Solving the crisis in psychopharmacological research: Cellular-membrane(s) pharmacology to the rescue?

There is an urgent need for the introduction of novel and better (i.e., improved risk-benefit profile) compounds for the treatment of major psychiatric disorders, in particular mood and psychotic disorders. However, despite increased societal awareness and a rising public and professional demand for such agents from patients and physicians, the pharmaceutical industry continues to close down its psychopharmacology research facilities in reaction to the lack of success with the search for new psychotropics. It is high time to stop this untoward trend and explore "new" lines of investigation to solve the current crisis in psychopharmacological research. In line with the prevailing molecular view in drug research in general, also in psychopharmacology mechanistic explanations for drug effects are "traditionally" looked for at the level of molecular targets, like receptors and transporters. Also, more recent approaches, although using so-called systems- and function-based approaches to model the multidimensional characteristics of psychiatric disorders and psychotropic drug action, still emphasize this search strategy for new therapeutic leads by identification of single molecules or molecular pathways. This "psychomolecular gaze" overlooks and disregards the fact that psychotropic agents usually are highly hydrophobic and amphipathic/amphiphilic agents that, in addition to their interaction with membrane-bound proteins in the form of e.g. receptors or transporters, also interact strongly with the lipid component of cellular membranes. Here we suggest to develop a program of systematic, whole-cell level based, investigation into the role of these physical-chemical cellular membrane interactions in the therapeutic action of known psychotherapeutics. This complementary yet conceptually different approach, in our opinion, will complement drug development in psychopharmacology and thereby assist in overcoming the current crisis. In this way the "old" physical theory of drug action, which antedates the current, primary molecular, paradigm may offer "new" options for lead discovery in psychopharmacological research.

The blood clotting Factor XIIIa forms unique complexes with amyloid-beta (Aß) and colocalizes with deposited Aß in cerebral amyloid angiopathy.

AIMS: Cerebral amyloid angiopathy (CAA) is a key pathological hallmark of Alzheimer's disease (AD) characterized by accumulation of amyloid-beta (Aß) protein in blood vessel walls. CAA impairs vessel functioning, affects blood brain barrier integrity and accelerates cognitive decline of AD patients. Unfortunately, mechanisms underlying Aß deposition in the vessel wall remain largely unknown. Factor XIIIa (FXIIIa) is a blood-derived transglutaminase crucial in blood coagulation by cross-linking fibrin molecules. Evidence is mounting that blood-derived factors are present in CAA and may play a role in protein deposition in the vessel wall. We therefore investigated whether FXIIIa is present in CAA and if FXIIIa cross-link activity affects Aß aggregation. METHODS: Using immunohistochemistry, we investigated the distribution of FXIIIa, its activator thrombin and in situ FXIIIa activity in CAA in post-mortem AD tissue. We used surface plasmon resonance and Western blot analysis to study binding of FXIIIa to Aß and the formation of FXIIIa-Aß complexes, respectively. In addition, we studied cytotoxicity of FXIIIa-Aß complexes to cerebrovascular cells. RESULTS: FXIIIa, thrombin and in situ FXIIIa activity colocalize with the Aß deposition in CAA. Furthermore, FXIIIa binds to Aß with a higher binding affinity for Aß1-42 compared with Aß1-40 . Moreover, highly stable FXIIIa-Aß complexes are formed independently of FXIIIa cross-linking activity that protected cerebrovascular cells from Aß-induced toxicity in vitro. CONCLUSIONS: Our data showed that FXIIIa colocalizes with Aß in CAA and that FXIIIa forms unique protein complexes with Aß that might play an important role in Aß deposition and persistence in the vessel wall.

Anti-inflammatory effect by lentiviral-mediated overexpression of IL-10 or IL-1 receptor antagonist in rat glial cells and macrophages.

