Paradoxical sleep deprivation in rats causes a selective reduction in the expression of type-2 metabotropic glutamate receptors in the hippocampus.

Paradoxical sleep deprivation in rats is considered as an experimental animal model of mania endowed with face, construct, and pharmacological validity. We induced paradoxical sleep deprivation by placing rats onto a small platform surrounded by water. This procedure caused the animal to fall in the water at the onset of REM phase of sleep. Control rats were either placed onto a larger platform (which allowed them to sleep) or maintained in their home cage. Sleep deprived rats showed a substantial reduction in type-2 metabotropic glutamate (mGlu2) receptors mRNA and protein levels in the hippocampus, but not in the prefrontal cortex or corpus striatum, as compared to both groups of control rats. No changes in the expression of mGlu3 receptor mRNA levels or mGlu1α and mGlu5 receptor protein levels were found with exception of an increase in mGlu1α receptor levels in the striatum of SD rats. Moving from these findings we treated SD and control rats with the selective mGlu2 receptor enhancer, BINA (30mg/kg, i.p.). SD rats were also treated with sodium valproate (300mg/kg, i.p.) as an active comparator. Both BINA and sodium valproate were effective in reversing the manic-like phenotype evaluated in an open field arena in SD rats. BINA treatment had no effect on motor activity in control rats, suggesting that our findings were not biased by a non-specific motor-lowering activity of BINA. These findings suggest that changes in the expression of mGlu2 receptors may be associated with the enhanced motor activity observed with mania.

Stress-related methylation of the catechol-O-methyltransferase Val 158 allele predicts human prefrontal cognition and activity.

DNA methylation at CpG dinucleotides is associated with gene silencing, stress, and memory. The catechol-O-methyltransferase (COMT) Val(158) allele in rs4680 is associated with differential enzyme activity, stress responsivity, and prefrontal activity during working memory (WM), and it creates a CpG dinucleotide. We report that methylation of the Val(158) allele measured from peripheral blood mononuclear cells (PBMCs) of Val/Val humans is associated negatively with lifetime stress and positively with WM performance; it interacts with stress to modulate prefrontal activity during WM, such that greater stress and lower methylation are related to reduced cortical efficiency; and it is inversely related to mRNA expression and protein levels, potentially explaining the in vivo effects. Finally, methylation of COMT in prefrontal cortex and that in PBMCs of rats are correlated. The relationship of methylation of the COMT Val(158) allele with stress, gene expression, WM performance, and related brain activity suggests that stress-related methylation is associated with silencing of the gene, which partially compensates the physiological role of the high-activity Val allele in prefrontal cognition and activity. Moreover, these results demonstrate how stress-related DNA methylation of specific functional alleles impacts directly on human brain physiology beyond sequence variation.

A liquid chromatography-tandem mass spectrometry method for the determination of 5-Fluorouracil degradation rate by intact peripheral blood mononuclear cells.

5-Fluorouracil (5-FU) is a major chemotherapy drug used for the treatment of tumors. It is catabolized mainly by dihydropyrimidine dehydrogenase, and patients with a complete or partial deficiency of dihydropyrimidine dehydrogenase activity are at risk of developing severe 5-FU-associated toxicity. The aim of this study was to demonstrate that intact peripheral blood mononuclear cells (PBMCs) can be an effective model to evaluate the degradation rate of 5-FU. We developed a sensitive and specific liquid chromatography-tandem mass spectrometry method to measure in vitro the rate of 5-FU degradation by intact PBMC. 5-FU degradation rate was determined by measuring the decrease of a fixed amount of 5-FU (10 microg/mL) added to a solution of PBMC, after 2 hours incubation, expressed as nanogram per milliliter of 5-FU degraded per minute x 10(6) cells. Freshly prepared intact PBMC can degrade efficiently in vitro-added 5-FU. The assay consists of 3 steps: (1) PBMC isolation from peripheral blood, (2) PBMC incubation with 5-FU in vitro for different times, and (3) determination of 5-FU amount to calculate the degradation rate. 5-FU was analyzed by a Q Trap 2000 triple quadrupole/ion trap mass spectrometer in the multiple-reaction-monitoring modes. The chromatographic separation was accomplished using a C18 column with a run time of 16 minutes. By analyzing samples from 39 patients with no 5-FU toxicity, the mean 5-FU degradation rate was 1.85 +/- 0.50 ng x mL(-1) x min(-1) x 10(6) cells. The assessment of a test to measure 5-FU degradation rate in PBMC of patients before 5-FU administration could represent a prescreening method for evaluating the possible toxicity of this drug as an aid to set up a personalized medicine approach for each patient.

