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1 In the central nervous system (CNS), glutamate rapidly upregulates the activities of different excitatory amino-acid transporter subtypes (EAATs) in order to help protect neurons from excitotoxicity. Since human platelets display a specific sodium-dependent glutamate uptake activity, and express the three major glutamate transporters, which may be affected in neurological disorders, we investigated whether platelets are subject to substrate-induced modulation as described for CNS. 2 A time- and dose-dependent upregulation of [H-3]-glutamate uptake ( up to two-fold) was observed in platelets preincubated with glutamate. There was an increase in maximal velocity rate without affinity changes. Glutamate receptor agonists and antagonists did not modulate this upregulation and preincubation with glutamate analogues failed to mimic the glutamate effect. Only aspartate preincubation increased the uptake, albeit similar to 35% less with respect to glutamate. 3 The effect of glutamate preincubation on the expression of the three major transporters was studied by Western blotting, showing an increase of similar to 70% in EAAT1 immunoreactivity that was completely blocked by cycloheximide (CEM). However, L-serine-O-sulphate, at a concentration (200 mu M) known to block EAAT1/3 selectively, did not completely inhibit the effect of glutamate stimulation, indicating the possible involvement of EAAT2. 4 In fact, glutamate stimulation was completely abolished only when, following CEM preincubation, the experiment was run in the presence of the selective EAAT2 inhibitor dihydrokainic acid. Since surface biotinylation experiments failed to show evidence of EAAT2 translocation, our results suggest the existence of a different way of regulating EAAT2 activity. 5 These findings indicate that human platelets display a substrate-dependent modulation of glutamate uptake mediated by different molecular mechanisms and confirm that ex vivo platelets are a reliable model to investigate the dysfunction of glutamate uptake regulation in patients affected by neurological disorders.

Several lines of evidence implicate a central role for alpha-synuclein (aSN) in the pathogenesis of Parkinson's disease (PD). Besides rare genetic mutations, post-translational mechanisms, such as oxidative stress-related nitration, may alter the protein properties in terms of propensity to aggregate or be degraded. Our group previously described increased reactive oxygen species (ROS) production within easily accessible peripheral blood mononuclear cells (PBMCs) in PD patients compared to healthy elderly subjects. In the present work, we demonstrated a significant induction of nitrotyrosine (NT)-modifications of aSN within PBMCs derived from individuals with idiopathic PD compared to controls, while aSN protein appeared similarly expressed in the two populations. The amount of NT-modified aSN within PBMCs was positively correlated with intracellular ROS concentration and inversely related to daily dosage of levodopa, making its measurement potentially relevant for disease-intervention studies. Neither aSN expression nor its NT-modifications showed any correlation to specific REP1 genotypes, polymorphic variants within aSN gene promoter whose association to PD susceptibility may occur through the modulation of aSN protein expression. Moreover, although NT-modified aSN has been linked to enhanced propensity to aggregate, we failed to detect an increased presence of insoluble aSN aggregates in PBMCs from PD subjects relative to controls, despite a lack of changes in the ubiquitin-proteasome expression or activity. Nonetheless, a significant activation of the autophagy response was identified within PBMCs from PD individuals, which could represent a protective mechanism against abnormal protein accumulation and may explain the lack of aSN aggregation. We discuss the relevance of these findings with respect to PD pathogenesis and biomarker development.

Oxidative stress, resulting from the imbalance between reactive oxygen species (ROS) formation and antioxidant defenses, plays a major role in the pathogenesis of Parkinson's disease (PD). However, the contribution of levodopa (LD) therapy to oxidative damage is still debated. We investigated oxidative stress in peripheral blood mononuclear cells (PBMCs) from LD-treated PD patients and healthy subjects. Increased ROS production associated with unaltered glutathione reductase activity was detected in PBMC from PD patients. LD daily dosage appeared to be inversely correlated with ROS levels and positively associated with GR activity, suggesting a protective role for LD on PBMCs redox status. Our data support the view of systemic oxidative stress involvement in PD and give further rationale for using PBMCs as an easily accessible ex-vivo dopaminergic model for exploring the biological effects of LD therapy.

