Diego Guidolin

{alpha}-Synuclein and Parkinson's disease

Alpha‐synuclein (α‐syn) is a small soluble protein expressed primarily at presynaptic terminals in the central nervous system. Interest in α‐syn has increased dramatically after the discovery of a relationship between its dysfunction and several neurodegenerative diseases, including Parkinsons disease (PD). The physiological functions of α‐syn remain to be fully defined, although recent data suggest a role in regulating membrane stability and neuronal plasticity. Various trigger factors, either environmental or genetic, can lead to a cascade of events involving misfolding or loss of normal function of α‐syn. In dopaminergic neurons, this may promote a vicious cycle in which elevation in cytoplasmic dopamine, oxidative stress, α‐syn dysfunction, and disruption of vesicle function lead to dopaminergic cell loss and PD. α‐Syn dysfunction appears to be a common feature of all forms of PD. The mechanism by which α‐syn induces neuronal cell toxicity may invoke multiple pathways, such as aggregation or interaction with other proteins and molecules, including synphilin‐1, chaperone 14‐3‐3 protein, and dopamine itself. This complexity has hindered the development of models to study PD. The available animal models of PD, each present distinct advantages and limits. Findings to date suggest that α‐syn‐based models represent a paradigm, which is closest to the human pathology.— Recchia, A., Debetto, P., Negro, A., Guidolin, D., Skaper, S. D., Giusti, P. α‐Synuclein and Parkinsons disease.

From the Golgi–Cajal mapping to the transmitter-based characterization of the neuronal networks leading to two modes of brain communication: Wiring and volume transmission ☆

After Golgi-Cajal mapped neural circuits, the discovery and mapping of the central monoamine neurons opened up for a new understanding of interneuronal communication by indicating that another form of communication exists. For instance, it was found that dopamine may be released as a prolactin inhibitory factor from the median eminence, indicating an alternative mode of dopamine communication in the brain. Subsequently, the analysis of the locus coeruleus noradrenaline neurons demonstrated a novel type of lower brainstem neuron that monosynaptically and globally innervated the entire CNS. Furthermore, the ascending raphe serotonin neuron systems were found to globally innervate the forebrain with few synapses, and where deficits in serotonergic function appeared to play a major role in depression. We propose that serotonin reuptake inhibitors may produce antidepressant effects through increasing serotonergic neurotrophism in serotonin nerve cells and their targets by transactivation of receptor tyrosine kinases (RTK), involving direct or indirect receptor/RTK interactions. Early chemical neuroanatomical work on the monoamine neurons, involving primitive nervous systems and analysis of peptide neurons, indicated the existence of alternative modes of communication apart from synaptic transmission. In 1986, Agnati and Fuxe introduced the theory of two main types of intercellular communication in the brain: wiring and volume transmission (WT and VT). Synchronization of phasic activity in the monoamine cell clusters through electrotonic coupling and synaptic transmission (WT) enables optimal VT of monoamines in the target regions. Experimental work suggests an integration of WT and VT signals via receptor-receptor interactions, and a new theory of receptor-connexin interactions in electrical and mixed synapses is introduced. Consequently, a new model of brain function must be built, in which communication includes both WT and VT and receptor-receptor interactions in the integration of signals. This will lead to the unified execution of information handling and trophism for optimal brain function and survival.

miR-17 family of microRNAs controls FGF10-mediated embryonic lung epithelial branching morphogenesis through MAPK14 and STAT3 regulation of E-Cadherin distribution

The miR-17 family of microRNAs has recently been recognized for its importance during lung development. The transgenic overexpression of the entire miR-17-92 cluster in the lung epithelium led to elevated cellular proliferation and inhibition of differentiation, while targeted deletion of miR-17-92 and miR-106b-25 clusters showed embryonic or early post-natal lethality. Herein we demonstrate that miR-17 and its paralogs, miR-20a, and miR-106b, are highly expressed during the pseudoglandular stage and identify their critical functional role during embryonic lung development. Simultaneous downregulation of these three miRNAs in explants of isolated lung epithelium altered FGF10 induced budding morphogenesis, an effect that was rescued by synthetic miR-17. E-Cadherin levels were reduced, and its distribution was altered by miR-17, miR-20a and miR-106b downregulation, while conversely, beta-catenin activity was augmented, and expression of its downstream targets, including Bmp4 as well as Fgfr2b, increased. Finally, we identified Stat3 and Mapk14 as key direct targets of miR-17, miR-20a, and miR-106b and showed that simultaneous overexpression of Stat3 and Mapk14 mimics the alteration of E-Cadherin distribution observed after miR-17, miR-20a, and miR-106b downregulation. We conclude that the mir-17 family of miRNA modulates FGF10-FGFR2b downstream signaling by specifically targeting Stat3 and Mapk14, hence regulating E-Cadherin expression, which in turn modulates epithelial bud morphogenesis in response to FGF10 signaling.

