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Alzheimer's disease (AD) is the most prevalent type of dementia affecting older people. The identification of biomarkers is increasingly important and would be crucial for future therapy. Here, we demonstrated that in AD erythrocytes: (i) the anion transporter band3 is highly phosphorylated; (ii) the lyn kinase is phosphorylated and activated; (iii) the tyrosine phosphatase activity is downregulated, with a significant inverse correlation between band3 phosphorylation and disease progression, as revealed by Mini Mental State Examination score. Finally, we showed that in normal erythrocytes, treated in vitro with Aβ1-42 peptide, both band3 phosphorylation and lyn activation occurs. These results suggest that modulation of tyrosine phosphorylation signaling may be evaluated as a potential peripheral marker in AD.

MLC1 is a membrane protein highly expressed by brain perivascular astrocytes. Mutations in the MLC1 gene account for megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, brain edema and cysts, myelin vacuolation and astrocyte swelling, causing cognitive and motor dysfunctions. It has been demonstrated that MLC1 mutations affect the swelling-activated Cl - currents (I Cl,swell ) mediated by volume-regulated anion channel (VRAC) and the consequent regulatory volume decrease (RVD) and lead to abnormal activation of intracellular signaling pathways linked to inflammation/osmotic stress. Despite this knowledge, the MLC1 physiological role and MLC molecular pathogenesis are still elusive. Following the observations that Ca 2+ regulates all the MLC1-modulated processes and that intracellular Ca 2+ homeostasis is altered in MLC1-defective cells, we applied a multidisciplinary approach including biochemistry, molecular biology, video imaging, electrophysiology and proteomic techniques on cultured astrocytes to uncover new Ca 2+ -dependent signaling pathways controlling MLC1 function. Here, we revealed that MLC1 binds the Ca 2+ effector proteins calmodulin (CaM) and Ca 2+ /CaM-dependent protein kinase II (CaMKII) and, as result, changes its assembly, localization and functional properties in response to Ca 2+ changes. Noteworthy, CaM binding to the COOH terminal promotes MLC1 trafficking to the plasma membrane, while CaMKII phosphorylation of the NH 2 -terminal potentiates MLC1 activation of I Cl,swell . Overall, these results revealed that MLC1 is a Ca 2+ -regulated protein linking VRAC function and, possibly, volume regulation to Ca 2+ signaling in astrocytes. These findings open new avenues of investigations aimed at clarifying the abnormal molecular pathways underlying MLC and other diseases characterized by astrocyte swelling and brain edema.

We recently demonstrated that a tonic activation of adenosine A2A receptors (A2A Rs) is required for cocaine-induced synaptic depression and increase in the activity of STriatal-Enriched protein tyrosine Phosphatase (STEP). In this study, we elaborated on the relationship between A2A R and STEP using genetic, pharmacological, and cellular tools. We found that the activities of protein tyrosine phosphatases (PTPs), and in particular of STEP, are significantly increased in the striatum and hippocampus of a transgenic rat strain over-expressing the neuronal A2A R (NSEA2A ) with respect to wild-type (WT) rats. Moreover the selective A2A R agonist 4-[2-[[6-Amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride up-regulates PTPs and STEP activities in WT but not in NSEA2A rats, while the selective A2A R antagonist 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol restores the tyrosine phosphatase activities in NSEA2A , having no effects in WT rats. In addition, while cocaine induced the activation of PTP and STEP in WT rats, it failed to increase phosphatase activity in NSEA2A rats. A2A Rs modulate STEP activity also in the SH-SY5Y neuroblastoma cell line, where a calcium-dependent calcineurin/PP1 pathway was found to play a major role. In summary, the present study identified a novel interaction between A2A R and STEP that could have important clinical implications, since STEP has emerged as key regulator of signaling pathways involved in neurodegenerative and neuropsychiatric diseases and A2A Rs are considered a promising target for the development of therapeutic strategies for different diseases of the central nervous system. Read the Editorial Highlight for this article on page 270.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 (MLC1) is a membrane protein expressed by perivascular astrocytes. MLC1 mutations cause MLC, an incurable leukodystrophy characterized by macrocephaly, brain edema, cysts, myelin vacuolation, and astrocytosis, leading to cognitive/motor impairment and epilepsy. Although its function is unknown, MLC1 favors regulatory volume decrease after astrocyte osmotic swelling and down-regulates intracellular signaling pathways controlling astrocyte activation and proliferation. By combining analysis of human brain tissues with in vitro experiments, here we investigated MLC1 role in astrocyte activation during neuroinflammation, a pathological condition exacerbating patient symptoms. MLC1 upregulation was observed in brain tissues from multiple sclerosis, Alzheimer’s, and Creutzfeld-Jacob disease, all pathologies characterized by strong astrocytosis and release of inflammatory cytokines, particularly IL-1β. Using astrocytoma lines overexpressing wild-type (WT) or mutated MLC1 and astrocytes from control and Mlc1 knock-out (KO) mice, we found that IL-1β stimulated WT-MLC1 plasma membrane expression in astrocytoma cells and control primary astrocytes. In astrocytoma, WT-MLC1 inhibited the activation of IL-1β–induced inflammatory signals (pERK, pNF-kB) that, conversely, were constitutively activated in mutant expressing cells or abnormally upregulated in KO astrocytes. WT-MLC1+ cells also expressed reduced levels of the astrogliosis marker pSTAT3. We then monitored MLC1 expression timing in a demyelinating/remyelinating murine cerebellar organotypic culture model where, after the demyelination and release of inflammatory cytokines, recovery processes occur, revealing MLC1 upregulation in these latter phases. Altogether, these findings suggest that by modulating specific pathways, MLC1 contributes to restore astrocyte homeostasis after inflammation, providing the opportunity to identify drug target molecules to slow down disease progression.

In fragile X syndrome (FXS) the lack of the fragile X mental retardation protein (FMRP) leads to exacerbated signaling through the metabotropic glutamate receptors 5 (mGlu5Rs). The adenosine A2A receptors (A2ARs), modulators of neuronal damage, could play a role in FXS. A synaptic colocalization and a strong permissive interaction between A2A and mGlu5 receptors in the hippocampus have been previously reported, suggesting that blocking A2ARs might normalize the mGlu5R-mediated effects of FXS. To study the cross-talk between A2A and mGlu5 receptors in the absence of FMRP, we performed extracellular electrophysiology experiments in hippocampal slices of Fmr1 KO mouse. The depression of field excitatory postsynaptic potential (fEPSPs) slope induced by the mGlu5R agonist CHPG was completely blocked by the A2AR antagonist ZM241385 and strongly potentiated by the A2AR agonist CGS21680, suggesting that the functional synergistic coupling between the two receptors could be increased in FXS. To verify if chronic A2AR blockade could reverse the FXS phenotypes, we treated the Fmr1 KO mice with istradefylline, an A2AR antagonist. We found that hippocampal DHPG-induced long-term depression (LTD), which is abnormally increased in FXS mice, was restored to the WT level. Furthermore, istradefylline corrected aberrant dendritic spine density, specific behavioral alterations, and overactive mTOR, TrkB, and STEP signaling in Fmr1 KO mice. Finally, we identified A2AR mRNA as a target of FMRP. Our results show that the pharmacological blockade of A2ARs partially restores some of the phenotypes of Fmr1 KO mice, both by reducing mGlu5R functioning and by acting on other A2AR-related downstream targets.