Neuroinflammation, as defined by activation of local glial cells and production of various inflammatory mediators, is an important feature of many neurological disorders. Expression of pro-inflammatory mediators produced by glial cells in the central nervous system (CNS) is considered to contribute to the neuropathology observed in those diseases. To diminish the production or action of pro-inflammatory mediators, we have used lentiviral (LV) vector-mediated encoding rat interleukin-10 (rIL-10) or rat interleukin-1 receptor antagonist (rIL-1ra) to direct the local, long-term expression of these anti-inflammatory cytokines in the CNS. We have shown that cultured macrophages or astroglia transduced with LV-rIL-10 or LV-rIL-1ra produced far less tumor necrosis factor (TNF)alpha or IL-6, respectively in response to pro-inflammatory stimuli. Moreover, intracerebroventricular (i.c.v.) administration of LV-rIL-10 or LV-rIL-1ra resulted in transduction of glial cells and macrophages and, subsequently reduced TNFalpha, IL-6 and inducible nitric oxide synthase (iNOS) expression in various brain regions induced by inflammatory stimuli, whereas peripheral expression of these mediators remained unaffected. In addition, expression levels of the anti-inflammatory cytokines IL-4 and transforming growth factor-beta were not altered in either brain or pituitary gland. Furthermore, i.c.v. administration of LV-rIL-10 or LV-rIL-1ra given during the remission phase of chronic-relapsing experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, improved the clinical outcome of the relapse phase. Thus, local application of LV vectors expressing anti-inflammatory cytokines could be of therapeutic interest to counteract pro-inflammatory processes in the brain without interfering with the peripheral production of inflammatory mediators.

The thiol antioxidant 1,2-dithiole-3-thione stimulates the expression of heat shock protein 70 in dopaminergic PC12 cells.

In Parkinson's disease (PD), the pathogenic factors oxidative stress and protein aggregation interact and culminate in the apoptotic death of (mainly catecholaminergic) neurons. The dithiolethiones comprise thiol antioxidants that are well known for their activation of the expression of a wide collection of cytoprotective genes, including genes coding for antioxidant enzymes. Given the observation that heat shock proteins (HSPs), in particular the heat shock protein 72 (HSP72), protects against cellular degeneration in various models of PD, the ability of the unsubstituted dithiolethione 1,2-dithiole-3-thione (D3T) to stimulate heat shock protein gene and protein expression was studied using the dopaminergic PC12 cell line. As anticipated, D3T stimulated the expression of the antioxidant enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1). Quantitative PCR analysis revealed that D3T stimulates the expression of the inducible, cytoplasmic HSP72. Moreover, D3T strongly potentiated HSP72 gene and protein expression in heat-stressed cells. Taken together, our data show that, in addition to antioxidant enzymes, D3T stimulates the expression of HSP72, a chaperone shown to be neuroprotective in various models of PD, in particular under conditions of cellular stress. Thus, the broad range manipulation of endogenous cellular defense mechanisms, through D3T, may represent an innovative approach to therapeutic intervention in PD.

Changed distribution pattern of the constitutive rather than the inducible HSP70 chaperone in neuromelanin-containing neurones of the Parkinsonian midbrain.

Aberrant protein aggregation has been recognized as an important factor in the degeneration of melanized dopaminergic neurones in Parkinson's disease (PD). The constitutive (HSP73) and (heat)-inducible (HSP72) proteins of the heat shock 70 family form a major defence system against pathological protein aggregation. However, the distribution patterns of these chaperones in nigral neuromelanin-laden neurones are largely unknown. The present study determined the distribution of HSP72 and HSP73 in control and Parkinsonian substantia nigra, using immunohistochemistry. In the neuromelanin-laden neurones of controls, HSP72 was nondetectable, whereas HSP73 was weakly expressed in both the cytosol and the nucleus. Surprisingly, in PD subjects, marked nuclear HSP73, but not HSP72 immunoreactivity was observed, while cytosolic immunoreactivity of the two chaperones resembled the labelling pattern observed in controls. Furthermore, HSP73 immunoreactivity was observed in a subset of the Lewy bodies (LBs) detected in the substantia nigra of PD subjects, whereas only few of these LBs were labelled with HSP72. Interestingly, HSP72 and to a lesser extent HSP73 immunoreactivity was much stronger in nonmelanized neurones as compared with melanized neurones in this area. Thus, we conclude that the distribution pattern of HSP73 rather than HSP72 is changed in the nigral neuromelanin-laden neurones of PD subjects as compared with control subjects. The impaired ability of aged, dopaminergic neurones to express high levels of chaperones, may contribute to the preferential vulnerability of the latter cells in PD.

