Marco Scarselli - Academia.edu (original) (raw)
Papers by Marco Scarselli
Encyclopedia of Molecular Pharmacology, 2020
Second-generation antipsychotics Definition The introduction of atypical antipsychotics (AAPs), s... more Second-generation antipsychotics Definition The introduction of atypical antipsychotics (AAPs), since the discovery of its prototypical drug clozapine in the 1970s, has been a milestone in psychopharmacology for treating schizophrenia, bipolar disorder, and other brain diseases that are characterized by psychotic features‚ as these drugs allow a significant recovery not only in terms of hospitalization and reduction in symptoms severity, but also in terms of safety, socialization, and better rehabilitation in the society. Compared to typical antipsychotics (TAPs), AAPs produce minimal motor side effects, such as parkinsonism and tardive dyskinesia, and seem to be more effective in improving cognitive symptoms (Meltzer 2013). Unfortunately, AAPs may cause weight gain and metabolic adverse reactions; hence‚ patients should be constantly monitored. Besides, each AAP has a unique pharmacological profile, which explains the clinical diversity between them.
Proceedings of the National Academy of Sciences, 2010
Therapeutic strategies that augment insulin release from pancreatic β-cells are considered benefi... more Therapeutic strategies that augment insulin release from pancreatic β-cells are considered beneficial in the treatment of type 2 diabetes. We previously demonstrated that activation of β-cell M 3 muscarinic receptors (M3Rs) greatly promotes glucose-stimulated insulin secretion (GSIS), suggesting that strategies aimed at enhancing signaling through β-cell M3Rs may become therapeutically useful. M3R activation leads to the stimulation of G proteins of the G q family, which are under the inhibitory control of proteins known as regulators of G protein signaling (RGS proteins). At present, it remains unknown whether RGS proteins play a role in regulating insulin release. To address this issue, we initially demonstrated that MIN6 insulinoma cells express functional M3Rs and that RGS4 was by far the most abundant RGS protein expressed by these cells. Strikingly, siRNA-mediated knockdown of RGS4 expression in MIN6 cells greatly enhanced M3R-mediated augmentation of GSIS and calcium release. We obtained similar findings using pancreatic islets prepared from RGS4-deficient mice. Interestingly, RGS4 deficiency had little effect on insulin release caused by activation of other β-cell GPCRs. Finally, treatment of mutant mice selectively lacking RGS4 in pancreatic β-cells with a muscarinic agonist (bethanechol) led to significantly increased plasma insulin and reduced blood glucose levels, as compared to control littermates. Studies with β-cell-specific M3R knockout mice showed that these responses were mediated by β-cell M3Rs. These findings indicate that RGS4 is a potent negative regulator of M3R function in pancreatic β-cells, suggesting that RGS4 may represent a potential target to promote insulin release for therapeutic purposes. knockout mice | muscarinic receptor | RGS proteins | G protein-coupled receptor T ype 2 diabetes (T2D) has emerged as a major threat to human health worldwide. Besides peripheral insulin resistance, T2D is usually associated with β-cell dysfunction (1). Thus, the development of new drugs aimed at improving β-cell function, including stimulation of insulin release, is the focus of many laboratories (2).
The Journal of Pharmacology and Experimental Therapeutics, 2003
The muscarinic M(2) receptor was split at the third cytoplasmic loop into two fragments: the one ... more The muscarinic M(2) receptor was split at the third cytoplasmic loop into two fragments: the one containing the first five transmembrane regions and the N-terminal part of the third cytoplasmic loop was named M(2trunk), while the other, which contained the last two transmembrane regions and the C-terminal part of the third cytoplasmic loop, was named M(2tail). As seen in many other G protein-coupled receptors, when these two fragments were transfected together in COS-7 cells they rescued the pharmacological profile and the functional activity of the wild-type M(2) receptor. Conversely, N-[(3)H]methylscopolamine ([(3)H]NMS) association binding experiments showed a substantial difference between the wild-type M(2) and the split M(2trunk)/M(2tail) receptors. The progression of the association binding kinetic of the M(2trunk)/M(2tail) receptor was strictly dependent upon the amount of the fragment DNA transfected. When the amount of transfected DNA was 4 microg/plate and the B(max) of [(3)H]NMS at equilibrium was around 200 fmol/mg protein the form of the association was that of classical saturation, but when the amount of transfected DNA was lower the [(3)H]NMS association reached a maximum binding point and then declined to a lower equilibrium binding level. The form of the association was temperature-dependent: as the temperature was lowered, the maximum binding point tended to be higher. We suggest that this peculiar form of the [(3)H]NMS association binding to the muscarinic M(2trunk)/M(2tail) receptor is attributable to a less stable interaction between the trunk and the tail fragments of the split receptor.
