Marco Parenti

PHARMACOLOGIC RESCUE OF IMPAIRED COGNITIVE FLEXIBILITY, SOCIAL DEFICITS, INCREASED AGGRESSION, AND SEIZURE SUSCEPTIBILITY IN OXYTOCIN RECEPTOR NULL MICE: A NEUROBEHAVIORAL MODEL OF AUTISM

BACKGROUNDnOxytocin (OT) has been suggested as a treatment to improve social behavior in autistic patients. Accordingly, the OT (Oxt(-/-)) and the OT receptor null mice (Oxtr(-/-)) display autistic-like deficits in social behavior, increased aggression, and reduced ultrasonic vocalization.nnnMETHODSnOxtr(-/-) mice were characterized for general health, sociability, social novelty, cognitive flexibility, aggression, and seizure susceptibility. Because vasopressin (AVP) and OT cooperate in controlling social behavior, learning, and aggression, they were tested for possible rescue of the impaired behaviors. Primary hyppocampal cultures from Oxtr(+/+) and Oxtr(-/-) mouse embryos were established to investigate the balance between gamma-aminobutyric acid (GABA) and glutamate synapses and the expression levels of OT and AVP (V1a) receptors were determined by autoradiography.nnnRESULTSnOxtr(-/-) mice display two additional, highly relevant, phenotypic characteristics: 1) a resistance to change in a learned pattern of behavior, comparable to restricted interests and repetitive behavior in autism, and 2) an increased susceptibility to seizures, a frequent and clinically relevant symptom of autism. We also show that intracerebral administration of both OT and AVP lowers aggression and fully reverts social and learning defects by acting on V1a receptors and that seizure susceptibility is antagonized by peripherally administered OT. Finally, we detect a decreased ratio of GABA-ergic versus total presynapses in hippocampal neurons of Oxtr(-/-) mice.nnnCONCLUSIONSnAutistic-like symptoms are rescued on administration of AVP and OT to young Oxtr(-/-) adult animals. The Oxtr(-/-) mouse is thus instrumental to investigate the neurochemical and synaptic abnormalities underlying autistic-like disturbances and to test new strategies of pharmacologic intervention.

G-protein-coupled receptors, cholesterol and palmitoylation: facts about fats

G-protein-coupled receptors (GPCRs) are integral membrane proteins, hence it is not surprising that a number of their structural and functional features are modulated by both proteins and lipids. The impact of interacting proteins and lipids on the assembly and signalling of GPCRs has been extensively investigated over the last 20-30 years, and a further impetus has been given by the proposal that GPCRs and/or their immediate signalling partners (G proteins) can partition within plasma membrane domains, termed rafts and caveolae, enriched in glycosphingolipids and cholesterol. The high content of these specific lipids, in particular of cholesterol, in the vicinity of GPCR transmembranes can affect GPCR structure and/or function. In addition, most GPCRs are post-translationally modified with one or more palmitic acid(s), a 16-carbon saturated fatty acid, covalently bound to cysteine(s) localised in the carboxyl-terminal cytoplasmic tail. The insertion of palmitate into the cytoplasmic leaflet of the plasma membrane can create a fourth loop, thus profoundly affecting GPCR structure and hence the interactions with intracellular partner proteins. This review briefly highlights how lipids of the membrane and the receptor themselves can influence GPCR organisation and functioning.

The Oxytocin Receptor Antagonist Atosiban Inhibits Cell Growth via a “Biased Agonist” Mechanism

In human myometrial cells, the promiscuous coupling of the oxytocin receptors (OTRs) to Gq and Gi leads to contraction. However, the activation of OTRs coupled to different G protein pathways can also trigger opposite cellular responses, e.g. OTR coupling to Gi inhibits, whereas its coupling to Gq stimulates, cell proliferation. Drug analogues capable of promoting a selective receptor-G protein coupling may be of great pharmacological and clinical importance because they may target only one specific signal transduction pathway. Here, we report that atosiban, an oxytocin derivative that acts as a competitive antagonist on OTR/Gq coupling, displays agonistic properties on OTR/Gi coupling, as shown by specific 35S-labeled guanosine 5′-3-O-(thio) trisphosphate ([35S]GTPγS) binding. Moreover, atosiban, by acting on a Gi-mediated pathway, inhibits cell growth of HEK293 and Madin-Darby canine kidney cells stably transfected with OTRs and of DU145 prostate cancer cells expressing endogenous OTRs. Notably, atosiban leads to persistent ERK1/2 activation and p21WAF1/CIP1 induction, the same signaling events leading to oxytocin-mediated cell growth inhibition via a Gi pathway. Finally, atosiban exposure did not cause OTR internalization and led to only a modest decrease (20%) in the number of high affinity cell membrane OTRs, two observations consistent with the finding that atosiban did not lead to any desensitization of the oxytocin-induced activation of the Gq-phospholipase C pathway. Taken together, these observations indicate that atosiban acts as a “biased agonist” of the human OTRs and thus belongs to the class of compounds capable of selectively discriminating only one among the multiple possible active conformations of a single G protein-coupled receptor, thereby leading to the selective activation of a unique intracellular signal cascade.