The antioxidant anethole dithiolethione inhibits monoamine oxidase-B but not monoamine oxidase A activity in extracts of cultured astrocytes.

Anethole dithiolethione (ADT) is a clinically available, pluripotent antioxidant proposed as a neuroprotectant for Parkinson's disease (PD). Here, using extracts from cultured astrocytes, containing both monoamine oxidase (MAO) A and B activity, we demonstrate that ADT concentration-dependently inhibits MAO-B activity in a clinically relevant concentration range (0.03-30 microM, IC-50 = 0.5 microM) without affecting MAO A activity. Considering the alleged contribution of MAO activity in general, and MAO-B in particular, to oxidative stress and neurodegeneration in PD, our data further support the neuroprotective potential of ADT.

Expression of NAD(P)H:quinone oxidoreductase in the normal and Parkinsonian substantia nigra.

Dopamine (DA) autooxidation, and consequent formation of neurotoxic DA-derived quinones and reactive oxygen species, has been implicated in dopaminergic cell death and, hence, in the pathogenesis of Parkinson's disease (PD). Stimulation of pathways involved in the detoxication of DA-quinones in the brain is hypothesized to be an effective means to limit oxidative stress and to confer neuroprotection in PD. In this respect, the inducible flavoprotein NAD(P)H:quinone oxidoreductase (NQO1) is of particular interest as it is directly implicated in the detoxication of DA-quinones and, in addition, has broad spectrum anti-oxidant properties. To study the potential pathophysiological role of NQO1 in PD, the cellular expression of NQO1 was examined in the mesencephalon of PD patients and age-matched controls. In the substantia nigra pars compacta (SNpc), NQO1 was found to be expressed in astroglial and endothelial cells and, albeit less frequently, also in dopaminergic neurons. Moreover, while overt NQO1 immunoreactivity was absent in the surrounding nervous tissue, in the Parkinsonian SNpc a marked increase in the astroglial and neuronal expression of NQO1 was consistently observed.

Loss of olfaction in de novo and treated Parkinson's disease: possible implications for early diagnosis.

Olfactory dysfunction is a common finding in patients with Parkinson's disease (PD). As most studies reported on odor identification in more advanced and treated PD, we administered an odor detection, discrimination, and identification test to a heterogeneous, partly de novo, group of patients. Forty-one non-demented PD patients, 24 of whom had untreated early PD, and 18 healthy controls, were examined. Odor identification and discrimination data were corrected for odor detection scores. PD patients scored significantly lower on all olfactory tests. Interestingly, the subgroup of de novo patients with early PD also showed significant olfactory disturbances compared with healthy subjects. Within the PD group, using multiple regression analysis, we found a significant, negative correlation between odor discrimination measures and disease The present study is the first to describe decreased performance of PD patients on odor discrimination, in addition to the already well-established deficits in odor detection and identification. Furthermore, odor discrimination measures were related to disease severity, possibly indicating that at least some aspects of olfactory dysfunction in PD may be secondary to ongoing degenerative processes in PD. As significant olfactory impairments were found in early, de novo PD, olfactory tests may be useful in the early diagnosis of PD.

Neuroprotection for Parkinson's disease: a new approach for a new millennium.

