DOPAL derived alpha-synuclein oligomers impair synaptic vesicles physiological function.

Parkinson's disease is a neurodegenerative disorder characterized by the death of dopaminergic neurons and by accumulation of alpha-synuclein (aS) aggregates in the surviving neurons. The dopamine catabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is a highly reactive and toxic molecule that leads to aS oligomerization by covalent modifications to lysine residues. Here we show that DOPAL-induced aS oligomer formation in neurons is associated with damage of synaptic vesicles, and with alterations in the synaptic vesicles pools. To investigate the molecular mechanism that leads to synaptic impairment, we first aimed to characterize the biochemical and biophysical properties of the aS-DOPAL oligomers; heterogeneous ensembles of macromolecules able to permeabilise cholesterol-containing lipid membranes. aS-DOPAL oligomers can induce dopamine leak in an in vitro model of synaptic vesicles and in cellular models. The dopamine released, after conversion to DOPAL in the cytoplasm, could trigger a noxious cycle that further fuels the formation of aS-DOPAL oligomers, inducing neurodegeneration.

Binding Interactions of Agents That Alter α-Synuclein Aggregation.

Further examination of peptides with well-folded antiparallel ß strands as inhibitors of amyloid formation from α-synuclein has resulted in more potent inhibitors. Several of these had multiple Tyr residues and represent a new lead for inhibitor design by small peptides that do not divert α-synuclein to non-amyloid aggregate formation. The most potent inhibitor obtained in this study is a backbone cyclized version of a previously studied ß hairpin, designated as WW2, with a cross-strand Trp/Trp cluster. The cyclization was accomplished by adding a d-Pro-l-Pro turn locus across strand termini. At a 2:1 peptide to α-synuclein ratio, cyclo-WW2 displays complete inhibition of ß-structure formation. Trp-bearing antiparallel ß-sheets held together by a disulphide bond are also potent inhibitors. 15N HSQC spectra of α-synuclein provided new mechanistic details. The time course of 15N HSQC spectral changes observed during ß-oligomer formation has revealed which segments of the structure become part of the rigid core of an oligomer at early stages of amyloidogenesis and that the C-terminus remains fully flexible throughout the process. All of the effective peptide inhibitors display binding-associated titration shifts in 15N HSQC spectra of α-synuclein in the C-terminal Q109-E137 segment. Cyclo-WW2, the most potent inhibitor, also displays titration shifts in the G41-T54 span of α-synuclein, an additional binding site. The earliest aggregation event appears to be centered about H50 which is also a binding site for our most potent inhibitor.

Climate change impact assessment on Veneto and Friuli Plain groundwater. Part I: an integrated modeling approach for hazard scenario construction.

Climate change impacts on water resources, particularly groundwater, is a highly debated topic worldwide, triggering international attention and interest from both researchers and policy makers due to its relevant link with European water policy directives (e.g. 2000/60/EC and 2007/118/EC) and related environmental objectives. The understanding of long-term impacts of climate variability and change is therefore a key challenge in order to address effective protection measures and to implement sustainable management of water resources. This paper presents the modeling approach adopted within the Life+ project TRUST (Tool for Regional-scale assessment of groUndwater Storage improvement in adaptation to climaTe change) in order to provide climate change hazard scenarios for the shallow groundwater of high Veneto and Friuli Plain, Northern Italy. Given the aim to evaluate potential impacts on water quantity and quality (e.g. groundwater level variation, decrease of water availability for irrigation, variations of nitrate infiltration processes), the modeling approach integrated an ensemble of climate, hydrologic and hydrogeologic models running from the global to the regional scale. Global and regional climate models and downscaling techniques were used to make climate simulations for the reference period 1961-1990 and the projection period 2010-2100. The simulation of the recent climate was performed using observed radiative forcings, whereas the projections have been done prescribing the radiative forcings according to the IPCC A1B emission scenario. The climate simulations and the downscaling, then, provided the precipitation, temperatures and evapo-transpiration fields used for the impact analysis. Based on downscaled climate projections, 3 reference scenarios for the period 2071-2100 (i.e. the driest, the wettest and the mild year) were selected and used to run a regional geomorphoclimatic and hydrogeological model. The final output of the model ensemble produced information about the potential variations of the water balance components (e.g. river discharge, groundwater level and volume) due to climate change. Such projections were used to develop potential hazard scenarios for the case study area, to be further applied within climate change risk assessment studies for groundwater resources and associated ecosystems. This paper describes the models' chain and the methodological approach adopted in the TRUST project and analyzes the hazard scenarios produced in order to investigate climate change risks for the case study area.

alpha-Glycerylphosphorylethanolamine rescues astrocytes from mitochondrial impairment and oxidative stress induced by amyloid beta-peptides.

