Homocysteine and electroencephalographic rhythms in Alzheimer disease: a multicentric study.

High plasma concentration of homocysteine is an independent risk factor for Alzheimer's disease (AD), due to microvascular impairment and consequent neural loss [Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D'Agostino RB, Wilson PW, Wolf PA (2002) Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. N Engl J Med 346(7):476-483]. Is high plasma homocysteine level related to slow electroencephalographic (EEG) rhythms in awake resting AD subjects, as a reflection of known relationships between cortical neural loss and these rhythms? To test this hypothesis, we enrolled 34 mild AD patients and 34 subjects with mild cognitive impairment (MCI). Enrolled people were then subdivided into four sub-groups of 17 persons: MCI and AD subjects with low homocysteine level (MCI- and AD-, homocysteine level <11 micromol/l); MCI and AD subjects with high homocysteine level (MCI+ and AD+, homocysteine level >or=11 micromol/l). Resting eyes-closed EEG data were recorded. EEG rhythms of interest were delta (2-4 Hz), theta (4-8 Hz), alpha 1 (8-10.5 Hz), alpha 2 (10.5-13 Hz), beta 1 (13-20 Hz), and beta 2 (20-30 Hz). EEG cortical sources were estimated by low-resolution brain electromagnetic tomography (LORETA). Results showed that delta (frontal and temporal), theta (central, frontal, parietal, occipital, and temporal), alpha 1 (parietal, occipital, and temporal), and alpha 2 (parietal and occipital) sources were stronger in magnitude in AD+ than AD- group. Instead, no difference was found between MCI- and MCI+ groups. In conclusion, high plasma homocysteine level is related to unselective increment of cortical delta, theta, and alpha rhythms in mild AD, thus unveiling possible relationships among that level, microvascular concomitants of advanced neurodegenerative processes, and synchronization mechanisms generating EEG rhythms.

Interleukin-1beta enhances NMDA receptor-mediated intracellular calcium increase through activation of the Src family of kinases.

Interleukin (IL)-1beta is a proinflammatory cytokine implicated in various pathophysiological conditions of the CNS involving NMDA receptor activation. Circumstantial evidence suggests that IL-1beta and NMDA receptors can functionally interact. Using primary cultures of rat hippocampal neurons, we investigated whether IL-1beta affects NMDA receptor function(s) by studying (1) NMDA receptor-induced [Ca2+]i increase and (2) NMDA-mediated neurotoxicity. IL1beta (0.01-0.1 ng/ml) dose-dependently enhances NMDA-induced [Ca2+]i increases with a maximal effect of approximately 45%. This effect occurred only when neurons were pretreated with IL-1beta, whereas it was absent if IL-1beta and NMDA were applied simultaneously, and it was abolished by IL-1 receptor antagonist (50 ng/ml). Facilitation of NMDA-induced [Ca2+]i increase by IL-1beta was prevented by both lavendustin (LAV) A (500 nm) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (1 microm), suggesting an involvement of tyrosine kinases. Increased tyrosine phosphorylation of NMDA receptor subunits 2A and 2B and coimmunoprecipitation of activated Src tyrosine kinase with these subunits was observed after exposure of hippocampal neurons to 0.05 ng/ml IL-1beta. Finally, 0.05 ng/ml IL-1beta increased by approximately 30% neuronal cell death induced by NMDA, and this effect was blocked by both lavendustin A and PP2. These data suggest that IL-1beta increases NMDA receptor function through activation of tyrosine kinases and subsequent NR2A/B subunit phosphorylation. These effects may contribute to glutamate-mediated neurodegeneration.

High-performance liquid chromatographic determination of total plasma homocysteine with or without internal standards.

Hyperhomocysteinemia is an independent risk factor for atherosclerosis and vascular occlusive disease. Assessment of total plasma concentration of homocysteine (tHcys) requires accurate and reproducible measurements. The aim of this study was to test a rapid isocratic HPLC method for tHcys analysis with an internal standard (I.S.) of alpha-mercaptopropionylglycine (MPG), 2-mercaptoethylamine (ME), or N-acetylcysteine (NAC) or without I.S., and to verify whether the use of an I.S. improves the precision. The method without I.S. showed an excellent linearity (y = 1.59x - 0.15, r = 1), recovery (100%) and a within-assay relative standard deviation (R.S.D.) of 1.2%. Instead, in our hands, the presence of I.S.s decreased the reproducibility (within-assay R.S.D. ranged from 4.5 to 6.5%) and lengthened the chromatogram by up to four to five times. In conclusion, HPLC measurement of plasma tHcys without I.S. improves accuracy with respect to determination with I.S.; moreover, this approach allows to routinely process larger amounts of plasma samples.

Screening of homocysteine from newborn blood spots by high-performance liquid chromatography with coulometric array detection.

Homocystinuria, due to a deficiency of cystationine-beta-synthase, refers to the rare inborn error of the metabolism of homocysteine. The identification and prompt treatment of homocystinuria during the neonatal period can prevent or greatly reduce the severity of the clinical consequences. We report a new method for homocystinuria diagnosis from dried blood spots on newborn screening cards, based on high-performance liquid chromatography with electrochemical coulometric array detection. This method shows an excellent linearity (y=10.36x+0.04; r=0.999), precision (RSDs ranged from 2.7 to 5.8%), recovery (87%) and appears to be a cost-effective approach, being simple, rapid, sensitive and cheap.

A new HPLC micromethod to measure total plasma homocysteine in newborn.