Parkinson's disease (PD) is the only neurodegenerative disorder in which pharmacological intervention has resulted in a marked decrease in morbidity and a significant delay in mortality. However, the medium to long-term efficacy of this pharmacotherapy, mainly consisting of dopaminomimetics like L -dopa and dopamine receptor agonists, suffers greatly from the unrelenting progression of the disease process underlying PD, i.e., the degeneration of neuromelanin-containing, dopaminergic neurones in the substantia nigra. Efforts concentrated on understanding the mechanisms of dopaminergic cell death in Parkinson's disease have led to identification of a large variety of pathogenetic factors, including excessive release of oxygen free radicals during enzymatic dopamine breakdown, impairment of mitochondrial function, production of inflammatory mediators, loss of trophic support, and apoptosis. Therapeutic approaches aimed at correcting these abnormalities are currently being evaluated on their efficacy as neuroprotectants for PD. Here, we focus on the process of dopamine auto-oxidation, the chain of reactions leading to the formation of neuromelanin, as an often overlooked, yet obvious pathogenetic factor. In particular, we discuss the option of drug-mediated stimulation of endogenous mechanisms responsible for the detoxification of dopamine auto-oxidation products as a novel means of neuroprotection in Parkinson's disease.

L-Dopa stimulates expression of the antioxidant enzyme NAD(P)H:quinone oxidoreductase (NQO) in cultured astroglial cells.

The autooxidation of L-Dopa, a catecholamine used in the symptomatic treatment of Parkinson's disease, generally yields reactive oxygen species and neurotoxic quinones. NAD(P)H:quinone oxidoreductase (NQO) is a flavoenzyme that is implicated in the detoxication of quinones, including those formed during L-Dopa autooxidation. Through the action of this enzyme, deleterious redox-labile quinones are turned into less toxic and more stable hydroquinones that are amenable to further detoxication and/or cellular excretion. In the present study, using primary rat astrocytes and C6 astroglioma as a model to evaluate the neuroprotective response of astroglial cells upon exposure to L-Dopa, we demonstrate that this compound, or more correctly its quinone (auto)oxidation products, up-regulates astroglial NQO in a time- and concentration-dependent way as assessed at the level of mRNA expression, protein level, and enzymatic activity. Moreover, under similar conditions cellular glutathione content was enhanced. It is concluded that, similar to glutathione, the oxidative stress limiting NQO is likely to contribute to the capacity of astroglial cells to protect dopaminergic neurons against L-Dopa, and, hence, may be considered as a potential target for the development of neuroprotective strategies for Parkinson's disease.

Dynorphin modulates dopamine D1-receptor mediated turning behavior in 6-hydroxydopamine-lesioned rats.

We investigated if the potentiated turning response to a challenge with the partial dopamine D1 receptor agonist SKF-38393, as seen after priming with L-dihydroxyphenylalanine (DOPA) of unilaterally 6-hydroxydopamine-lesioned rats, can be modulated by infusion of dynorphin A (1-17) in the striatum. Seventeen days after the 6-hydroxydopamine lesion, rats received intrastriatal dynorphin (0. 08 or 3.85 microg) followed by L-DOPA (50 mg/kg i.p.) and were challenged 3 days later with SKF-38393 (3.0 mg/kg s.c.). Compared to controls, the lower dose of dynorphin caused an earlier onset of turning, while the higher dose decreased the response to SKF-38393. These findings suggest a dose-dependent modulatory role for striatal dynorphin in L-DOPA-priming of a D1-mediated behavioral response.

Drug treatment of Parkinson's disease. Time for phase II.

Parkinson's disease (PD) is a neurodegenerative syndrome for which at present no cure is available; therapy consists mainly of amelioration of the symptoms with L-Dopa and/or dopamine (DA) agonists. Development of an effective causal therapy should be focussed on preventing or at least retarding the neurodegenerative process underlying the disease. At the cellular level, PD is characterized by degeneration of neuromelanin-containing dopaminergic neurons in the substantia nigra. Neuromelanin formation is the outcome of a process generally known as DA autooxidation, a chain of oxidation reactions in which highly neurotoxic DA-quinones are produced. The level of these DA-quinones, as estimated by the occurrence of their cysteinyl conjugates, is reported to be increased in the Parkinsonian substantia nigra. Hence, stimulation of pathways implicated in the detoxication of DA-quinones in the brain may provide neuroprotection in PD. Besides their inactivation through non-enzymatic antioxidants such as ascorbic acid and glutathione, DA-quinones are efficiently inactivated enzymatically by NAD(P)H:quinone oxidoreductase (NQO) and glutathione transferase(s), both of which are expressed in the human substantia nigra. The activity of these enzymes, which belong to the group of phase II biotransformation enzymes, can be up-regulated by a large variety of compounds. These compounds, including dithiolethiones, phenolic anti-oxidants, and isothiocyanates, have been shown to be active both in vitro and in vivo. Thus, considering the role of phase II biotransformation enzymes, in particular NQO and glutathione transferase(s), in the detoxication of DA-quinones, we propose that phase II enzyme inducers warrant evaluation on their neuroprotective potential in PD.