Altered glutamate transport and aberrant EAAT1 expression were shown in Alzheimer’s disease (AD) brains. It is presently unknown whether these modifications are a consequence of neurodegeneration or play a pathogenetic role. However, recent findings of decreased glutamate uptake, EAAT1 protein and mRNA in AD platelets suggest that glutamate transporter modifications may be systemic and might explain the decreased glutamate uptake. We now used primary fibroblast cultures from 10 AD patients to further investigate the specific involvement of glutamate transporters in this disorder and in normal aging. Decreased glutamate uptake (p < 0.001), EAAT1 expression (p < 0.05) and mRNA (p < 0.01) were observed in aged people, compared to younger controls. In AD fibroblasts, compared to age-matched controls, we observed further reductions of glutamate uptake (p < 0.0005) and EAAT1 expression (p < 0.005), while EAAT1 mRNA increase (p < 0.001) was shown. EAAT1 parameters were mutually correlated (p < 0.01) and correlations were shown with dementia severity (p < 0.05 MMSE-expression, p < 0.005 MMSE-mRNA). We suggest fibroblast cultures as possible ex vivo peripheral model to study the glutamate involvement and possible molecular and therapeutic targets in AD.

Neurotransmitter ligand binding in blood cells was assessed in borderline personality disorder (BDP) patients, testing the possibility that different biochemical endophenotypes might lie beneath a specific clinical presentation. The density of peripheral benzodiazepine receptors (PBR) and serotonin transporters were assessed in peripheral blood mononuclear cells (PBMC) and platelets, respectively, showing a decrease of both parameters. Moreover, a further significant decrease of PBR in PBMC was shown for those patients with a depressive trait. Further confirmation of the presence of different molecular endophenotypes underlying the dissimilar clinical presentations in BPD may advance our possibility of successfully treating these patients. © 2009 Elsevier Ireland Ltd. All rights reserved.

In this study we employed an ELISA assay to measure alpha-synuclein protein in lymphomonocytes from 78 PD patients and 78 controls. We correlated protein levels with demographic and clinical characteristics and with the chymotryptic and tryptic activities of the 20S proteasome. Alpha-synuclein levels were not significantly different between patients and controls. In control subjects, alpha-synuclein protein levels increased significantly with age and were significantly higher in men compared to women. Proteasome activity was not significantly different between cases and controls. In control group, the 20S chymotryptic activity tended to decrease significantly with increasing age, though it was not correlated to alpha-synuclein levels. The 20S tryptic activity was not significantly correlated to age, but was inversely correlated to alpha-synuclein levels. Our findings suggest that alpha-synuclein levels in lymphomonocytes are affected by age, gender, and by the 20S proteasome activity in control subjects, but they are not useful as a diagnostic biomarker for PD.

Oxidative stress has been demonstrated in Alzheimer's disease (AD) brain and may affect glutamate transport (GT), thereby leading to excitotoxic neuronal death. Since oxidative stress markers have been shown also in peripheral tissues, we investigated possible GT alterations in fibroblast cultures obtained from 18 patients with AD and 15 control patients and analyzed the effects of the lipoperoxidation product 4-hydroxynonenal (4-HNE) and antioxidants. Basal GT was decreased by 60% in fibroblasts from patients with AD versus control patients. Exposure to HNE did not affect GT in control patients, but it reduced GT by 50% in patients with AD, without any concomitant change in cell viability; conversely, HNE exposure induced a larger increase in ROS intracellular levels in AD than in control fibroblasts. Glutathione and N-acetylcysteine completely blocked 4-HNE effects and also increased basal uptake in AD cells. Moreover, inhibition of glutathione synthesis in control fibroblasts by pretreatment with buthionine sulfoximine resulted in GT reduction (40%) and an increase in ROS levels after exposure to 4-HNE. Nevertheless, since there are no differences between GSH basal level in controls and patients with AD, the alteration of other antioxidant systems cannot be excluded. Our study supports the hypothesis of a systemic impairment of GT in AD, possibly linked to oxidative stress and to reduced antioxidant defenses, which may be partially reversed by antioxidant treatment. Therefore, we suggest fibroblast cultures as a tool for exploring pathogenetic mechanisms and possible therapeutic strategies in patients with AD. (C) 2004 Elsevier Inc. All rights reserved.

