Homocysteine and A2A-D2 Receptor-Receptor Interaction at Striatal Astrocyte Processes.

The interaction between adenosine A2A and dopamine D2 receptors in striatal neurons is a well-established phenomenon and has opened up new perspectives on the molecular mechanisms involved in Parkinson's disease. However, it has barely been investigated in astrocytes. Here, we show by immunofluorescence that both A2A and D2 receptors are expressed in adult rat striatal astrocytes in situ, and investigate on presence, function, and interactions of the receptors in the astrocyte processes-acutely prepared from the adult rat striatum-and on the effects of homocysteine on the A2A-D2 receptor-receptor interaction. We found that A2A and D2 receptors were co-expressed on vesicular glutamate transporter-1-positive astrocyte processes, and confirmed that A2A-D2 receptor-receptor interaction controlled glutamate release-assessed by measuring the [3H]D-aspartate release-from the processes. The complexity of A2A-D2 receptor-receptor interaction is suggested by the effect of intracellular homocysteine, which reduced D2-mediated inhibition of glutamate release (homocysteine allosteric action on D2), without interfering with the A2A-mediated antagonism of the D2 effect (maintained A2A-D2 interaction). Our findings indicate the crucial integrative role of A2A-D2 molecular circuits at the plasma membrane of striatal astrocyte processes. The fact that homocysteine reduced D2-mediated inhibition of glutamate release could provide new insights into striatal astrocyte-neuron intercellular communications. As striatal astrocytes are recognized to be involved in Parkinson's pathophysiology, these findings may shed light on the pathogenic mechanisms of the disease and contribute to the development of new drugs for its treatment.

A2A-D2 receptor-receptor interaction modulates gliotransmitter release from striatal astrocyte processes.

Evidence for striatal A2A-D2 heterodimers has led to a new perspective on molecular mechanisms involved in schizophrenia and Parkinson's disease. Despite the increasing recognition of astrocytes' participation in neuropsychiatric disease vulnerability, involvement of striatal astrocytes in A2A and D2 receptor signal transmission has never been explored. Here, we investigated the presence of D2 and A2A receptors in isolated astrocyte processes prepared from adult rat striatum by confocal imaging; the effects of receptor activation were measured on the 4-aminopyridine-evoked release of glutamate from the processes. Confocal analysis showed that A2A and D2 receptors were co-expressed on the same astrocyte processes. Evidence for A2A-D2 receptor-receptor interactions was obtained by measuring the release of the gliotransmitter glutamate: D2 receptors inhibited the glutamate release, while activation of A2A receptors, per se ineffective, abolished the effect of D2 receptor activation. The synthetic D2 peptide VLRRRRKRVN corresponding to the receptor region involved in electrostatic interaction underlying A2A-D2 heteromerization abolished the ability of the A2A receptor to antagonize the D2 receptor-mediated effect. Together, the findings are consistent with heteromerization of native striatal astrocytic A2A-D2 receptors that via allosteric receptor-receptor interactions could play a role in the control of striatal glutamatergic transmission. These new findings suggest possible new pathogenic mechanisms and/or therapeutic approaches to neuropsychiatric disorders.

On the role of the extracellular space on the holistic behavior of the brain.

Multiple players are involved in the brain integrative action besides the classical neuronal and astrocyte networks. In the past, the concept of complex cellular networks has been introduced to indicate that all the cell types in the brain can play roles in its integrative action. Intercellular communication in the complex cellular networks depends not only on well-delimited communication channels (wiring transmission) but also on diffusion of signals in physically poorly delimited extracellular space pathways (volume transmission). Thus, the extracellular space and the extracellular matrix are the main players in the intercellular communication modes in the brain. Hence, the extracellular matrix is an 'intelligent glue' that fills the brain and, together with the extracellular space, contributes to the building-up of the complex cellular networks. In addition, the extracellular matrix is part of what has been defined as the global molecular network enmeshing the entire central nervous system, and plays important roles in synaptic contact homeostasis and plasticity. From these premises, a concept is introduced that the global molecular network, by enmeshing the central nervous system, contributes to the brain holistic behavior. Furthermore, it is suggested that plastic 'brain compartments' can be detected in the central nervous system based on the astrocyte three-dimensional tiling of the brain volume and on the existence of local differences in cell types and extracellular space fluid and extracellular matrix composition. The relevance of the present view for neuropsychiatry is discussed. A glossary box with terms and definitions is provided.

