Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures.

Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A(2A) and D(2) receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D(2)R-CFP or A(2A)R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D(2)R-CFP and A(2A)R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A(2A)R positive MVs were treated with the adenosine A(2A) receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A(2A)Rs were functionally competent in target cells. These findings demonstrate that A(2A) receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

Aspects on the integrative actions of the brain from neural networks to "brain-body medicine".

"Integration" is a key term in describing how nervous system can perform high level functions. A first condition to have "integration" is obviously the presence of efficient "communication processes" among the parts that have to be combined into the harmonious whole. In this respect, two types of communication processes, called wiring transmission (WT) and volume transmission (VT), respectively, were found to play a major role in the nervous system, allowing the exchange of signals not only between neurons, but rather among all cell types present in the central nervous system (CNS). A second fundamental aspect of a communication process is obviously the recognition/decoding process at target level. As far as this point is concerned, increasing evidence emphasizes the importance of supramolecular complexes of receptors (the so called receptor mosaics) generated by direct receptor-receptor interactions. Their assemblage would allow a first integration of the incoming information already at the plasma membrane level. Recently, evidence of two new subtypes of WT and VT has been obtained, namely the tunnelling nanotubes mediated WT and the microvesicle (in particular exosomes) mediated VT allowing the horizontal transfer of bioactive molecules, including receptors, RNAs and micro-RNAs. The physiological and pathological implications of these types of communication have opened up a new field that is largely still unexplored. In fact, likely unsuspected integrative actions of the nervous system could occur. In this context, a holistic approach to the brain-body complex as an indissoluble system has been proposed. Thus, the hypothesis has been introduced on the existence of a brain-body integrative structure formed by the "area postrema/nucleus tractus solitarius" (AP/NTS) and the "anteroventral third ventricle region/basal hypothalamus with the median eminence" (AV3V-BH). These highly interconnected regions operate as specialized interfaces between the brain and the body integrating brain-borne and body-borne neural and humoral signals.

Common key-signals in learning and neurodegeneration: focus on excito-amino acids, beta-amyloid peptides and alpha-synuclein.

In this paper a hypothesis that some special signals ("key-signals" excito-amino acids, beta-amyloid peptides and alpha-synuclein) are not only involved in information handling by the neuronal circuits, but also trigger out substantial structural and/or functional changes in the Central Nervous System (CNS) is introduced. This forces the neuronal circuits to move from one stable state towards a new state, but in doing so these signals became potentially dangerous. Several mechanisms are put in action to protect neurons and glial cells from these potentially harmful signals. However, in agreement with the Red Queen Theory of Ageing (Agnati et al. in Acta Physiol Scand 145:301-309, 1992), it is proposed that during ageing these neuroprotective processes become less effective while, in the meantime, a shortage of brain plasticity occurs together with an increased need of plasticity for repairing the wear and tear of the CNS. The paper presents findings supporting the concept that such key-signals in instances such as ageing may favour neurodegenerative processes in an attempt of maximizing neuronal plasticity.

Structural plasticity in G-protein coupled receptors as demonstrated by the allosteric actions of homocysteine and computer-assisted analysis of disordered domains.

