Mice treated with the psychostimulant methamphetamine (MA) showed the appearance of intracellular inclusions in the nucleus of medium sized striatal neurones and cytoplasm of neurones of the substantia nigra pars compacta but not in the frontal cortex. All inclusions contained ubiquitin, the ubiquitin activating enzyme (E1), the ubiquitin protein ligase (E3-like, parkin), low and high molecular weight heat shock proteins (HSP 40 and HSP 70). Inclusions found in nigral neurones stained for a-synuclein, a proteic hallmark of Lewy bodies that are frequently observed in Parkinson's disease and other degenerative disorders. However, differing from classic Lewy bodies, MA-induced neuronal inclusions appeared as multilamellar bodies resembling autophagic granules. Methamphetamine reproduced this effect in cultured PC12 cells, which offered the advantage of a simple cellular model for the study of the molecular determinants of neuronal inclusions. PC12 inclusions, similar to those observed in nigral neurones, were exclusively localized in the cytoplasm and stained for a-synuclein. Time-dependent experiments showed that inclusions underwent a progressive fusion of the external membranes and developed an electrodense core. Inhibition of dopamine synthesis by a-methyl-p-tyrosine (aMpT), or administering the antioxidant S-apomorphine largely attenuated the formation of inclusions in PC12 cells exposed to MA. Inclusions were again observed when aMpTtreated cells were loaded with L-DOPA, which restored intracellular dopamine levels.

The involvement of mitogen-activated protein (MAP) kinases in the mitogenic effect of thyrotropin (TSH) is not fully elucidated. In FRTL-5 cells, we found that the MAP kinase kinase (MEK) inhibitors UO126 and PD98059 substantially decreased TSH-induced DNA synthesis, indicating that MAP kinases are involved in the TSH-stimulated proliferative response. Accordingly, TSH, forskolin (FSK) and 8-bromo-cAMP induced a rapid (3 min) and transient activation of ERK1/2, as assessed by phosphorylation of myelin basic protein and ERK1/2. This effect was cAMP-dependent and protein kinase A (PKA)-independent. The activation of Rap1 and B-Raf was involved in the mechanism of MAP kinase stimulation by TSH. TSH induced rapid (3 min) GDP/GTP exchange and activation of Rap1. After a 3-min exposure to FSK, B-Raf was recruited to a vesicular compartment, where it colocalized with Rap1. Both activation of Rap1 and translocation of B-Raf were PKA-independent. The Rap1 dominant negative Rap1N17 significantly reduced TSH-stimulated but not insulin-like growth factor 1-stimulated ERK1/2 phosphorylation, whereas the Ras dominant negative RasN17 inhibited the effect of both agonists. In conclusion, our results document that TSH increases intracellular cAMP, which rapidly stimulates MAP kinase cascade independent of PKA. This novel mechanism could integrate other pathways involved in TSH-stimulated proliferative response.

The metabotropic glutamate 1 (mGlu 1 ) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G proteincoupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu 1 receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu 1 receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated G␣ q . In cells transfected with GRK4 and exposed to agonist, ␤-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu 1 receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of ␤-arrestin V53D dominant negative mutant, which did not affect the mGlu 1 receptor internalization, reduced by 70 -80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu 1 receptor. The agonist-stimulated differential sorting of the mGlu 1 receptor and ␤-arrestin as well as the activation of MAP kinases by mGlu 1 agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of ␤-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing ␤-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu 1 receptor by different mechanisms and that ␤-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule.Metabotropic glutamate (mGlu) 1 receptors, which are activated by the excitatory amino acid glutamate, are part of an original family of G protein-coupled receptors (GPCR) called the family 3 GPCRs (1-3). These include all the mGlu receptor subtypes, Ca 2ϩ -sensing and GABA B receptors, and some putative olfactory, pheromone, and taste receptors. Eight subtypes of mGlu receptors have been identified, which are implicated in different aspects of physiology and pathology of the central nervous system. Group I mGlu receptors (mGlu 1 and mGlu 5 ), which stimulate polyphosphoinositide hydrolysis by coupling to G q , are localized in the peripheral parts of postsynaptic dendrites and contribute to the regulation of synaptic plasticity. For example, mGlu 1 receptor present in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Similar to many other GPCRs, the signal transduction of the mGlu 1 receptor is strictly regulated by multiple mechanisms acting at different levels of signal propagation (1). After prolonged or repeated stimulation, receptors are profoundly desensitized. Protein kinase C is clearly involved in this process, although a protein kinase C-independent component of mGlu 1 receptor desensitization was also observed (4). The activated ␣ subunit of the G q (G␣ q ) ca...

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Alzheimer's disease (AD) has been recently associated with vascular risk factors. ␤-amyloid peptides (A␤P), the main component of senile plaques typical of AD, circulate in soluble globular form in bloodstream. Interestingly, A␤P is able to induce endothelial dysfunction, and this effect may represent the link between vascular and neuronal pathophysiological factors involved in AD. We aimed to clarify the molecular mechanisms underlying globular A␤P-induced vascular toxicity. Using several methodological approaches, we have observed that in vascular tissues globular A␤P is unable to induce oxidative stress, one of the mechanisms hypothesized involved in ␤-amyloid toxicity. More important, we have demonstrated that globular A␤P is able to localize on vascular endothelium, where it inhibits eNOS enzymatic activity. In particular, A␤P enhances eNOS phosphorylation on threonine 495 and serine 116 and reduces acetylcholine-induced phosphorylation on serine 1177. Such an effect depends on a PKC signaling pathway, as suggested by its phosphorylation on serine 660. In fact, selective inhibition of the calcium-dependent group of PKC is able to rescue ␤-amyloid-induced alteration of eNOS phosphorylation, NO production, and endothelial vasorelaxation. The activation of these Ca 2؉ -dependent pathways is probably due to the ability of A␤P to evoke Ca 2؉ leakage from inositol 1,4,5-triphosphate receptors on endoplasmic reticulum. Our data demonstrate that globular A␤P-induced endothelial NO dysfunction can be attributed to an alteration of intracellular Ca 2؉ homeostasis, which could lead to the activation of calcium-dependent group of PKC with a consequent change of the eNOS phosphorylation pattern. These mechanisms could contribute to shed further light on the toxic effect of ␤-amyloid in vascular tissues. Alzheimer's disease (AD)1 is a progressive neurodegenerative disorder characterized by irreversible cognitive and physical deterioration. It is a major cause of death and a growing public health problem as life expectancy in the general population increases (1).Typical features of AD are the senile plaques present in the brain, cerebral blood vessels, and other tissues (2). The plaques are composed mainly of fibrillar amyloid ␤ peptides (A␤P) generated from the amyloid precursor protein, a ubiquitously expressed transmembrane glycoprotein. The amyloid peptides, constituted primarily of 39 -42 residues (A␤P 1-39, 1-40, and 1-42), are released continuously during cellular metabolism. These peptides circulate in soluble globular form in the bloodstream (3, 4) and accumulate on the vascular wall of AD patients (5). In the brain, soluble ␤-amyloid monomers are able to deposit and transform into insoluble and fibrillar aggregates, forming amyloid plaques (6, 7).Although AD has been considered for long to be of nonvascular origin, a growing body of recent studies has indicated the possibility that vascular risk factors could be involved in the pathophysiology of AD (8). In particular, patients with AD have morphological alterat...