Leads for the development of neuroprotective treatment in Parkinson's disease and brain imaging methods for estimating treatment efficacy.

Patients suffering from Parkinson's disease display severe and progressive deficits in motor behavior, predominantly as a consequence of the degeneration of dopaminergic neurons, located in the mesencephalon and projecting to striatal regions. The cause of Parkinson's disease is still an enigma. Consequently, the pharmacotherapy of Parkinson's disease consists of symptomatic treatment, with in particular L-dihydroxyphenylalanine (L-DOPA) and/or dopamine receptor agonists. These induce a dramatic initial improvement. However, serious problems gradually develop during long-term treatment. Therefore, a more rational, c.q. causal treatment is needed which requires the introduction of compounds ameliorating the disease process itself. The development of such compounds necessitates (1) more information on the etiopathogenesis, i.e., the cascade of events that ultimately leads to degeneration of the dopaminergic neurons, and (2) brain imaging methods, to estimate the extent of the degeneration of the dopaminergic neurons in the living patient. This is not only important for the early diagnosis, but will also allow to monitor the effectiveness of alleged neuroprotective compounds on a longitudinal base. In this paper, etiopathogenic mechanisms are highlighted along the line of the oxidative stress hypothesis and within this framework, attention is mainly focused on the putative role of glutathione, dopamine auto-oxidation and phase II biotransformation enzymes. Especially, drugs able to increase the activity of phase II biotransformation enzymes seem to elicit a broad-spectrum (neuro)protective response and look very promising leads for the development of neuroprotective treatment strategies in Parkinson's disease. New developments in brain imaging methods (single photon emission computed tomography (SPECT) and positron emission tomography (PET)) to visualize the integrity of the striatal dopaminergic neurons in humans are highlighted as well. Especially, the introduction of radioligands that bind selectively to the dopamine transporter seems to be a significant step forward for the early diagnosis of Parkinson's disease. Performing these brain imaging studies with fixed time intervals does not only create the possibility to follow the degeneration rate of the dopaminergic neurons in Parkinson's disease but also provides the opportunity to estimate therapeutic effects of putative neuroprotective agents in the individual patient.

The alleged dopamine D1 receptor agonist SKF 83959 is a dopamine D1 receptor antagonist in primate cells and interacts with other receptors.

So far, no clear correlation has been found between the effects of dopamine D1 receptor agonists on motor behavior in primate models of Parkinson's disease and their ability to stimulate adenylate cyclase in rats, the benzazepine SKF 83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-]H- 3-benzazepine) being the most striking example. Since this discrepancy might be attributed to: (A) the different species used to study these effects or (B) the interaction of SKF 83959 with other catecholamine receptors, the aims of this study were: (1) to study the ability of SKF 83959 to stimulate adenylate cyclase in cultured human and monkey glial cells equipped with dopamine D1 receptors and (2) to evaluate the affinity for and the functional interaction of SKF 83959 with other catecholamine receptors. Binding studies revealed that SKF 83959 displayed the highest affinity for the dopamine D1 receptor (pKi=6.72) and the alpha2-adrenoceptor (pKi=6.41) and moderate affinity for the dopamine D2 receptor and the noradrenaline transporter. In monkey and human cells, SKF 83959 did not stimulate cyclic adenosine monophosphate (cAMP) formation to a significant extent, but antagonized very potently the dopamine-induced stimulation of cAMP formation in both cell types. The compound stimulated basal dopamine outflow and inhibited depolarization-induced acetylcholine release only at concentrations > 10 microM. Finally, SKF 83959 concentration dependently increased electrically evoked noradrenaline release, indicating that it had alpha2-adrenoceptor blocking activity and interfered with the noradrenaline transporter. In conclusion, SKF 83959 is a potent dopamine D1 receptor and alpha2-adrenoceptor antagonist. Thus, the anti-parkinsonian effects of SKF 83959 in primates are not mediated by striatal dopamine D1 receptors coupled to adenylate cyclase in a stimulatory way.