Acute isoproterenol induces anxiety-like behavior in rats and increases plasma content of extracellular vesicles.

Several clinical observations have demonstrated a link between heart rate and anxiety or panic disorders. In these patients, ß-adrenergic receptor function was altered. This prompted us to investigate whether the ß-adrenergic receptor agonist isoproterenol, at a dose that stimulates peripheral ß-adrenergic system but has no effects at the central nervous system, can induce anxiety-like behavior in rats. Moreover, some possible messengers involved in the peripheral to brain communication were investigated. Our results showed that isoproterenol (5 mg kg(-1) i.p.) increased heart rate, evoked anxiety-like behavior, did not result in motor impairments and increased extracellular vesicle content in the blood. Plasma corticosterone level was unmodified as well as vesicular Hsp70 content. Vesicular miR-208 was also unmodified indicating a source of increased extracellular vesicles different from cardiomyocytes. We can hypothesize that peripheral extracellular vesicles might contribute to the ß-adrenergic receptor-evoked anxiety-like behavior, acting as peripheral signals in modulating the mental state.

In vitro effects of cocaine on tunneling nanotube formation and extracellular vesicle release in glioblastoma cell cultures.

The effects of cocaine (150 nM, 300 nM, and 150 µM) on human glioblastoma cell cultures were studied on tunneling nanotube formation (1-h cocaine treatment) and extracellular vesicle release (1-, 3-, and 8-h cocaine treatment). Cocaine significantly increased the number of tunneling nanotubes only at the lowest concentration used. The release of extracellular vesicles (mainly exosomes) into the medium was stimulated by cocaine at each concentration used with a maximum effect at the highest concentration tested (150 µM). Moreover, cocaine (150 nM) significantly increased the number of vesicles with 61-80 nm diameter while at concentrations of 300 nM and 150 µM, and the smaller vesicles (30-40 nm diameter) were significantly increased with a reduction of the larger vesicles (41-60 nm diameter). A time dependence in the release of extracellular vesicles was observed. In view of the proposed role of these novel intercellular communication modes in the glial-neuronal plasticity, it seems possible that they can participate in the processes leading to cocaine addiction. The molecular target/s involved in these cocaine effects could be specific molecular components of plasma membrane lipid rafts and/or cocaine-induced modifications in cytoplasmic lipid composition.

"Neuro-semeiotics" and "free-energy minimization" suggest a unified perspective for integrative brain actions: focus on receptor heteromers and Roamer type of volume transmission.

Two far-reaching theoretical approaches, namely "Neuro-semeiotics" (NS) and "Free-energy Minimization" (FEM), have been recently proposed as frames within which to put forward heuristic hypotheses on integrative brain actions. In the present paper these two theoretical approaches are briefly discussed in the perspective of a recent model of brain architecture and information handling based on what we suggest calling Jacob's tinkering principle, whereby "to create is to recombine!". The NS and FEM theoretical approaches will be discussed from the perspective both of the Roamer-Type Volume Transmission (especially exosome-mediated) of intercellular communication and of the impact of receptor oligomers and Receptor-Receptor Interactions (RRIs) on signal recognition/decoding processes. In particular, the Bio-semeiotics concept of "adaptor" will be used to analyze RRIs as an important feature of NS. Furthermore, the concept of phenotypic plasticity of cells will be introduced in view of the demonstration of the possible transfer of receptors (i.e., adaptors) into a computational network via exosomes (see also Appendix). Thus, Jacob's tinkering principle will be proposed as a theoretical basis for some learning processes both at the network level (Turing-like type of machine) and at the molecular level as a consequence of both the plastic changes in the adaptors caused by the allosteric interactions in the receptor oligomers and the intercellular transfer of receptors. Finally, on the basis of NS and FEM theories, a unified perspective for integrative brain actions will be proposed.

