Biomedical subjects

Stefano Ruggieri

Publications and source records attributed to Stefano Ruggieri.

At least 19 recordsLinked to original sources

PubMed2006

Fine ultrastructure and biochemistry of PC12 cells: a comparative approach to understand neurotoxicity.

The PC12 cell line is commonly used as a tool to understand the biochemical mechanisms underlying the physiology and degeneration of central dopamine neurons. Despite the broad use of this cell line, there are a number of points differing between PC12 cells and dopamine neurons in vivo which are missed out when translating in vitro data into in vivo systems. This led us to compare the PC12 cells with central dopamine neurons, aiming at those features which are predictors of in vivo physiology and degeneration of central dopamine neurons. We carried out this comparison, either in baseline conditions, following releasing or neurotoxic stimuli (i.e. acute or chronic methamphetamine), to end up with therapeutic agents which are suspected to produce neurotoxicity (l-DOPA). Although the neurotransmitter pattern of PC12 cells is close to dopamine neurons, ultrastructural morphometry demonstrates that, in baseline conditions, PC12 cells possess very low vesicles density, which parallels low catecholamine levels. Again, compartmentalization of secretory elements in PC12 cells is already pronounced in baseline conditions, while it is only slightly affected following catecholamine-releasing stimuli. This low flexibility is caused by the low ability of PC12 cells to compensate for sustained catecholamine release, due both to non-sufficient dopamine synthesis and poor dopamine storage mechanisms. This contrasts markedly with dopamine-containing neurons in vivo lending substance to opposite findings between these compartments concerning the sensitivity to a number of neurotoxins.

Adrenal Medulla↗

PubMed2006

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.

Amphetamine-Related Disorders↗

PubMed2006

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.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2006

Effects of methamphetamine on the cerebellar cortex: a preliminary study.

Methamphetamine (METH) targets monoamine nerve terminals and produces motor effects, which are related to changes in catecholamine activity within the basal ganglia. Cerebellum plays an important role in motor control, nonetheless only a few studies investigated the effects of METH in this area. In this article, we report preliminary results on protein expression in the cerebellum following METH administration. In particular, we focused on the rate-limiting catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). By using immunoblotting, we found that METH administration produces a dose-dependent increase of TH within the cerebellar cortex of mice, which is opposite to the decrease of TH within the striatum. Further investigations are needed in order to determine the time course, the cerebellar regions, the cellular (and subcellular) compartments, and the functional role related to these effects.

Animals↗

PubMed2006

In PC12 cells neurotoxicity induced by methamphetamine is related to proteasome inhibition.

Neurodegenerative disorders are featured by a variety of pathological hallmarks, and very often they are characterized by neuronal inclusions in specific brain nuclei. Occurrence of neuronal inclusions has been often related to the onset of cell death. Recent studies demonstrated that amphetamine derivatives produce intracellular inclusions, which are reminiscent of those occurring in degenerative disorders. In the present article, we analyzed the correlation between neuronal inclusions and cell death using methamphetamine (METH) in PC12 cell cultures. We found that the dose necessary to induce cell death is higher compared with that required to induce inclusions formation. Our results demonstrate a dissociation between formation of inclusion bodies and cell death suggesting that neuronal inclusions do not necessarily lead to cell death. The conclusions of the present article suggest that the onset of inclusion bodies represents a slight consequence of toxicity, which requires a prolonged cell viability to take place.

Animals↗

PubMed2006

Inclusion dynamics in PC12 is comparable between amphetamines and MPTP.

In previous studies it was demonstrated that amphetamine derivatives and 1-methyl article-4-phenylpyridinium produce neuronal cell bodies. In the present work, we compared the fine ultrastructure of the intracellular inclusions induced by these different neurotoxic treatments. In particular, we compared the dynamical changes occurring when a mild toxic stimulus acts for different time intervals. For this purpose, we exposed catecholamine-synthesizing PC12 cells to different amphetamine derivatives (methamphetamine and 3,4-methylenedioxymethamphetamine), or 1-methyl-4-phenylpyridinium ion, which represents the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,3,4,6-tetrahydropyridine. Despite inclusions that are elicited by different mechanisms depending on the specific neurotoxin, their ultrastructural features are similar and there is a high parallelism in their temporal evolution. This suggests that formation of inclusions is a multi-step process that might be elicited by different stimuli and, once triggered, leads to the same final effect.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2006

Dopamine stimulation via infusion in the lateral ventricle.

