Ruolo fisiopatologico del trasporto ionico di membrana

Diabetes leads to a number of complications involving brain function, including cognitive decline and depression. It is widely accepted that depression is the consequence of an impaired adult neurogenesis in the gyrus dentatus of hippocampus, but the molecular mechanisms leading to this phenomenon are still poorly understood. The process of neurogenesis consists of the proliferation of neural progenitors and differentiation of new neurons, and volume-regulated anion channels (VRAC), responsible for the activation of a chloride conductance (IClswell) after anisosmotic cell swelling during cell volume regulation, are also essential in cell proliferation. In diabetes, an increased O-GlcNAcylation (O- GlcNAc) of cellular proteins is often seen. O-GlcNAc is a reversible post-translational conjugation of N-acetylglucosamine to Serine or Threonine residues of nuclear and cytoplasmic proteins, regulated by two enzymes called O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Recently, the protein ICln, which is crucial in the activation of the chloride conductance IClswell, has been found to be O-GlcNacylated. Mass spectrometry and bioinformatics analysis of ICln amino acid sequence indicate several O-GlcNAc modification regions, including a putative Yin O Yang site on Serine 67, i.e. a site that could be either phosphorylated or O-GlcNAc modified in a mutually exclusive manner. In the present study, we investigated the behaviour of the swelling-activated chloride current IClswell following O-GlcNAc elevation, with focus on the role of ICln protein in neuronal-like SH-SY5Y and in HEK 293 Phoenix cells. O-GlcNAc elevation was obtained by incubation of cells with PUGNAc (a pharmacological inhibitor of OGA) and Glucosamine (an O-GlcNAc precursor). Basal or swelling- activated IClswell was measured by patch-clamp in whole-cell configuration in native cells or cells overexpressing either the wild type ICln or non-glycosilable forms of ICln (IClnT223A, IClnS193X and IClnS67A). The results show that exposure of SH-SY5Y or HEK 293 Phoenix cells to PUGNAc and Glucosamine increases the global O-GlcNAc levels and, in parallel, significantly reduces cell viability. In SH-SY5Y cells, O-GlcNAc elevation significantly suppresses the basally activated IClswell current. In HEK 293 Phoenix cells, O-GlcNAc elevation inhibits the swelling-activated IClswell current following ICln overexpression, with no effect on the endogenous swelling-activated current, thus indicating that O- GlcNAc modification can impair ICln function. The IClnT223A-induced current is similar to the wild type ICln-induced current and is similarly suppressed after O-GlcNAc elevation. The IClnS193X loses most of its activity, though the residual current is sensitive to O-GlcNAc elevation. Finally, the IClnS67A-induced current is similar to the wild type ICln-induced current, but insensitive to O-GlcNAc elevation. Therefore, Threonine 223 is not involved in the suppression of ICln function following O-GlcNAc modification, and the O-GlcNAc modification site responsible for the suppression of ICln-induced current must be located upstream residue 193. Serine 67 is a strong candidate for O-GlcNAc modification. These results underscore the essential role of O-GlcNAc modification in governing the chloride current IClswell through VRAC channels. We conclude that O-GlcNAcylation of the VRAC channels activator ICln in the context of hyperglycaemic conditions may lead to VRAC inhibition in brain, thus resulting in reduced neurogenesis, eventually leading to the depression disorder. The protein ICln may represent a novel therapeutic target in the prevention or treatment of pathological states characterized by chronically elevated O-GlcNAcylation of cellular proteins, including mood disorders linked to uncontrolled or poorly controlled hyperglycaemia.