Neural damage biomarkers during open carotid surgery versus endovascular approach.

BACKGROUND: Carotid endarterectomy (CEA) is the gold standard for treating severe carotid artery stenosis, whereas carotid artery stenting (CAS) represents an endovascular alternative. The objective of this study was to assess the potential neural damage following open or endovascular carotid surgery measured by peripheral blood concentration of 3 biomarkers: S100ß, matrix metalloproteinase-9 (MMP-9), and d-dimer. METHODS: Data for this prospective investigation were obtained from the Carotid Markers study (January 2010-2011), which sought to measure the levels of specific biomarkers of neuronal damage and thrombosis on candidates to CEA or CAS presenting at the Department of Vascular Surgery of the Nuovo Ospedale S. Agostino Estense of Modena (Italy) at baseline and at 24 hr after surgery. Relevant medical comorbidities were noted. RESULTS: A total of 113 consecutive patients were enrolled in the study, 41 in the endarterectomy group and 72 in the endovascular group. The baseline levels of the studied biomarkers did not show any statistically significant difference between the groups with the exception of MMP-9, which showed higher concentrations in the endovascular group (median 731 vs. 401, P = 0.0007), while 24 hr after surgery the endarterectomy group featured significantly higher peripheral blood concentrations of MMP-9, S100ß, and d-dimer. Conversely, no significant difference was detected in the endovascular group except the d-dimer level. CONCLUSIONS: Neural damage biomarkers demonstrated a substantial difference between open and endovascular carotid surgery, which, if performed in selected patients, may become a less invasive alternative to CEA.

Information handling by the brain: proposal of a new "paradigm" involving the roamer type of volume transmission and the tunneling nanotube type of wiring transmission.

The current view on the organization of the central nervous system (CNS) is basically anchored to the paradigm describing the brain as formed by networks of neurons interconnected by synapses. Synaptic contacts are a fundamental characteristic for describing CNS operations, but increasing evidence accumulated in the last 30 years pointed to a refinement of this view. A possible overcoming of the classical "neuroscience paradigm" will be here outlined, based on the following hypotheses: (1) the basic morpho-functional unit in the brain is a compartment of tissue (functional module) where different resident cells (not only neurons) work as an integrated unit; (2) in these complex networks, a spectrum of intercellular communication processes is exploited, that can be classified according to a dichotomous criterion: wiring transmission (occurring through physically delimited channels) and volume transmission (exploiting diffusion in the extracellular space); (3) the connections between cells can themselves be described as a network, leading to an information processing occurring at different levels from cell network down to molecular level; (4) recent evidence of the existence of specialized structures (microvesicles and tunneling nanotubes) for intercellular exchange of materials, could allow a further type of polymorphism of the CNS networks based on at least transient changes in cell phenotype. When compared to the classical paradigm, the proposed scheme of cellular organization could allow a strong increase of the degrees of freedom available to the whole system and then of its plasticity. Furthermore, long range coordination and correlation can be more easily accommodated within this framework.

A new interpretative paradigm for Conformational Protein Diseases.

Conformational Protein Diseases (CPDs) comprise over forty clinically and pathologically diverse disorders in which specific altered proteins accumulate in cells or tissues of the body. The most studied are Alzheimerß's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, prion diseases, inclusion body myopathy, and the systemic amyloidoses. They are characterised by three dimensional conformational alterations, which are often rich in ß- structure. Proteins in this non-native conformation are highly stable, resistant to degradation, and have an enhanced tendency to aggregate with like protein molecules. The misfolded proteins can impart their anomalous properties to soluble, monomeric proteins with the same amino acid sequence by a process that has been likened to seeded crystallization. However, these potentially pathogenic proteins also have important physiological actions, which have not completely characterized. This opens up the question of what process transforms physiological actions into pathological actions and most intriguing, is why potentially dangerous proteins have been maintained during evolution and are present from yeasts to humans. In the present paper, we introduce the concept of mis-exaptation and of mis-tinkering since they may help in clarifying some of the double edged sword aspects of these proteins. Against this background an original interpretative paradigm for CPDs will be given in the frame of the previously proposed Red Queen Theory of Aging.