G protein-coupled receptor oligomerization provides the framework for signal discrimination.

The idea that G protein‐coupled receptors (GPCRs) may undergo homo‐ or hetero‐oligomerization, although highly controversial up to a few years ago, has recently gained wide acceptance. The recognition that GPCRs may exhibit either dimeric or oligomeric structures is based upon a large body of biochemical and biophysical evidence. While much effort has been spent to demonstrate the mechanism(s) by which GPCRs interact with each other, the physiological relevance of this phenomenon remains rather elusive. GPCR oligomerization has been proposed to play a role in receptor ontogeny by either chaperoning protein folding or controlling trafficking to the cell surface. However, the acquisition of these roles does not rule out the possibility that oligomeric receptors may have additional functions, once they are brought to the cell surface. Herein, we propose that protein–protein as well as protein–lipid interactions may provide the structural basis for organizing distinct cell compartments along the plasma membrane where different extracellular signals may be perceived and discriminated.

Dual modulation of inward rectifier potassium currents in olfactory neuronal cells by promiscuous G protein coupling of the oxytocin receptor

J. Neurochem. (2010) 114, 1424–1435.

Sphingolipid Metabolism and Caveolin Expression in Gonadotropin-Releasing Hormone-Expressing GN11 and Gonadotropin-Releasing Hormone-Secreting GT1-7 Neuronal Cells

In this paper, we show that caveolin-1 is abundantly present in a cell line of immortalized gonadotropin-releasing hormone-expressing neurons (GN11). In contrast to GN11, caveolin is undetectable in a cognate cell line of immortalized gonadotropin-releasing hormone-secreting neurons (GT1-7). These two cell lines are characterized by a radically different sphingolipid metabolism. After incubation in the presence of tracer amount of [1-3H]sphingosine, GN11 and GT1-7 neurons incorporated similar amounts of radioactivity. In GT1-7 neurons, [1-3H]sphingosine metabolism was markedly oriented toward the biosynthesis of complex sphingolipids. In fact, almost all the radioactivity in the lipid extracts from GT1-7 cells was associated with biosynthetic products (ceramide, sphingomyelin, and glycosphingolipids). In particular glycosphingolipids represented more than 65% of total lipid radioactivity in these cells, and the main glycosphingolipid was GM3 ganglioside (about 47% of total lipid radioactivity). In the case of GN11 neurons, a high portion of [1-3H]sphingosine underwent complete degradation, as indicated by the formation of high levels of radioactive phosphatidylethanolamine (about 23% of lipid radioactivity). Moreover, the main complex sphingolipid in GN11 neurons was not a glycolipid, but sphingomyelin (its level in these cells, about 54% of lipid radioactivity, was two-fold higher than in GT1-7). Glycolipids, gangliosides in particular, were present in low amount (9.5% of lipid radioactivity) if compared with the cognate GT1-7 cell line, and GM3 was almost absent in GN11 neurons. Despite the radical differences in ganglioside and caveolin content, from both cell types a membrane fraction similarly enriched in sphingolipids was prepared. In the case of GN11 cells, this fraction was also enriched in caveolin. The presence of caveolin or GM3 may correlate with different functional properties linked to the stage of neuronal maturation, since GN11 and GT1-7 are representative, respectively, of immature, migrating, and differentiated, postmigratory gonadotropin-releasing hormone-positive neurons.

Light on the structure of thromboxane A2 receptor heterodimers

The structure-based design of a mutant form of the thromboxane A2 prostanoid receptor (TP) was instrumental in characterizing the structural determinants of the hetero-dimerization process of this G protein coupled receptor (GPCR). The results suggest that the hetero-dimeric complexes between the TPα and β isoforms are characterized by contacts between hydrophobic residues in helix 1 from both monomers. Functional characterization confirms that TPα–TPβ hetero-dimerization serves to regulate TPα function through agonist-induced internalization, with important implications in cardiovascular homeostasis. The integrated approach employed in this study can be adopted to gain structural and functional insights into the dimerization/oligomerization process of all GPCRs for which the structural model of the monomer can be achieved at reasonable atomic resolution.