In fragile X syndrome (FXS) the lack of the fragile X mental retardation protein (FMRP) leads to exacerbated signaling through the metabotropic glutamate receptors 5 (mGlu5Rs). The adenosine A2A receptors (A2ARs), modulators of neuronal damage, could play a role in FXS. A synaptic colocalization and a strong permissive interaction between A2A and mGlu5 receptors in the hippocampus have been previously reported, suggesting that blocking A2ARs might normalize the mGlu5R-mediated effects of FXS. To study the cross-talk between A2A and mGlu5 receptors in the absence of FMRP, we performed extracellular electrophysiology experiments in hippocampal slices of Fmr1 KO mouse. The depression of field excitatory postsynaptic potential (fEPSPs) slope induced by the mGlu5R agonist CHPG was completely blocked by the A2AR antagonist ZM241385 and strongly potentiated by the A2AR agonist CGS21680, suggesting that the functional synergistic coupling between the two receptors could be increased in FXS. To verify if chronic A2AR blockade could reverse the FXS phenotypes, we treated the Fmr1 KO mice with istradefylline, an A2AR antagonist. We found that hippocampal DHPG-induced long-term depression (LTD), which is abnormally increased in FXS mice, was restored to the WT level. Furthermore, istradefylline corrected aberrant dendritic spine density, specific behavioral alterations, and overactive mTOR, TrkB, and STEP signaling in Fmr1 KO mice. Finally, we identified A2AR mRNA as a target of FMRP. Our results show that the pharmacological blockade of A2ARs partially restores some of the phenotypes of Fmr1 KO mice, both by reducing mGlu5R functioning and by acting on other A2AR-related downstream targets.

Astrocytes, the most numerous cells of the central nervous system, exert critical functions for brain homeostasis. To this purpose, astrocytes generate a highly interconnected intercellular network allowing rapid exchange of ions and metabolites through gap junctions, adjoined channels composed of hexamers of connexin (Cx) proteins, mainly Cx43. Functional alterations of Cxs and gap junctions have been observed in several neuroinflammatory/neurodegenerative diseases. In the rare leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), astrocytes show defective control of ion/fluid exchanges causing brain edema, fluid cysts, and astrocyte/myelin vacuolation. MLC is caused by mutations in MLC1, an astrocyte-specific protein of elusive function, and in GlialCAM, a MLC1 chaperon. Both proteins are highly expressed at perivascular astrocyte end-feet and astrocyte-astrocyte contacts where they interact with zonula occludens-1 (ZO-1) and Cx43 junctional proteins. To investigate the possible role of Cx43 in MLC pathogenesis, we studied Cx43 properties in astrocytoma cells overexpressing wild type (WT) MLC1 or MLC1 carrying pathological mutations. Using biochemical and electrophysiological techniques, we found that WT, but not mutated, MLC1 expression favors intercellular communication by inhibiting extracellular-signal-regulated kinase 1/2 (ERK1/2)-mediated Cx43 phosphorylation and increasing Cx43 gap-junction stability. These data indicate MLC1 regulation of Cx43 in astrocytes and Cx43 involvement in MLC pathogenesis, suggesting potential target pathways for therapeutic interventions.

We recently demonstrated that a tonic activation of adenosine A

The effects of the anti-Vascular Endothelial Growth Factor (VEGF) drugs ranibizumab and aflibercept were studied in Müller glia in primary mixed cultures from rat neonatal retina. Treatment with both agents induced activation of Müller glia, demonstrated by increased levels of Glial Fibrillary Acidic Protein. In addition, phosphorylated Extracellular-Regulated Kinase 1/2 (ERK 1/2) showed enhanced immunoreactivity in activated Müller glia. Treatment with aflibercept induced an increase in K(+) channel (Kir) 4.1 levels and both drugs upregulated Aquaporin 4 (AQP4) in activated Müller glia. The results show that VEGF-antagonizing drugs influence the homeostasis of Müller cells in primary retinal cultures, inducing an activated phenotype. Upregulation of Kir4.1 and AQP4 suggests that Müller glia activation following anti-VEGF drugs may not depict a detrimental gliotic reaction. Indeed, it could represent one of the mechanisms able to contribute to the therapeutic effects of these drugs, particularly in the presence of macular edema.

Curcumin is one of the major compounds contained in turmeric, the powdered rhizome of Curcuma longa. Results obtained in various experimental models indicate that curcumin has the potential to treat a large variety of neuronal diseases. Excitotoxicity, the toxicity due to pathological glutamate receptors stimulation, has been considered to be involved in several ocular pathologies including ischemia, glaucoma, and diabetic retinopathy. The NMDA receptor (NMDAR), a heteromeric ligand-gated ion channel, is composed of GluN1 and GluN2 subunits. There are four GluN2 subunits (GluN2A-D), which are major determinants of the functional properties of NMDARs. It is widely accepted that GluN2B has a pivotal role in excitotoxicity while the role of GluN2A remains controversial. We previously demonstrated that curcumin is neuroprotective against NMDA-induced excitotoxicity with a mechanism involving an increase of GluN2A subunit activity. In this paper, we investigate the mechanisms involved in curcumin-induced GluN2A increase in retinal cultures. Our results show that curcumin treatment activated CaMKII with a time-course that paralleled those of GluN2A increase. Moreover, KN-93, a CaMKII inhibitor, was able to block the effect of curcumin on GluN2A expression. Finally, in our experimental model, curcumin reduced ser/thr phosphatases activity. Using okadaic acid, a specific PP1 and PP2A blocker, we observed an increase in GluN2A levels in cultures. The ability of okadaic acid to mimic the effect of curcumin on GluN2A expression suggests that curcumin might regulate GluN2A expression through a phosphatase-dependent mechanism. In conclusion, our findings indicate curcumin modulation of CaMKII and/or ser/thr phosphatases activities as a mechanism involved in GluN2A expression and neuroprotection against excitotoxicity.