Intramembrane receptor-receptor interactions: a novel principle in molecular medicine

Summary.In 1980/81 Agnati and Fuxe introduced the concept of intramembrane receptor–receptor interactions and presented the first experimental observations for their existence in crude membrane preparations. The second step was their introduction of the receptor mosaic hypothesis of the engram in 1982. The third step was their proposal that the existence of intramembrane receptor–receptor interactions made possible the integration of synaptic (WT) and extrasynaptic (VT) signals. With the discovery of the intramembrane receptor–receptor interactions with the likely formation of receptor aggregates of multiple receptors, so called receptor mosaics, the entire decoding process becomes a branched process already at the receptor level in the surface membrane. Recent developments indicate the relevance of cooperativity in intramembrane receptor–receptor interactions namely the presence of regulated cooperativity via receptor–receptor interactions in receptor mosaics (RM) built up of the same type of receptor (homo-oligomers) or of subtypes of the same receptor (RM type1). The receptor–receptor interactions will to a large extent determine the various conformational states of the receptors and their operation will be dependent on the receptor composition (stoichiometry), the spatial organization (topography) and order of receptor activation in the RM. The biochemical and functional integrative implications of the receptor–receptor interactions are outlined and long-lived heteromeric receptor complexes with frozen RM in various nerve cell systems may play an essential role in learning, memory and retrieval processes. Intramembrane receptor–receptor interactions in the brain have given rise to novel strategies for treatment of Parkinson’s disease (A2A and mGluR5 receptor antagonists), schizophrenia (A2A and mGluR5 agonists) and depression (galanin receptor antagonists). The A2A/D2, A2A/D3 and A2A/mGluR5 heteromers and heteromeric complexes with their possible participation in different types of RM are described in detail, especially in the cortico-striatal glutamate synapse and its extrasynaptic components, together with a postulated existence of A2A/D4 heteromers. Finally, the impact of intramembrane receptor–receptor interactions in molecular medicine is discussed outside the brain with focus on the endocrine, the cardiovascular and the immune systems.

Aspects of neural plasticity in the central nervous system-I. Computer-assisted image analysis methods.

Microdensitometric and morphometric techniques have been developed to quantitatively characterize cell groups and terminal populations of transmitter-identified neuronal systems. Various microdensitometric methods implemented on the image analyzer or on the scanning microdensitometer were introduced and compared. On this basis a technique to assess the half-life of dopamine stores determined by quantitative immunocytochemistry has been developed. The problem of relative and absolute quantification of microdensitometric analysis of immunocytochemical preparations is discussed here. A method has been developed for the study, both in 2- and 3-dimensions, of the overall features of the profile distribution in a defined neuroanatomical area. An approach to determine the degree of uniformity of a certain profile distribution is also proposed. Furthermore, methods for the evaluation of the codistribution of two or more different types of profiles and to characterize the morphometric features of patches of profiles in a certain region are presented. All these quantitative morphological approaches were tested in relevant preparations of the central nervous system.

Volume transmission and wiring transmission from cellular to molecular networks: history and perspectives.

The present paper deals with a fundamental issue in neuroscience: the inter‐neuronal communication. The paper gives a brief account of our previous and more recent theoretical contributions to the subject and also reports new recent data that support some aspects of our proposal on two major modes of communication in the central nervous system: the wiring and the volume transmission. There exist two competing theories on inter‐neuronal communication: the neuron doctrine and the theory of the diffuse nerve network, supported by Cajal and Golgi, respectively (see their respective Nobel Lectures). The present paper gives a brief account of a view on inter‐neuronal communication in the brain, the volume and wiring transmission concept that to a great extent reconcile these two theories. Thus, the theory of volume and wiring transmission are summarized and its recent developments that allow to extend these two modes of communication from the cellular network to the molecular network level is also briefly illustrated. The explanatory value of this broadened view is further enhanced by our recent proposal on the existence of a Global Molecular Network enmeshing the entire central nervous system. It may be interesting to note that also the Global Molecular Network theory is reminiscent of the old reticular theory of Apathy. Finally, the so‐called ‘tide hypothesis’ for diffusion of signals in the brain is briefly discussed and its possible extension to the molecular level is for the first time introduced. Early indirect evidence supporting volume transmission in the brain was the discovery of transmitter‐receptor mismatches. Thus, as an experimental part of the present paper a new approach to evaluate transmitter‐receptor mismatches is given and evidence for inter‐relationships between temperature micro‐gradients and mismatches is provided.