Neuronal correlates to consciousness. The "Hall of Mirrors" metaphor describing consciousness as an epiphenomenon of multiple dynamic mosaics of cortical functional modules.

Humans share the common intuition of a self that has access to an inner 'theater of mind' (Baars, 2003). The problem is how this internal theater is formed. Moving from Cook's view (Cook, 2008), we propose that the 'sentience' present in single excitable cells is integrated into units of neurons and glial cells transiently assembled into "functional modules" (FMs) organized as systems of encased networks (from cell networks to molecular networks). In line with Hebb's proposal of 'cell assemblies', FMs can be linked to form higher-order mosaics by means of reverberating circuits. Brain-level subjective awareness results from the binding phenomenon that coordinates several FM mosaics. Thus, consciousness may be thought as the global result of integrative processes taking place at different levels of miniaturization in plastic mosaics. On the basis of these neurobiological data and speculations and of the evidence of 'mirror neurons' the 'Hall of Mirrors' is proposed as a significant metaphor of consciousness. This article is part of a Special Issue entitled: Brain Integration.

Possible new targets for GPCR modulation: allosteric interactions, plasma membrane domains, intercellular transfer and epigenetic mechanisms.

It has been estimated that at least 50% of the drugs available on the market act on G-protein coupled receptors (GPCRs) and most of these are basically or agonists or antagonists of this type of receptors. Herein, we propose new putative targets for drug development based on recent data on GPCR allosterism and on the existence of receptor mosaics (RMs). The main target for drug development is still GPCRs, but the focus is not the orthosteric binding pocket. According to the mosaic model of the plasma membrane, we mainly discuss the possibility of indirect modulatory pharmacological actions on expression/function of GPCRs. In particular, the following two new targets will be analyzed: a) The possibility of pharmacological interventions on the roamer-type of volume transmission (VT), which allow the intercellular transfer of set of signal molecules such as GPCRs, tetraspanins and ribonucleic acids. Thus, there is the possibility of pharmacological interventions on the decoding capabilities of neurons and/or glial cells by means of an action on composition and release of micro-vesicles. b) The possibility of pharmacological interventions on epigenetic mechanisms by taking into account their inter-relationships with GPCRs. As a matter of fact, there are epigenetic changes that are characteristic of periods of developmental plasticity that could provide a target for therapeutic intervention in the event of brain damage. We believe that almost all the biochemical knowledge presently available on GPCRs can be used in the development of these new pharmacological approaches.

Bioinformatics and mathematical modelling in the study of receptor-receptor interactions and receptor oligomerization: focus on adenosine receptors.

The concept of intra-membrane receptor-receptor interactions (RRIs) between different types of G protein-coupled receptors (GPCRs) and evidence for their existence was introduced by Agnati and Fuxe in 1980/81 through the biochemical analysis of the effects of neuropeptides on the binding characteristics of monoamine receptors in membrane preparations from discrete brain regions and functional studies of the interactions between neuropeptides and monoamines in the control of specific functions such as motor control and arterial blood pressure control in animal models. Whether GPCRs can form high-order structures is still a topic of an intense debate. Increasing evidence, however, suggests that the hypothesis of the existence of high-order receptor oligomers is correct. A fundamental consequence of the view describing GPCRs as interacting structures, with the likely formation at the plasma membrane of receptor aggregates of multiple receptors (Receptor Mosaics) is that it is no longer possible to describe signal transduction simply as the result of the binding of the chemical signal to its receptor, but rather as the result of a filtering/integration of chemical signals by the Receptor Mosaics (RMs) and membrane-associated proteins. Thus, in parallel with experimental research, significant efforts were spent in bioinformatics and mathematical modelling. We review here the main approaches that have been used to assess the interaction interfaces allowing the assembly of GPCRs and to shed some light on the integrative functions emerging from the complex behaviour of these RMs. Particular attention was paid to the RMs generated by adenosine A(2A), dopamine D(2), cannabinoid CB(1), and metabotropic glutamate mGlu(5) receptors (A(2A), D(2), CB(1) and mGlu(5), respectively), and a possible approach to model the interplay between the D(2)-A(2A)-CB(1) and D(2)-A(2A)-mGlu(5) trimers is proposed.