Advances in Neurology, Feb 1, 2001
FEBS Journal, 2015
The introduction of super resolution fluorescence microscopy has allowed to visualize single prot... more The introduction of super resolution fluorescence microscopy has allowed to visualize single proteins in their biological environment. Recently, these techniques have been applied to determine the organization of class A G protein-coupled receptors (GPCRs), and to determine whether they exist as monomers, dimers and/or higher-order oligomers. On this subject, this review highlights recent evidence coming from Photoactivated Localization Microscopy (PALM) that allows the visualization of single molecules in dense samples, and Single-Molecule Tracking (SMT) that determines how GPCRs move and interact in living cells in the presence of different ligands. PALM has demonstrated that GPCR oligomerization depends on the receptor subtype, cell-type, actin cytoskeleton and other proteins. Conversely, SMT has revealed the transient dynamics of dimer formation, where receptors display a monomer-dimer equilibrium characterized by rapid association and dissociation. At steady state, depending on the subtype, approximately 30-50% of receptors are part of dimeric complexes. Notably, the existence of many GPCR di-/oligomers is also supported by using well-known techniques, such as Resonance Energy Transfer (RET) methodologies, and by approaches that exploit fluorescence fluctuations, such as Fluorescence Correlation Spectroscopy (FCS). Future research using single-molecule methods will deepen our knowledge related to function and druggability of homo- and hetero-oligomers. This article is protected by copyright. All rights reserved.
Nature methods, 2007
G protein-coupled receptors (GPCRs) comprise one of the largest protein families found in nature.... more G protein-coupled receptors (GPCRs) comprise one of the largest protein families found in nature. Here we describe a new experimental strategy that allows rapid identification of functionally critical amino acids in the rat M(3) muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR. This approach involves low-frequency random mutagenesis of the entire M3R coding sequence, followed by the application of a new yeast genetic screen that allows the recovery of inactivating M3R single point mutations. The vast majority of recovered mutant M3Rs also showed substantial functional impairments in transfected mammalian (COS-7) cells. A subset of mutant receptors, however, behaved differently in yeast and mammalian cells, probably because of the specific features of the yeast expression system used. The screening strategy described here should be applicable to all GPCRs that can be expressed functionally in yeast.
European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology, Jan 23, 2014
The brain׳s complexity derives not only from the way the intricate network of neurons is wired, b... more The brain׳s complexity derives not only from the way the intricate network of neurons is wired, but also by protein complexes that recognize and decode chemical information. G protein-coupled receptors (GPCRs) represent the most abundant family of proteins mediating neurotransmission in the brain, and their ability to form homo- and heteromers which amplifies the scope for synaptic communication and fine-tuning. Dopamine receptors are important drug targets and members of both the D1/D5 and D2/D3/D4 receptor families form homo- and heteromers. The present article focuses on D3 receptor homo- and heteromers, in particular, those formed in association with their D2 counterparts. We highlight the binding profiles and mechanisms of interaction with D3-D3 homomers and D3-D2 heteromers of: first, the PET ligand and potent agonist [(11)C]-(+)-PHNO; second, the novel, bitopic/allosteric dopamine D3 receptor antagonist, SB269,652; and third, diverse partial agonists like antipsychotic and ar...
Plos One, 2011
In this work we discuss how to use photophysical information for improved quantitative measuremen... more In this work we discuss how to use photophysical information for improved quantitative measurements using Photo Activated Localization Microscopy (PALM) imaging. We introduce a method that reliably estimates the number of photoblinking molecules present in a biological sample and gives a robust way to quantify proteins at the single-cell level from PALM images. We apply this method to determine the amount of β2 adrenergic receptor, a prototypical G Protein Coupled Receptor, expressed on the plasma membrane of HeLa cells.
Frontiers in Behavioral Neuroscience, 2013
The present review update the relationship between acetaldehyde (ACE) and parkinsonism with a spe... more The present review update the relationship between acetaldehyde (ACE) and parkinsonism with a specific focus on the role of P450 system and CYP 2E1 isozyme particularly. We have indicated that ACE is able to enhance the parkinsonism induced in mice by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin able to damage the nigrostriatal dopaminergic pathway. Similarly diethyldithiocarbamate, the main metabolite of disulfiram, a drug widely used to control alcoholism, diallylsulfide (DAS) and phenylisothiocyanate also markedly enhance the toxin-related parkinsonism. All these compounds are substrate/inhibitors of CYP450 2E1 isozyme. The presence of CYP 2E1 has been detected in the dopamine (DA) neurons of rodent Substantia Nigra (SN), but a precise function of the enzyme has not been elucidated yet. By treating CYP 2E1 knockout (KO) mice with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the SN induced lesion was significantly reduced when compared with the lesion observed in wild-type animals. Several in vivo and in vitro studies led to the conclusion that CYP 2E1 may enhance the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice by increasing free radical production inside the dopaminergic neurons. ACE is a good substrate for CYP 2E1 enzyme as the other substrate-inhibitors and by this way may facilitate the susceptibility of dopaminergic neurons to toxic events. The literature suggests that ethanol and/or disulfiram may be responsible for toxic parkinsonism in human and it indicates that basal ganglia are the major targets of disulfiram toxicity. A very recent study reports that there are a decreased methylation of the CYP 2E1 gene and increased expression of CYP 2E1 mRNA in Parkinson's disease (PD) patient brains. This study suggests that epigenetic variants of this cytochrome contribute to the susceptibility, thus confirming multiples lines of evidence which indicate a link between environmental toxins and PD.