Degeneration of cortical and spinal motor neurons is the typical feature of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease for which a pathogenetic role for the Cu/Zn superoxide dismutase (SOD1) has been demonstrated. Mice overexpressing a mutated form of the SOD1 gene (SOD1G93A) develop a syndrome that closely resembles the human disease. The SOD1 mutations confer to this enzyme a “gain-of-function,” leading to increased production of reactive oxygen species. Several oxidants induce tyrosine phosphorylation through direct stimulation of kinases and/or phosphatases. In this study, we analyzed the activities of src and fyn tyrosine kinases and of protein tyrosine phosphatases in synaptosomal fractions prepared from the motor cortex and spinal cord of transgenic mice expressing SOD1G93A. We found that (i) protein phosphotyrosine level is increased, (ii) src and fyn activities are upregulated, and (iii) the activity of tyrosine phosphatases, including the striatal-enriched tyrosine phosphatase (STEP), is significantly decreased. Moreover, the NMDA receptor (NMDAR) subunit GluN2B tyrosine phosphorylation was upregulated in SOD1G93A. Tyrosine phosphorylation of GluN2B subunits regulates the NMDAR function and the recruitment of downstream signaling molecules. Indeed, we found that proline-rich tyrosine kinase 2 (Pyk2) and ERK1/2 kinase are upregulated in SOD1G93A mice. These results point out an involvement of tyrosine kinases and phosphatases in the pathogenesis of ALS.

Experimental models of diabetic retinopathy (DR) have had a crucial role in the comprehension of the pathophysiology of the disease and the identification of new therapeutic strategies. Most of these studies have been conductedin vivo, in animal models. However, a significant contribution has also been provided by studies on retinal cultures, especially regarding the effects of the potentially toxic components of the diabeticmilieuon retinal cell homeostasis, the characterization of the mechanisms on the basis of retinal damage, and the identification of potentially protective molecules. In this review, we highlight the contribution given by primary retinal cultures to the study of DR, focusing on early neuroglial impairment. We also speculate on possible themes into which studies based on retinal cell cultures could provide deeper insight.

In recent years, citicoline has been the object of remarkable interest as a possible neuroprotectant. The aim of this study was to investigate if citicoline affected cell survival in primary retinal cultures and if it exerted neuroprotective activity in conditions modeling retinal neurodegeneration. Primary retinal cultures, obtained from rat embryos, were first treated with increasing concentrations of citicoline (up to 1000 µM) and analyzed in terms of apoptosis and caspase activation and characterized by immunocytochemistry to identify neuronal and glial cells. Subsequently, excitotoxic concentration of glutamate or High Glucose-containing cell culture medium (HG) was administered as well-known conditions modeling neurodegeneration. Glutamate or HG treatments were performed in the presence or not of citicoline. Neuronal degeneration was evaluated in terms of apoptosis and loss of synapses. The results showed that citicoline did not cause any damage to the retinal neuroglial population up to 1000 µM. At the concentration of 100 µM, it was able to counteract neuronal cell damage both in glutamate- and HG-treated retinal cultures by decreasing proapoptotic effects and contrasting synapse loss. These data confirm that citicoline can efficiently exert a neuroprotective activity. In addition, the results suggest that primary retinal cultures, under conditions inducing neurodegeneration, may represent a useful system to investigate citicoline neuroprotective mechanisms.

Objective Advanced glycation end-products (AGEs) constitute a highly heterogeneous family of compounds, relevant in the pathogenesis of diabetic complications, which could represent efficient biomarkers of disease progression and drug response. Unfortunately, due to their chemical heterogeneity, no method has been validated to faithfully monitor their levels in the course of the disease. In this study, we refine a procedure to quantitatively analyze fluorescent AGEs (fAGEs), a subset considered remarkably representative of the entire AGE family, and measure them in in vitro glycated BSA (gBSA) and in plasma and vitreous of diabetic rats, for testing its use to possibly quantify circulating AGEs in patients, as markers of metabolic control. Methods fAGE levels were evaluated by spectrofluorimetric analysis in in vitro and in vivo experimental models. BSA was glycated in vitro with increasing D-glucose concentrations for a fixed time or with a fixed D-glucose concentration for increasing time. In in vivo experiments, streptozotocin-induced diabetic rats were studied at 1, 3, 6 and 12 weeks to analyze plasma and vitreous. To confirm the presence of AGEs in our models, non-diabetic rat retinal explants were exposed to high glucose (HG), to reproduce short-term effects, or in vitro gBSA, to reproduce long-term effects of elevated glucose concentrations. Rat retinal explants and diabetic retinal tissues were evaluated for the receptor for advanced glycation end-product (RAGE) by Western blot analysis. Results In in vitro experiments, fluorescence emission showed glucose concentration- and time-dependent increase of fAGEs in gBSA (p ≤ 0.05). In streptozotocin-induced diabetic rats, fAGE in plasma and vitrei showed an increase at 6 (p ≤ 0.005) and 12 (p ≤ 0.05) weeks of diabetes, with respect to control. RAGE was time-dependently upregulated in retinas incubated with gBSA, but not with HG, and in diabetic retinal tissue, substantiating exposure to AGEs. Conclusions Applying the proposed technique, we could show that fAGEs levels increase with glucose concentration and time of exposure in vitro. Furthermore, in diabetic rats, it showed that circulating fAGEs are similarly upregulated as those in vitreous, suggesting a correlation between circulating and tissue AGEs. These results support the use of this method as a simple and reliable test to measure circulating fAGEs and monitor diabetes progression.

Mutations in the MLC1 gene, which encodes a protein expressed in brain astrocytes, are the leading cause of MLC, a rare leukodystrophy characterized by macrocephaly, brain edema, subcortical cysts, myelin and astrocyte vacuolation. Although recent studies indicate that MLC1 protein is implicated in the regulation of cell volume changes, the exact role of MLC1 in brain physiology and in the pathogenesis of MLC disease remains to be clarified. In preliminary experiments, we observed that MLC1 was poorly expressed in highly proliferating astrocytoma cells when compared with primary astrocytes, and that modulation of MLC1 expression influenced astrocyte growth. Because volume changes are key events in cell proliferation and during brain development MLC1 expression is inversely correlated to astrocyte progenitor proliferation levels, we investigated the possible role for MLC1 in the control of astrocyte proliferation. We found that overexpression of wild type but not mutant MLC1 in human astrocytoma cells hampered cell growth by favoring epidermal growth factor receptor (EGFR) degradation and by inhibiting EGF-induced Ca(+) entry, ERK1/2 and PLCγ1 activation, and calcium-activated KCa3.1 potassium channel function, all molecular pathways involved in astrocyte proliferation stimulation. Interestingly, MLC1 did not influence AKT, an EGFR-stimulated kinase involved in cell survival. Moreover, EGFR expression was higher in macrophages derived from MLC patients than from healthy individuals. Since reactive astrocytes proliferate and re-express EGFR in response to different pathological stimuli, the present findings provide new information on MLC pathogenesis and unravel an important role for MLC1 in other brain pathological conditions where astrocyte activation occurs.

Progranulin (PGRN) is a widely expressed multifunctional protein, involved in regulation of cell growth and cell cycle progression with a possible involvement in neurodegeneration. We looked for PGRN regulation in three different human neuroblastoma cell lines, following exposure to two different stimuli commonly associated to neurodegeneration: hypoxia and oxidative stress. For gene and protein expression analysis we carried out a quantitative RT-PCR and western blotting analysis. We show that PGRN is strongly up-regulated by hypoxia, through the mitogen-actived protein kinase (MAPK)/extracellular signal-regulated kinase (MEK) signaling cascade. PGRN is not up-regulated by H2O2-induced oxidative stress. These results suggest that PGRN in the brain could exert a protective role against hypoxic stress, one of principal risk factors involved in frontotemporal dementia pathogenesis.