Structural plasticity of G-protein coupled receptors (GPCRs) is of basic importance for their interactions with ligands, in particular with other proteins such as receptors or receptor-modifying proteins that can lead to different functions for the same GPCR. In the present paper, structural plasticity of GPCRs has been investigated discussing allosteric modulatory actions of Homocysteine (Hcy) on D2 receptors together with data obtained by computer-assisted analysis of the presence of disordered domains in GPCRs. Previous evidence for a modulatory action of Hcy on D2 receptors has been further extended by means of experiments on the effects of Hcy local intrastriatal injection on rotational behaviour. Altogether the present data allow considering under a new angle the well known proposal of A2A antagonists as new therapeutic agents in Parkinson's disease (PD). Furthermore, they point out to not only the importance of drugs capable of reducing Hcy brain levels, but also to the potential therapeutic impact of drugs capable of regionally blocking (for PD) or enhancing (for some schizophrenic syndromes) Hcy allosteric action on D2 receptors. As far as the investigations on GPCR plastic domains, extracellular, intracellular and transmembrane domains of 14 GPCRs have been considered and propensity of each of these domains for a structured or unstructured conformation has been evaluated by means of ad hoc computer programs. It has been shown that the N- and C-terminals as well as intracellular loop 3 have a high propensity towards an unstructured conformation, hence they are potentially very plastic domains, which can undergo easily to interactions with other ligands, particularly with other protein domains. This aspect is obviously of the greatest importance not only for the function of single GPCRs, but also for their interactions either with other receptors (receptor-receptor interactions) or, more generally, for formation of clusters of membrane associated proteins, hence of "protein mosaics", where the GPCRs could represent the input unit of the supra-molecular device.

Receptor-receptor interactions within receptor mosaics. Impact on neuropsychopharmacology.

Future therapies for diseases associated with altered dopaminergic signaling, including Parkinson's disease, schizophrenia and drug addiction or drug dependence may substantially build on the existence of intramembrane receptor-receptor interactions within dopamine receptor containing receptor mosaics (RM; dimeric or high-order receptor oligomers) where it is believed that the dopamine D(2) receptor may operate as the 'hub receptor' within these complexes. The constitutive adenosine A(2A)/dopamine D(2) RM, located in the dorsal striato-pallidal GABA neurons, are of particular interest in view of the demonstrated antagonistic A(2A)/D(2) interaction within these heteromers; an interaction that led to the suggestion and later demonstration that A(2A) antagonists could be used as novel anti-Parkinsonian drugs. Based on the likely existence of A(2A)/D(2)/mGluR5 RM located both extrasynaptically on striato-pallidal GABA neurons and on cortico-striatal glutamate terminals, multiple receptor-receptor interactions within this RM involving synergism between A(2A)/mGluR5 to counteract D(2) signaling, has led to the proposal of using combined mGluR5 and A(2A) antagonists as a future anti-Parkinsonian treatment. Based on the same RM in the ventral striato-pallidal GABA pathways, novel strategies for the treatment of schizophrenia, building on the idea that A(2A) agonists and/or mGluR5 agonists will help reduce the increased dopaminergic signaling associated with this disease, have been suggested. Such treatment may ensure the proper glutamatergic drive from the mediodorsal thalamic nucleus to the prefrontal cortex, one which is believed to be reduced in schizophrenia due to a dominance of D(2)-like signaling in the ventral striatum. Recently, A(2A) receptors also have been shown to counteract the locomotor and sensitizing actions of cocaine and increases in A(2A) receptors have also been observed in the nucleus accumbens after extended cocaine self-administration, probably representing a compensatory up-regulation to counteract the cocaine-induced increases in dopamine D(2) and D(3) signaling. Therefore, A(2A) agonists, through antagonizing D(2) and D(3) signaling within A(2A)/D(2) and A(2A)/D(3) RM heteromers in the nucleus accumbens, may be found useful as a treatment for cocaine dependence. Furthermore, antagonistic cannabinoid CB(1)/D(2) interactions requiring A(2A) receptors have also been discovered and possibly operate in CB(1)/D(2)/A(2A) RM located principally on striatal glutamate terminals but also on some ventral striato-pallidal GABA neurons, thereby opening up a new mechanism for the integration of endocannabinoid, DA and adenosine mediated signals. Thus, A(2A), mGluR5 and/or CB(1) receptors can form integrative units with D(2) receptors within RM displaying different compositions, topography and localization. Also galaninR/5-HT(1A) RM probably participates in the transmission of the ascending 5-hydroxytryptamine neurons, where galanin receptors antagonize 5-HT(1A) recognition and signaling. Subtype specific galanin receptor antagonists may therefore represent novel antidepressant drugs. These results suggest the importance of a complete understanding of the function of these RM with regard to disease. Ultimately receptor-receptor interactions within RM that modify dopaminergic and serotonergic signaling may give new strategies for treatment of a wide range of diseases associated with altered dopaminergic and serotonergic signaling.