An integrated view on the role of receptor mosaics at perisynaptic level: focus on adenosine A(2A), dopamine D(2), cannabinoid CB(1), and metabotropic glutamate mGlu(5) receptors.

The available evidence for receptor-receptor interactions between adenosine A(2A), dopamine D(2), cannabinoid CB(1), and metabotropic glutamate mGlu(5) receptors (A(2A), D(2), CB(1), and mGlu(5), respectively) is revised under the "receptor mosaic" perspective. Furthermore, the concept of "hub receptor" is defined in accordance with informatics and it is tentatively illustrated in the case of the hypothesized tetramer formed by the above mentioned receptors. On the basis of some biochemical features of the four receptors and of a bioinformatics analysis, an objective deduction of their "similarity" has been obtained. To this aim the Canberra, Euclidean and Chebyshev multivariate distance metrics have been used. It is interesting to note that A(2A) and D(2) are the most different ones, while CB(1) and mGlu(5) are the most similar ones among the four receptors analyzed. Finally, by means of a bioinformatics analysis based on different approaches the possible binding sites mediating G-protein-coupled receptor (GPCR) interactions have been indicated. It is interesting to note that in some instances accordance has been found between the bioinformatics indications and the available experimental data.

Understanding wiring and volume transmission.

The proposal on the existence of two main modes of intercellular communication in the central nervous system (CNS) was introduced in 1986 and called wiring transmission (WT) and volume transmission (VT). The major criterion for this classification was the different characteristics of the communication channel with physical boundaries well delimited in the case of WT (axons and their synapses; gap junctions) but not in the case of VT (the extracellular fluid filled tortuous channels of the extracellular space and the cerebrospinal fluid filled ventricular space and sub-arachnoidal space). The basic dichotomic classification of intercellular communication in the brain is still considered valid, but recent evidence on the existence of unsuspected specialized structures for intercellular communication, such as microvesicles (exosomes and shedding vesicles) and tunnelling nanotubes, calls for a refinement of the original classification model. The proposed updating is based on criteria which are deduced not only from these new findings but also from concepts offered by informatics to classify the communication networks in the CNS. These criteria allowed the identification also of new sub-classes of WT and VT, namely the "tunnelling nanotube type of WT" and the "Roamer type of VT." In this novel type of VT microvesicles are safe vesicular carriers for targeted intercellular communication of proteins, mtDNA and RNA in the CNS flowing in the extracellular fluid along energy gradients to reach target cells. In the tunnelling nanotubes proteins, mtDNA and RNA can migrate as well as entire organelles such as mitochondria. Although the existence and the role of these new types of intercellular communication in the CNS are still a matter of investigation and remain to be fully demonstrated, the potential importance of these novel types of WT and VT for brain function in health and disease is discussed.

Differential sensitivity of A2A and especially D2 receptor trafficking to cocaine compared with lipid rafts in cotransfected CHO cell lines. Novel actions of cocaine independent of the DA transporter.