The validity of the pretreated, unilaterally MPTP-treated monkeys as a model of Parkinson's disease: a detailed behavioural analysis of the therapeutic and undesired effects of the D2 agonist quinpirole and the D1 agonist SKF 81297.

The goal of this study was to evaluate the validity of the pretreated, unilaterally MPTP-treated monkey as an animal model of Parkinson's disease (PD). For that purpose, a detailed ethogram was developed and assessed in four male rhesus monkeys that had received MPTP (2.5 mg) in the carotid artery contralateral to the dominant limb. Subsequently, the behavioural effects of the dopamine D2 agonist quinpirole and the dopamine D1 agonist SKF 81297 were studied. The ethogram was found to allow a clear-cut and objective separation of drug-induced behaviours into therapeutic and undesired effects in the MPTP-treated monkeys. Saline-treated monkeys predominantly displayed ipsilateral goal-directed fore-limb movements, and distinct types of ipsilaterally directed rotations. Although quinpirole and SKF 81297 increased motor behaviours, such as body displacement, contralateral fore-limb movements and contralateral rotational behaviours, assessment of the new detailed ethogram revealed that this increase was completely due to the activation of abnormal, non-goal-directed behaviours, such as dyskinetic fore-limb movements, pivoting and shuffling. Moreover, the new ethogram made clear that the drug treatments induced not only dyskinesia and dystonia, but also epileptoid behaviour, which was confirmed by EEG analysis. In summary, the detailed behavioural analysis showed that this model does not adequately predict the clinical effects of the D2 agonist. It is concluded that the pretreated, unilaterally MPTP-treated monkey is not a valid model to predict the therapeutic and undesired effects of dopaminergic drugs in humans.

The predictive validity of the drug-naive bilaterally MPTP-treated monkey as a model of Parkinson's disease: effects of L-DOPA and the D1 agonist SKF 82958.

The aim of this study was twofold: (1) to study the predictive validity of the drug-naive, bilaterally MPTP-treated monkey as an animal model of Parkinson's disease (PD), and (2) to investigate the therapeutic and undesired effects of the D1 agonist SKF 82958 as compared to L-DOPA treatment, in drug-naive and L-DOPA pretreated monkeys. A detailed ethogram was used, allowing the separation of therapeutic and undesired effects. Eight weeks after bilateral intracarotid MPTP administration, SKF 82958 (1 mg/kg, n = 4, SKF 82958, naive group) or methyl-L-DOPA + carbi-dopa (10 + 2.5 mg/kg, n = 4, L-DOPA group) was administered intramuscularly for 22 days. After a drug-free period of eight weeks, the L-DOPA group was treated with SKF 82958 for 22 days (SKF 82959, 1 mg/kg, n=4, pretreated). All drug treatments increased the parameters used classically to evaluate dopaminergic drugs, namely body displacement, dyskinesia and dystonia. However, the new detailed analysis revealed that L-DOPA, but not SKF 82958, had therapeutic effects, reflected by an increase in goal-directed fore-limb use. SKF 82958, but not L-DOPA, induced additional undesired effects; including epileptoid behaviours in both drug-naive and drug-pretreated monkeys. In one L-DOPA-unresponsive monkey, SKF 82958 did induce minor therapeutic effects, as well as undesired effects. Although the effects of SKF 82958 on fore-limb movements, rotational behaviours and body displacement were comparable in the naive and pretreated group, SKF 82958 re-initiated undesired effects in the L-DOPA pretreated group from day one. It is concluded that the bilaterally MPTP-treated monkey is an animal model with predictive validity for PD: it adequately predicts the therapeutic effects and undesired effects of L-DOPA. Furthermore, it is concluded that SKF 82958 is less effective than L-DOPA in the treatment of PD, because it did not induce therapeutic effects, but instead elicited several undesired effects.