Role of cooperativity in protein folding and protein mosaic assemblage relevance for protein conformational diseases.

Biological systems are organized in intricate and highly structured networks with hierarchies and multiple scales. Cells can be considered as "meso-scale level" systems placed between the "macro-scale level" (systems of cellular networks) and the "micro-scale level" (systems of molecular networks). In fact, cells represent complex biochemical machineries made by networks of molecules connected by biochemical reactions. Thus, the brain should be studied as a system of "networks of networks". Recently, the existence of a Global Molecular Network (GMN) enmeshing the entire CNS was proposed. This proposal is based on the evidence that the extra-cellular matrix is a dynamic molecular structure capable of storing and releasing signals and of interacting with receptors and proteins on the cell membranes. Proteins have a special role in molecular networks since they can be assembled into high-order molecular complexes, which have been defined as Protein Mosaics (PM). Protein monomers in a PM (the "tesserae" of the mosaic) can interact via classical and non-classical cooperativity behaviour involving allosteric interactions. In the present paper, new features of allostery and cooperativity for protein folding, assemblage and topological features of PM will be discussed. Against this background, alterations in PM via allosteric modulations and non-classical cooperativity mechanisms may lead to protein aggregates like beta amyloid fibrils. Such aggregates cause pathological changes in the GMN structure and function leading to neurodegenerative diseases such as Alzheimer's disease. Thus, a novel view of the so called Protein Conformational Diseases (PCD) is proposed.

On the existence of a global molecular network enmeshing the whole central nervous system: physiological and pathological implications.

Proteins are endowed with the "Lego property", i.e., the capability of steric fitting with other proteins to form high molecular weight complexes with emergent functions. These interactions may occur both as horizontal molecular networks at the plasma membrane level and as vertical molecular networks, i.e., towards the extra- and/or intracellular side of the cell. The present paper broadens this view by proposing the existence of three dimensional molecular networks, mainly made by proteins and carbohydrates, which might interact with each other at boundaries of compartments such as plasma membranes to form a "global molecular network" (GMN) that pervades the intra- as well as the extra-cellular environment of the entire central nervous system. The GMN is a potentially plastic structure regulated through several means. For example, its extra-cellular part is under the remodeling action of the matrix metalloproteinases. The proposal of a GMN has physiological and pathological implications. In primis, classical synaptic transmission, gap junctions and volume transmission signals by modulating GMN could importantly contribute to the "binding phenomenon", i.e. the phase synchronization of firing rates in far-located neuronal cortical groups. Secondly, alterations in protein conformation could alter the GMN organization and hence the neuronal network morphology and function. This could lead to the formation of abnormal protein aggregates such as amyloid plaques and neurofibrillary tangles, which, in turn, might affect the GMN function and/or the reciprocal interactions between its parts especially at the boundaries between compartments.

The nigrostriatal DA pathway and Parkinson's disease.

The discovery of the nigrostriatal DA system in the rat was made possible by the highly specific and sensitive histochemical fluorescence method of Falck and Hillarp in combinations with electrolytic lesions in the substantia nigra and removal of major parts of the neostriatum. Recent work on DA neuron evolution shows that in the Bottlenose Dolphin the normal DA cell groups of the substantia nigra are very cell sparse, while there is a substantial expansion of the A9 medial and A10 lateral subdivisions forming an impressive "ventral wing" in the posterior substantia nigra. The nigrostriatal DA pathway mainly operates via Volume Transmission. Thus, DA diffuses along concentration gradients in the ECF to reach target cells with high affinity DA receptors. A novel feature of the DA receptor subtypes is their physical interaction in the plasma membrane of striatal neurons forming receptor mosaics (RM) with the existence of two types of RM. The "functional decoding unit" for DA is not the single receptor, but rather the RM that may affect not only the integration of signals in the DA neurons but also their trophic conditions. In 1991 A2A receptor antagonists were indicated to represent novel antiparkinsonian drugs based on the existence of A2A/D2 receptor-receptor interactions and here P2X receptor antagonists are postulated to be neuroprotective drugs in treatment of Parkinson's Disease.