The present work shows that alpha-glycerylphosphorylethanolamine (alpha-GPE) is effective in recovering astrocytes from mitochondrial membrane integrity and potential derangement and cellular oxidative stress that occur under amyloid beta-peptides-induced reactive gliosis.alpha-Glycerylphosphorylethanolamine (alpha-GPE), a new compound with nootropic properties, known to improve in vivo the learning and memory processes, has been tested for its protective properties on an in vitro model of degeneration. Rat primary astrocytic cultures treated with two amyloid-derived peptides, Abeta((1-40)) and Abeta(3(pE)-42), showed a marked reduction of the mitochondrial redox activity and membrane potential, together with an increase of oxidative species production. Plasma membrane lipid peroxidation (LPO) as well as generation of peroxides is greatly increased under Abeta-peptides toxicity. These features, typical of the reactive gliosis that accompanies neuronal degeneration, were readily recovered by pretreatment with alpha-GPE. alpha-GPE, likely improving the fluidity of cell membrane, has the potential to recover astrocytes from the general redox derangement induced by different amyloid fragments and possibly to protect from inflammation, gliosis and neurodegeneration. This is the first evidence of an antioxidant effect of the ethanolamine derivative on a rat model of chronic gliosis.

Acetaminophen protects hippocampal neurons and PC12 cultures from amyloid beta-peptides induced oxidative stress and reduces NF-kappaB activation.

The present findings show that an atypical non-steroidal anti-inflammatory drug, such as acetaminophen, retains the ability to recover amyloid beta-peptides driven neuronal apoptosis through the impairment of oxidative stress. Moreover, this compound reduces the increased NF-kappaB binding activity, which occurs in these degenerative conditions. Therapeutic interventions aimed at reducing the inflammatory response in Alzheimer's disease (AD) recently suggested the application of non-steroidal anti-inflammatory drugs. Although the anti-inflammatory properties of acetaminophen are controversial, it emerged that in an amyloid-driven astrocytoma cell degeneration model acetaminophen proved to be effective. On these bases, we analyzed the role of acetaminophen against the toxicity exerted by different Abeta-peptides on rat primary hippocampal neurons and on a rat pheochromocytoma cell line. We found a consistent protection from amyloid beta-fragments 1-40 and 1-42-induced impairment of mitochondrial redox activity on both cell cultures, associated with a marked reduction of apoptotic nuclear fragmentation. An antioxidant component of the protective activity emerged from the analysis of the reduction of phospholipid peroxidation, and also from a significant reduction of cytoplasmic accumulation of peroxides in the pheochromocytoma cell line. Moreover, activation of NF-kappaB by amyloid-derived peptides was greatly impaired by acetaminophen pre-treatment in hippocampal cells. This evidence points out antioxidant and anti-transcriptional properties of acetaminophen besides the known capability to interfere with inflammation within the central nervous system, and suggests that it can be exploited as a possible therapeutic approach against AD.

Inhibition of nuclear factor-kappaB activation induces apoptosis in cerebellar granule cells.

The nuclear factor (NF)-kappaB family of transcription factors plays important roles in the regulation of many activities of neuronal cells, such as synaptic transmission, inflammation, neuroprotection, and neurotoxicity. In resting cells, NF-kappaB activity is present both in the cytoplasm, as an inducible-inactive complex, and in the nucleus, as a constitutive form. Regulation of its inducible activity relies on processing of IkappaB(s), which occurs through the proteasome. Here we show that in cerebellar granule cells (CGC) the induction of apoptosis, by potassium withdrawal (5 mM KCl), decreases the amount of nuclear NF-kappaB. To understand whether NF-kappaB was required for CGC survival, these cells, maintained under depolarizing conditions (25 mM KCl and serum), were treated with proteasome inhibitors. The results show that these treatments reduce the nuclear amount of NF-kappaB and increase p65 cytoplasmic levels, a process partially regulated via IkappaBalpha degradation. These events are also associated with an impairment in CGC survival, with changes in nuclear morphology, induction of DNA laddering, and oligonucleosome formation, consistent with apoptosis. According to the K+ deprivation model, PSI-induced apoptosis is reversed by inhibitors of transcription and translation as well as by specific caspase inhibitors. Together our results show an important role for NF-kappaB in maintaining CGC survival. Indeed, under conditions of mild depolarization (K25) necessary for CGC survival, NF-kappaB is distributed between cytosol and nucleus, whereas, under apoptotic conditions (K5), it is depleted from the nucleus, such as after proteasome inhibitor treatment. Therefore, NF-kappaB nuclear deprivation is involved in the induction of CGC apoptosis.