Continuous dopamine (DA) stimulation is a therapeutic approach that applies to the treatment of motor fluctuations due to pulsatile DA stimulation in Parkinson's disease (PD), to cure the abuse of drugs, such as cocaine or amphetamine (which produce short-lasting peaks of extracellular DA), and as a safe therapeutic approach to avoid hedonistic homeostatic dysregulation (which sometime develops as an abuse pattern in PD patients receiving a pulsatile DA replacement therapy). However, systemic continuous delivery of DA agonists leads to a variety of side effects. In search for an alterative approach, in the present study we evaluated the possibility of delivering intracerebroventricularly (i.c.v.), a DA agonist: lisuride that was already shown to be effective when administered continuously subcutaneously (s.c.). In particular, we were interested in examining whether lisuride infused within the lateral ventricle was still able to stimulate DA receptor by inducing contralateral turning behavior in hemiparkinsonian rats. We found that lisuride, when infused in the lateral ventricle was effective in reducing the threshold for stimulating DA receptors. These results offer a more reliable and safe therapeutic approach to deliver continuous DA selectively in the brain.

Animals↗

PubMed2006

Nigrostriatal damage with 6-OHDA: validation of routinely applied procedures.

The 6-hydroxydopamine (6-OHDA) model of Parkinson's disease in the rat represents a fundamental tool for investigating the pathophysiology of dopamine denervation. Nevertheless, 6-OHDA can induce also noradrenergic lesions; therefore desmethylimipramine (DMI) is co-administrated as a selective inhibitor of noradrenergic reuptake to protect noradrenaline (NA) fibers neighboring DA neurons and/or axons. The neurotoxin 6-OHDA must be microinfused selectively into the substantia nigra pars compacta (SNpc) or into the medial forebrain bundle (MFB) to determine the nigrostriatal lesion. However, this experimental procedure is invasive and always produces a certain amount of mechanical damage that cannot be prevented by pharmacological approaches. For this reason, we have compared two types of experimental design in which we tested critical steps of the procedures, such as the flow rate. We microinfused rats in MFB with 8 microL of total volume of a solution containing the neurotoxin (infusion rate 2 microL/min in 4 min) according with general practice, and rats microinfused with an amount of 2 microL of total volume with a slower rate (0.2 microL/min in 10 min) of infusion. Rats infused with a higher flow rate of infusion underwent striatal NA loss in spite of the administration of DMI. On the contrary, rats infused with a slow infusion flow rate had spared NA axons following DMI. These results suggest that the flow rate and the volume of 6-OHDA infusion are critical to prevent the occurrence of nonspecific mechanical effects.

Adrenergic Agents↗

PubMed2006

MDMA and seizures: a dangerous liaison?

In the past decades, there was a massive increase in the abuse of methylenedioxymethamphetamine (MDMA) in the Western countries. Seizure onset after MDMA is considered to be related mainly to its acute systemic effects (e.g., hyponatremia and hyperthermia). However, additional mechanisms might concur to it as well. Experiments aimed at disclosing the basis for such an acute effect have the advantage of profiting of controlled conditions and the "pure" compounds, as opposed to the limits of clinical data which are biased by several confounding factors. Amphetamines exert profound effect on different monoaminergic systems, which might participate to lowering of seizure threshold. Chronic effects of MDMA abuse on seizure threshold have not been explored in detail so far. Recent data showed that in mice receiving small, repeated doses of MDMA, a persisting pro-convulsant effect toward limbic seizures and metabolic hyperexcitability can be observed. In the present article, we reviewed these studies and we report our preliminary experimental data documenting the lack of mossy fiber sprouting at short time intervals following MDMA, when seizure susceptibility is already present.

Acute Disease↗

PubMed2005

Occurrence of neuronal inclusions combined with increased nigral expression of alpha-synuclein within dopaminergic neurons following treatment with amphetamine derivatives in mice.