The early effects of the diabetic milieu on retinal tissue and their relation to the Renin–Angiotensin system (RAS) activation are poorly known. Here we investigated RAS signaling in retinas explanted from adult rats exposed for 48 h to high glucose (HG), with or without the Angiotensin Converting Enzyme inhibitor enalaprilat, which blocks RAS. HG was observed to i) initiate a phosphotyrosine-dependent signaling cascade; ii) up-regulate Angiotensin1 Receptor (AT1R); iii) activate src tyrosine kinase and increase phosphorylation of Pyk2, PLCγ1 and ERK1/2; and iv) activate Akt and the transcription factor CREB. In the presence of enalaprilat, tyrosine phosphorylation signal and AT1R upregulation decreased and activation of PLCγ1 and CREB reverted, showing their relation to RAS signaling. In line with Akt activation, no apoptosis or synapse degeneration was found. Muller glia was activated, but in a RAS-independent manner. Our results suggest that, in early phases of HG exposure, a pro-survival cell program may be induced in the retina.

The Nef protein of the human immunodeficiency virus type 1 (HIV-1) plays a crucial role in AIDS pathogenesis by modifying host cell signaling pathways. We investigated the effects of Nef on the NADPH oxidase complex, a key enzyme involved in the generation of reactive oxygen species during the respiratory burst in human monocyte/macrophages. We have recently shown that the inducible expression of HIV-1 Nef in human macrophages cell line modulates in bi-phasic mode the superoxide anion release by NADPH oxidase, inducing a fast increase of the superoxide production, followed by a delayed strong inhibition mediated by Nef-induced soluble factor(s). Our study is focused on the molecular mechanisms involved in Nef-mediated activation of NADPH oxidase and superoxide anion release. Using U937 cells stably transfected with different Nef alleles, we found that both Nef membrane localization and intact SH3-binding domain are needed to induce superoxide release. The lack of effect during treatment with a specific MAPK pathway inhibitor, PD98059, demonstrated that Nef-induced superoxide release is independent of Erk1/2 phosphorylation. Furthermore, Nef induced the phosphorylation and then the translocation of the cytosolic subunit of NADPH oxidase complex p47(phox) to the plasma membrane. Adding the inhibitor PP2 prevented this process, evidencing the involvement of the Src family kinases on Nef-mediated NADPH oxidase activation. In addition, LY294002, a specific inhibitor of phosphoinositide 3-kinase (PI3K) inhibited both the Nef-induced p47(phox) phosphorylation and the superoxide anion release. These data indicate that Nef regulates the NADPH oxidase activity through the activation of the Src kinases and PI3K.

Protein serine/threonine phosphorylation is a significant component of the intracellular signal that together with tyrosine phosphorylation regulates several processes, including cell-cycle progression, muscle contraction, transcription, and neuronal signaling. Cross-talk between phosphoserine/threonine- and phosphotyrosine-mediated pathways is not yet well understood. In this study we found that peroxynitrite, a physiological oxidant formed by the fast radical-radical reaction between the nitric oxide and the superoxide anion, induced tyrosine phosphorylation of the serine/threonine protein phosphatase 1alpha (PP1alpha) in human erythrocytes through activation of src family kinases. We have previously shown in mouse red cells that upregulation of the src kinase fgr phosphorylates PP1alpha, acting as an upstream negative regulator of PP1alpha, and downregulates K-Cl cotransport. Here we found that PP1alpha is a selective substrate of peroxynitrite-activated fgr and that tyrosine phosphorylation of PP1alpha corresponds to an inhibition of its enzymatic activity. Despite fgr activation and PP1alpha downregulation, peroxynitrite stimulated in a dose-dependent fashion the function of the K-Cl cotransporter. In an attempt to understand the mechanism of K-Cl cotransport activation, we found that the effect of peroxynitrite is completely reversed by dithriothreitol, suggesting that peroxynitrite acts as an oxidizing agent by an SH-dependent and PP1alpha-independent mechanism. These findings highlight a novel function of peroxynitrite in regulating the intracellular signal transduction pathways involving serine/threonine phosphorylation and the functional role of proteins that are targets of these phosphatases.

The hypothesis that peroxynitrite may act as a signaling molecule able to upregulate protein tyrosine phosphorylation is discussed. This article focuses on the mechanisms for activating kinases of the src family, an important class of nonreceptor tyrosine kinases implicated in the regulation of cell communication, proliferation, migration, differentiation, and survival. Recent in vitro findings show that in erythrocytes, synaptosomes, and cerebellar primary culture cells peroxynitrite is able to inhibit phosphatases and to activate different members of the src kinase family through different mechanisms involving cysteine-dependent and -independent processes. The ability of nitrotyrosine-containing peptides with SH2 binding affinity to activate src kinases is also discussed.

The reactive species peroxynitrite, formed via the near diffusion-limited reaction of nitric oxide and superoxide anion, is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. Peroxynitrite readily nitrates tyrosine residues in proteins, producing a permanent modification that can be immunologically detected. We have previously demonstrated that in the nerve terminal, nitrotyrosine immunoreactivity is primarily associated with synaptophysin. Here we identify two other presynaptic proteins nitrated by peroxynitrite, Munc-18 and SNAP25, both of which are involved in sequential steps leading to vesicle exocytosis. To investigate whether peroxynitrite affects vesicle exocytosis, we used the fluorescent dye FM1-43 to label a recycling population of secretory vesicles within the synaptosomes. Bolus addition of peroxynitrite stimulated exocytosis and glutamate release. Notably, these effects were strongly reduced in the presence of NaHCO3, indicating that peroxynitrite acts mainly intracellularly. Furthermore, peroxynitrite enhanced the formation of the sodium dodecyl sulfate-resistant SNARE complex in a dose-dependent manner (100–1000 µm) and induced the formation of 3-nitrotyrosine in proteins of SNARE complex. These data suggest that modification(s) of synaptic vesicle proteins induced by peroxynitrite may affect protein–protein interactions in the docking/fusion steps, thus promoting exocytosis, and that, under excessive production of superoxide and nitric oxide, neurons may up-regulate neuronal signaling.

