Proteolytic cleavage of the Na þ /Ca 2 þ exchanger (NCX) by calpains impairs calcium homeostasis, leading to a delayed calcium overload and excitotoxic cell death. However, it is not known whether reversal of the exchanger contributes to activate calpains and trigger neuronal death. We investigated the role of the reversal of the NCX in Ca 2 þ dynamics, calpain activation and cell viability, in a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-stimulated hippocampal neurons. The sodium-calcium exchanger (NCX) plays a fundamental role in controlling Na þ and Ca 2 þ homeostasis. 1,2 NCX primarily extrudes Ca 2 þ in exchange for Na þ , whereas upon neuronal depolarization, Na þ is pumped out by NCX, while Ca 2 þ is pumped in. In pathophysiological conditions, overactivation of glutamate receptors can cause the reversal of NCX, leading to Ca 2 þ entry into the cell. 3 Three NCX genes have been identified, NCX1, 4 NCX2 5 and NCX3. 6 In excitotoxic cell death, an increase in intracellular free calcium concentration ([Ca 2 þ ] i ) may directly cause activation of Ca 2 þ -dependent cysteine proteases, the calpains. Calpains modulate a variety of physiological processes, 7 and are important mediators of cell death. 8,9 Calpains mediate the neurotoxic effect of N-methyl-D-aspartate (NMDA) in cultured hippocampal neurons by a caspase-independent cell death mechanism of excitotoxicity. 10 Calpains are also involved in the neurotoxic effect caused by a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor activation in cultured hippocampal neurons, 11 and in hippocampal slice cultures. 12 During excitotoxic neurodegeneration, calpains are responsible for the proteolysis of several cytoskeletal and associated proteins, kinases and phosphatases, membrane receptors and transporters. 13 Recently, the involvement of calpains in the cleavage of NCX was described in cultured cerebellar granule neurons exposed to glutamate and following brain ischemia. 14 The NCX3 subtype is inactivated by proteolytic cleavage by calpains, and is no longer able to pump Ca 2 þ out of the cell, thus enhancing cell death. Furthermore, NCX3 was shown to be more relevant for cell survival than NCX1 or NCX2, namely in cultured cerebellar granule neurons. 14, 15 We recently reported that neurotoxicity induced by activation of AMPA receptors is characterized by calpain activation, lack of caspase activation, nuclear condensation/fragmentation, release of cytochrome c from mitochondria, decreased intracellular ATP levels, production of nitric oxide, moderate superoxide production and increased levels of peroxynitrite. [16][17][18] In this in vitro model of excitotoxicity, the cell

Purpose Animal models are valuable tools to study the pathogenic mechanisms of many diseases. Diabetic retinopathy is a low‐grade chronic inflammatory disease. Nitric oxide (NO) is involved in leukostasis and blood‐retinal barrier (BRB) breakdown in the early stages of the disease. However, the role of the different NO synthase (NOS) isoforms was not elucidated. We aimed to clarify the involvement of constitutive (eNOS, nNOS) and inducible NOS (iNOS) isoforms and the mechanisms underlying NO‐mediated leukostasis and BRB breakdown, by using an experimental model of diabetes and iNOS KO mice. Methods Diabetes was induced by streptozotocin in normal and KO mice (2 weeks of diabetes). Normal mice were treated with L‐NAME (NOS inhibitor). Vessel leakage was assessed with Evans blue. Leukostasis was quantified in flat‐mounted retinas and in vivo. ICAM‐1, occludin, ZO‐1 and nitrotyrosine levels were assessed by Western blotting or immunohistochemistry. Results Diabetes increased the permeability of BRB and leukostasis, which were reduced by L‐NAME. Similar effects were observed in diabetic iNOS KO mice. In diabetic mice, the immunoreactivity of tight junction proteins, occludin and ZO‐1, decreased, whereas ICAM‐1 protein levels increased. Those effects were prevented by L‐NAME and in diabetic iNOS KO mice. Diabetes also upregulated all NOS isoforms and increased nitrotyrosine levels in normal mice, but did not significantly increase eNOS and nNOS, and nitrotyrosine levels, in iNOS KO mice. Conclusion These data demonstrate that iNOS plays a predominant role in leukostasis and BRB breakdown. The mechanism involves ICAM‐1 upregulation and tight junction proteins downregulation. Support: FCT, Portugal