Polarization of caveolins and caveolae during migration of immortalized neurons.

During CNS development neurons undergo directional migration to achieve their adult localizations. To study neuronal migration, we used a model cell line of immortalized murine neurons (gonadotropin‐releasing hormone expressing neurons; GN11), enriched with caveolins and caveolae invaginations that show in vitro chemotaxis upon serum exposure. Cholesterol depletion with methyl‐β‐cyclodextrin induced the loss of caveolae and the inhibition of chemotaxis, thus suggesting that GN11 migration depends upon the structural integrity of caveolae. Polarization of proteins and organelles is a hallmark of cell migration. Accordingly, GN11 cells transmigrating through filter pores exhibited a polarized distribution of caveolin‐1 isoform (cav‐1) in the leading processes. In contrast, during two‐dimensional migration cav‐1 and caveolae polarized at the trailing edge. As caveolae are enriched with signaling molecules, we suggest that asymmetrical localization of caveolae may spatially orient GN11 neurons to incoming migratory signals thereby transducing them into directional migration.

Presence of delta opioid receptors on a subset of hypothalamic gonadotropin releasing hormone (GnRH) neurons

Opioid peptides exert an inhibitory effect on hypothalamic gonadotropin releasing hormone (GnRH) secretion mainly by interacting with mu-opioid receptors. Although a direct role for opioids via delta-opioid receptors (DORs) has been suggested, the presence of these receptors on GnRH neurons has never been demonstrated. In the present study, we determined the distribution of DORs in the basal hypothalamus of rat with special focus on their relation to GnRH neurons. Double-labelling immunofluorescence and confocal microscopy revealed that DORs are exclusively present in a subpopulation of GnRH nerve terminals, with the highest density in the external layer of the median eminence. We then studied the functional characteristics of DORs in an immortalized GnRH-secreting neuronal cell line (GT1-1) known to endogenously express this receptor. Here, pertussis toxin pretreatment abolished the delta-agonist (DPDPE) inhibitory effect on cAMP accumulation. We also analyzed the type of G proteins involved in the signal transduced by the DOR and showed that GT1-1 cells express the inhibitory Go and Gi2 alpha-subunits. However, only Go was down-regulated under chronic DPDPE exposure. Finally, since DOR is expressed postnatally in brain, we compared GnRH neuronal cells immortalized at different developmental stages (the more mature GT1-1 and GT1-7 cells, versus the more immature GN11 cells), evidencing that only mature neurons express DOR. In conclusion, our study indicates that a direct control of opioids via delta-receptors occurs on GnRH neurons and validates the use of GT1 cells to further investigate the nature of the DOR present on GnRH neurons.

Mesenchymal stem cells enhance GABAergic transmission in co-cultured hippocampal neurons

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent stem cells endowed with neurotrophic potential combined with immunological properties, making them a promising therapeutic tool for neurodegenerative disorders. However, the mechanisms through which MSCs promote the neurological recovery following injury or inflammation are still largely unknown, although cell replacement and paracrine mechanisms have been hypothesized. In order to find out what are the mechanisms of the trophic action of MSCs, as compared to glial cells, on CNS neurons, we set up a co-culture system where rat MSCs (or cortical astrocytes) were used as a feeding layer for hippocampal neurons without any direct contact between the two cell types. The analysis of hippocampal synaptogenesis, synaptic vesicle recycling and electrical activity show that MSCs were capable to support morphological and functional neuronal differentiation. The proliferation of hippocampal glial cells induced by the release of bioactive substance(s) from MSCs was necessary for neuronal survival. Furthermore, MSCs selectively increased hippocampal GABAergic pre-synapses. This effect was paralleled with a higher expression of the potassium/chloride KCC2 co-transporter and increased frequency and amplitude of mIPSCs and sIPSCs. The enhancement of GABA synapses was impaired by the treatment with K252a, a Trk/neurotrophin receptor blocker, and by TrkB receptor bodies hence suggesting the involvement of BDNF as a mediator of such effects. The results obtained here indicate that MSC-secreted factors induce glial-dependent neuronal survival and trigger an augmented GABAergic transmission in hippocampal cultures, highlighting a new effect by which MSCs could promote CNS repair. Our results suggest that MSCs may be useful in those neurological disorders characterized by an impairment of excitation versus inhibition balance.