ACTH and its role in immune-neuroendocrine functions. A comparative study.

Adrenocorticotropic hormone (ACTH) belongs to the melanocortine group of related peptides which share a common precursor, the pro-opiomelanocortin (POMC). Melanocortin expresses its functional effects by the stimulation of specific G-protein coupled receptors. To date, five receptor subtypes have been cloned. The POMC gene has been highly conserved during evolution, and ACTH has been found in different cells from invertebrates to vertebrates to vertebrates, including man. With regards the immune system, the presence of melanocortin receptors and the production of ACTH have been reported in invertebrate and vertebrate immuno-competent cells. Among its various physiological effects, ATCH has been shown to play a central role in immmune responses, such as chemotaxis and phagocytosis, in lower and higher forms of life. Moreover, ACTH is a key actor in stress response, and the complex cascade of events observed in vertebrates is reproduced and concentrated in intvertebrate immunocytes. On the basis of the present findings, ACTH should be considered an important immuno-regulator, forming part of the complex mosaic of relationships between the immune and neuroendocrine system which appears to have been substantially maintained over the course of evolution.

Influence of S-adenosyl-L-methionine on chronic mild stress-induced anhedonia in castrated rats.

1. S-adenosyl-L-methionine (SAMe) is the most important methyl donor in the brain and is essential for polyamine synthesis. Methyl group deficiency in the brain has been implicated in depression; on the other hand, polyamines enhance phosphorylation processes, and phosphorylation of functional proteins in neurons in involved in the therapeutic mechanisms of antidepressants. 2. The effect of SAMe in an animal model of 'depression', the chronic mild stress-induced anhedonia, was studied using long-term castrated male and female Lister hooded rats. 3. Chronic daily exposure to an unpredictable sequence of mild stressors produced, within 3 weeks, a significant reduction of the consumption of a sucrose solution. SAMe (100, 200 or 300 mg kg-1 daily i.m.) while having no influence on sucrose intake in non-stressed animals, dose-dependently reinstated sucrose consumption within the first week of treatment, both in male and in female stressed rats. Imipramine (10 mg kg-1 daily i.p.) produced a similar effect after a 3 week treatment. 4. Similarly, a palatable food reward-induced place preference conditioning was developed in SAMe (200 or 300 mg kg-1 daily i.m.)--and in imipramine (10 mg kg-1 daily i.p.)--treated chronically stressed animals (males and females), whilst it could not be obtained in vehicle-treated rats. 5. Moreover, the same doses of SAMe (but not of imipramine) restored the exploratory activity and curiosity for the environment (rearing), in the open-field test. 6. While imipramine caused a blockade of the growth throughout the treatment, SAMe produced only a transient growth arrest during the first week of treatment. 7. These results show that SAMe reverses an experimental condition of 'depression-like' behaviour in rats, the effect being more rapid and complete than that of imipramine, and without apparent side effects.

Effect of castration and testosterone in experimental models of depression in mice.

In the behavioral despair (forced swimming) test and in the tail-suspension test, long-term (30-32 days) castration significantly increased the duration of immobility in mice. Testosterone propionate (1 or 10 mg.kg-1.day sc for 4 days), although not affecting the duration of immobility in sham-operated mice, reduced the duration of immobility in castrated mice to within normal limits. Desipramine (20 mg/kg ip) decreased the duration of immobility both in sham-operated and in castrated animals. These results indicate that castration favors an inactive behavior and that testosterone, although having no "antidepressant" effect per se, is necessary for the male animal to cope normally with adverse environmental situations.