The effects of low and high concentrations of cocaine have been studied in vitro on the trafficking of plasma membrane A(2A) and D(2) immunoreactivities in previously characterized A(2A)-D(2) CHO cell lines. Receptor double immunofluorescence staining was performed with D(2) and A(2A) antibodies, planar lipid rafts immunolabeling with biotinylated cholera toxin subunit B and membrane invaginations with an anti-caveolin-1 antibody. A computer-assisted image analysis demonstrated a substantial and highly significant rise of membrane-associated D(2) immunoreactivity (IR) after 8 h of exposure to a low concentration of cocaine (150 nM). At this low concentration of cocaine, there was also an increase of membrane associated A(2A) immunoreactivity but smaller and less significant. However, this increase became considerably larger and highly significant at 150 microM at which concentration the rise of D(2) immunoreactivity had begun to disappear. It may be suggested that an allosteric action of cocaine at 150 nM on the D(2) receptors may primarily increase the insertion of D(2) monomers, homomers and also of a subpopulation of A(2A)-D(2) heteromers from the cytoplasm into the plasma membrane due to the conformational change induced by cocaine in the D(2) receptor. The planar lipid rafts and the caveolae are only affected by the higher concentrations of cocaine. It is proposed that changes in D(2) and A(2A)-D(2) trafficking induced by allosteric actions of cocaine at D(2) receptors may contribute to the alterations of D(2) signaling found in cocaine abusers.

Astrocytes and Glioblastoma cells release exosomes carrying mtDNA.

Cells can exchange information not only by means of chemical and/or electrical signals, but also via microvesicles released into the intercellular space. The present paper, for the first time, provides evidence that Glioblastoma and Astrocyte cells release microvesicles, which carry mitochondrial DNA (mtDNA). These microvesicles have been characterised as exosomes in view of the presence of some protein markers of exosomes, such as Tsg101, CD9 and Alix. Thus, the important finding has been obtained that bonafide exosomes, constitutively released by Glioblastoma cells and Astrocytes, can carry mtDNA, which can be, therefore, transferred between cells. This datum may help the understanding of some diseases due to mitochondrial alterations.

Receptor-receptor interactions: A novel concept in brain integration.

A brief historical presentation of the hypothesis on receptor-receptor interactions as an important integrative mechanism taking place at plasma membrane level is given. Some concepts derived from this integrative mechanism especially the possible assemblage of receptors in receptor mosaics (high-order receptor oligomers) and their relevance for the molecular networks associated with the plasma membrane are discussed. In particular, the Rodbell's disaggregation theory for G-proteins is revisited in the frame of receptor mosaic model. The paper also presents some new indirect evidence on A2A;D2 receptor interactions obtained by means of Atomic Force Microscopy on immunogold preparations of A2A and D2 receptors in CHO cells. These findings support previous data obtained by means of computer-assisted confocal laser microscopy. The allosteric control of G-protein coupled receptors is examined in the light of the new views on allosterism and recent data on a homocysteine analogue capable of modulating D2 receptors are shown. Finally, the hypothesis is introduced on the existence of check-points along the amino acid pathways connecting allosteric and orthosteric binding sites of a receptor and their potential importance for drug development.

Influence of f-MLP, ACTH(1-24) and CRH on in vitro chemotaxis of monocytes from centenarians.