Molecular pharmacology, 2011
The M₃ muscarinic acetylcholine receptor (M3R) regulates many fundamental physiological functions... more The M₃ muscarinic acetylcholine receptor (M3R) regulates many fundamental physiological functions. To identify novel M3R-interacting proteins, we used a recently developed yeast two-hybrid screen (split ubiquitin method) to detect interactions among membrane proteins. This screen led to the identification of many novel M3R-associated proteins, including the putative membrane protein transmembrane protein 147 (Tmem147). The amino acid sequence of Tmem147 is highly conserved among mammals, but its physiological roles are unknown at present. We initially demonstrated that Tmem147 could be coimmunoprecipitated with M3Rs in cotransfected mammalian cells (COS-7 cells). Confocal imaging studies showed that Tmem147 was localized to endoplasmic reticulum (ER) membranes and that the Tmem147/M3R interaction occurred in the ER of cotransfected COS-7 cells, resulting in impaired trafficking of the M3R to the cell surface. To study the role of Tmem147 in modulating M3R function in a more physiolo...
European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology, Jan 18, 2014
Clozapine is the most effective antipsychotic to date, but its benefits are counterbalanced by th... more Clozapine is the most effective antipsychotic to date, but its benefits are counterbalanced by the risk of severe hematological effects. In this study, we analyzed whether clozapine inhibits polymorphonuclear (PMN) leukocyte chemotaxis. We found that clozapine, within the therapeutic concentration range, potently and selectively inhibits PMN chemotaxis induced by interleukin 8 (IL-8), a chemokine inducing neutrophil migration. The effect was not due to its action at dopamine, serotonin and muscarinic receptors, or to a direct antagonism to IL-8 receptors. Furthermore, clozapine did not inhibit PMN chemotaxis by its presumed toxic mechanism. In fact, after an overnight incubation in cell culture, the drug did not increase the physiological PMN apoptosis. An interference of clozapine with the autocrine release of leukotriene B4 (LTB4), a secondary chemoattractant secreted by neutrophils in response to the primary chemoattractant IL-8, was hypothesized. In agreement with this hypothesi...
Methods in enzymology, 2013
Before the molecular biology era, functional experiments on isolated organs and radioligand bindi... more Before the molecular biology era, functional experiments on isolated organs and radioligand binding and biochemical experiments on animal tissues were widely used to characterize G protein-coupled receptors (GPCRs). The introduction of recombinant cell lines expressing a single GPCR type has been a big step forward for studying both drug-receptor interactions and signal transduction. Before the introduction of the concept of receptor oligomerization, all data generated were attributed to the interaction of drugs with receptor monomers. Now, considerable data must be reinterpreted in light of receptor homo- and heteromerization. In this chapter, we will review some of the methods used to study radioligand binding and signal transduction modifications induced by GPCR homo- and heteromerization.
Proceedings of the National Academy of Sciences, 2009
Impaired functioning of pancreatic  cells is a key hallmark of type 2 diabetes.  cell function ... more Impaired functioning of pancreatic  cells is a key hallmark of type 2 diabetes.  cell function is modulated by the actions of different classes of heterotrimeric G proteins. The functional consequences of activating specific  cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that  cell G protein-coupled receptors (GPCRs) are also expressed by many other tissues. To circumvent these difficulties, we developed a chemicalgenetic approach that allows for the conditional and selective activation of specific  cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR in  cells only. Importantly, the two designer receptors differed in their G protein-coupling properties (Gq/11 versus Gs). They were unable to bind endogenous ligand(s), but could be efficiently activated by an otherwise pharmacologically inert compound (clozapine-N-oxide), leading to the conditional activation of either  cell Gq/11 or Gs G proteins. Here we report the findings that conditional and selective activation of  cell Gq/11 signaling in vivo leads to striking increases in both first-and second-phase insulin release, greatly improved glucose tolerance in obese, insulin-resistant mice, and elevated  cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of  cell Gs triggered qualitatively similar in vivo metabolic effects. Thus, this developed chemical-genetic strategy represents a powerful approach to study G protein regulation of  cell function in vivo.