L-Glutamic acid acts as the major excitatory neurotransmitter and, at the same time, represents a potential neurotoxin for the mammalian central nervous system (CNS). The termination of excitatory transmission and the maintenance of physiologic levels of extracellular glutamate, which is necessary to prevent excitotoxicity, are prominently mediated by a family of high-affinity sodium-dependent excitatory amino acid transporters (EAATs). Five subtypes of EAATs have been cloned, possessing distinct pharmacology, localization, sensitivity to transport inhibitors and modulatory mechanisms. Expression and activity of EAATs have been shown to be amenable to fine endogenous and, potentially, pharmacological regulation by substrate itself, growth factors, second messengers, hormones, biological oxidants, inflammatory mediators and pathological conditions. The present review describes basic pharmacological studies, mostly performed on animal models or cell preparations, in order to obtain an updated picture of the known regulatory mechanisms of single EAAT expression and activity. New insight into molecular pathways involved in EAAT regulation will allow pharmacological manipulation of excitatory CNS activity, possibly avoiding adverse effects of glutamate receptor blockade.

Overexpression of APP and SOD induces beta-amyloid deposition and oxidative stress in Down syndrome (DS) patients. Both phenomena may impair glutamate transport and decreased glutamate uptake sites have been demonstrated in patient brains at autopsy. Since alterations of APP metabolism and oxidative damage are systemic, we investigated glutamate uptake in platelets and fibroblasts from DS patients to explore whether abnormalities in this process are inherent properties of DS cells and not secondary to neurodegeneration. Glutamate uptake was significantly decreased in platelets (P

Decreased glutamate uptake and a loss of the astrocytic glutamate transporter EAAT2 (GLT-1) have been shown in spinal cord and motor cortex of patients with ALS. Because platelets express the three major glutamate transporter subtypes, including GLT-1, and possess a high-affinity glutamate uptake, the authors investigated glutamate uptake in platelets from patients with ALS and controls. A 43% reduction of high-affinity glutamate uptake rate (p < 0.0001) was observed in patients with ALS compared with normal controls and chronic neurologic disorder patients, suggesting a systemic impairment of glutamate uptake in ALS

Defects in mitochondrial enzymes have been found not only in substantia nigra, but also in platelets from Parkinson's Disease (PD) patients, suggesting a systemic impairment of energy metabolism. Since platelets present an energy-dependent glutamate uptake similar to that described in central nervous system, glutamate uptake was determined in platelets from 34PD patients and 21 age-related normal controls, as Na+-dependent [3H]glutamate influx; glutamate level was also analyzed by reverse-phase HPLC. A 50% reduction of glutamate uptake (p < 0.001) was observed in idiopathic PD patients, respect to controls and secondary parkinsonian syndromes. The decrease correlated with the severity of PD, measured by the UPDRS (r = -0.54; P < 0.05). Glutamate level was increased in platelets of PD patients, but was not correlated to the uptake decrease. Both phoenomena may be explained by the modifications of mitochondrial enzymes described in platelets, which could be used as a peripheral model of glutamatergic function in PD

Genetic risk factors seem to play a role in sporadic Parkinson's disease (PD), maybe triggering oxidative stress and excitotoxicity within substantia nigra. However, genetic factors act at systemic level: reduced activity of mitochondrial enzymes and decreased glutamate uptake have been shown in platelets from PD patients. In this study we investigated glutamate uptake in platelets from 38 sporadic PD patients, 13 patients with parkinsonian syndromes and 28 controls and assessed polymorphisms of alpha -synuclein and ApoE genes. A 48% reduction of glutamate uptake (p

Objective To investigate possible associations between serotonin transporter (5-HTT) promoter genotypic variants ( I/I, I/s , and s/s ) and differential regulation of platelet 5-HTT functionality parameters in a group of drug-naive depressed children and adolescents and healthy controls. Method Children and adolescents with major depression ( n = 18) defined by DSM-III-R criteria and normal controls ( n = 21) were assessed both for platelet serotonin functionality and for genotypic variants on 5-HTT promoter region. Four parameters were considered: (1) serotonin uptake rate (V max ); (2) serotonin dissociation constant (K m ); (3) paroxetine binding and density of site (B max ); and (4) paroxetine dissociation constant (K d ). Results Depressed children had lower V max and K m. Control subjects with I/I genotype had significantly higher V max than control subjects with I/s and s/s genotype. Control subjects with I/I genotype also had significantly higher V max than their depressed homologs. In contrast, V max was not significantly different between depressed and nondepressed subjects who carried the other 2 genotypes. The 5-HTT promoter genotype, diagnoses, or their interaction had no effect on the other serotonin parameters. Conclusions While showing a significant decrease of V max and K m in a group of drug-naive depressed children and adolescents, these data suggest that I/I genotype has a substantial effect on the decrease of V max during a depressive episode.