Sex-linked differences in avoidance learning in the offspring of rats treated with nicotine during pregnancy.

The offspring of rats treated with nicotine (0.5 mg/kg/day SC) on days 1-20 of gestation, were trained for active avoidance conditioning when 60 days old. Although learning was similar in both control groups of males and females, nicotine exposure during fetal life improved learning in females but reduced it in males, the difference between these two groups being statistically significant from day 17 until the end of the training period (day 25).

Pharmacological manipulation of brain galaninergic system and sexual behavior in male mice.

RATIONALE: Available data suggest a complex role for the brain galaninergic system in male sexual behavior; however, the results so far obtained in animals with either galanin or galanin antagonists are conflicting. OBJECTIVE: To define the better influence of galanin on male sexual behavior by studying, in mice, (i) the effect of galanin and of the chimeric galanin peptide M40 on the copulatory performance, and (ii) galanin mRNA levels in hypothalamic arcuate and dorso-medial nuclei. METHODS: For the behavioral testing, only sexually sluggish male mice were used. Galanin mRNA levels were studied in both sexually potent and impotent mice by means of in situ hybridization. Standard behavioral parameters for sexual behavior were recorded or calculated. Synthetic galanin (0.05, 0.1 or 1 microg/mouse) and M40 (5 or 20 microg/mouse) were intracerebroventricularly (ICV) injected, 15 min before the copulatory test. Galanin mRNA levels were evaluated. RESULTS: In sexually sluggish male mice, both galanin (0.1 and 1 microg/mouse ICV) and M40 (20 microg/mouse ICV), significantly increased intromission frequency and ejaculation latency; M40 also improved copulatory efficacy. On the other hand, in the hypothalamic arcuate and dorso-medial nuclei, the levels of galanin mRNA were not significantly different in sexually potent and impotent male mice. CONCLUSIONS: These results show that in sexually sluggish male mice the ICV injection of either galanin or the chimeric analogue M40 greatly prolongs the duration of the copulation; without a reduction of the sexual drive or of the copulatory performance. On the other hand, the hybridization experiments seem to rule out an important physiological role of the brain galaninergic system in the regulation of male sexual behavior, at least in mice.

Influence of SAMe on the modifications of brain polyamine levels in an animal model of depression.

The mechanism(s) of the antidepressant activity of S-adenosyl-L-methionine (SAMe) have not yet been elucidated. SAMe is essential for the synthesis of polyamines, which have a key role in protein synthesis, cell proliferation, and neuronal plasticity. On the other hand, accumulating data indicate that depression is associated with a reduction in regional brain volume and that antidepressants increase neurogenesis in defined brain regions and also influence neuronal plasticity. Here we show that in a validated rat model of depression (chronic unpredictable mild stress-induced anhedonia) there is a significant reduction of putrescine, spermidine and spermine in the hippocampus, and of only putrescine in the nucleus accumbens septi. SAMe, at a fully antidepressant dose (300 mg/kg i.m., daily for 7 days), completely restores the levels of putrescine in the nucleus accumbens, and restores in part the levels of both spermidine and spermine in the hippocampus. These results may suggest (i) a role for brain polyamines in depression and in reward processes, and (ii) that the antidepressant effect of SAMe may be due, at least in part, to a normalization of putrescine levels in the nucleus accumbens septi.

ACTH(1-24) stimulates the migration of human monocytes in vitro.

In view of the increasing evidence that a variety of stresses can influence immune responses, the direct effect of adrenocorticotropic hormone on the migration of human monocytes was studied in vitro. ACTH(1-24) significantly increased the number of migrating cells when placed in the same or the opposite compartment of the chemotaxis chamber, maximum activity being obtained at 10(-14) and 10(-8) M. The results indicate that ACTH(1-24) directly and potently stimulates the migration of human monocytes by means of a chemokinetic effect.