Peroxynitrite, the product of the radical-radical reaction between nitric oxide and superoxide anion, is a potent oxidant involved in tissue damage in neurodegenerative disorders. We investigated the modifications induced by peroxynitrite in tyrosine residues of proteins from synaptosomes. Peroxynitrite treatment (> or =50 microM) induced tyrosine nitration and increased tyrosine phosphorylation. Synaptophysin was identified as one of the major nitrated proteins and pp60src kinase as one of the major phosphorylated substrates. Further fractionation of synaptosomes revealed nitrated synaptophysin in the synaptic vesicles, whereas phosphorylated pp60src was enriched in the postsynaptic density fraction. Tyrosine phosphorylation was increased by treatment with 50-500 microM peroxynitrite and decreased by higher concentrations, suggesting a possible activation/inactivation of kinases. Immunocomplex kinase assay proved that peroxynitrite treatment of synaptosomes modulated the pp60src autophosphorylation activity. The addition of bicarbonate (CO2 1.3 mM) produced a moderate enhancing effect on some nitrated proteins but significantly protected the activity of pp60src against peroxynitrite-mediated inhibition so that at 1 mM peroxynitrite, the kinase was still more active than in untreated synaptosomes. The phosphotyrosine phosphatase activity of synaptosomes was inhibited by peroxynitrite (> or =50 microM) but significantly protected by CO2. Thus, the increase of phosphorylation cannot be attributed to peroxynitrite-mediated inhibition of phosphatases. We suggest that peroxynitrite may regulate the posttranslational modification of tyrosine residues in pre- and postsynaptic proteins. Identification of the major protein targets gives insight into the pathways possibly involved in neuronal degeneration associated with peroxynitrite overproduction.

Exposure to Stimulating Environments (SE) during development may improve neuroplasticity in central nervous system, protect against neurotoxic damage, and promote neuronal recovery in adult life. While biochemical mechanisms of SE-promoted neuronal plasticity are well known in the brain, much less is known on the signaling cascade governing plasticity and neuroprotection in the retina. In order to investigate if in the retina signaling molecules involved in neuronal plasticity are affected by SE, neonatal CD-1 mice were exposed to moderate corticosterone levels (NC), supplemented through maternal milk during the first postnatal week, or to environmental enrichment (EE) conditions (physical and social stimuli) from early adolescence. Our results showed that both NC and EE increased the phosphorylation level of Extracellularly Regulated Kinase 1/2 (ERK1/2) and cAMP response element-binding protein (CREB) in the adult retinal tissue. Furthermore, we observed that activated ERK1/2 was restricted to Muller cells, while pCREB was mostly present in the nuclei of retinal neurons. Neither NC, nor EE modified the expression of GFAP, a marker of Muller cells activation. In conclusion our results indicate that both NC and EE activate ERK1/2 and CREB in the retina and provide a biochemical background for the neuroprotective activity exerted by SE against retinal damage. Furthermore, they support the role of Muller glia as a key cell determinant of retinal neuroplasticity.

Muller cell activation is an early finding in diabetic retinopathy (DR), but its physiopathologic role in the disease is still unclear, especially in the early phases. We investigated on Muller glial activation in primary rat retinal cultures, exposed to High Glucose (HG), and in retinas from streptozotocin (stz)-induced diabetic rats. First of all, we checked if the presence of Muller glia influenced HG neurotoxicity. In mixed glial/neuronal retinal cultures, a single HG administration (sHG) for 48 h induced activation of Muller glia, in absence of neuronal damage. In contrast, in pure neuronal cultures, a marked neurotoxic effect was detected, suggesting that in this cell model Muller glia protect neurons from HG neurotoxicity. To better mimic the diabetic milieu, where retinal cells are constantly bathed in hyperglycemic fluid, and to further characterize astrocytic neuroprotective ability, mixed retinal cultures were exposed to repeated daily replacement of HG (rHG). In this paradigm, starting from 48 h, increased apoptosis and synaptic loss were observed, even in the presence of Muller cells. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whose activation triggers a prosurvival pathway, was increased by sHG, while it was down-regulated by rHG, suggesting that ERK1/2 activation is involved in neuroprotection. Consistently, in presence of ERK1/2 inhibitor PD98059, sHG exerted a proapoptotic effect also in glial/neuronal retinal cultures. In line with the in vitro data, early changes in diabetic retinas from stz-injected rats included Muller cell activation and increased pERK1/2 levels, but no signs of neuronal damage. These results suggest that, in the early phases of DR, Muller glial activation does not contribute to neurodegeneration, but may indeed have a neuroprotective activity against HG-induced neurotoxicity through a mechanism involving pERK1/2.

Peroxynitrite, the product of superoxide and nitric oxide radicals, is considered one of the major oxidants formed in vivo under intense oxidative stress. We have previously reported the upregulation by peroxynitrite of src kinase activity in red blood cells. In this study, we investigated the mechanisms of peroxynitrite action and we demonstrate that two src kinases (lyn and hck) are activated through different pathways involving cysteine-dependent or -independent oxidations. Activation of hck by peroxynitrite or by hydrogen peroxide could be explained by reversible SH redox changes, whereas lyn was unaffected by hydrogen peroxide and its direct activation by peroxynitrite occurred through a still unknown modification(s) not reverted by SH reduction or inhibited by SH alkylation. Moreover, lyn could be activated also downstream by peroxynitrite-activated hck. The cross talk between lyn and hck was selective, since activated hck did not activate the non-src kinase syk. This study illustrates the complexity of redox-dependent src regulation and suggests that one reason for src heterogeneity may be a peculiar difference in their sensitivity to physiological oxidants. Irrespectively of the activation pathway, the final effect of peroxynitrite is the amplification of tyrosine-dependent signaling, a finding of general interest in nitric oxide-related pathophysiology.

The striatum is a brain area implicated in the pharmacological action of drugs of abuse. Adenosine A2A receptors (A2ARs) are highly expressed in the striatum and mediate, at least in part, cocaine-induced psychomotor effects in vivo. Here we studied the synaptic mechanisms implicated in the pharmacological action of cocaine in the striatum and investigated the influence of A2ARs. We found that synaptic transmission was depressed in corticostriatal slices after perfusion with cocaine (10 μM). This effect was reduced by the A2AR antagonist ZM241385 and almost abolished in striatal A2AR-knockout mice (mice lacking A2ARs in striatal neurons, stA2ARKO). The effect of cocaine on synaptic transmission was also prevented by the protein tyrosine phosphatases (PTPs) inhibitor sodium orthovanadate (Na3VO4). In synaptosomes prepared from striatal slices, we found that the activity of striatal-enriched protein tyrosine phosphatase (STEP) was upregulated by cocaine, prevented by ZM241385, and absent in synaptosomes from stA2ARKO. The role played by STEP in cocaine modulation of synaptic transmission was investigated in whole-cell voltage clamp recordings from medium spiny neurons of the striatum. We found that TAT-STEP, a peptide that renders STEP enzymatically inactive, prevented cocaine-induced reduction in AMPA- and NMDA-mediated excitatory post-synaptic currents, whereas the control peptide, TAT-myc, had no effect. These results demonstrate that striatal A2ARs modulate cocaine-induced synaptic depression in the striatum and highlight the potential role of PTPs and specifically STEP in the effects of cocaine.