Involvement of dopamine receptors and beta-arrestin in metamphetamine-induced inclusions formation in PC12 cells.

Exposure of PC12 cells to metamphetamine (MA) induces the formation of multilamellar structures (whorls) resembling autophagic granules that subsequently develop as intracellular inclusions. These inclusions stain for a variety of antigens belonging to the ubiquitin proteasome pathway. Since MA-induced intracellular bodies require the presence of dopamine in the present study we analyzed the role of dopamine (DA) receptors in producing neuronal inclusions. Moreover, we investigated potential signaling pathways which could lead to ubiquitination in the presence of MA. Based on recent reports that ubiquitination of beta-adrenergic receptors is promoted by beta-arrestin which shuttles proteins from the plasma membrane to the ubiquitin proteasome system we investigated whether beta-arrestin is involved in MA-induced inclusion formation. Our experiments document that (i) beta-arrestin was associated with MA-induced intracellular bodies; (ii) MA induced a rapid and reversible ubiquitination of beta-arrestin; (iii) dopamine antagonists reduced both MA-induced beta-arrestin ubiquitination and intracellular whorls formation; (iv) the number of MA-induced intracellular bodies was reduced in cells transfected with the beta-arrestin dominant negative mutant, betaarrV53D and was increased by the persistently ubiquitinated beta-arrestin-ubiquitin fusion protein. In conclusion, the present study demonstrates the involvement of beta-arrestin in MA-induced intracellular bodies and the participation of dopamine receptors in this process.

Tyrosine phosphorylation of G-protein-coupled-receptor kinase 2 (GRK2) by c-Src modulates its interaction with Galphaq.

G-protein-coupled-receptor kinase 2 (GRK2) plays a key role in the modulation of G-protein-coupled-receptor (GPCR) signaling by both phosphorylating agonist-occupied GPCRs and by directly binding to activated Galphaq subunits, inhibiting downstream effectors activation. The GRK2/Galphaq interaction involves the N-terminal region of the kinase that displays homology to regulators of G-protein signaling (RGS) proteins. We have previously reported that upon GPCR stimulation, GRK2 can be phosphorylated by c-Src on tyrosine residues that are present in the RGS-homology (RH) region of this kinase. Here, we demonstrate that c-Src kinase activity increases the interaction between GRK2 and Galphaq. Tyrosine phosphorylation of GRK2 appears to be critically involved in the modulation of this interaction since the stimulatory effect of c-Src is not observed with a GRK2 mutant with impaired tyrosine phosphorylation (GRK2 Y13,86,92F), whereas a mutant that mimics GRK2 tyrosine phosphorylation in these residues displays an increased interaction with Galphaq. As evidence for a physiological role of this modulatory mechanism, activation of the muscarinic receptor M1, a Galphaq-coupled receptor, promotes an increase in GRK2/Galphaq co-immunoprecipitation that parallels the enhanced GRK2 phosphorylation on tyrosine residues. Moreover, c-Src activation enhances inhibition of the Galphaq/phospholipase Cbeta signaling pathway in intact cells, in a GRK2-tyrosine-phosphorylation-dependent manner. Our results suggest a feedback mechanism by which phosphorylation of GRK2 by c-Src increases both GRK2 kinase activity towards GPCRs and its specific interaction with Galphaq subunits, leading to a more rapid switch off of Galphaq-mediated signaling.

Type-7 metabotropic glutamate receptors negatively regulate α1-adrenergic receptor signalling.