Cell composition of the human pulmonary valve : A comparative study with the aortic valve : The VESALIO* project

BACKGROUND Cell populations present in human semilunar valves have not been investigated thoroughly. The aim of this study was to characterize the cell phenotypes in pulmonary valve leaflets (PVL) in comparison with aortic (AVL) valve leaflets. METHODS AVL and PVL were dissected from hearts (n = 4) harvested from transplanted patients. Leaflets were processed for immunocytochemistry analysis and Western blotting procedures using a panel of monoclonal antibodies specific for cytoskeletal/contractile antigens. RESULTS The fibrosa and the ventricularis layers of AVL had a higher cellularity than PVL. In PVL and AVL most cells were reactive for vimentin and nonmuscle (NM) myosin, though vimentin-positive cells were more abundant in AVL than in PVL. Sparse cells positive to anti-smooth muscle (SM) alpha-actin, calponin, and anti-SM myosin antibodies were found only at the outer edge of fibrosa. In Western blotting, AVL and PVL extracts were shown to be equally reactive for vimentin, SM alpha-actin, and NM myosin, whereas both valves were negative for SM myosin and SM22. CONCLUSIONS Three distinct cell phenotypes have been identified in both valves: fibroblasts, myofibroblasts, and fetal-type SM cells whose distribution is specifically related to the valve layers. Although PVL and AVL cell populations differ quantitatively, some minor qualitative differences exist for vimentin and NM myosin distribution. These data are essential for studies aimed at repopulating valve scaffolds by using tissue engineering technology.

Glucocorticoids depress activity-dependent expression of Bdnf mrna in hippocampal neurones

Glucocorticoid hormones are important regulators of brain development and ageing, and can impair the capacity of hippocampal neurones to survive various neurological insults. Here we show that dexamethasone, a synthetic glucocorticoid, prevents activity-dependent increases of brain-derived neurotrophic factor (BDNF) mRNA in cultures of rat hippocampal neurones. In situ hybridization was used to evaluate the levels of BDNF mRNA. Up-regulation of BDNF mRNA triggered by depolarization with high potassium, or exposure to the glutamate receptor agonist kainic acid, resulted both from higher levels of expression in neurones and from new recruitment of cells. These data suggest that the known ability of glucocorticoids to exacerbate neuronal injury following ischaemia and other metabolic insults could be due to antagonism of regulatory mechanisms governing neurotrophin levels in the brain.

Receptor-receptor interactions, receptor mosaics, and basic principles of molecular network organization: possible implications for drug development.

The phenomenon of receptor-receptor interactions was hypothesized by Agnati and Fuxe in the 1980s, and several indirect proofs were provided in the following years by means of in vitro binding experiments and in vivo experiments in physiological and pathological animal models. This paper aims to outline some of the most important features and consequences of this phenomenon in the frame of the structural and functional aspects of molecular networks. In particular, the concepts of receptor mosaic (RM), and of horizontal and vertical molecular networks (HMNs, VMNs, respectively) are illustrated. To discuss some aspects of the functional organization of molecular networks, not only new data on protein-protein interactions but also the biochemical mechanism of cooperativity will be used. On this basis, some theoretical deductions can be drawn that allow a tentative classification of the RMs and the proposal of the extension of the concept of branching point introduced for enzymes to the possible switching role of some RMs in directing signals to various VMNs. Finally, the cooperativity phenomenon and the so-called symmetry rule will be used to introduce a proper mathematical approach that characterizes RMs as to their receptor composition, receptor topography, and order of receptor activation inside the RM. These new data on G protein-coupled receptors and molecular network organization indicate possible new approaches for drug development.

Ghrelin inhibits FGF-2-mediated angiogenesis in vitro and in vivo.

Recent evidence indicates that ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), is highly expressed in the cardiovascular system, and in this study we addressed the possibility that ghrelin may affect angiogenesis in vitro and in vivo. Reverse transcription-polymerase chain reaction showed that human umbilical vein endothelial cells (HUVECs) express ghrelin and GHS-R mRNAs. Ghrelin inhibited FGF-2-induced proliferation of HUVECs cultured in vitro, the maximal effective concentration being 10(-8) M, and this effect was annulled by the GHS-R antagonist D-Lys3-growth hormone releasing peptide-6. FGF-2 stimulated HUVEC cultured on Matrigel to form capillary-like structures, and ghrelin (10(-8) M) suppressed this effect. In the chick embryo chorioallantoic membrane in vivo assay, FGF-2 induced a strong angiogenic response, which was counteracted by ghrelin (500 ng). Taken together, these findings suggest that ghrelin acts as an angiostatic molecule and indicate that its activity is comparable to that of a well-known angiostatic agent, i.e., vinblastine. The antiangiogenic activity of ghrelin deserves further investigations, alone or together with other antiangiogenic agents, for the treatment of pathological conditions characterized by enhanced angiogenesis.