We compared the nitric oxide (.NO)-releasing characteristics of two NO donors, the S-nitroso adduct of bovine serum albumin (BSANO) and the S-nitroso adduct of L-glutathione (GSNO). In oxygenated phosphate buffer (pH 7.4) and in hemoglobin solution, both NO donors released .NO only in the presence of a low molecular weight thiol (the most active was L-cysteine). The requirement of thiol to release .NO strongly suggests that a transnitrosation reaction occurs between the S-nitroso adduct of the NO donor and the sulfhydryl group of the NO acceptor. The reaction produced a labile S-nitroso-L-cysteine intermediate that released .NO. As shown by spin-trapping experiments, the transnitrosation reaction involved the formation of .NO (trapped by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide) and .S radicals (trapped by 5,5'-dimethyl-1-pyrroline N-oxide) of both the NO donors and the NO acceptor (L-cysteine). The reaction leading to .S radical formation was distinct from the transnitrosation reaction, since it was oxygen-dependent. We suggest that .S radicals are formed from oxidizing species produced after a reaction between .NO and molecular oxygen (.NO2 is a likely candidate). As for pure .NO gas, the major oxidation product of NO donors, in phosphate buffer (pH 7.4), was NO2-, with no formation of NO3-. In the presence of oxyhemoglobin, both NO donors produced only NO3-. BSANO and GSNO showed distinct patterns of .NO release both in phosphate buffer and in the presence of hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the role of free radicals in hemoglobin (Hb) oxidation and denaturation. To generate free radicals, we used two azocompounds, the hydrophilic 2,2′-azobis(2-amidinopropane hydrochloride and the hydrophobic 2,2′-azobis(2,4-dimethylvaleronitrile) and a drug of the quinone family, phenazine methosulfate. The radical species involved were analyzed by direct EPR and spin trapping with 5,5-dimethyl-1-pyrroline N-oxide, and N-t-butyl-α-phenyl-nitrone. The free radicals generated by the azocompounds were carbon radicals and, in the presence of molecular oxygen, peroxyl/alkoxyl radicals. The reaction of phenazine with Hb produced a nitrogen-centered semiquinoid radical detectable by EPR only under N2 and reactive oxygen species (O.−2 and H2O2) in the presence of molecular oxygen. Azocompounds oxidized Hb to methemoglobin, hemichromes, and choleglobin while phenazine produced methemoglobin and ferrylhemoglobin. For all three drugs, low oxygen tensions (pO2 62 mm Hg) increased the formation of Hb oxidation products, whereas high oxygen tensions (pO2 540 mm Hg) reduced Hb oxidation. The formation of irreversible Hb oxidation products (irreversible hemichromes and Hb cross-linking) was observed only with the azocompounds and was reduced at high pO2. Spin traps and thiourea protected Hb from the oxidative damage induced by the azocompounds, whereas enzymes scavenging reactive oxygen species, such as superoxide dismutase and catalase, affected Kb oxidation induced by phenazine and that induced by the hydrophobic azocompound. These results indicate distinct patterns of oxidation and denaturation with each agent. Damage induced by phenazine was dependent on the formation of reactive oxygen species, whereas the damage induced by the azocompounds was due mainly to carbon-centered radicals with some involvement by reactive oxygen species only for the hydrophobic azocompound. The preferential interaction of Hb with drug radicals scavenged by molecular oxygen indicates that this protein may be more reactive under hypoxic conditions and led to the view that a good supply of oxygen can provide an important defense against drug-induced Hb oxidation.

Sixty-day-old virgin female Swiss CD1 mice were treated with 1.5% 2,5-hexanedione in their drinking water; control mice received tap water; duration of treatment was either 4 or 6 weeks. Under these conditions the treated mice did not show any clinical symptoms although electromyography revealed some signs of polyneuropathy. Protein and DNA content per mg of ovarian tissue in treated mice were not significantly different from controls. Histological examination of ovarian sections at the light microscope level showed no significant alterations after exposure. A morphometric study revealed a statistically significant reduction in the number of growing oocytes after 6 weeks of treatment. For fertility studies three groups of 15 female mice each were treated for 0, 4 or 6 weeks as above and then permanently housed with untreated proven breeder male mice (one male per female); cages were checked daily for newly born mice. All litters appeared normal by gross examination. During the first 14 weeks of continuous mating the mean litter size (number of newborns per litter) remained about 11.4 in all groups; this number subsequently began to decrease. Control and 4-week treatment regression curves did not differ statistically, while the slope of the 6-week line was significantly steeper, indicating a faster decrease in litter size over time and a shortening of fertile life in the latter group of treated females.

Peroxynitrite is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. We have previously reported that treatment of rat brain synaptosomes with peroxynitrite induced post-translational modifications in pre- and post-synaptic proteins and stimulated soluble N-ethylmaleimide sensitive fusion proteins attachment receptor complex formation and endogenous glutamate release. In this study we show that, following peroxynitrite treatment, the synaptic vesicle protein synaptophysin (SYP) can be both phosphorylated and nitrated in a dose-dependent manner. We found that tyrosine-phosphorylated, but not tyrosine-nitrated, SYP bound to the src tyrosine kinase and enhanced its catalytic activity. These effects were mediated by direct and specific binding of the SYP cytoplasmic C-terminal tail with the src homology 2 domain. Using mass spectrometry analysis, we mapped the SYP C-terminal tail tyrosine residues modified by peroxynitrite and found one nitration site at Tyr250 and two phosphorylation sites at Tyr263 and Tyr273. We suggest that peroxynitrite-mediated modifications of SYP may be relevant in modulating src signalling of synaptic terminal in pathophysiological conditions.

The dystrophin-related and -associated protein dystrobrevin is a component of the dystrophin-associated protein complex, which directly links the cytoskeleton to the extracellular matrix. It is now thought that this complex also serves as a dynamic scaffold for signaling proteins, and dystrobrevin may play a role in this context. Since dystrobrevin involvement in signaling pathways seems to be dependent on its interaction with other proteins, we sought new insights and performed a two-hybrid screen of a mouse brain cDNA library using beta-dystrobrevin, the isoform expressed in non-muscle tissues, as bait. Among the positive clones characterized after the screen, one encodes the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA). We confirmed the interaction by in vitro and in vivo association assays, and mapped the binding site of beta-dystrobrevin on RIalpha to the amino-terminal region encompassing the dimerization/docking domain of PKA regulatory subunit. We also found that the domain of interaction for RIalpha is contained in the amino-terminal region of beta-dystrobrevin. We obtained evidence that beta-dystrobrevin also interacts directly with RIIbeta, and that not only beta-dystrobrevin but also alpha-dystrobrevin interacts with PKA regulatory subunits. We show that both alpha and beta-dystrobrevin are specific phosphorylation substrates for PKA and that protein phosphatase 2A (PP2A) is associated with dystrobrevins. Our results suggest a new role for dystrobrevin as a scaffold protein that may play a role in different cellular processes involving PKA signaling.