Introduction Age-related macular degeneration (AMD) is a degenerative retinal disease that affects central vision. Most of their phenotypical features are believed to be associated with the dysfunction of retinal pigment epithelium (RPE). The accumulation of damaged proteins in aged RPE is associated with disruption of proteolytic pathways and exocytic activity, with release of intracellular proteins via exosomes (Exo), that are important players in intercellular communication and can contribute to disease progression. However, the impact of their secretion by polarized RPE on outer blood retinal barrier (oBRB) breakdown remains largely elusive Objectives Our aim was to explore the role of inflammation on the loss of RPE integrity and to understand the relative role of directional secretion of Exo by RPE in the loss of polarity and barrier disruption Methodology We used a human RPE cell line (ARPE-19), highly polarized RPE primary cultures (pRPE) and porcine eyecups. To mimic the inflammatory conditions present in AMD, cells were treated with two inflammatory stimuli, TNF (10 ng/mL) or LPS (100 ng/mL) Results TNF and LPS do not affect the viability of the RPE cells. RPE cells developed a confluent monolayer and reached a relatively constant TER of about 40 Ω/cm2 (ARPE-19) or higher than 150 Ω/cm2 (pRPE). Treatment with TNF significantly reduces the TER, decreased immunoreactivity and co-localization of the TJ proteins ZO-1 and occludin and increases MMP-2/-9 activity in the medium. Apical Exo isolated from the RPE cells are enriched in CD63 compared to the basolateral Exo, that are enriched in CD81. The Exo isolated from porcine eyecups, especially with the LPS stimulus, are enriched in CD81 and MMP-2 but have similar levels of CD63 Conclusion Overall, our results show that inflammation induces loss of RPE integrity and release of different populations of Exo. The unravelling of novel drug targets paves the way for development of new therapeutic strategies for AMD.

Purpose: The recreational drug 3,4‐methylenedioxymethamphetamine (MDMA; ecstasy) causes neurotoxicity in the brain. Ecstasy consumers may experience visual effects, and significant levels of MDMA were detected in the human vitreous after lethal doses. It is also known that Neuropeptide Y (NPY) can be a neuroprotective agent. In this study, we investigated the putative neurotoxic effect of MDMA in retina neural cells, and the potential neuroprotective effect of NPY. Methods: Rat retina neural cell cultures were prepared from newborn Wistar rats. The cells were exposed to MDMA (100‐1600 μM) for 24 or 48 h, at 37 ºC or 40 ºC. Cell viability was evaluated by MTT assay and propidium iodide staining. Retinal cells were identified by immunocytochemistry. Results: MDMA decreased retinal cells viability in a concentration‐, time‐ and temperature‐dependent manner. The toxic effect of MDMA non‐selectively affected different retinal cells (neurons and glial cells) and was dependent on caspase activation. In addition, the toxic effect was significantly reduced by ketanserin, a 5‐HT2A antagonist, and by NPY, but the protective effect of NPY was more pronounced. Conclusions: These results show that MDMA is neurotoxic to retina neural cells, and the toxic effect can be exacerbated by hyperthermia. The toxicity of MDMA is partially mediated by the activation of the 5‐HT2A receptor. Also, NPY is a potent neuroprotective agent against MDMA‐induced retina cell degeneration. These findings extend previous findings about the dangers of MDMA consumption. Further studies and a closer follow‐up on the visual status of human consumers are evidently necessary. Support: FCT (SFRH/BD/12900/2003) and Fac. Medicine, Univ. Coimbra (GAI 01/07), Portugal