In recent years several clinical and research findings have demonstrated the involvement of the presynaptic protein alpha-synuclein in a variety of neurodegenerative disorders which are known as synucleinopathies. Although the function of this protein in the physiology of the cell remains unknown, it is evident that both genetic alterations or a mere overexpression of the native molecule produces a degeneration of nigral dopamine-containing neurons leading to movement disorders, as demonstrated in inherited Parkinson's disease. In the present study, we investigated whether widely abused drugs such as methamphetamine and methylenedioxymethamphetamine (ecstasy), which are known to damage the nigrostriatal dopamine pathway of mice, increase the expression of alpha-synuclein within dopamine neurons of the substantia nigra pars compacta. The results of this study demonstrate that nigrostriatal dopamine denervation and occurrence of intracellular inclusions in nigral neurons produced by amphetamine derivatives are related to increased expression of alpha-synuclein within dopamine neurons of the substantia nigra. This lends substance to the hypothesis that increased amounts of native alpha-synuclein may be per se a detrimental factor for the dopamine neurons.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2005

Neuronal inclusions in degenerative disorders Do they represent static features or a key to understand the dynamics of the disease?

This brief paper analyzes a few degenerative diseases expressing as movement disorders and featuring at sub-cellular level the presence of neuronal inclusions in selective brain regions. We will first draw a short draft of representative neurological diseases featuring inclusion bodies by describing the type of inclusions occurring in various disorders and analyzing both common features and distinctive aspects. As a further step, we move from the bed to the bench side discussing recent developments obtained from experimental models of these disorders which shed new light into the cause and progression of neuronal inclusions, thus helping to understand the pathophysiology of neuronal degeneration underlying movement disorders. In line with this, we will focus on recent studies which led to reproduce neuronal inclusions in vivo and in vitro by manipulating selective cellular structures/enzymatic pathways. In this way, we will try to encompass the dynamics of inclusion formation based on their fine ultrastructure and the analysis of the molecular components as well as their subcellular compartmentalization trying to relate the dynamics of inclusion formation and the pathophysiology of the disease process. An emphasis will be made on the ubiquitin proteasome system and Parkinson's disease where the analysis of neuronal inclusions enlightened potential therapeutic strategies to occlude the progression of this neuronal degeneration featured by movement disorders.

Animals↗

PubMed2005

CAPS1 regulates catecholamine loading of large dense-core vesicles.

CAPS1 is thought to play an essential role in mediating exocytosis from large dense-core vesicles (LDCVs). We generated CAPS1-deficient (KO) mice and studied exocytosis in a model system for Ca2+-dependent LDCV secretion, the adrenal chromaffin cell. Adult heterozygous CAPS1 KO cells display a gene dosage-dependent decrease of CAPS1 expression and a concomitant reduction in the number of docked vesicles and secretion. Embryonic homozygous CAPS1 KO cells show a strong reduction in the frequency of amperometrically detectable release events of transmitter-filled vesicles, while the total number of fusing vesicles, as judged by capacitance recordings or total internal reflection microscopy, remains unchanged. We conclude that CAPS1 is required for an essential step in the uptake or storage of catecholamines in LDCVs.

Animals↗

PubMed2005

Parkinson-like syndrome induced by continuous MPTP infusion: convergent roles of the ubiquitin-proteasome system and alpha-synuclein.

In animals, sporadic injections of the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively damage dopaminergic neurons but do not fully reproduce the features of human Parkinson's disease. We have now developed a mouse Parkinson's disease model that is based on continuous MPTP administration with an osmotic minipump and mimics many features of the human disease. Although both sporadic and continuous MPTP administration led to severe striatal dopamine depletion and nigral cell loss, we find that only continuous administration of MPTP produced progressive behavioral changes and triggered formation of nigral inclusions immunoreactive for ubiquitin and alpha-synuclein. Moreover, only continuous MPTP infusions caused long-lasting activation of glucose uptake and inhibition of the ubiquitin-proteasome system. In mice lacking alpha-synuclein, continuous MPTP delivery still induced metabolic activation, but induction of behavioral symptoms and neuronal cell death were almost completely alleviated. Furthermore, the inhibition of the ubiquitinproteasome system and the production of inclusion bodies were reduced. These data suggest that continuous low-level exposure of mice to MPTP causes a Parkinson-like syndrome in an alpha-synuclein-dependent manner.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2005

Intermittent vs continuous levodopa administration in patients with advanced Parkinson disease: a clinical and pharmacokinetic study.

