Beta-amyloid accumulation in brain is a driving force for Alzheimer's disease pathogenesis. Apolipoprotein E (ApoE) represents a critical player in betaamyloid homeostasis, but its role in disease progression is controversial. We previously reported that the acute-phase protein haptoglobin binds ApoE and impairs its function in cholesterol homeostasis. The major aims of this study were to characterize the binding of haptoglobin to beta-amyloid, and to evaluate whether haptoglobin affects ApoE binding to beta-amyloid. Haptoglobin is here reported to form a complex with beta-amyloid as shown by immunoblotting experiments with purified proteins, or by its immunoprecipitation in brain tissues from patients with Alzheimer's disease. The interaction between ApoE and beta-amyloid was previously shown to be crucial for limiting beta-amyloid neurotoxicity and for promoting its clearance. We demonstrate that haptoglobin, rather than impairing ApoE binding to beta-amyloid, promotes to a different extent the formation of the complex between beta-amyloid and ApoE2 or ApoE3 or ApoE4. Our data suggest that haptoglobin and ApoE functions in brain should be evaluated taking into account their mutual interaction with beta-amyloid. Hence, the risk of developing Alzheimer's disease might not only be linked to the different ApoE isoforms, but also rely on the level of critical ligands, such as haptoglobin.

Alteration in cholesterol metabolism has been implicated in the pathogenesis of several neurodegenerative disorders. Apolipoprotein E (ApoE) is the major component of brain lipoproteins supporting cholesterol transport. We previously reported that the acute-phase protein Haptoglobin (Hpt) binds ApoE, and influences its function in blood cholesterol homeostasis. Major aim of this study was to investigate whether Hpt influences the mechanisms by which cholesterol is shuttled from astrocytes to neurons. In detail it was studied Hpt effect on ApoE-dependent cholesterol efflux from astrocytes and ApoE-mediated cholesterol incorporation in neurons. We report here that Hpt impairs ApoE-mediated cholesterol uptake in human neuroblastoma cell line SH-SY5Y, and limits the toxicity of a massive concentration of cholesterol for these cells, while it does not affect cholesterol efflux from the human glioblastoma-astrocytoma cell line U-87 MG. As aging is the most important non-genetic risk factor for various neurodegenerative disorders, and our results suggest that Hpt modulates ApoE functions, we evaluated the Hpt and ApoE expression profiles in cerebral cortex and hippocampus of adolescent (2 months), adult (5 and 8 months), and middle-aged (16 months) rats. Hpt mRNA level was higher in hippocampus of 8 and 16 month-old than in 2-month old rats (p < 0.05), and Hpt concentration increased with the age from adolescence to middle-age (p < 0.001). ApoE concentration, in hippocampus, was higher (p < 0.001) in 5 month-old rats compared to 2 month but did not further change with aging. No age-related changes of Hpt (protein and mRNA) were found in the cortex. Our results suggest that aging is associated with changes, particularly in the hippocampus, in the Hpt/ApoE ratio. Age-related changes in the concentration of Hpt were also found in human cerebrospinal fluids. The age-related changes might affect neuronal function and survival in brain, and have important implications in brain pathophysiology.

Haptoglobin is an acute phase glycoprotein, secreted by hepatocytes and other types of cells including keratinocytes. Haptoglobin has been suggested to impair the immune response, inhibit gelatinases in the extracellular matrix and promote angiogenesis, but its role in psoriasis is obscure to date. Changes in haptoglobin glycan structure were observed in several diseases. The aim of this study was to investigate whether haptoglobin displays glycan variations in psoriasis. We found that the pattern of plasma haptoglobin glycoforms, following two-dimensional electrophoresis, exhibited significant quantitative differences in spot intensities between patients and controls. Quantitative and qualitative differences in glycan mass, between patients and controls, were found by mass spectrometry of glycopeptides from tryptic digests of protein isolated from both patients and controls. The number of distinct fucosylated glycoforms of peptides NLFLNHSENATAK and MVSHHNLTTGATLINEQWLLTTAK was higher in patients than in controls, but no fucosylated glycan was detected on peptide VVLHPNYSQ-VDIGLIK in either case. The number of peptides with distinct triantennary and tetraantennary glycans was higher in patients than in controls. Abundance or structure of specific glycans, which are present in haptoglobin from patients and are different or missing in normal haptoglobin, might be associated with disease activity.

A number of commercial software packages are currently available to perform digital two-dimensional electrophoresis (2D-GE) gel analysis. However, both the high cost of the commercial packages and the unavailability of a standard data analysis workflow, have prompted several groups to develop freeware systems to perform certain steps of gel analysis. Unfortunately, to the best of our knowledge none of them offer a package that performs all the steps envisaged in a 2D-GE gel analysis. Here we describe an ImageJ-based procedure, able to manage all the steps of a 2D-GE gel analysis. ImageJ is a free available image processing and analysis application developed by National Institutes of Health (NIH) and widely used in different life sciences fields as medical imaging, microscopy, western blotting and PAGE. Nevertheless no one has yet developed a procedure enabled to compare spots on 2D-GE gels. We collected all used ImageJ tools in a plug-in that allows us to perform the whole 2D-GE analysis. To test it, we performed a set of 2D-GE experiments on plasma samples from 9 patients victims of acute myocardial infarction and 8 controls, and we compared the results obtained by our procedure to those obtained using a widely diffuse commercial package, finding similar performances.

Obesity and dietary fats are well known risk factors for the pathogenesis of neurodegenerative diseases. The analysis of specific markers, whose brain level can be affected by diet, might contribute to unveil the intersection between inflammation/obesity and neurodegeneration. Haptoglobin (Hpt) is an acute phase protein, which acts as antioxidant by binding free haemoglobin (Hb), thus neutralizing its pro-oxidative action. We previously demonstrated that Hpt plays critical functions in brain, modulating cholesterol trafficking in neuroblastoma cell lines, beta-amyloid (Aβ) uptake by astrocyte, and limiting Aβ toxicity on these cells. A major aim of this study was to evaluate whether a long term (12 or 24 weeks) high-fat diet (HFD) influences Hpt and Hb expression in rat hippocampus. We also assessed the development of obesity-induced inflammation by measuring hippocampal level of TNF-alpha, and the extent of protein oxidation by titrating nitro-tyrosine (N-Tyr). Hpt concentration was lower (p < 0.001) in hippocampus of HFD rats than in control animals, both in the 12 and in the 24 weeks fed groups. HFD was also associated in hippocampus with the increase of Hb level (p < 0.01), inflammation and protein oxidative modification, as evidenced by the increase in the concentration of TNF-alpha and nitro-tyrosine. In fact, TNF-alpha concentration was higher in rats receiving HFD for 12 (p < 0.01) or 24 weeks (p < 0.001) compared to those receiving the control diet. N-Tyr concentration was more elevated in hippocampus of HFD than in control rats in both 12 weeks (p = 0.04) and 24 weeks groups (p = 0.01), and a positive correlation between Hb and N-Tyr concentration was found in each group. Finally, we found that the treatment of the human glioblastoma-astrocytoma cell line U-87 MG with cholesterol and fatty acids, such as palmitic and linoleic acid, significantly impairs (p < 0.001) Hpt secretion in the extracellular compartment. We hypothesize that the HFD-dependent decrease of Hpt in hippocampus, as associated with Hb increase, might enhance the oxidative stress induced by free Hb. Altogether our data, identifying Hpt as a molecule modulated in the brain by dietary fats, may represent one of the first steps in the comprehension of the molecular mechanisms underlying the diet-related effects in the nervous system.