Quinolinic acid (QA) is an endogenous excitotoxin acting on N-methyl-d-aspartate receptors (NMDARs) that leads to the pathologic and neurochemical features similar to those observed in Huntington's disease (HD). The mechanism of QA toxicity also involves free radicals formation and oxidative stress. NMDARs are particularly vulnerable to the action of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can act as modulators of the activity of protein tyrosine kinases (PTKs) and phosphotyrosine phosphatases (PTPs). Because QA is able to activate neuronal nitric oxide synthase (nNOS) as well as to stimulate the NMDARs, we evaluated the effect of Nω-Nitro-l-arginine-methyl ester (l-NAME), a selective nNOS inhibitor, on QA-induced neurotoxicity in rat corticostriatal slices. In electrophysiologic experiments we observed that slice perfusion with QA induced a strong reduction of field potential (FP) amplitude, followed by a partial recovery at the end of the QA washout. In the presence of l-NAME the recovery of FP amplitude was significantly increased with respect to QA alone. In synaptosomes, prepared from corticostriatal slices after the electrophysiologic recordings, we observed that l-NAME pre-incubation reversed the QA-mediated inhibitory effects on protein tyrosine phosphorylation pattern, c-src, lyn, and fyn kinase activities and tyrosine phosphorylation of NMDAR subunit NR2B, whereas the PTP activity was not recovered in the presence of l-NAME. These findings suggest that NO plays a key role in the molecular mechanisms of QA-mediated excitotoxicity in experimental model of HD. © 2007 Wiley-Liss, Inc.

Quinolinic acid (QA) has been shown to evoke neurotoxic events via NMDA receptor (NMDAR) overactivation and oxidative stress. NMDARs are particularly vulnerable to free radicals, which can modulate protein tyrosine kinase (PTK) and phosphotyrosine phosphatase (PTP) activities. The src family of tyrosine kinases are associated with the NMDAR complex and regulate NMDA channel function. Because QA is an NMDAR agonist as well as a pro-oxidant agent, we investigated whether it may affect the activity of PTKs and PTPs in vivo and in vitro. In synaptosomes prepared from striata dissected 15 min, 30 min or 15 days after bilateral injection of QA we observed modulation of the phosphotyrosine pattern; a significant decrease in PTP activity; and a sustained increase in c-src and lyn activity at 15 and 30 min after treatment with QA, followed by a decrease 2 weeks later. Striatal synaptosomes treated in vitro with QA showed time- and dose-dependent modulation of c-src and lyn kinase activities. Moreover, the nitric oxide synthase inhibitor NG-nitro-L-arginine-methyl ester, the NMDAR antagonist d-2-amino-5-phosphonovaleric acid and pyruvate suppressed the QA-induced modulation of c-src activity. These findings suggest a novel feature of QA in regulating src kinase activity through the formation of reactive radical species and/or NMDAR overactivation.

Several studies have demonstrated that treatment of cells with oxidants, and in particular with peroxynitrite, may cause the upregulation of tyrosine phosphorylation signaling. In erythrocytes, peroxynitrite induces tyrosine phosphorylation of the major intrinsic membrane protein, band 3. A closer look at the enzymes involved revealed that the effect of peroxynitrite was due to the inhibition of phosphotyrosine phosphatases and/or to the activation of src kinases. The activity of src kinases is modulated not only by phosphatases and other kinases but also through redox modification of cysteine residues: Peroxynitrite can, thus, affect src kinase activity by means of direct and indirect mechanisms. In this chapter, we describe the different pathways leading to src kinase activation and the experimental procedures that can be performed to reveal the activation mechanism. The aim is to provide a more general strategy adaptable to different cell types and different oxidants.

Several studies have demonstrated that treatment of cells with oxidants, and in particular with peroxynitrite, may cause the upregulation of tyrosine phosphorylation signaling. In erythrocytes, peroxynitrite induces tyrosine phosphorylation of the major intrinsic membrane protein, band 3. A closer look at the enzymes involved revealed that the effect of peroxynitrite was due to the inhibition of phosphotyrosine phosphatases and/or to the activation of src kinases. The activity of src kinases is modulated not only by phosphatases and other kinases but also through redox modification of cysteine residues: Peroxynitrite can, thus, affect src kinase activity by means of direct and indirect mechanisms. In this chapter, we describe the different pathways leading to src kinase activation and the experimental procedures that can be performed to reveal the activation mechanism. The aim is to provide a more general strategy adaptable to different cell types and different oxidants.

The protective effects of eating fruits and vegetables in the prevention of several degenerative pathologies have been attributed at least in part to the antioxidant and anti-inflammatory properties of polyphenols. In this study, we investigated the effects of two polyphenols, quercetin and resveratrol, on red blood cell Band 3 tyrosine phosphorylation signalling activated by peroxynitrite. Peroxynitrite is a physiological oxidant scavenged largely by the erythrocyte and formed by the reaction between nitrogen monoxide and superoxide anion. Quercetin and its structurally analogous (+)-catechin inhibited the peroxynitrite-dependent upregulation of Band 3 tyrosine phosphorylation. Quercetin was found to downregulate the activity of syk, which is upstream in the Band 3 tyrosine phosphorylation cascade, and partially prevented peroxynitrite-mediated phosphotyrosine phosphatase inhibition. Resveratrol and hydroxytyrosol, unexpectedly, amplified peroxynitrite-dependent upregulation of Band 3 tyrosine phosphorylation through the activation of lyn, a kinase of the src family. The present results clearly indicate that polyphenols may activate cell transduction pathways in different and sometimes opposite ways.

The nitration of tyrosine residues in protein occurs through the action of reactive oxygen and nitrogen species and is considered a marker of oxidative stress under pathological conditions. The most active nitrating species so far identified is peroxynitrite, the product of the reaction between nitric oxide and superoxide anion. Previously, we have reported that in erythrocytes peroxynitrite irreversibly upregulates lyn, a tyrosine kinase of the src family. In this study we investigated the possible role of tyrosine nitration in the mechanism of lyn activation. We found that tyrosine containing peptides modelled either on the C-terminal tail of src kinases or corresponding to the first 15 amino acids of human erythrocyte band 3 were able to activate lyn when the tyrosine was substituted with 3-nitrotyrosine. The activity of nitrated peptides was shared with phosphorylated but not with unphosphorylated, chlorinated or scrambled peptides. Recombinant lyn src homology 2 (SH2) domain blocked the capacity of the band 3-derived nitrotyrosine peptide to activate lyn and we demonstrated that this peptide specifically binds the SH2 domain of lyn. We propose that nitropeptides may activate src kinases through the displacement of the phosphotyrosine in the tail from its binding site in the SH2 domain. These observations suggest a new mechanism of peroxynitrite-mediated signalling that may be correlated with the upregulation of tyrosine phosphorylation observed in several pathological conditions.