BACKGROUND: Levodopa-related motor complications can be an important source of disability for patients with advanced Parkinson disease. Current evidence suggests that these motor complications are related to the relatively short half-life of levodopa and its potential to induce pulsatile stimulation of striatal dopamine receptors. Motor complications can be diminished with a continuous infusion of levodopa. OBJECTIVE: To investigate the specific pharmacokinetic changes associated with the benefits of levodopa infusion. DESIGN: We performed an open-label study in 6 patients with Parkinson disease who experienced severe motor complications while receiving standard oral formulations of levodopa/carbidopa. Patients were subsequently treated for 6 months with continuous daytime intraintestinal infusions of levodopa methyl ester. Levodopa pharmacokinetic studies were performed at baseline and 6 months in 3 of these patients. RESULTS: Compared with treatment with intermittent doses of a standard oral formulation of levodopa, continuous infusion provided significant improvement in both "off periods" and dyskinesia. Results of plasma pharmacokinetic studies demonstrated that compared with oral administration, continuous levodopa infusion was associated with a significant increase in the levodopa area under the curve and avoided the low plasma trough levels seen with oral drug administration. CONCLUSIONS: This study confirms that a continuous levodopa infusion is associated with reduced motor complications compared with the standard oral formulation of the drug in patients with advanced PD. Pharmacokinetic studies demonstrate that reduced motor complications are associated with avoiding low plasma levodopa trough levels and are not adversely affected by relatively high plasma levodopa concentrations. We propose that if levodopa/carbidopa could be administered orally in a manner that mirrors the pharmacokinetic pattern of the infusion, it might lead to a similar reduction in motor complications.

Administration, Oral↗

PubMed2003

Fine structure and biochemical mechanisms underlying nigrostriatal inclusions and cell death after proteasome inhibition.

Mutation of genes encoding for various components of a metabolic pathway named the ubiquitin-proteasome system (UP) leads to inherited forms of Parkinson's disease (PD), whereas various components of the UP are constantly present within neuronal inclusions, Lewy bodies, that characterize most genetic and sporadic forms of PD. It has been hypothesized that impairment of this metabolic pathway might be a common mechanism for the onset of PD, and a recent study demonstrated a dysfunction of the UP system within the substantia nigra of patients affected by sporadic PD. In search for the mechanisms underlying the selective toxicity for nigral neurons after inhibition of the UP system, we explored the selective effects after striatal microinfusions of lactacystin or epoxomycin and potential retrograde changes within the ipsilateral substantia nigra. We found that neurotoxicity was selective for striatal dopamine (DA) components and led to retrograde apoptosis within nigral DA cells, which developed neuronal inclusions staining for antigens of the UP system. We found the same ultrastructural features characterizing inclusions obtained in vivo and in vitro after UP inhibition. In vivo, lactacystin-epoxomycin-induced toxicity was suppressed by inhibiting DA synthesis. Similarly, in vitro inclusions and apoptosis were prevented by reducing endogenous DA. On the other hand, toxicity of proteasome inhibition was enhanced by drugs augmenting DA availability: l-3,4-dihydroxyphenylalanine, monoamine oxidase blockers, and DA beta-hydroxylase blockers. These findings demonstrate that impairment of the UP system produces cell death and neuronal inclusions selectively for DA-containing neurons that depend on the occurrence of endogenous DA.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2003

Dual dopamine agonist treatment in Parkinson's disease.