We studied the interaction between mGlu7 and α1-adrenergic receptors in heterologous expression systems, brain slices, and living animals. L-2-Amino-4-phosphonobutanoate (L-AP4), and l-serine-O-phosphate (L-SOP), which activate group III mGlu receptors, restrained the stimulation of polyphosphoinositide (PI) hydrolysis induced by the α1-adrenergic receptor agonist, phenylephrine, in HEK 293 cells co-expressing α1-adrenergic and mGlu7 receptors. The inibitory action of L-AP4 was abrogated by (i) the mGlu7 receptor antagonist, XAP044; (ii) the C-terminal portion of type-2 G protein coupled receptor kinase; and (iii) the MAP kinase inhibitors, UO126 and PD98059. This suggests that the functional interaction between mGlu7 and α1-adrenergic receptors was mediated by the ßγ-subunits of the Gi protein and required the activation of the MAP kinase pathway. Remarkably, activation of neither mGlu2 nor mGlu4 receptors reduced α1-adrenergic receptor-mediated PI hydrolysis. In mouse cortical slices, both L-AP4 and L-SOP were able to attenuate norepinephrine- and phenylephrine-stimulated PI hydrolysis at concentrations consistent with the activation of mGlu7 receptors. L-AP4 failed to affect norepinephrine-stimulated PI hydrolysis in cortical slices from mGlu7-/- mice, but retained its inhibitory activity in slices from mGlu4-/- mice. At behavioural level, i.c.v. injection of phenylephrine produced antidepressant-like effects in the forced swim test. The action of phenylephrine was attenuated by L-SOP, which was inactive per se. Finally, both phenylephrine and L-SOP increased corticosterone levels in mice, but the increase was halved when the two drugs were administered in combination. Our data demonstrate that α1-adrenergic and mGlu7 receptors functionally interact and suggest that this interaction might be targeted in the treatment of stress-related disorders.

Convergent roles of alpha-synuclein, DA metabolism, and the ubiquitin-proteasome system in nigrostriatal toxicity.

Recent studies disclosed the relevance of specific molecules for the onset of Parkinson's disease (PD) and for the composition of neuronal inclusions. The scenario which is now emerging leads to identify a potential common pathway named the ubiquitin-proteasome (UP) system. In line with this, striatal or systemic inhibiton of the UP system causes experimental Parkinsonism characterized by the formation of neuronal inclusions. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is also a complex I inhibitor, has been used for decades to produce experimental Parkinsonism with no evidence for neuronal inclusions in rodents. This leaves open the question whether neuronal inclusions need an alternative mechanism or the inhibition of complex I needs to be carried out continuously to build up inclusions. In the present article, we administered continuously MPTP. In these experimental conditions we compared the neurological consequence of intermittent versus continuous MPTP. In both cases we observed a severe dopamine (DA) denervation and cell loss. However, when MPTP was delivered continuously, spared DA nigral neurons develop ubiquitin, parkin, and alpha-synuclein positive inclusions, which are not detectable after intermittent dosing. The onset of Parkinsonism is associated with inhibition of the UP system. We compared these results with those obtained with amphetamine derivative in vivo and in vitro in which occurrence of neuronal inclusions was associated with inhibition of the UP system and we evaluated the role of DA metabolism in inducing these effects.

The platelet maximum number of A2A-receptor binding sites (Bmax) linearly correlates with age at onset and CAG repeat expansion in Huntington's disease patients with predominant chorea.

Huntington's disease (HD) is caused by an expanded CAG mutation and may show a heterogeneous clinical presentation. To date, although the age at onset mostly depends on the expanded CAG repeat number, no validated easy-to-test biomarkers exist either for following up patients progression rate or for exactly predicting age at onset (defined as the time when motor clinical manifestations first became noticeable). We tested the function of A(2A) receptor, strongly expressed in the brain striatum and peripheral cells, in patients' blood platelets and confirmed a maximum number of binding sites (B(max)) higher than in controls (216 +/- 9 versus 137 +/- 7; p=0.0001). We found a linear correlation between the receptor B(max) and the expanded CAG repeat number (n=52, r(2)=0.19, p=0.0011). When we selected the patients according to their clinical presentation (according to the predominating motor manifestations) and plotted the receptor B(max) against patients' age at onset, we found a significant linear correlation only when considering those subjects with chorea predominant on all other motor symptoms (n=26, r(2)=0.39, p=0.0007). Because the typical chorea may depend on early dysfunction of the striatum in HD, peripheral A(2A) amplification in blood platelets might reflect a central dysfunction in this part of the brain. Further studies on a larger sample size should confirm whether the analysis of A(2A)-receptor binding in patients' blood could be a useful clinical marker according to the patients' phenotype.

BDNF rs6265 methylation and genotype interact on risk for schizophrenia.