Neurotoxicity Research, 1999
Epidemiological studies have shown a reduced incidence of cancer in Parkinson's disease. Since ne... more Epidemiological studies have shown a reduced incidence of cancer in Parkinson's disease. Since nearly all parkinsonian patients with clinical impairment are treated with L-l~-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine (DA)ergic agonists, a possibility exists that these therapeutic agents can influence the risk of cancer. We studied the antiproliferative effect of these therapeutic agents (and substances structurally correlated) on Chinese hamster ovary (CHO)-K1 cell growth. Among the compounds tested, apomorphine proved to be the most potent inhibitor of CHO-K1 cell growth, with an EC5o of 3.35 • 0.12 ~M. The apomorphine analogues, apocodeine and hydroxyethylnorapomorphine, were less active as inhibitors of CHO-K1 cell growth. The activity of DA, 6-hydroxydopamine (6-OHDA), phenylethylamine (PEA), L-DOPA and bromocriptine as antiproliferative was one order of magnitude lower than that of apomorphine while pergolide was ineffective. To test whether or not the oxidative potential of these compounds was important for their antiproliferative effect, several antioxidants were assayed. Among them, glutathione (GSH) and dithiothreitol (DTT) were effective in reversing the antiproliferative effect of apomorphine, DA, 6-OHDA and PEA, conversely they did not work with bromocriptine. GSH and DTT are sulphydryl-reducing agents; while their effect could explain the efficacy against apomorphine, DA and 6-OHDA, it is difficult to understand why they should have any effect on PEA as this substance does not react with sulphydryl groups. The oxidative potential as a mechanism of action was also questioned by the results obtained with dihydrorhodamine 123, a probe that changes its fluorescent emission wave when oxidized. None of the compounds, with the exception of 6-OHDA, had any effect on the fluorescent emission wave of the probe at the maximal concentrations used to inhibit CHO-K1 cell growth. At concentrations five times higher, apomorphine and DA generated reactive oxygen species but PEA and bromocriptine did not. These data demonstrate that the antiproliferative effect of these compounds is not due to their oxidative potential, but another mechanism must be postulated.
Nature Methods, 2007
G protein-coupled receptors (GPCRs) comprise one of the largest protein families found in nature.... more G protein-coupled receptors (GPCRs) comprise one of the largest protein families found in nature. Here we describe a new experimental strategy that allows rapid identification of functionally critical amino acids in the rat M(3) muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR. This approach involves low-frequency random mutagenesis of the entire M3R coding sequence, followed by the application of a new yeast genetic screen that allows the recovery of inactivating M3R single point mutations. The vast majority of recovered mutant M3Rs also showed substantial functional impairments in transfected mammalian (COS-7) cells. A subset of mutant receptors, however, behaved differently in yeast and mammalian cells, probably because of the specific features of the yeast expression system used. The screening strategy described here should be applicable to all GPCRs that can be expressed functionally in yeast.
The Journal of Physical Chemistry Letters, 2010
Illumination with 405 nm light can recover the emission for single green fluorescent protein (GFP... more Illumination with 405 nm light can recover the emission for single green fluorescent protein (GFP) mutants that have gone into a long-lived dark state. The reported behavior is the standard for the reverse photoswitchable protein Dronpa and its mutants. However, conventional knowledge regarding the mEos2 photoactivatable fluorescent protein (PA-FP) is that, once bleached, this fluorophore is hardly reactivated, aside from a minority population that might display this behavior. Here we show that, in a typical experiment, approximately 50% of the investigated single molecule time traces display multiple reactivations, making this a seemingly inherent feature of the mEos2 PA-FP. These results hint to some similarities between mEos2 and other reversibly photoactivatable probes such as Dronpa. We investigate the consequences of this phenomenon in the framework of photoactivated localization microscopy (PALM) experiments.
Journal of Pharmacology and Experimental Therapeutics, 2003
The muscarinic M(2) receptor was split at the third cytoplasmic loop into two fragments: the one ... more The muscarinic M(2) receptor was split at the third cytoplasmic loop into two fragments: the one containing the first five transmembrane regions and the N-terminal part of the third cytoplasmic loop was named M(2trunk), while the other, which contained the last two transmembrane regions and the C-terminal part of the third cytoplasmic loop, was named M(2tail). As seen in many other G protein-coupled receptors, when these two fragments were transfected together in COS-7 cells they rescued the pharmacological profile and the functional activity of the wild-type M(2) receptor. Conversely, N-[(3)H]methylscopolamine ([(3)H]NMS) association binding experiments showed a substantial difference between the wild-type M(2) and the split M(2trunk)/M(2tail) receptors. The progression of the association binding kinetic of the M(2trunk)/M(2tail) receptor was strictly dependent upon the amount of the fragment DNA transfected. When the amount of transfected DNA was 4 microg/plate and the B(max) of [(3)H]NMS at equilibrium was around 200 fmol/mg protein the form of the association was that of classical saturation, but when the amount of transfected DNA was lower the [(3)H]NMS association reached a maximum binding point and then declined to a lower equilibrium binding level. The form of the association was temperature-dependent: as the temperature was lowered, the maximum binding point tended to be higher. We suggest that this peculiar form of the [(3)H]NMS association binding to the muscarinic M(2trunk)/M(2tail) receptor is attributable to a less stable interaction between the trunk and the tail fragments of the split receptor.