OBJECTIVE: The lifelong exposure to a variety of stressors activates a plethora of defense mechanisms, including the hypothalamic-pituitary-adrenal axis which releases neuropeptides affecting the immune responses. Here, we report data on the capability of monocytes from young subjects and centenarians to migrate towards chemotactic stimuli (formyl-methionyl-leucyl-phenylalanine, f-MLP; adrenocorticotropic hormone, ACTH, and corticotrophin-releasing hormone, CRH). Plasma levels of ACTH, CRH and cortisol were measured as an index of ongoing stress response. METHODS: Monocyte chemotaxis towards f-MLP (10(-8)M), ACTH(1-24) (10(-14) and 10(-8)M) and CRH (10(-14) and 10(-8)M) was evaluated in vitro in young subjects (n = 8, age range 25-35 years) and centenarians (n = 9, age >100 years) and expressed as chemotactic index. In 9 young subjects and 6 centenarians, plasma levels of cortisol, ACTH and CRH were measured. RESULTS: Monocyte chemotaxis towards f-MLP, ACTH(1-24) and CRH (10(-8)M) was well preserved in centenarians, except when the lowest concentration of CRH was used. CRH, ACTH and cortisol plasma levels were significantly higher in centenarians than in young subjects. CONCLUSIONS: The capability of monocytes from centenarians to respond to chemotactic neuropeptides is well preserved. The decreased responsiveness to the lowest concentration of CRH might be due to downregulation of CRH receptors or to defects in the intracellular signal transduction pathway. The high plasma levels of cortisol, CRH and ACTH in centenarians indicate an activation of the entire stress axis, likely counteracting the systemic inflammatory process occurring with age. This activation fits with the hypothesis that lifelong low-intensity stressors activate ancient, hormetic defense mechanisms, favoring healthy aging and longevity.

Understanding neuronal molecular networks builds on neuronal cellular network architecture.

The central nervous system (CNS) is a nested network at all levels of its organization. In particular, neuronal cellular networks (the neuronal circuits), interconnected to form neuronal systems, are formed by neurons, which operate thanks to their molecular networks. Proteins are the main components of the molecular networks and via protein-protein interactions can be assembled in multimeric complexes, which can work as micro-devices. On this basis, we have introduced the term "fractal logic" to describe networks of networks where at the various levels of the nested organization the same rules (logic) to perform operations are used. If this assumption is true, the description of the information handling at one of the nested levels sheds light on the way in which similar operations are carried out at other levels. This conceptual frame has been used to deduce from some features of neuronal networks the features of the molecular networks as far as modes for inter-node communication and their architecture. It should be noted that these features are such to allow a highly regulated cross-talk between signalling pathways, hence preserving selectivity and privacy. To investigate these aspects, the protein-protein interactions in beta2 Adrenergic Receptor (beta2AR) and Epidermal growth factor receptor (EGFR) signalling pathways have been analysed. The presence of disordered sequences in interacting domains can favour via the "fly-casting mechanism" protein-protein interactions, in addition it favours an induced-fitting rather than a lock-key type of interactions. Thus, by means of a computer assisted analysis the presence of disorder sequences in the main streams of the molecular networks that have beta2AR and EGFR as input proteins leading to MAP kinase activation has been evaluated.

Hyper-homocysteinemia alters amyloid peptide-clusterin interactions and neuroglial network morphology and function in the caudate after intrastriatal injection of amyloid peptides.

Amyloid peptides (Abeta) are fragments of the Amyloid Precursor Protein (APP), an integral membrane protein. Abeta peptides are continuously generated by neurons and non-neuronal cells via sequential cleavage of APP by secretases. In particular, Abeta1-42 is the main component of the senile plaques associated with Alzheimer's disease (AD). Glial cells participate in the uptake of soluble extra-cellular Abeta and in the clearance of this material at localized sites where the Abeta are concentrated. It has been shown that clusterin (Apo J) and apolipoprotein E (ApoE) exert important additive effects in reducing Abeta deposition. In agreement with the fact that homocysteine (Hcy) potentiates Abeta peptide neurotoxicity, and Hcy brain levels increase with age, it has been demonstrated that high plasma levels of Hcy are a risk factor for AD. In the present paper, we used animals subjected to chronic intake of methionine (1 g/kg/day) in the drinking water, since this treatment can increase plasma Hcy levels by 30%. By means of this animal model, interactions between the Abeta beta-sheet rich fibrils and clusterin, have been evaluated in striata of animals after Abeta injection. Furthermore, it has been demonstrated that Abeta peptides are not only signals capable of activating astrocytes but also capable of reducing tyrosine-hydroxylase immunoreactivity in the basal ganglia probably leading to a reduction of volume transmission. These alterations in the neuroglial network morphology and function can, at least in part, explain the enhanced pain threshold observed in the Abeta intra-striatally injected animals.