Epigenetic mechanisms can mediate gene-environment interactions relevant for complex disorders. The BDNF gene is crucial for development and brain plasticity, is sensitive to environmental stressors, such as hypoxia, and harbors the functional SNP rs6265 (Val(66)Met), which creates or abolishes a CpG dinucleotide for DNA methylation. We found that methylation at the BDNF rs6265 Val allele in peripheral blood of healthy subjects is associated with hypoxia-related early life events (hOCs) and intermediate phenotypes for schizophrenia in a distinctive manner, depending on rs6265 genotype: in ValVal individuals increased methylation is associated with exposure to hOCs and impaired working memory (WM) accuracy, while the opposite is true for ValMet subjects. Also, rs6265 methylation and hOCs interact in modulating WM-related prefrontal activity, another intermediate phenotype for schizophrenia, with an analogous opposite direction in the 2 genotypes. Consistently, rs6265 methylation has a different association with schizophrenia risk in ValVals and ValMets. The relationships of methylation with BDNF levels and of genotype with BHLHB2 binding likely contribute to these opposite effects of methylation. We conclude that BDNF rs6265 methylation interacts with genotype to bridge early environmental exposures to adult phenotypes, relevant for schizophrenia. The study of epigenetic changes in regions containing genetic variation relevant for human diseases may have beneficial implications for the understanding of how genes are actually translated into phenotypes.

Selective blockade of mGlu5 metabotropic glutamate receptors is protective against methamphetamine neurotoxicity.

Methamphetamine (MA), a widely used drug of abuse, produces oxidative damage of nigrostriatal dopaminergic terminals. We examined the effect of subtype-selective ligands of metabotropic glutamate (mGlu) receptors on MA neurotoxicity in mice. MA (5 mg/kg, i.p.; injected three times, every 2 hr) induced, 5 d later, a substantial degeneration of striatal dopaminergic terminals associated with reactive gliosis. MA toxicity was primarily attenuated by the coinjection of the noncompetitive mGlu5 receptor antagonists 2-methyl-6-(phenylethynyl)pyridine and (E)-2-methyl-6-styrylpyridine both at 10 mg/kg, i.p.). In contrast, the mGlu1 receptor antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (10 mg/kg, i.p.), and the mGlu2/3 receptor agonist (-)-2-oxa-4-aminocyclo[3.1.0]hexane-4,6-dicarboxylic acid (1 mg/kg, i.p.), failed to affect MA toxicity. mGlu5 receptor antagonists reduced the production of reactive oxygen species but did not reduce the acute stimulation of dopamine release induced by MA both in striatal synaptosomes and in the striatum of freely moving mice. We conclude that endogenous activation of mGlu5 receptors enables the development of MA neurotoxicity and that mGlu5 receptor antagonists are neuroprotective without interfering with the primary mechanism of action of MA.

Interferon beta-1a counteracts effects of activation on the expression of G-protein-coupled receptor kinases 2 and 3, beta-arrestin-1, and regulators of G-protein signalling 2 and 16 in human mononuclear leukocytes.

Activation regulates the responsiveness of G-protein-coupled receptors (GPCRs) on T cells, and modifications in the activity of GPCRs characterize lymphocytes from some immune disorders such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Some lines of evidence suggest that such an effect is connected with the altered expression of some GPCRs regulatory proteins. Herein we demonstrate that phitoemagglutinin (PHA)-induced activation leads to differential expression of G-protein-coupled receptor kinase (GRK) 2, GRK3, beta-arrestin-1, regulators of G-protein signalling (RGS) 2, and RGS16 and decreases responsiveness of mononuclear leukocytes (MNL) to the beta-adrenergic agonist isoproterenol. Interferon beta-1a (IFN beta-1a), which is known to ameliorate the course of MS, counteracts the activation-induced effects on the expression of these GPCR regulatory proteins in MNL. Furthermore, IFN beta-1a quenches the effects of PHA on the isoproterenol-induced accumulation of cyclic AMP (cAMP). We suggest that regulation of GPCRs responsiveness may be a relevant property of IFN beta-1a in MS.

Xanthurenic Acid Activates mGlu2/3 Metabotropic Glutamate Receptors and is a Potential Trait Marker for Schizophrenia.

The kynurenine pathway of tryptophan metabolism has been implicated in the pathophysiology of psychiatric disorders, including schizophrenia. We report here that the kynurenine metabolite, xanturenic acid (XA), interacts with, and activates mGlu2 and mGlu3 metabotropic glutamate receptors in heterologous expression systems. However, the molecular nature of this interaction is unknown, and our data cannot exclude that XA acts primarily on other targets, such as the vesicular glutamate transporter, in the CNS. Systemic administration of XA in mice produced antipsychotic-like effects in the MK-801-induced model of hyperactivity. This effect required the presence of mGlu2 receptors and was abrogated by the preferential mGlu2/3 receptor antagonist, LY341495. Because the mGlu2 receptor is a potential drug target in the treatment of schizophrenia, we decided to measure serum levels of XA and other kynurenine metabolites in patients affected by schizophrenia. Serum XA levels were largely reduced in a large cohort of patients affected by schizophrenia, and, in patients with first-episode schizophrenia, levels remained low after 12 months of antipsychotic medication. As opposed to other kynurenine metabolites, XA levels were also significantly reduced in first-degree relatives of patients affected by schizophrenia. We suggest that lowered serum XA levels might represent a novel trait marker for schizophrenia.