Encyclopedia of Molecular Pharmacology, 2020
Second-generation antipsychotics Definition The introduction of atypical antipsychotics (AAPs), s... more Second-generation antipsychotics Definition The introduction of atypical antipsychotics (AAPs), since the discovery of its prototypical drug clozapine in the 1970s, has been a milestone in psychopharmacology for treating schizophrenia, bipolar disorder, and other brain diseases that are characterized by psychotic features‚ as these drugs allow a significant recovery not only in terms of hospitalization and reduction in symptoms severity, but also in terms of safety, socialization, and better rehabilitation in the society. Compared to typical antipsychotics (TAPs), AAPs produce minimal motor side effects, such as parkinsonism and tardive dyskinesia, and seem to be more effective in improving cognitive symptoms (Meltzer 2013). Unfortunately, AAPs may cause weight gain and metabolic adverse reactions; hence‚ patients should be constantly monitored. Besides, each AAP has a unique pharmacological profile, which explains the clinical diversity between them.
Proceedings of the National Academy of Sciences, 2010
Therapeutic strategies that augment insulin release from pancreatic β-cells are considered benefi... more Therapeutic strategies that augment insulin release from pancreatic β-cells are considered beneficial in the treatment of type 2 diabetes. We previously demonstrated that activation of β-cell M 3 muscarinic receptors (M3Rs) greatly promotes glucose-stimulated insulin secretion (GSIS), suggesting that strategies aimed at enhancing signaling through β-cell M3Rs may become therapeutically useful. M3R activation leads to the stimulation of G proteins of the G q family, which are under the inhibitory control of proteins known as regulators of G protein signaling (RGS proteins). At present, it remains unknown whether RGS proteins play a role in regulating insulin release. To address this issue, we initially demonstrated that MIN6 insulinoma cells express functional M3Rs and that RGS4 was by far the most abundant RGS protein expressed by these cells. Strikingly, siRNA-mediated knockdown of RGS4 expression in MIN6 cells greatly enhanced M3R-mediated augmentation of GSIS and calcium release. We obtained similar findings using pancreatic islets prepared from RGS4-deficient mice. Interestingly, RGS4 deficiency had little effect on insulin release caused by activation of other β-cell GPCRs. Finally, treatment of mutant mice selectively lacking RGS4 in pancreatic β-cells with a muscarinic agonist (bethanechol) led to significantly increased plasma insulin and reduced blood glucose levels, as compared to control littermates. Studies with β-cell-specific M3R knockout mice showed that these responses were mediated by β-cell M3Rs. These findings indicate that RGS4 is a potent negative regulator of M3R function in pancreatic β-cells, suggesting that RGS4 may represent a potential target to promote insulin release for therapeutic purposes. knockout mice | muscarinic receptor | RGS proteins | G protein-coupled receptor T ype 2 diabetes (T2D) has emerged as a major threat to human health worldwide. Besides peripheral insulin resistance, T2D is usually associated with β-cell dysfunction (1). Thus, the development of new drugs aimed at improving β-cell function, including stimulation of insulin release, is the focus of many laboratories (2).
The Journal of Pharmacology and Experimental Therapeutics, 2003
The muscarinic M(2) receptor was split at the third cytoplasmic loop into two fragments: the one ... more The muscarinic M(2) receptor was split at the third cytoplasmic loop into two fragments: the one containing the first five transmembrane regions and the N-terminal part of the third cytoplasmic loop was named M(2trunk), while the other, which contained the last two transmembrane regions and the C-terminal part of the third cytoplasmic loop, was named M(2tail). As seen in many other G protein-coupled receptors, when these two fragments were transfected together in COS-7 cells they rescued the pharmacological profile and the functional activity of the wild-type M(2) receptor. Conversely, N-[(3)H]methylscopolamine ([(3)H]NMS) association binding experiments showed a substantial difference between the wild-type M(2) and the split M(2trunk)/M(2tail) receptors. The progression of the association binding kinetic of the M(2trunk)/M(2tail) receptor was strictly dependent upon the amount of the fragment DNA transfected. When the amount of transfected DNA was 4 microg/plate and the B(max) of [(3)H]NMS at equilibrium was around 200 fmol/mg protein the form of the association was that of classical saturation, but when the amount of transfected DNA was lower the [(3)H]NMS association reached a maximum binding point and then declined to a lower equilibrium binding level. The form of the association was temperature-dependent: as the temperature was lowered, the maximum binding point tended to be higher. We suggest that this peculiar form of the [(3)H]NMS association binding to the muscarinic M(2trunk)/M(2tail) receptor is attributable to a less stable interaction between the trunk and the tail fragments of the split receptor.