C60 carboxyfullerene exerts a protective activity against oxidative stress-induced apoptosis in human peripheral blood mononuclear cells.

C60 carboxyfullerene is a novel buckminsterfullerene-derived compound that behaves as a free-radical scavenger. In the present report, we investigated whether this drug exerts a protective activity against oxidative stress-induced apoptosis. Human peripheral blood mononuclear cells (PBMCs) were challenged by 2-deoxy-d-ribose (dRib) or TNF-alpha plus cycloheximide as agents that trigger apoptosis by interfering with the redox status of cell and mitochondrial membrane potential. We found that carboxyfullerene was able to protect quiescent PBMCs from apoptosis caused either by 2-deoxy-d-ribose or TNF-alpha plus cycloheximide by a mechanism partially involving the mitochondrial membrane potential integrity, known to be associated with early stages of apoptosis. These results represent the first indication for a target activity of buckminsterfullerenes on cells of the immune system and their mitochondria.

C3-fullero-tris-methanodicarboxylic acid protects cerebellar granule cells from apoptosis.

Buckminsterfullerenols were recently investigated for their protective properties in different models of acute and chronic neurodegeneration. We tested C3-fullero-tris-methanodicarboxylic acid in our in vitro model of apoptotic neuronal death, which consists of shifting the culture K+ concentration from 25 to 5 mM for rat cerebellar granule cells. The impairment of mitochondrial respiratory function as well as chromatin derangement and fragmentation of DNA in apoptotic oligonucleosomes that occur in these conditions were protected by this compound in a concentration-dependent way. To assess whether antioxidant activity could account for the rescue of cerebellar granule cells from apoptosis, we tested the fullerene derivative under FeSO4-induced oxidative stress and found significant protection. Thus, we visualized membrane and cytoplasmic peroxides and reactive oxygen species and found a significant reduction of the species after 24 h in 5 mM K+ with the fullerene derivative. Such evidence suggests that this compound exerts a protective role in cerebellar granule cell apoptosis, likely reducing the oxidative stress.

C3-fullero-tris-methanodicarboxylic acid protects epithelial cells from radiation-induced anoikia by influencing cell adhesion ability.

Anoikia is a type of apoptotic cell death that occurs in cells that are substrate-restricted in their growth. Buckminsterfullerenes represent a new class of chemical compounds with wide potential pharmacological antioxidant activity. In this report we provide the first demonstration that a water-soluble fullerene derivative, C3-fullero-tris-methanodicarboxylic acid, synthesized in our laboratories, is capable of inducing anoikia resistance in epithelial cells by a mechanism involving a 'trophic' effect on cell spreading-associated cytoskeletal components, i.e. on actin microfilaments.

D-serine modulates the NMDA receptor/nitric oxide/cGMP pathway in the rat cerebellum during in vivo microdialysis.

Several lines of investigation indicate that D-serine may be an endogenous ligand for the glycine site of N-methyl-D-aspartate (NMDA) receptors in some CNS regions. We here studied the in vivo effects of D-serine on the NMDA receptor/nitric oxide/cGMP pathway by monitoring extracellular cGMP in the cerebellum of freely-moving rats subjected to transcerebral microdialysis. Local application of NMDA (200, 500 microM) through the dialysis probe for 20 min evoked transient, concentration-dependent cGMP responses which peaked in the fraction of drug administration, the nucleotide levels returning to basal values after 40 min. The NMDA-induced elevation of the extracellular nucleotide was completely inhibited by the selective receptor channel blocker dizocilpine (MK-801) locally co-perfused at the concentration of 10 microM. The non-competitive antagonist had no effect on its own suggesting that endogenous glutamic acid does not tonically activate NMDA receptors. The effect of 200 microM NMDA was largely attenuated by 30 microM 7-chloro-kynurenic acid and completely abrogated when the concentration of the strychnine-insensitive glycine receptor antagonist was raised to 100 microM. D-serine (300 microM), perfused in the presence of 7-chloro-kynurenate (30 microM), was able to fully restore the NMDA (200 microM)-induced increase of cGMP extracellular levels. On the other hand, the D-amino acid directly potentiated in a concentration-dependent manner (0.3, 1 and 10 mM) the NMDA (200 microM)-evoked cGMP production whereas it was inactive on its own. These data show that in vivo the activation of the strychnine-insensitive glycine site is essential for the functioning of the NMDA receptor complex and can be activated by the selective agonist D-serine. They also confirm that cerebellar NMDA receptors do not have their glycine sites saturated.