Benjamin Drukarch

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 Parkinsons 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 severity.

Astrocyte-mediated enhancement of neuronal survival is abolished by glutathione deficiency

Astrocytes promote the survival of neurons. Conditions characterized by loss of neurons, such as aging and aging-related neurodegenerative disorders, are accompanied by both disturbances in astrocyte-neuron interactions and signs of oxidative damage. Neuronal glutathione, a major antioxidant in the brain, is maintained by astrocytes and brain levels of glutathione are reduced in named conditions. Therefore, we focused on a possible link between glutathione deficiency and loss of astrocyte-derived neuronal support. For this purpose, we used a coculture system consisting of rat striatal astrocytes and mesencephalic, dopaminergic (DAergic) neurons. Using tyrosine hydroxylase immunocytochemistry and radiolabeled dopamine uptake as parameters, an increase in the number and outgrowth of DAergic neurons was noted in cocultures as compared to cultures of mesencephalic neurons alone. This enhanced survival of DAergic neurons in cocultures was abolished following depletion of glutathione with buthionine sulfoximine. As demonstrated by glial fibrillary acidic protein immunocytochemistry and a microtiter tetrazolium assay, under these conditions no change in astrocyte survival occurred. However, glutathione depletion in cocultures was accompanied by loss of astrocyte-mediated neuroprotection against hydrogen peroxide toxicity. Thus, our results indicate that glutathione is important for the maintenance of the neuronal support function of astrocytes and that glutathione deficiency in the brain may lead to enhanced vulnerability of neurons to (oxidative) damage.

Cultured rat striatal and cortical astrocytes protect mesencephalic dopaminergic neurons against hydrogen peroxide toxicity independent of their effect on neuronal development

Reactive oxygen species (ROS), including hydrogen peroxide, are supposed to be involved in the degeneration of dopaminergic neurons in Parkinsons disease. The potential role of astrocytes against neurotoxic effects of ROS was studied in cocultures of rat mesencephalic neurons and rat striatal or cortical astrocytes. Neuronal [3H]dopamine uptake, a marker of dopaminergic neuron integrity, was enhanced by striatal astrocytes, but not by cortical astrocytes, compared to uptake in mesencephalic neurons cultured alone. Whereas hydrogen peroxide at concentrations up to 100 microM reduced the [3H]dopamine uptake in neuronal cultures, no reduction of the uptake was observed in cocultures, regardless of the origin of the supporting astrocytes. These results suggest that astrocyte mediated protection of neurons against hydrogen peroxide induced toxicity is not directly related to a region-specific neurotrophic effect.

Drug treatment of Parkinson’s disease: Time for phase II

Parkinsons 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.

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 Parkinsons 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 Neuroprotective Antioxidant α-lipoic Acid Induces Detoxication Enzymes in Cultured Astroglial Cells

f -Lipoic acid (LA), an antioxidant with broad neuroprotective capacity, is thought to act by scavenging reactive oxygen species and stimulation of glutathione synthesis. LA shows structural resemblance to dithiolethiones, like anethole dithiolethione (ADT). ADT protects against oxidative damage, primarily by induction of phase II detoxication enzymes, in particular NAD(P)H:quinone oxidoreductase (NQO1) and glutathione- S -transferase (GST). Therefore, we investigated whether LA, like ADT, is capable also of inducing these protective enzymes. Our data show that LA, like ADT, induces a highly significant, time- and concentration dependent, increase in the activity of NQO1 and GST in C6 astroglial cells. The LA or ADT mediated induction of NQO1 was further confirmed by quantitative PCR and western blot analysis. This work for the first time unequivocally demonstrates LA mediated upregulation of phase II detoxication enzymes, which may highly contribute to the compounds neuroprotective potential. Moreover, the data support the notion of a common mechanism of action of LA and ADT.

Amantadine as N- methyl- d -aspartic acid receptor antagonist: new possibilities for therapeutic applications?

The N-methyl-D-aspartic acid (NMDA) receptor is an intriguing target for the development of drugs with anti-Parkinsonian activity as well as with protective actions against degenerative processes induced by brain ischemia. Amantadine is used in the treatment of Parkinsons disease without a well established mechanism of action. We show here that amantadine inhibits, in a non-competitive way, the NMDA receptor-mediated stimulation of acetylcholine release from rat neostriatum in vitro in therapeutic (i.e., low micromolar) concentrations. This indicates that amantadine might exert its anti-Parkinsonian effect via blockade of NMDA receptors. Sustained stimulation of NMDA receptors induces so-called excitotoxicity. Recently, it was demonstrated that amantadine is able to inhibit NMDA induced cell death in a neuronal culture. On the basis of these findings it seems worth investigating if amantadine is also able to protect against neurodegenerative processes caused by brain ischemia in vivo.

Anethole dithiolethione prevents oxidative damage in glutathione-depleted astrocytes.

Astrocytes protect neurons against reactive oxygen species such as hydrogen peroxide, a capacity which reportedly is abolished following loss of the antioxidant glutathione. Anethole dithiolethione, a sulfur-containing compound which is used in humans, is known to increase cellular glutathione levels and thought thereby to protect against oxidative damage. In the present study we found that anethole dithiolethione increased the glutathione content of cultured rat striatal astrocytes. This effect was abolished by coincubation with the glutathione synthesis inhibitor buthionine sulfoximine. Nevertheless, in the presence of buthionine sulfoximine, despite the lack of an increase in the lowered glutathione level, anethole dithiolethione fully protected the astrocytes against the enhanced toxicity of hydrogen peroxide. Thus, apparently other mechanisms than stimulation of glutathione synthesis are involved in the compounds protective action in astrocytes. Considering the occurrence of lowered glutathione levels in neurodegenerative syndromes, we conclude that further evaluation of the therapeutic potential of anethole dithiolethione is warranted.

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

Patients suffering from Parkinsons 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 Parkinsons disease is still an enigma. Consequently, the pharmacotherapy of Parkinsons 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 Parkinsons 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 Parkinsons 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 Parkinsons disease but also provides the opportunity to estimate therapeutic effects of putative neuroprotective agents in the individual patient.

Dopamine D1 receptor agonists display a different intrinsic activity in rat, monkey and human astrocytes

Measuring dopamine D1 receptor stimulated cyclic AMP production in cultured astrocytes from rat, monkey and human brain, we demonstrate that the classical drug SKF 38393 (7,8-dihydroxy-1- phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine) is a partial agonist with particularly low intrinsic activity in primates. Furthermore, its analogue SKF 81297 (6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5- tetrahydro-1 H-3-benzazepine) is shown to be a full agonist in rats but a partial, albeit more efficacious, agonist in primates, whereas the benzopyran A 68930 ((1R,3S)-1-aminomethyl-5,6- dihydroxy-3-phenyl-isochroman HCl) displays full efficacy in both species. The data suggest that cultured astrocytes provide a good model to study species differences in the pharmacological characteristics of dopamine D1 receptor agonists and indicate that SKF 38393 is not suited to study dopamine D1 receptor function in primates.