Total plasma homocysteine (tHcy) in children may be an useful biochemical marker for genetic risk of premature cardiovascular disease. We reported a rapid, isocratic HPLC method able to process very small amount of newborn plasma samples. A blood sample from heel capillary circulation was collected, using a heparinized capillary glass tube. Plasma sample from 1 to 10 microl was derivatized with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate after reduction with tri-n-butylphosphine and analyzed on Discovery C18 column, with a solution of acetonitrile-dihydrogenphosphate 0.1 M (8:92 v/v pH*2.1). This assay ensures a good recovery (95%), precision (CV 4.5%) and linearity (y=2.41x + 0.31, r=1). Due to its simplicity and reliability, our method is suitable for routine analysis of tHcy and other aminothiols (Cys, Cys-Gly, GSH) assessed for clinical and research purposes. With this HPLC method we have assayed tHcy levels in 1400 apparently healthy newborn babies (tHcy mean value=4.9+/-2.7 microM). In conclusion, this accurate and linear HPLC method allows measurement of tHcy in newborn during the routinary capillary blood collection in the fourth living day without any other invasive procedure.

The autophagy-associated factors DRAM1 and p62 regulate cell migration and invasion in glioblastoma stem cells.

The aggressiveness of glioblastoma multiforme (GBM) is defined by local invasion and resistance to therapy. Within established GBM, a subpopulation of tumor-initiating cells with stem-like properties (GBM stem cells, GSCs) is believed to underlie resistance to therapy. The metabolic pathway autophagy has been implicated in the regulation of survival in GBM. However, the status of autophagy in GBM and its role in the cancer stem cell fraction is currently unclear. We found that a number of autophagy regulators are highly expressed in GBM tumors carrying a mesenchymal signature, which defines aggressiveness and invasion, and are associated with components of the MAPK pathway. This autophagy signature included the autophagy-associated genes DRAM1 and SQSTM1, which encode a key regulator of selective autophagy, p62. High levels of DRAM1 were associated with shorter overall survival in GBM patients. In GSCs, DRAM1 and SQSTM1 expression correlated with activation of MAPK and expression of the mesenchymal marker c-MET. DRAM1 knockdown decreased p62 localization to autophagosomes and its autophagy-mediated degradation, thus suggesting a role for DRAM1 in p62-mediated autophagy. In contrast, autophagy induced by starvation or inhibition of mTOR/PI-3K was not affected by either DRAM1 or p62 downregulation. Functionally, DRAM1 and p62 regulate cell motility and invasion in GSCs. This was associated with alterations of energy metabolism, in particular reduced ATP and lactate levels. Taken together, these findings shed new light on the role of autophagy in GBM and reveal a novel function of the autophagy regulators DRAM1 and p62 in control of migration/invasion in cancer stem cells.

Expression of sterol 27-hydroxylase in glial cells and its regulation by liver X receptor signaling.

Cholesterol is required in the brain for synaptogenesis and its turnover is critical for cerebral functions. Several proteins involved in cholesterol handling and metabolism are transcriptionally regulated by the nuclear liver X receptor (LXR) alpha and beta. Sterol 27-hydroxylase (CYP27) is a ubiquitously expressed enzyme involved in cholesterol metabolism. Notably, its deficiency causes a disease characterized by progressive neurologic impairment. With the final goal to understand the pathophysiological role of CYP27A1 in the CNS, we studied the expression pattern of Cyp27a1 and other related genes in primary cultures of rat glia and neurons. Secondly, given the pivotal role of LXR in the regulation of cholesterol homeostasis, we investigated the effects of its activation on the expression of Cyp27a1.We found that primary astrocytes express different sterol hydroxylases and are able to uptake exogenous 27-hydroxycholesterol. We found that both microglia and astrocytes express preferentially Lxrbeta. However, despite this similarity, we observed cell-specific responsiveness of known and novel (including Cyp27a1) target genes to LXR activation. The increase of mRNA and protein levels in treated astrocytes is paralleled by transactivation of the proximal Cyp27a1 promoter in transfected astrocytes. We suggest that the astrocyte-restricted up-regulation of Cyp27a1 may be ascribable to differential expression of transcriptional co-activators. Given the role of astrocytes in maintaining brain homeostasis, we hypothesize that impairment of CYP27 activity in these cells may alter critical features of the astrocytes, from the handling and delivery of cholesterol to neurons to the release of signaling molecules.

Protein tyrosine nitration in hydrophilic and hydrophobic environments.

In this review we address current concepts on the biological occurrence, levels and consequences of protein tyrosine nitration in biological systems. We focused on mechanistic aspects, emphasizing on the free radical mechanisms of protein 3-nitrotyrosine formation and critically analyzed the restrictions for obtaining large tyrosine nitration yields in vivo, mainly due to the presence of strong reducing systems (e.g. glutathione) that can potently inhibit at different levels the nitration process. Evidence is provided to show that the existence of metal-catalyzed processes, the assistance of nitric oxide-dependent nitration steps and the facilitation by hydrophobic environments, provide individually and/or in combination, feasible scenarios for nitration in complex biological milieux. Recent studies using hydrophobic tyrosine analogs and tyrosine-containing peptides have revealed that factors controlling nitration in hydrophobic environments such as biomembranes and lipoproteins can differ to those in aqueous compartments. In particular, exclusion of key soluble reductants from the lipid phase will more easily allow nitration and lipid-derived radicals are suggested as important mediators of the one-electron oxidation of tyrosine to tyrosyl radical in proteins associated to hydrophobic environments. Development and testing of hydrophilic and hydrophobic probes that can compete with endogenous constituents for the nitrating intermediates provide tools to unravel nitration mechanisms in vitro and in vivo; additionally, they could also serve to play cellular and tissue protective functions against the toxic effects of protein tyrosine nitration.