COINVOLGIMENTO DELLA PROTEINA CLIC1 NELL¿INSORGENZA E NEL PROCESSO NEURODEGENERATIVO DEL GLAUCOMA

CLIC1 is a member of an intracellular chloride channel family, CLIC, highly conserved in vertebrates. It has both soluble and membrane-integrated forms. Different studies demonstrate that several stimuli, like oxidation or pH changes, promote CLIC1 insertion in the membrane and a consequent generation of a chloride current. It has been reported that CLIC1 has an important role in different cell functions, such as migration, swelling or division of the cell, endocytosis and exocytosis, intracellular vesicles fusion, and apoptosis. CLIC1 and other CLIC family members are involved in cellular function not directly correlated with the ion channel activity in the plasma membrane. Recent studies have shown that CLIC1 may be involved in mediating neurotoxicity in Alzheimer’s disease. A previous work has shown that CLIC1 is upregulated and translocated in plasma-membrane in microglial cell (BV2) after treatment with amyloid beta peptide (Aβ) and in a triple mutant AD mouse brain. Inhibition of CLIC1 activity with the specific blocker (IAA 94) or expression by siRNA reduced reactive oxygen species (ROS), nitric oxide (NO) and TNFalpha production in microglial cells, leading to a neuroprotective effect in microglia-neuron co-cultures. The hypothesis is that CLIC1-dependent chloride current is required for Aβ-mediated ROS production by microglial cells, maintainig NADPH oxidase activity and providing the charge compensation to avoid membrane depolarization. Enhanced levels of Aβ in fibrillar or soluble forms, have been shown to cause neurotoxicity in neurodegenerative disorders. The accumulation of Aβ, has recently been critically involved, as well as widely descripted in Alzheimer’s disease, in the pathogenesis of ocular disease as age-related macular degeneration (ARMD) and glaucoma. Aβ is associated with impaired mitochondrial function, activation of cellular oxidases and generation of reactive oxygen species (ROS), leading to oxidative injury and alterations in neuronal ionic homeostasis. To date the molecular and cellular mechanisms involved in Aβ-mediated retinal neurotoxicity are still not well understood. Among all the other factors that participate to the glaucoma pathogenesis, oxidative damage caused by the production of reactive oxygen species (ROS) is certainly one of the prominent causes of retinal ganglion cell (RGC) death and optic nerve degeneration. Previous studies have shown that glaucoma has all the characteristics of a neurodegenerative disease: microglial activation, state of chronic inflammation, increase of extracellular Aβ, oxidative stress and neuronal death. In the last past years similarities have been discovered between glaucoma and Alzheimer’s disease. Starting from the data of CLIC1 involvement in mediating inflammatory response under Aβ over-expression condition in microglial cells and in AD brain, our aim was to characterize this protein in the retina, which is part of the CNS and that is more easily accessible. Hence the importance of our work is not just limited to understanding the possible involvement of CLIC1 in retinal neurodegeneration, but also gives us insight on what happens in the brain. In this work we characterize CLIC1 expression in the retinal layers. Our attention was focused primarily on glial cells, microglia and macroglia, in order to define the correlation between CLIC1, reactive oxygen species (ROS) production and Aβ. We have used different animal and cellular models in condition of amyloid over-expression. The characterization of CLIC1 in Alzheimer’s and glaucoma models has allowed us to define its expression in neurodegenerative conditions. CLIC1 is expressed mainly in RGC and INL (Inner Nuclear Layer) layer in the retina. We used a mice model of Aβ over-expression, obtained with an intravitreal injection of Aβ in the eye of C57Bl6 mice. In this model we have an increase of microglial activation after injection. In agreement with literature we have also observed a growing apoptosis signal, mainly in the RGC layer. CLIC1 expression is up-regulated after intravitreal injection of Aβ, and it is localized mainly in RGC and INL as in the control retina, with an increased staining in Müller cells ramifications through the IPL. Although previous data report a localization of CLIC1 in the microglial cells of the brain, we did not observe any overlapping signal in our retinal samples. CLIC1 seems to be not expressed in retinal microglia. We proceeded with a characterization of a mouse model of AD (APPswe/PS1DE9). Once established an increase of amyloid expression in the AD retinal lysates, we analyzed the morphological changes that occur in the retina. The most visible changes are the decrease in the GCL’s cell number and a reduction in the total retinal thickness. We also observed a higher levels of ROS and an increase in the levels of hydroperoxides. CLIC1 is up-regulated in AD samples compared to controls. Knowing that CLIC1 is involved in ROS production and that reactive oxygen species in the retina are mainly observed in the INL and OPL layers, where Müller cells are located, we analyzed CLIC1 expression in this cell type. Aβ induces an increase of CLIC1 expression in Müller cells at different treatment times. In response to Aβ stimulation we observe also a CLIC1 intracellular mobilization from the cytoplasm to the perinuclear area, that co-localizes with gamma-tubulin, marker of the microtubule organization center (MTOC). DCF staining revealed that Aβ treatment stimulates ROS production in Müller cells. This effect was significantly inhibited by blockade of CLIC1 activity with IAA94. As we previously observed in retinas injected with the amyloid peptide, in cell culture experiments we confirm an increased activation of Müller cells after exposure to Aβ. This activation seems to be reduced with CLIC1 inhibition. CLIC1 also appears to mediate Aβ phagocytosis, whose intracellular levels are greatly increased as a results of its inhibition. In a mice model of glaucoma (DBA2) we observed an up-regulation of CLIC1 expression, glial activation and an increase of amyloid peptide. In DBA2 mice CLIC1 co-localizes with Müller cells, stained with GFAP. Experiments on RGC5 cells confirmed the increased expression of CLIC1 after Aβ exposure, its perinuclear localization and its role in the regulation of oxidative stress. All these results confirmed the involvement of CLIC1 in retinal ROS production. CLIC1 is clearly involved in the activation of Müller cells induced by Aβ. Recently has been shown that Müller cells appear to play a role in the mechanism of RGC death due to Aβ. The Müller cell dysfunction induced by amyloid aggregates, together with changes in retinal vasculature, have a massive effect on neurons. Our studies suggest that CLIC1 could be a novel mediator of Aβ-induced glial activation in the retina. Probably the protein acts by regulating the activation of several stress response, such as ROS production, activation of Müller cells and interaction with the MTOC. It is known that MTOC has an important role in misfolded and aggregate protein transport and their disposal to the aggresome. CLIC1, as shown in the results obtained, could be involved in the phagocytosis of Aβ and its intracellular transport. In several animal models over-expressing Aβ we observed an up-regulation of CLIC1 expression, related to increased oxidative stress and glial activation. Therefore, CLIC1 is potentially involved in ocular diseases that have been implicated Aβ activity, such as glaucoma and age-related macular degeneration (AMD).