Advances in Neurology, Feb 1, 2001
FEBS Journal, 2015
The introduction of super resolution fluorescence microscopy has allowed to visualize single prot... more The introduction of super resolution fluorescence microscopy has allowed to visualize single proteins in their biological environment. Recently, these techniques have been applied to determine the organization of class A G protein-coupled receptors (GPCRs), and to determine whether they exist as monomers, dimers and/or higher-order oligomers. On this subject, this review highlights recent evidence coming from Photoactivated Localization Microscopy (PALM) that allows the visualization of single molecules in dense samples, and Single-Molecule Tracking (SMT) that determines how GPCRs move and interact in living cells in the presence of different ligands. PALM has demonstrated that GPCR oligomerization depends on the receptor subtype, cell-type, actin cytoskeleton and other proteins. Conversely, SMT has revealed the transient dynamics of dimer formation, where receptors display a monomer-dimer equilibrium characterized by rapid association and dissociation. At steady state, depending on the subtype, approximately 30-50% of receptors are part of dimeric complexes. Notably, the existence of many GPCR di-/oligomers is also supported by using well-known techniques, such as Resonance Energy Transfer (RET) methodologies, and by approaches that exploit fluorescence fluctuations, such as Fluorescence Correlation Spectroscopy (FCS). Future research using single-molecule methods will deepen our knowledge related to function and druggability of homo- and hetero-oligomers. This article is protected by copyright. All rights reserved.
Nature methods, 2007
G protein-coupled receptors (GPCRs) comprise one of the largest protein families found in nature.... more G protein-coupled receptors (GPCRs) comprise one of the largest protein families found in nature. Here we describe a new experimental strategy that allows rapid identification of functionally critical amino acids in the rat M(3) muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR. This approach involves low-frequency random mutagenesis of the entire M3R coding sequence, followed by the application of a new yeast genetic screen that allows the recovery of inactivating M3R single point mutations. The vast majority of recovered mutant M3Rs also showed substantial functional impairments in transfected mammalian (COS-7) cells. A subset of mutant receptors, however, behaved differently in yeast and mammalian cells, probably because of the specific features of the yeast expression system used. The screening strategy described here should be applicable to all GPCRs that can be expressed functionally in yeast.
European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology, Jan 23, 2014
The brain׳s complexity derives not only from the way the intricate network of neurons is wired, b... more The brain׳s complexity derives not only from the way the intricate network of neurons is wired, but also by protein complexes that recognize and decode chemical information. G protein-coupled receptors (GPCRs) represent the most abundant family of proteins mediating neurotransmission in the brain, and their ability to form homo- and heteromers which amplifies the scope for synaptic communication and fine-tuning. Dopamine receptors are important drug targets and members of both the D1/D5 and D2/D3/D4 receptor families form homo- and heteromers. The present article focuses on D3 receptor homo- and heteromers, in particular, those formed in association with their D2 counterparts. We highlight the binding profiles and mechanisms of interaction with D3-D3 homomers and D3-D2 heteromers of: first, the PET ligand and potent agonist [(11)C]-(+)-PHNO; second, the novel, bitopic/allosteric dopamine D3 receptor antagonist, SB269,652; and third, diverse partial agonists like antipsychotic and ar...
Plos One, 2011
In this work we discuss how to use photophysical information for improved quantitative measuremen... more In this work we discuss how to use photophysical information for improved quantitative measurements using Photo Activated Localization Microscopy (PALM) imaging. We introduce a method that reliably estimates the number of photoblinking molecules present in a biological sample and gives a robust way to quantify proteins at the single-cell level from PALM images. We apply this method to determine the amount of β2 adrenergic receptor, a prototypical G Protein Coupled Receptor, expressed on the plasma membrane of HeLa cells.
Frontiers in Behavioral Neuroscience, 2013
The present review update the relationship between acetaldehyde (ACE) and parkinsonism with a spe... more The present review update the relationship between acetaldehyde (ACE) and parkinsonism with a specific focus on the role of P450 system and CYP 2E1 isozyme particularly. We have indicated that ACE is able to enhance the parkinsonism induced in mice by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin able to damage the nigrostriatal dopaminergic pathway. Similarly diethyldithiocarbamate, the main metabolite of disulfiram, a drug widely used to control alcoholism, diallylsulfide (DAS) and phenylisothiocyanate also markedly enhance the toxin-related parkinsonism. All these compounds are substrate/inhibitors of CYP450 2E1 isozyme. The presence of CYP 2E1 has been detected in the dopamine (DA) neurons of rodent Substantia Nigra (SN), but a precise function of the enzyme has not been elucidated yet. By treating CYP 2E1 knockout (KO) mice with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the SN induced lesion was significantly reduced when compared with the lesion observed in wild-type animals. Several in vivo and in vitro studies led to the conclusion that CYP 2E1 may enhance the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice by increasing free radical production inside the dopaminergic neurons. ACE is a good substrate for CYP 2E1 enzyme as the other substrate-inhibitors and by this way may facilitate the susceptibility of dopaminergic neurons to toxic events. The literature suggests that ethanol and/or disulfiram may be responsible for toxic parkinsonism in human and it indicates that basal ganglia are the major targets of disulfiram toxicity. A very recent study reports that there are a decreased methylation of the CYP 2E1 gene and increased expression of CYP 2E1 mRNA in Parkinson's disease (PD) patient brains. This study suggests that epigenetic variants of this cytochrome contribute to the susceptibility, thus confirming multiples lines of evidence which indicate a link between environmental toxins and PD.