In this study, we demonstrate that the phosphorylation activity of five tyrosine kinases of the src family from both human erythrocytes (lyn, hck and c-fgr) and bovine synaptosomes (lyn and fyn) was stimulated by treatment with 30-250 microM peroxynitrite. This effect was not observed with syk, a non-src family tyrosine kinase. Treatment of kinase immunoprecipitates with 0.01-10 microM peroxynitrite showed that the interaction of these enzymes with the oxidant also activated the src kinases. Higher concentrations of peroxynitrite inhibited the activity of all kinases, indicating enzyme inactivation. The addition of bicarbonate (1.3 mM CO2) did not modify the upregulation of src kinases but significantly protected the kinases against peroxynitrite-mediated inhibition. Upregulation of src kinase activity by 1 microM peroxynitrite was 3.5-5-fold in erythrocytes and 1.2-2-fold in synaptosomes, but this could be the result, at least in part, of the higher basal level of src kinase activity in synaptosomes. Our results indicate that peroxynitrite can upregulate the tyrosine phosphorylation signal through the activation of src kinases.

Bilirubin is a bile pigment that may have an important role as an antioxidant. Its antioxidant potential is attributed mainly to the scavenging of peroxyl radicals. We investigated the reaction of bilirubin with peroxynitrite in phosphate buffer and in blood plasma. In phosphate buffer bilirubin was rapidly oxidized by micromolar concentrations of peroxynitrite, and its oxidation yield was higher at alkaline pH with an apparent p K a = 6.9. In contrast, the major oxidation product of bilirubin in plasma was biliverdin, and the pH profile of its oxidation yield showed a slightly increased oxidation at acidic pH without a clear inflection point. The addition of NaHCO 3 to bilirubin decreased the peroxynitrite-dependent oxidation, suggesting that the reactive intermediates formed in the reaction between CO 2 and peroxynitrite are less efficient oxidants of bilirubin. The antioxidant role of bilirubin was investigated in some peroxynitrite-mediated plasma protein modifications that are enhanced by CO 2 (tryptophan oxidation and protein tyrosine nitration) or slightly decreased by CO 2 (protein carbonyl groups). Bilirubin in the micromolar concentration range afforded a significant protection against all these oxidative modifications and, notably, protected plasma proteins even when the pigment was added 5 s after peroxynitrite (i.e., when peroxynitrite is completely decomposed). The loss of tryptophan fluorescence triggered by peroxynitrite was a relatively slow process fulfilled only after a few minutes. After this time, bilirubin was unable to reduce the tryptophan loss, and it was unable to reduce previously formed nitrated albumin or previously formed carbonyls. We deduce that bilirubin in plasma cannot react to a significant extent with peroxynitrite, and we suggest that bilirubin, through a hydrogen donation mechanism, participates as a scavenger of secondary oxidants formed in the oxidative process.

Purpose Curcumin, a phenolic compound extracted from the rhizome of Curcuma longa, was found to attenuate NMDA-induced excitotoxicity in primary retinal cultures. This study was conducted to further characterize curcumin neuroprotective ability and analyze its effects on NMDA receptor (NMDAr). Methods NMDAr modifications were analyzed in primary retinal cell cultures using immunocytochemistry, whole-cell patch-clamp recording and western blot analysis. Cell death was evaluated with the TUNEL assay in primary retinal and hippocampal cultures. Optical fluorometric recordings with Fura 2-AM were used to monitor [Ca(2+)](i). Results Curcumin dose- and time-dependently protected both retinal and hippocampal neurons against NMDA-induced cell death, confirming its anti-excitotoxic property. In primary retinal cultures, in line with the observed reduction of NMDA-induced [Ca(2+)](i) rise, whole-cell patch-clamp experiments showed that a higher percentage of retinal neurons responded to NMDA with low amplitude current after curcumin treatment. In parallel, curcumin induced an increase in NMDAr subunit type 2A (NR2A) level, with kinetics closely correlated to time-course of neuroprotection and decrease in [Ca(2+)](i). The relation between neuroprotection and NR2A level increase was also in line with the observation that curcumin neuroprotection required protein synthesis. Electrophysiology confirmed an increased activity of NR2A-containing NMDAr at the plasma membrane level. Conclusions These results confirm the neuroprotective activity of curcumin against NMDA toxicity, possibly related to an increased level of NR2A, and encourage further studies for a possible therapeutic use of curcumin based on neuromodulation of NMDArs.

The effects of 2,5-hexanedione (2,5 HD) on skeletal proteins of red blood cells (RBCs) were investigated both in vitro (human RBCs) and in vivo in male Sprague-Dawley rats which had been treated with the drug for several days. We found that 2,5 HD induced the following major changes in the electrophoretic pattern of the skeletal proteins: (i) the appearance of high-molecular weight bands, (ii) a dose-dependent decrease in spectrin Bands 1 and 2, and (iii) a dose-dependent increase in the amount of hemoglobin (Hb) associated with the membrane. Membranoskeletons, prepared from resealed ghosts which had been previously treated with 2,5 HD, were able to bind an increased amount of Hb from untreated RBCs, thus suggesting a drug-induced modification of the membrane. Extraction of spectrin and actin from ghosts did not remove the membrane-bound Hb and, furthermore, Hb bound to 2,5 HD-treated membranes mainly bearing Band 3 and free of peripheral proteins. These data suggested a 2,5 HD-induced modification of an intrinsic membrane protein, probably Band 3. This hypothesis was consistent with the observation that 2,5 HD also induced a modification of Band 3 aminogroups, as evidenced by a dose-dependent decrease in the binding of eosin probes. Furthermore, RBCs treated in vitro with 2,5 HD bound an increased amount of autologous immunoglobulins (IgG). As reported by Kay and Low et al. the binding of autologous IgG is a phenomenon associated with the aging process of RBCs and may involve a modification of Band 3. Our data show that RBCs treated with 2,5 HD acquired various characteristics of senescent cells such as spectrin cross-linking, Hb-membrane binding and increased IgG binding, and suggest that 2,5 HD treatment might affect RBC survival.

Adult male rats were subjected to 4 weeks' respiratory treatment with n-hexane (5000 ppm, 16h/day, 6 days/week); motor conduction velocity was significantly decreased in tail nerves at all weekly intervals and did not approach normal values in the 4 weeks following interruption of treatment. Plasma acetylcholinesterase (AChE) levels were significantly increased at all weekly intervals during treatment (25-40%); 2 weeks after the end of treatment they had returned to baseline. Oral treatment with 2,5-hexanedione (HD) (1% in drinking water) caused a similar increase in plasma levels; this increase was statistically significant also when compared with pair-fed (PF) control rats. A sucrose density gradient analysis showed only one peak of AChE activity at approximately 10 S (as in normal plasma). The levels of butyrylcholinesterase were unaltered in plasma of both n-hexane-and HD-treated rats. Both the fast-contracting EDL and the slow-contracting soleus muscles lost weight in HD-treated rats with respect to free-fed (AL) and PF controls. AChE levels responded differently to HD treatment in the two muscle types: in EDL total extracts, AChE activity increased considerably with respect to AL controls (+ 70%, p less than 0.001), while the levels of the 16 S and 4 S molecular forms were unaltered. The increased levels of AChE found in plasma of rats intoxicated with n-hexane or with its metabolite HD may originate from muscle and correspond to an increased secretion of this molecular form.