity that may be effective in treating Rb patients.—Theodoropoulou, S., Kolovou, P. E., Morizane, Y., Kayama, M., Nicolaou, F., Miller, J. W., Gragoudas, E., Ksander, B. R., Vavvas, D. G. Retinoblastoma cells are inhibited by aminoimidazole carboxamide ribonucleotide (AICAR) partially through activation of AMP‐dependent kinase. FASEB J. 24, 2620–2630 (2010). www.fasebj.org

AMP-dependent kinase inhibits oxidative stress-induced caveolin-1 phosphorylation and endocytosis by suppressing the dissociation between c-Abl and Prdx1 proteins in endothelial cells

Background: Oxidative stress increases vascular permeability though caveolin-1 phosphorylation. The exact role of AMPK is unknown. Results: AMP-dependent kinase (AMPK) inhibits caveolin-1 phosphorylation by stabilizing the interaction between c-Abl and Prdx-1. Conclusion: AMPK activation inhibits oxidant induced-vascular permeability. Significance: The present study shows a novel protective role of AMPK in the vascular homeostasis. Caveolin-1 is the primary structural component of endothelial caveolae that is essential for transcellular trafficking of albumin and is also a critical scaffolding protein that regulates the activity of signaling molecules in caveolae. Phosphorylation of caveolin-1 plays a fundamental role in the mechanism of oxidant-induced vascular hyper permeability. However, the regulatory mechanism of caveolin-1 phosphorylation remains unclear. Here we identify a previously unexpected role for AMPK in inhibition of caveolin-1 phosphorylation under oxidative stress. A pharmacological activator of AMPK, 5-amino-4-imidazole carboxamide riboside (AICAR), inhibited oxidative stress-induced phosphorylation of both caveolin-1 and c-Abl, which is the major kinase of caveolin-1, and endocytosis of albumin in human umbilical vein endothelial cell. These effects were abolished by treatment with two specific inhibitors of AICAR, dipyridamole, and 5-iodotubericidin. Consistently, knockdown of the catalytic AMPKα subunit by siRNA abolished the inhibitory effect of AICAR on oxidant-induced phosphorylation of both caveolin-1 and c-Abl. Pretreatment with specific c-Abl inhibitor, imatinib mesylate, and knock down of c-Abl significantly decreased the caveolin-1 phosphorylation after H2O2 exposure and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Interestingly, knockdown of Prdx-1, an antioxidant enzyme associated with c-Abl, increased phosphorylation of both caveolin-1 and c-Abl and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Furthermore, co-immunoprecipitation experiment showed that AICAR suppressed the oxidant-induced dissociation between c-Abl and Prdx1. Overall, our results suggest that activation of AMPK inhibits oxidative stress-induced caveolin-1 phosphorylation and endocytosis, and this effect is mediated in part by stabilizing the interaction between c-Abl and Prdx-1.

Aminoimidazole carboxamide ribonucleotide ameliorates experimental autoimmune uveitis

PURPOSE To investigate the anti-inflammatory effect of an adenosine monophosphate (AMP) analog, aminoimidazole carboxamide ribonucleotide (AICAR), in experimental autoimmune uveoretinitis (EAU). METHODS C57BL/6 mice were injected daily with AICAR (200 mg/kg, intraperitoneally [IP]) from day 0, the day of interphotoreceptor retinoid-binding protein (IRBP) immunization, until day 21. The severity of uveitis was assessed clinically and histopathologically. T-cell proliferation and cytokine production of IFN-γ, IL-17, and IL-10 in response to IRBP stimulation were determined. In addition, regulatory T-cell (Treg) populations were measured. Co-stimulatory molecule expression (CD40, 80, 86, and I-Ab) on dendritic cells (DCs) in EAU and on bone marrow-derived dendritic cells (BMDCs) treated with AICAR was measured. RESULTS AICAR treatment significantly reduced clinical and histologic severity of EAU as well as ocular cytokine production. An anti-inflammatory effect associated with the inhibition of T-cell proliferation and Th1 and Th17 cytokine production was observed. Increases in the Th2 response and Treg population were not observed with AICAR treatment. AICAR did significantly inhibit BMDC maturation by reducing co-stimulatory molecule expression. CONCLUSIONS AICAR attenuates EAU by preventing generation of Ag-specific Th1 and Th17 cells. Impaired DC maturation may be an underlying mechanism for this anti-inflammatory effect observed with AICAR.