Molecular pharmacology, 2011
The M₃ muscarinic acetylcholine receptor (M3R) regulates many fundamental physiological functions... more The M₃ muscarinic acetylcholine receptor (M3R) regulates many fundamental physiological functions. To identify novel M3R-interacting proteins, we used a recently developed yeast two-hybrid screen (split ubiquitin method) to detect interactions among membrane proteins. This screen led to the identification of many novel M3R-associated proteins, including the putative membrane protein transmembrane protein 147 (Tmem147). The amino acid sequence of Tmem147 is highly conserved among mammals, but its physiological roles are unknown at present. We initially demonstrated that Tmem147 could be coimmunoprecipitated with M3Rs in cotransfected mammalian cells (COS-7 cells). Confocal imaging studies showed that Tmem147 was localized to endoplasmic reticulum (ER) membranes and that the Tmem147/M3R interaction occurred in the ER of cotransfected COS-7 cells, resulting in impaired trafficking of the M3R to the cell surface. To study the role of Tmem147 in modulating M3R function in a more physiolo...
European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology, Jan 18, 2014
Clozapine is the most effective antipsychotic to date, but its benefits are counterbalanced by th... more Clozapine is the most effective antipsychotic to date, but its benefits are counterbalanced by the risk of severe hematological effects. In this study, we analyzed whether clozapine inhibits polymorphonuclear (PMN) leukocyte chemotaxis. We found that clozapine, within the therapeutic concentration range, potently and selectively inhibits PMN chemotaxis induced by interleukin 8 (IL-8), a chemokine inducing neutrophil migration. The effect was not due to its action at dopamine, serotonin and muscarinic receptors, or to a direct antagonism to IL-8 receptors. Furthermore, clozapine did not inhibit PMN chemotaxis by its presumed toxic mechanism. In fact, after an overnight incubation in cell culture, the drug did not increase the physiological PMN apoptosis. An interference of clozapine with the autocrine release of leukotriene B4 (LTB4), a secondary chemoattractant secreted by neutrophils in response to the primary chemoattractant IL-8, was hypothesized. In agreement with this hypothesi...
Methods in enzymology, 2013
Before the molecular biology era, functional experiments on isolated organs and radioligand bindi... more Before the molecular biology era, functional experiments on isolated organs and radioligand binding and biochemical experiments on animal tissues were widely used to characterize G protein-coupled receptors (GPCRs). The introduction of recombinant cell lines expressing a single GPCR type has been a big step forward for studying both drug-receptor interactions and signal transduction. Before the introduction of the concept of receptor oligomerization, all data generated were attributed to the interaction of drugs with receptor monomers. Now, considerable data must be reinterpreted in light of receptor homo- and heteromerization. In this chapter, we will review some of the methods used to study radioligand binding and signal transduction modifications induced by GPCR homo- and heteromerization.
Proceedings of the National Academy of Sciences, 2009
Impaired functioning of pancreatic  cells is a key hallmark of type 2 diabetes.  cell function ... more Impaired functioning of pancreatic  cells is a key hallmark of type 2 diabetes.  cell function is modulated by the actions of different classes of heterotrimeric G proteins. The functional consequences of activating specific  cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that  cell G protein-coupled receptors (GPCRs) are also expressed by many other tissues. To circumvent these difficulties, we developed a chemicalgenetic approach that allows for the conditional and selective activation of specific  cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR in  cells only. Importantly, the two designer receptors differed in their G protein-coupling properties (Gq/11 versus Gs). They were unable to bind endogenous ligand(s), but could be efficiently activated by an otherwise pharmacologically inert compound (clozapine-N-oxide), leading to the conditional activation of either  cell Gq/11 or Gs G proteins. Here we report the findings that conditional and selective activation of  cell Gq/11 signaling in vivo leads to striking increases in both first-and second-phase insulin release, greatly improved glucose tolerance in obese, insulin-resistant mice, and elevated  cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of  cell Gs triggered qualitatively similar in vivo metabolic effects. Thus, this developed chemical-genetic strategy represents a powerful approach to study G protein regulation of  cell function in vivo.