Analysis of differential modulatory activities of GRK2 and GRK4 on Galphaq-coupled receptor signaling.

G-protein-coupled receptor kinases (GRK) contain a regulator of G-protein signaling (RGS)-like domain located at the N terminus (GRK-Nter) of their sequence. This domain is present in all the GRK subtypes, but the RGS-like domain of GRK2 was documented to be functionally active, as it is able to interact selectively with Galphaq (both in vitro and in cells) and to inhibit Galphaq-dependent signaling. In contrast GRK4, GRK5, and GRK6 are unable to interact with Galphaq. This article describes the methodology to investigate the modulatory activity of GRK2 and GRK4 on GPCR-stimulated Galphaq signaling. This analysis is essentially based on three types of experiments: (a) study of the effect of the GRK-Nter on GPCR-dependent signaling; (b) analysis of the binding of GRK-Nter to Galphaq in vitro; and (c) analysis of the interaction of GRK with Galphaq in cells.

Differential regulation of membrane bound and soluble ICAM 1 in human endothelium and blood mononuclear cells: effects of interferon beta-1a.

The membrane-associated Intercellular Adhesion Molecule 1 (mICAM 1) is fundamental for adhesion of leukocytes to endothelial cells. A soluble form of ICAM 1 (sICAM 1) exists in the human serum, and is seen as marker of disease activity in patients suffering from Multiple Sclerosis (MS). High levels of sICAM 1 have been detected in MS patients benefiting from interferon beta (IFNbeta) treatment, but little is known on the molecular origins of sICAM 1. This study investigated the interrelationship and the mechanisms of production of sICAM 1 and mICAM 1 in human endothelium (Human Umbilical Vein Endothelial Cells, HUVECs) and mononuclear leukocytes (MNL) upon stimulation with IFNbeta-1a and other inducers. We found that the expression of mICAM 1 and the release of sICAM 1 are differentially regulated in both these cytotypes. HUVECs and MNL express specific mRNA for both mICAM 1 and sICAM 1, and modification of the content of each of these transcripts results in regulation of both the ICAM 1 isoforms. We show that IFNbeta-1a is strong regulator of the ICAM 1 RNA splicing machinery. Effect of IFNbeta-1a over expression of the ICAM 1 isoforms might have a relevant immunomoregulatory role in Multiple Sclerosis.

Expression of OP4 (ORL1, NOP1) receptors in vascular endothelium.

Endothelial cells from rat brain microvessels, human aortic artery and human umbilical vein were examined, together with ex vivo rat brain capillaries and rat aortic ring sections, for the expression of opioid receptor-like OP-4 mRNA and protein. High levels of mRNA expression and an immunopositive reaction for the receptor protein were detected in the endothelial cells from primary and from established in vitro cultures, as well as in the intima of ex vivo rat aortic rings, where the signal was limited to the endothelial layer. Interaction of the OP4 receptor with its physiological ligand nociceptin caused, in cultured endothelial cells, the activation of a mitogen-activated protein (MAP) kinase cascade. Taken together, these results show that the OP4 receptor is synthesised and functionally expressed in endothelial cells, presumably as a starting point for some vasoactive mechanism(s).

Viral infection protocols.

This chapter describes the protocol for preparation of recombinant adenoviruses and infection of target cells to express transiently G-protein-coupled receptors or other proteins of interest. Adenoviruses are nonenveloped viruses containing a linear double-stranded DNA genome. Their life cycle does not normally involve integration into the host genome, rather they replicate as episomal elements in the nucleus of the host cell and consequently there is no risk of insertional mutagenesis. The wild-type adenovirus genome is approx 35 kb, of which up to 30 kb can be replaced by foreign DNA. Adenoviral vectors are very efficient at transducing the gene of interest in target cells in vitro and in vivo and can be produced at high titers (>10(11)/mL). The viral infection has a number of useful features: (1) the efficiency of gene transduction is very high (up to 100% in sensitive cells). (2) The infection is easy and does not alter physically the cell membrane for gene transduction. (3) It is possible to infect cells that are resistant to transfection with plasmids (including nondividing cells).