Differential modulation of invertebrate hemocyte motility by CRF, ACTH, and its fragments.

Various reports have shown that invertebrate hemocytes are responsive to mammalian neuropeptides and cytokines. In the present study, we demonstrate that corticotropin-releasing factor (CRF) and adrenocorticotropin (ACTH) fragments (1-24), (1-4), (4-9), (1-13), (1-17), and (11-24) significantly stimulate molluscan hemocyte migration, and the whole sequence (1-39) and the fragment (4-11) have an inhibitory effect. Differences between species were found with respect to the response to individual fragments. Additionally, the (4-11) fragment was able to antagonize some of the stimulatory fragments (4-9) as well as tumor necrosis factor (TNF-alpha)-induced chemotaxis. Our results suggest that invertebrate hemocytes are able to respond to CRF and ACTH fragments that in turn provide further evidence of the complexity of intercellular signaling within the immune system in relatively primitive animals. Thus, auto- and neuroimmunoregulatory activities in mammals must have had an earlier beginning than previously believed.

ACTH-(11-24) antagonizes ACTH-(1-24)-induced behavioral syndrome.

The possible involvement of the ACTH-(11-24) fragment on the stretchings, yawnings and penile erections induced by the intracerebroventricular injection of ACTH-(1-24) in rats, was studied. The results indicate that this C-terminal fragment is devoid of any behavioral activity, but inhibits the behavioral syndrome induced by ACTH-(1-24). This suggests that the fragment-(11-24) of the ACTH molecule may contain or represent an address sequence for brain ACTH receptors and may be involved in the termination of the behavioral response to melanocortins.

Effect of alpha-difluoromethylornithine (DFMO) on the behavioral syndrome induced by intracerebroventricular injection of ACTH 1-24, in rats.

Stretching-yawning syndrome (SYS) and penile erections (PE) are typical components of the behavioral picture induced by intracerebroventricular (i.c.v.) injection of ACTH 1-24. The present study shows that pretreatment with alpha-difluoromethylornithine (DFMO), and irreversible inhibitor of mammalian ornithine decarboxylase (ODC), antagonizes these behavioral effects in a dose-dependent way, in rats. These results suggest that ODC and polyamines may play a role in the transduction and transmission of receptor-mediated signals.

Influence of CRF and alpha-MSH on the migration of human monocytes in vitro.

The effects of stress in the modulation of immune responses are increasingly reported by a rapidly growing body of experimental and clinical data. Here we show that corticotropin releasing factor (CRF) stimulates 'in vitro' the migration of human monocytes, the maximum effect being obtained at 10(-14) M. On the other hand, another important neuropeptide of the stress response, alpha-melanocyte stimulating hormone (alpha-MSH), has no significant effect on the migration of monocytes. These findings show that one of the oldest immune responses is directly modulated by a key mediator of the stress response.

Influence of antineoplastic drugs on morphine analgesia and on morphine tolerance.

The possible influence of cisplatin, methotrexate, adriamycin and vincristine on thermal pain threshold, morphine analgesia and development of morphine tolerance was investigated in mice. In the hot-plate test, the nociceptive threshold was not affected by acute or repeated administration of any of the antineoplastic drugs used. The analgesic activity of morphine was significantly reduced by pretreatment with cisplatin, intraperitoneally (i.p.) injected at the dose of 2 mg/kg. In contrast, methotrexate, subcutaneously (s.c.) injected at the dose of 1 and 5 mg/kg, adriamycin (1 and 3 mg/kg s.c.), vincristine (0.25 and 0.5 mg/kg i.p.) and a lower dose of cisplatin (1 mg/kg i.p.) had no effect. The development of tolerance to morphine analgesia was delayed by adriamycin but was not influenced by the other antineoplastic drugs used. These data show that, of the four antineoplastic agents used in this study, cisplatin may interfere in the mechanism of action of morphine, and that adriamycin may delay the development of opiate tolerance.