Priming with L-DOPA differently affects dynorphin and substance P mRNA levels in the striatum of 6-hydroxydopamine-lesioned rats after challenge with dopamine D1-receptor agonist.

In unilaterally 6-hydroxydopamine-lesioned rats, potentiation of D1-agonist-induced turning behavior by priming with l-DOPA was correlated with changes in striatal neuropeptide mRNA levels. In non-primed rats, administration of the D1-agonist SKF-38393 markedly increased dynorphin and substance P mRNA levels in the lesioned striatum. Priming with l-DOPA dissociated the response of the two neuropeptides to the D1-agonist, with higher dynorphin and reduced substance P mRNA levels.

Astrocyte-enhanced neuronal survival is mediated by scavenging of extracellular reactive oxygen species.

The survival of cultured neurons is promoted by the presence of antioxidants or astrocytes. This indicates that extracellular reactive oxygen species (ROS) impair neuronal survival and suggests that astrocytes exert their survival-enhancing effect through inactivation of these toxicants. However, to our knowledge, data supporting this hypothesis are lacking. Previously, we showed that loss of the antioxidant glutathione abolishes the neuronal survival-stimulating action of astrocytes in cocultures, consisting of rat striatal astrocytes and mesencephalic, dopaminergic neurons. Using uptake of [3H]dopamine as marker of neuronal survival, we presently investigated whether this effect of glutathione depletion is mediated by extracellular ROS. For this purpose, we incubated glutathione-depleted cocultures with superoxide dismutase, catalase or both. Whereas superoxide dismutase had no effect and catalase only partially protected, addition of the enzymes together completely prevented the impairment of neuronal survival caused by glutathione loss. No change in neuronal survival occurred upon exposure of control cocultures to superoxide dismutase and/or catalase. These data strongly implicate scavenging of extracellular ROS in astrocyte-stimulated neuronal survival and moreover suggest a crucial role for glutathione in this process.

Sustained pharmacological inhibition of nitric oxide synthase does not affect the survival of intrastriatal rat fetal mesencephalic transplants.

The objective of the present study was to investigate the potential role of the free radical nitric oxide (NO) in the development of fetal rat mesencephalic neurons grafted in a 6-hydroxydopamine (6-OHDA) lesioned rat model of Parkinson's disease. First, using nitric oxide synthase (NOS)-immunocytochemistry and reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, we investigated the presence of the neuronal isoform of NOS (nNOS) in intrastriatal mesencephalic grafts. During the course of the experiment (16 weeks) an increase in the staining intensity and the number of nNOS/NADPH-d positive cells within the grafts was observed, as well as a gradual maturation of dopaminergic neurons. In addition, within both the host striatal and grafted mesencephalic tissue, a NO-dependent accumulation of cyclic guanosine monophosphate (cGMP) was detected, indicating the presence of guanylate cyclase, i.e., the target-enzyme for NO. Secondly, to determine the impact of NO on the survival of grafted dopaminergic neurons, 6-OHDA lesioned rats received mesencephalic grafts and were subsequently treated with the competitive NOS-inhibitor Nomega-nitro-l-arginine methylester (l-NAME). After chronic treatment for 4 weeks, tyrosine hydroxylase immunocytochemistry revealed no apparent differences between the survival of grafted dopaminergic neurons in control- or l-NAME treated animals, respectively. As the maturation of grafted dopaminergic neurons coincides with a gradual increase in the expression of nNOS within the graft and since dopaminergic cell numbers are not changed upon administration of l-NAME, it is concluded that endogenously produced and potentially toxic NO does not affect the survival of grafted fetal dopaminergic neurons.