Molecular mechanisms that desensitize metabotropic glutamate receptor signaling: an overview.

The purpose of the present article is to review our actual knowledge on the desensitization of metabotropic glutamate receptors based on the literature available so far, with the attempt to emphasize all converging data and to give a possible explanation to those evidences that still remain controversial. 1. We review our knowledge on the regulation of mGlu receptors based on the available literature 2. We report converging data and we comment on issues that still remain controversial. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Viral infection for GPCR expression in eukaryotic cells.

This chapter describes the protocol for the preparation of recombinant adenoviruses and infection of target cells to transiently express G protein-coupled receptors (GPCRs) or other proteins of interest. Adenoviruses are non-enveloped viruses containing a linear double-stranded DNA genome. Their life cycle does not normally involve integration into the host genome, rather they replicate as episomal -elements in the nucleus of the host cell, and consequently there is no risk of insertional mutagenesis. Up to 30 kb out of the 35 kb of the wild-type adenovirus genome can be replaced by foreign DNA. Adenoviral vectors are very efficient in transducing target cells in vitro and in vivo and can be produced at high titers (>10¹¹/mL). The viral infection has a number of useful features: (1) the efficiency of gene transduction is very high (up to 100% in sensitive cells); (2) the infection is easy and does not physically alter the cell membrane for gene transduction; (3) it is possible to infect cells that are resistant to transfection with plasmids (including nondividing cells); and (4) the viral vectors can be used for infection in vivo (including gene therapy) and can potentially be targeted cell-specifically.

Reduced insulin-induced phosphatidylinositol-3-kinase activation in peripheral blood mononuclear leucocytes from patients with Alzheimer's disease.

The epidemiological finding of an increased risk of dementia in patients with diabetes mellitus has raised the hypothesis that a dysfunction of the insulin receptors plays a role in the pathogenesis of Alzheimer's disease (AD). A possible link is suggested by the evidence that the insulin-stimulated phosphatidylinositol-3-kinase (PI-3-K)/phospho-Akt pathway negatively controls the glycogen synthase kinase-3beta. The activation of this enzyme mediates the hyperphosphorylation of the tau protein, a relevant step in the formation of the neurofibrillary tangles associated with AD. We hypothesized that the neurodegeneration associated with AD is related to an impairment of the intracellular signalling stimulated by insulin receptors. To test this hypothesis we assessed the PI-3-K/phospho-Akt pathway following in-vitro challenge with insulin in peripheral blood mononuclear cells from subjects with AD (n = 20) and controls (n = 20). We found that the stimulation of PI-3-K is blunted in patients with AD with respect to control. The reduction did not correlate with the extent of cognitive decline or with scores at neuropsychological tests exploring attention, memory, language or visuospatial abilities. The study supports the hypothesis that an impaired control of glycogen synthase kinase-3beta activity by insulin receptor-mediated signalling plays a role in the pathogenesis of AD, facilitating tau protein phosphorylation and neurofibrillary tangle formation.

The neurotoxicity of amphetamines: bridging drugs of abuse and neurodegenerative disorders.

Amphetamine derivatives are the most commonly abused drugs. These compounds have been known for many years to induce neurotoxicity. However, recent findings have highlighted novel alterations produced by amphetamines in the central nervous system consisting of neuronal inclusions and the involvement of proteins belonging to a multi-enzymatic complex known as the ubiquitin-proteasome system. These ultrastructural and molecular changes are similar to those that occur during degenerative processes that affect the basal ganglia, and in particular Parkinson's disease, which is characterized by ubiquitin-containing neuronal inclusions in the subtantia nigra. This is recently confirmed by the occurrence of ubiquitin immunoreactive structures in the substantia nigra of humans abusing methamphetamines. In this article, we propose that the neurotoxicity of amphetamines and degenerative disorders share a number of steps in their mechanism of action involving the ubiquitin-proteasome system. The fine tuning of this ubiquitous proteolytic pathway is now being elucidated because G-protein-coupled receptors and signaling proteins such as beta-arrestin regulate access to this catalytic machinery. The identification of the ubiquitin-proteasome pathway and beta-arrestin as molecular targets of neurotoxicity is expected to provide novel therapeutic strategies both for the treatment of drug addiction and the treatment of neurodegenerative disorders.