We studied the effectiveness of cabergoline as an adjunct in patients with early Parkinson's disease receiving another dopamine agonist (pramipexole or ropinirole), at the maximal permitted dose. The study enrolled 47 patients: 35 had motor fluctuations and were receiving a dopamine agonist with levodopa; and 12, without motor fluctuations, were receiving a dopamine agonist without levodopa. These medications had in all cases failed to achieve adequate symptom control. In the 35 patients with motor fluctuations, when cabergoline was added to therapy, the time spent in the OFF period decreased by 65.6% (from 3.14+/-1.11 to 1.08+/-1.07 hours p<0.0001); and the Unified Parkinson's Disease Rating Scale (UPDRS) motor score decreased by 19.24% (from 41.68+/-12.6 to 33.66+/-10.22; (p<0.0001) during the OFF condition, and by 7.11% (from 17.01+/-6.63 to 15.8+/-7.22; p<0.001) during the ON condition. Nocturnal akinesia improved in all the patients except one. In the 12 patients without motor fluctuations, when cabergoline was added, the UPDRS score improved by 34.4% (from 23.5+/-5.3 to 15.5+/-4.7). This open study shows that dual dopamine agonist therapy can be useful in the symptomatic treatment of patients with early or more advanced Parkinson's disease receiving therapy with or without levodopa.

Adult↗

PubMed2003

Protective role of group-II metabotropic glutamate receptors against nigro-striatal degeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice.

To examine how mGlu2/3 metabotropic glutamate receptors affect nigro-striatal degeneration, we used the agonist, LY379268, and the antagonist, LY341495, in mice challenged with the nigro-striatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In control mice, high doses of MPTP (20 mg/kg, i.p., injected four times with 2 h of interval) induced a nearly total degeneration of the nigro-striatal pathway, as shown by measurements of striatal dopamine (DA) levels and by immunohistochemical analysis of tyrosine hydroxylase, high affinity dopamine transporter, and glial fibrillary acidic protein in the corpus striatum and substantia nigra. Lower cumulative doses of MPTP (30 mg/kg, i.p., injected only once) produced a partial lesion of the nigro-striatal pathway (about 50% reduction of striatal DA content). Systemic injection of LY379268 (1 mg/kg, i.p., 30 min prior to each injection of MPTP) partially reduced the extent of nigro-striatal degeneration induced by high doses of MPTP. Similar results were obtained by continuously delivering LY379268 (1 mg/kg/d for 7 d) by means of a subcutaneous osmotic minipump. The protective effect of LY379268 was antagonized by LY341495 (also delivered by the osmotic minipump). In mice challenged with the lower cumulative dose of MPTP, injection of LY379268 did not produce a significant neuroprotective effect. In contrast, the lesion was amplified by the antagonist, LY341495. Neither LY379268 nor LY341495 influenced the central bioavailability and the local half-life of MPTP, as shown by measurements of the toxin and its active metabolite, MPP(+), in the striatum. We conclude that mGlu2/3 receptors play a protective role against MPTP toxicity, and that the efficacy of the agonist, LY379268, critically depends on the extent of the nigro-striatal lesion.

Amino Acids↗

PubMed2002

Human herpesvirus 6 and multiple sclerosis: a study of T cell cross-reactivity to viral and myelin basic protein antigens.

Several reports have suggested an association of human herpesvirus 6 (HHV-6) with multiple sclerosis. Autoreactive T lymphocytes directed against myelin components seem to contribute to the pathogenesis of the disease. It has been suggested that molecular mimicry between viral and self-antigens might be one of the mechanisms that determine the onset of several autoimmune diseases. Following this hypothesis, the purpose of the present study was to evaluate if HHV-6 could play a role in activating T cells capable of cross-reaction with an important myelin component, the myelin basic protein. T cell lines were established from 22 multiple sclerosis patients and from 16 healthy controls, and their capability to react to both virus and myelin basic protein antigens was compared. The analysis of T cell cross-reactivity in patients and controls did not show significant differences in the HHV-6 ability to activate myelin basic protein-reactive T cells. Similarly, the evaluation of the humoral immune response to HHV-6 in patients and controls did not mirror any abnormality in the HHV-6 status in multiple sclerosis patients. Therefore, although the findings of activity in vitro of T cell lines with dual specificity are consistent with the hypothesis of molecular mimicry, the lack of differences in cross-reactivity between patients and controls do not support molecular mimicry as an important mechanism in the physiopathology of this disease.

Adolescent↗