Aminoimidazole Carboxamide Ribonucleotide (AICAR) Inhibits the Growth of Retinoblastoma In Vivo by Decreasing Angiogenesis and Inducing Apoptosis

5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), an analog of AMP is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy change. Recently, we showed that AICAR-induced AMPK activation inhibits the growth of retinoblastoma cells in vitro by decreasing cyclins and by inducing apoptosis and S-phase arrest. In this study, we investigated the effects of AMPK activator AICAR on the growth of retinoblastoma in vivo. Intraperitoneal injection of AICAR resulted in 48% growth inhibition of Y79 retinoblastoma cell tumors in mice. Tumors isolated from mice treated with AICAR had decreased expression of Ki67 and increased apoptotic cells (TUNEL positive) compared with the control. In addition, AICAR treatment suppressed significantly tumor vessel density and macrophage infiltration. We also showed that AICAR administration resulted in AMPK activation and mTOR pathway inhibition. Paradoxically observed down-regulation of p21, which indicates that p21 may have a novel function of an oncogene in retinoblastoma tumor. Our results indicate that AICAR treatment inhibited the growth of retinoblastoma tumor in vivo via AMPK/mTORC1 pathway and by apoptogenic, anti-proliferative, anti-angiogenesis mechanism. AICAR is a promising novel non-chemotherapeutic drug that may be effective as an adjuvant in treating Retinoblastoma.

Evidence for baseline retinal pigment epithelium pathology in the Trp1-Cre mouse

The increasing popularity of the Cre/loxP recombination system has led to the generation of numerous transgenic mouse lines in which Cre recombinase is expressed under the control of organ- or cell-specific promoters. Alterations in retinal pigment epithelium (RPE), a multifunctional cell monolayer that separates the retinal photoreceptors from the choroid, are prevalent in the pathogenesis of a number of ocular disorders, including age-related macular degeneration. To date, six transgenic mouse lines have been developed that target Cre to the RPE under the control of various gene promoters. However, multiple lines of evidence indicate that high levels of Cre expression can be toxic to mammalian cells. In this study, we report that in the Trp1-Cre mouse, a commonly used transgenic Cre strain for RPE gene function studies, Cre recombinase expression alone leads to RPE dysfunction and concomitant disorganization of RPE layer morphology, large areas of RPE atrophy, retinal photoreceptor dysfunction, and microglial cell activation in the affected areas. The phenotype described herein is similar to previously published reports of conditional gene knockouts that used the Trp1-Cre mouse, suggesting that Cre toxicity alone could account for some of the reported phenotypes and highlighting the importance of the inclusion of Cre-expressing mice as controls in conditional gene targeting studies.

Inhibitory effect of aminoimidazole carboxamide ribonucleotide (AICAR) on endotoxin-induced uveitis in rats.

PURPOSE. To investigate the anti-inflammatory effect of aminoimidazole carboxamide ribonucleotide (AICAR), an analog of adenosine monophosphate (AMP), in endotoxin-induced uveitis (EIU). METHODS. EIU was induced by subcutaneous injection of lipopolysaccharide (LPS) (200 μg) in Lewis rats. AICAR (50 mg/kg, intraperitoneally) was given 6 hours prior and at the same time as LPS injection. Clinical uveitis scores, number of anterior chamber (AC) infiltrating cells, anterior chamber protein concentration, retinal vessel leukocyte adhesion, and protein leakage were measured 24 hours later. Protein levels of C-C chemokine ligand-2 (CCL-2)/monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) in aqueous humor and retina and nuclear translocation of nuclear factor-κB (NF-κB) in the retina were determined by enzyme-linked immunosorbent assay (ELISA). Both mRNA and protein levels of CD14 in peripheral blood mononuclear cells were also measured. RESULTS. AICAR treatment significantly reduced EIU clinical severity as well as inflammatory cell infiltration and protein concentration in aqueous humor. Similarly, the number of retinal vessel-adherent leukocytes and protein leakage were decreased by AICAR treatment. Protein levels of TNF-α, CCL-2/MCP-1, and ICAM-1 in aqueous humor and CCL-2/MCP-1 and ICAM-1 levels in retina were suppressed with AICAR treatment. AICAR also reduced NF-κB translocation and CD14 expression. CONCLUSIONS. AICAR reduces systemic LPS susceptibility and attenuates intraocular inflammation in a rat EIU model by limiting infiltration of leukocytes, suppressing inflammatory mediators, and inhibiting the NF-κB pathway.