Neurotoxicity Research, 1999
Epidemiological studies have shown a reduced incidence of cancer in Parkinson's disease. Since ne... more Epidemiological studies have shown a reduced incidence of cancer in Parkinson's disease. Since nearly all parkinsonian patients with clinical impairment are treated with L-l~-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine (DA)ergic agonists, a possibility exists that these therapeutic agents can influence the risk of cancer. We studied the antiproliferative effect of these therapeutic agents (and substances structurally correlated) on Chinese hamster ovary (CHO)-K1 cell growth. Among the compounds tested, apomorphine proved to be the most potent inhibitor of CHO-K1 cell growth, with an EC5o of 3.35 • 0.12 ~M. The apomorphine analogues, apocodeine and hydroxyethylnorapomorphine, were less active as inhibitors of CHO-K1 cell growth. The activity of DA, 6-hydroxydopamine (6-OHDA), phenylethylamine (PEA), L-DOPA and bromocriptine as antiproliferative was one order of magnitude lower than that of apomorphine while pergolide was ineffective. To test whether or not the oxidative potential of these compounds was important for their antiproliferative effect, several antioxidants were assayed. Among them, glutathione (GSH) and dithiothreitol (DTT) were effective in reversing the antiproliferative effect of apomorphine, DA, 6-OHDA and PEA, conversely they did not work with bromocriptine. GSH and DTT are sulphydryl-reducing agents; while their effect could explain the efficacy against apomorphine, DA and 6-OHDA, it is difficult to understand why they should have any effect on PEA as this substance does not react with sulphydryl groups. The oxidative potential as a mechanism of action was also questioned by the results obtained with dihydrorhodamine 123, a probe that changes its fluorescent emission wave when oxidized. None of the compounds, with the exception of 6-OHDA, had any effect on the fluorescent emission wave of the probe at the maximal concentrations used to inhibit CHO-K1 cell growth. At concentrations five times higher, apomorphine and DA generated reactive oxygen species but PEA and bromocriptine did not. These data demonstrate that the antiproliferative effect of these compounds is not due to their oxidative potential, but another mechanism must be postulated.
Nature Methods, 2007
G protein-coupled receptors (GPCRs) comprise one of the largest protein families found in nature.... more G protein-coupled receptors (GPCRs) comprise one of the largest protein families found in nature. Here we describe a new experimental strategy that allows rapid identification of functionally critical amino acids in the rat M(3) muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR. This approach involves low-frequency random mutagenesis of the entire M3R coding sequence, followed by the application of a new yeast genetic screen that allows the recovery of inactivating M3R single point mutations. The vast majority of recovered mutant M3Rs also showed substantial functional impairments in transfected mammalian (COS-7) cells. A subset of mutant receptors, however, behaved differently in yeast and mammalian cells, probably because of the specific features of the yeast expression system used. The screening strategy described here should be applicable to all GPCRs that can be expressed functionally in yeast.
The Journal of Physical Chemistry Letters, 2010
Illumination with 405 nm light can recover the emission for single green fluorescent protein (GFP... more Illumination with 405 nm light can recover the emission for single green fluorescent protein (GFP) mutants that have gone into a long-lived dark state. The reported behavior is the standard for the reverse photoswitchable protein Dronpa and its mutants. However, conventional knowledge regarding the mEos2 photoactivatable fluorescent protein (PA-FP) is that, once bleached, this fluorophore is hardly reactivated, aside from a minority population that might display this behavior. Here we show that, in a typical experiment, approximately 50% of the investigated single molecule time traces display multiple reactivations, making this a seemingly inherent feature of the mEos2 PA-FP. These results hint to some similarities between mEos2 and other reversibly photoactivatable probes such as Dronpa. We investigate the consequences of this phenomenon in the framework of photoactivated localization microscopy (PALM) experiments.
Journal of Pharmacology and Experimental Therapeutics, 2003
The muscarinic M(2) receptor was split at the third cytoplasmic loop into two fragments: the one ... more The muscarinic M(2) receptor was split at the third cytoplasmic loop into two fragments: the one containing the first five transmembrane regions and the N-terminal part of the third cytoplasmic loop was named M(2trunk), while the other, which contained the last two transmembrane regions and the C-terminal part of the third cytoplasmic loop, was named M(2tail). As seen in many other G protein-coupled receptors, when these two fragments were transfected together in COS-7 cells they rescued the pharmacological profile and the functional activity of the wild-type M(2) receptor. Conversely, N-[(3)H]methylscopolamine ([(3)H]NMS) association binding experiments showed a substantial difference between the wild-type M(2) and the split M(2trunk)/M(2tail) receptors. The progression of the association binding kinetic of the M(2trunk)/M(2tail) receptor was strictly dependent upon the amount of the fragment DNA transfected. When the amount of transfected DNA was 4 microg/plate and the B(max) of [(3)H]NMS at equilibrium was around 200 fmol/mg protein the form of the association was that of classical saturation, but when the amount of transfected DNA was lower the [(3)H]NMS association reached a maximum binding point and then declined to a lower equilibrium binding level. The form of the association was temperature-dependent: as the temperature was lowered, the maximum binding point tended to be higher. We suggest that this peculiar form of the [(3)H]NMS association binding to the muscarinic M(2trunk)/M(2tail) receptor is attributable to a less stable interaction between the trunk and the tail fragments of the split receptor.