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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.

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.

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.

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.

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.

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.

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.